11401507>Molecular dissection of the <n>importin beta1</n>-recognized nuclear targeting signal of <n>parathyroid hormone-related protein</n>. Produced by various types of solid tumors, <n>parathyroid hormone-related protein</n> (<n>PTHrP</n>) is the causative agent of humoral hypercalcemia of malignancy. The similarity of <n>PTHrP's</n> amino-terminus to that of <n>parathyroid hormone</n> enables it to share some of the latter's signalling properties, but its carboxy-terminus confers distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. <n>PTHrP</n> nuclear import occurs via a novel <n>importin beta1</n>-mediated pathway. The present study uses several different direct binding assays to map the interaction of <n>PTHrP</n> with <n>importin beta</n> using a series of alanine mutated <n>PTHrP</n> peptides and truncated human <n>importin beta1</n> derivatives. Our results indicate that <n>PTHrP</n> amino acids 83-93 (KTPGKKKKGK) are absolutely essential for <n>importin beta1</n> recognition with residues 71-82 (TNKVETYKEQPL) additionally required for high affinity binding; residues 380-643 of <n>importin beta1</n> are required for the interaction. Binding of <n>importin beta1</n> to <n>PTHrP</n> is reduced in the presence of the GTP-bound but not GDP-bound form of the guanine nucleotide binding protein <n>Ran</n>, consistent with the idea that <n>Ran</n>GTP binding to <n>importin beta</n> is involved in the release of <n>PTHrP</n> into the nucleus following translocation across the nuclear envelope. This study represents the first detailed examination of a modular, non-arginine-rich <n>importin beta1</n>-recognized nuclear targeting signal. Copyright 2001 Academic Press.
11327761>Functional mutagenesis of AML1/RUNX1 and PEBP2 beta/CBF beta define distinct, non-overlapping sites for DNA recognition and heterodimerization by the Runt domain. The Runt domain family of transcription factors play key roles in transcriptional regulation of definitive hematopoiesis and osteogenesis. This transcription factor family is characterized by a DNA-binding alpha-subunit harboring the Runt domain and a secondary subunit, beta, which binds to the Runt domain and enhances its interaction with DNA. Missense mutations in the Runt domain from either the blood or bone-related gene product are associated with the onset of acute human leukemia as well as a disease of skeletal patterning known as cleidocranial dysplasia. NMR "footprinting" analysis of Runt domain/beta/DNA ternary complexes in solution previously identified the likely residues that form the heterodimerization and DNA-binding surfaces of the Runt domain. Functional mutagenesis at 37 positions in the Runt domain or beta confirms the original identification of these interaction surfaces and reveals that the heterodimerization and DNA-binding surfaces of the Runt domain occur at distinct, non-overlapping sites within the domain. The analysis of an additional 21 disease-related missense mutations identified from patients with either blood or bone disease demonstrates that the primary defect in these patients is a failure in DNA-recognition by the Runt domain. The molecular basis for the DNA-binding defect is analyzed in the context of the three-dimensional structure of the Runt domain in binary and ternary protein/DNA complexes. Copyright 2001 Academic Press.
11279204>Inhibitory effect of bovine milk <n>lactoferrin</n> on the interaction between a streptococcal surface protein antigen and human <n>salivary agglutinin</n>. Human whole saliva induces aggregation of Streptococcus mutans cells via an interaction between a surface protein antigen (<n>PAc</n>) of the organism and <n>salivary agglutinin</n>. Bovine milk inhibits the saliva-induced aggregation of S. mutans. In this study, the milk component that possesses inhibitory activity against this aggregation was isolated and found to be <n>lactoferrin</n>. Surface plasmon resonance analysis indicated that bovine <n>lactoferrin</n> binds more strongly to <n>salivary agglutinin</n>, especially to high molecular mass glycoproteins, which is a component of the <n>agglutinin</n>, than to recombinant <n>PAc</n>. The binding of bovine <n>lactoferrin</n> to salivary <n>agglutinin</n> was thermostable, and the optimal pH for binding was 4.0. To identify the saliva-binding region of bovine <n>lactoferrin</n>, 11 truncated bovine <n>lactoferrin</n> fragments were constructed. A fragment corresponding to the C-terminal half of the <n>lactoferrin</n> molecule had a strong inhibitory effect on the saliva-induced aggregation of S. mutans, whereas a fragment corresponding to the N-terminal half had a weak inhibitory effect. Seven shorter fragments corresponding to <n>lactoferrin</n> residues 473-538 also showed a high ability to inhibit the aggregation of S. mutans. These results suggest that residues 473-538 of bovine <n>lactoferrin</n> are important in the inhibition of saliva-induced aggregation of S. mutans.
11259620>Direct and differential interaction of beta-arrestins with the intracellular domains of different opioid receptors. Beta-arrestins have been shown to play important roles in regulation of signaling and desensitization of <n>opioid receptors</n> in many in vivo studies. The current study was carried out to measure the direct interaction of beta-arrestins with two functional intracellular domains, the third intracellular loop (I3L) and the carboxyl terminus (CT), of <n>delta-</n>, <n>mu-</n>, and <n>kappa-opioid receptors</n> (<n>DOR</n>, <n>MOR</n>, and <n>KOR</n>, respectively). Results from the pull-down assay using <n>glutathione S-transferase</n> fusion proteins demonstrated that beta-arrestins (1 and 2) were able to bind to the I3L of <n>DOR</n> and to the CT of <n>DOR</n> and <n>KOR</n>. Surface plasmon resonance measurement gave similar results with typical dissociation equilibrium constant (K(D)) values in the micromolar range. The site-directed mutagenesis experiment further revealed that certain specific serine/threonine residues in these receptor domains play a critical role in their interaction with beta-arrestins. Taken together, our data clearly indicated that beta- arrestins interact differentially with the functional domains of different opioid receptors; this may provide a possible molecular basis for differential regulation of opioid receptors by beta-arrestins.
11233172>Wavelength-dependent spectral changes accompany <n>CN-hemin</n> binding to human <n>apohemoglobin</n>. The interaction of <n>apohemoglobin</n> with two heme derivatives, <n>CN-protohemin</n> and <n>CN-deutero-hemin</n>, was monitored at multiple Soret wavelengths (417-423 and 406-412 nm, respectively) in 0.05 M potassium phosphate buffer, pH 7.0, at 10 degrees C and revealed, as previously reported, a multiphasic kinetic reaction. Wavelength-dependent reactions were observed for both <n>CN-protohemin</n> and <n>CN-deuterohemin</n> derivatives with the alpha chain (bathochromic entity) displaying faster (4- to 7-fold) rates throughout the courses of both heme-binding reactions. The basis of this spectrally heterogeneous kinetic phenomenon could be deduced from molecular modeling studies of alpha- and beta-chain structures. Key differences in the number of stabilizing contacts of the two chains with the peripheral alpha propionyl 45(CE3); 58(E7); 61(E10) as well as the beta vinyl 38(C4); 71(E15); 106(G8) groups were found. Furthermore, RMS plots comparing apo- and heme-containing subunits reveal substantial structural disparities in the C-CD-F-FG helical regions of the alphabeta dimer interface.
11181178>Mitotic checkpoint proteins <n>HsMAD1</n> and <n>HsMAD2</n> are associated with nuclear pore complexes in interphase. <n>Mad1</n> was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly in mitosis and prevents premature anaphase onset. Using antibodies to the human homologue of <n>Mad1</n> (<n>HsMAD1</n>), we have begun to characterize this protein in mammalian cells. <n>HsMad1</n> is found localized at kinetochores in mitosis. The labeling is brightest in prometaphase and is absent from kinetochores at metaphase and anaphase. In cells where most chromosomes have reached the metaphase plate, those aligned at the plate show no labeling while remaining, unaligned chromosomes are still brightly labeled. We find <n>HsMad1</n> associated with <n>HsMad2</n>. Association with <n>p55CDC</n>, a protein previously shown to bind <n>HsMad2</n>, was not detected. Surprisingly, unlike any other known mitotic checkpoint proteins, <n>HsMad1</n> and <n>HsMAD2</n> were found localized at nuclear pores throughout interphase. This was confirmed by co-labeling with an antibody to known nuclear pore complex proteins and by their co-purification with enriched nuclear envelope fractions. <n>HsMad1</n> was identified serendipitously by its binding to a viral protein, HTLV-1 <n>Tax</n>, which affects transcription of viral and human proteins. The localization of <n>HsMad1</n> to nuclear pore complexes suggests an alternate, non-mitotic role for the <n>Mad1</n>/<n>Tax</n> interaction in the viral transformation of cells.
11168528><n>p35</n>/<n>cdk5</n> binds and phosphorylates <n>beta-catenin</n> and regulates <n>beta-catenin</n>/<n>presenilin-1</n> interaction. The neuronal cyclin-dependent kinase <n>p35</n>/<n>cdk5</n> comprises a catalytic subunit (<n>cdk5</n>) and an activator subunit (<n>p35</n>). To identify novel <n>p35</n>/<n>cdk5</n> substrates, we utilized the yeast two-hybrid system to screen for human <n>p35</n> binding partners. From one such screen, we identified <n>beta-catenin</n> as an interacting protein. Confirmation that <n>p35</n> binds to <n>beta-catenin</n> was obtained by using <n>glutathione S-transferase</n> (<n>GST</n>)-<n>beta-catenin</n> fusion proteins that interacted with both endogenous and transfected <n>p35</n>, and by showing that <n>beta-catenin</n> was present in <n>p35</n> immunoprecipitates. <n>p35</n> and <n>beta-catenin</n> also displayed overlapping subcellular distribution patterns in cells including neurons. Finally, we demonstrated that <n>p35</n>/<n>cdk5</n> phosphorylates <n>beta-catenin</n>. <n>beta-catenin</n> also binds to <n>presenilin-1</n> and altered <n>beta-catenin</n>/<n>presenilin-1</n> interactions may be mechanistic in Alzheimer's disease (AD). Abnormal <n>p35</n>/<n>cdk5</n> activity has also been suggested to contribute to AD. We therefore investigated how modulation of <n>p35</n>/<n>cdk5</n> activity influenced <n>beta-catenin</n>/<n>presenilin-1</n> interactions. Inhibition of <n>p35</n>/<n>cdk5</n> with roscovitine did not alter the steady state levels of either <n>beta-catenin</n> or <n>presenilin-1</n> but reduced the amount of <n>presenilin-1</n> bound to <n>beta-catenin</n>. Thus, <n>p35</n>/<n>cdk5</n> binds and phosphorylates <n>beta-catenin</n> and regulates its binding to <n>presenilin-1</n>. The findings reported here therefore provide a novel molecular framework to connect <n>p35</n>/<n>cdk5</n> with <n>beta-catenin</n> and <n>presenilin-1</n> in AD.
11157096>Association of Trk <n>neurotrophin</n> receptors with components of the cytoplasmic <n>dynein</n> motor. <n>nerve growth factor</n> (<n>NGF</n>) initiates its trophic effects by long-range signaling through binding, internalization, and transport of a ligand-receptor complex from the axon terminal to the cell body. However, the mechanism by which retrograde transport of <n>NGF</n> takes place has not been elucidated. Here we describe an interaction between the Trk receptor tyrosine kinase and a 14 kDa light chain of cytoplasmic <n>dynein</n>. After transfection in human embryonic kidney 293 cells, this 14 kDa <n>dynein</n> light chain was found to bind to <n>TrkA</n>, <n>TrkB</n>, and <n>TrkC receptors</n>. Mapping experiments indicated that the 14 kDa <n>dynein</n> light chain binds to the distal region of the <n>TrkA</n> juxtamembrane domain. Coimmunoprecipitation experiments in vivo indicate that Trk receptors are in a complex with the 14 kDa light chain and 74 kDa intermediate chain of <n>dynein</n>. Confirming the physiological relevance of this association, a marked accumulation of Trk with the 14 kDa and the 74 kDa <n>dynein</n> components was observed after ligation of the sciatic nerve. The association of Trk receptors with components of cytoplasmic <n>dynein</n> suggests that transport of <n>neurotrophins</n> during vesicular trafficking may occur through a direct interaction of the Trk receptor with the <n>dynein</n> motor machinery.
11135361>Characterization of <n>Cre</n>-loxP interaction in the major groove: hint for structural distortion of mutant <n>Cre</n> and possible strategy for HIV-1 therapy. Although the crystal structure of <n>Cre recombinase</n> complexed with DNA, named loxA, was elucidated a couple of years ago, it has not yet been determined which amino acids of the protein are involved in the specific <n>Cre</n>-loxP interaction. Arg259 and Gln90 interact with DNA substrate in the major groove from which the specificity of protein-DNA interaction comes. In this study, we substituted these residues for other amino acids. Also, two mutated DNA substrates were constructed. In each mutant, one of the bases that interact with Arg259 or Gln90 was changed into another base. In vitro binding assays and recombination assays of variant lox sites with wild-type and mutant-type <n>Cre</n> revealed that Arg259 plays a key role in <n>Cre</n>-loxP binding but Gln90 does not. However, the recombination activity still remained intact, although the binding between <n>Cre</n> and DNA substrate was not ensured. Copyright 2001 Wiley-Liss, Inc.
11111045>Identification of upstream stimulatory factor as an activator of the human <n>dipeptidyl peptidase IV</n> gene in Caco-2 cells. The 5' upstream region (-448/-443) of the human <n>dipeptidyl peptidase IV</n> gene promoter containing a consensus E-box (CACGTG) was shown to bind upstream stimulatory factor using nuclear extracts from mouse (3T3) fibroblasts and the human intestinal and hepatic epithelial cell lines Caco-2 and HepG2. Supershift analysis with specific antibodies to <n>USF-1</n> and <n>USF-2</n> indicates that <n>USF-1</n> is the primary isoform binding to the E-box in nuclear extracts from these cells. Using cell culture, transient cotransfection of USF expression vectors with <n>dipeptidyl peptidase IV</n> promoter constructs revealed that both <n>USF-1</n> and <n>USF-2</n> caused an approximately tenfold increase in reporter gene expression in Caco-2 cells. Mutant forms of <n>USF-1</n> and <n>USF-2</n> lacking the DNA binding or transcriptional activation domains were unable to stimulate reporter gene expression. Mutation of the E-box prevented binding of USF, although stimulation of reporter gene expression by cotransfection with USF was reduced by only 50%. By using a series of deletion constructs in cotransfection experiments, a second possible site of USF interaction with the <n>dipeptidyl peptidase IV</n> promoter was localized to the -119/-88 region.
11090287>Search for intermediate structures in <n>transthyretin</n> fibrillogenesis: soluble tetrameric Tyr78Phe <n>TTR</n> expresses a specific epitope present only in amyloid fibrils. Familial Amyloidotic Polyneuropathy (FAP) is caused by the assembly of <n>TTR</n> into an insoluble beta-sheet. The <n>TTR</n> tetramer is thought to dissociate into monomeric intermediates and subsequently polymerise into the pathogenic amyloid form. The biochemical mechanism behind this transformation is unknown. We characterised intermediate <n>TTR</n> structures in the in vitro amyloidogenesis pathway by destabilising the AB loop through substitution of residue 78. Changes at this residue, should destabilise the <n>TTR</n> tetrameric fold, based on the known crystallographic structure of a Leu55Pro <n>transthyretin</n> variant. We generated a soluble tetrameric form of <n>TTR</n> that is recognised by a monoclonal antibody, previously reported to react only with highly amyloidogenic mutant proteins lacking the tetrameric native fold and with amyloid fibrils. BIAcore system analysis showed that Tyr78Phe had similar binding properties as synthetic fibrils. The affinity of this interaction was 10(7) M(-1). We suggest that the tetrameric structure of Tyr78Phe is altered due to the loosening of the AB loops of the tetramer, leading to a structure that might represent an early intermediate in the fibrillogenesis pathway.
11073994>Novel <n>Upf2p</n> orthologues suggest a functional link between translation initiation and nonsense surveillance complexes. Transcripts harboring premature signals for translation termination are recognized and rapidly degraded by eukaryotic cells through a pathway known as nonsense-mediated mRNA decay (NMD). In addition to protecting cells by preventing the translation of potentially deleterious truncated peptides, studies have suggested that NMD plays a broader role in the regulation of the steady-state levels of physiologic transcripts. In Saccharomyces cerevisiae, three trans-acting factors (<n>Upf1p</n> to <n>Upf3p</n>) are required for NMD. Orthologues of <n>Upf1p</n> have been identified in numerous species, showing that the NMD machinery, at least in part, is conserved through evolution. In this study, we demonstrate additional functional conservation of the NMD pathway through the identification of <n>Upf2p</n> homologues in Schizosaccharomyces pombe and humans (<n>rent2</n>). Disruption of S. pombe UPF2 established that this gene is required for NMD in fission yeast. <n>rent2</n> was demonstrated to interact directly with <n>rent1</n>, a known trans-effector of NMD in mammalian cells. Additionally, fragments of <n>rent2</n> were shown to possess nuclear targeting activity, although the native protein localizes to the cytoplasmic compartment. Finally, novel functional domains of <n>Upf2p</n> and <n>rent2</n> with homology to <n>eukaryotic initiation factor 4G</n> (<n>eIF4G</n>) and other translational regulatory proteins were identified. Directed mutations within these so-called <n>eIF4G</n> homology (4GH) domains were sufficient to abolish the function of S. pombe <n>Upf2p</n>. Furthermore, using the two-hybrid system, we obtained evidence for direct interaction between <n>rent2</n> and human <n>eIF4AI</n> and <n>Sui1</n>, both components of the translation initiation complex. Based on these findings, a novel model in which <n>Upf2p</n> and <n>rent2</n> effects decreased translation and accelerated decay of nonsense transcripts through competitive interactions with <n>eIF4G</n>-binding partners is proposed.
11056215>An acidic amino acid cluster regulates the nucleolar localization and ribosome assembly of human <n>ribosomal protein L22</n>. The control of human <n>ribosomal protein L22</n> (<n>rpL22</n>) to enter into the nucleolus and its ability to be assembled into the ribosome is regulated by its sequence. The nuclear import of <n>rpL22</n> depends on a classical nuclear localization signal of four lysines at positions 13-16. <n>RpL22</n> normally enters the nucleolus via a compulsory sequence of KKYLKK (I-domain, positions 88-93). An acidic residue cluster at the C-terminal end (C-domain) plays a nuclear retention role. The retention is concealed by the N-domain (positions 1-9), which weakly interacts with the C-domain as demonstrated in the yeast two-hybrid system. Once it reaches the nucleolus, the question of whether <n>rpL22</n> is assembled into the ribosome depends upon the presence of the N-domain. This suggests that the N-domain, on dissociation from its interaction with the C-domain, binds to a specific region of the 28S rRNA for ribosome assembly.
11027346>Selective <n>CXCR4</n> antagonism by <n>Tat</n>: implications for in vivo expansion of coreceptor use by HIV-1. Chemokines and chemokine receptors play important roles in HIV-1 infection and tropism. <n>CCR5</n> is the major macrophage-tropic coreceptor for HIV-1 whereas <n>CXC chemokine receptor 4</n> (<n>CXCR4</n>) serves the counterpart function for T cell-tropic viruses. An outstanding biological mystery is why only R5-HIV-1 is initially detected in new seroconvertors who are exposed to R5 and X4 viruses. Indeed, X4 virus emerges in a minority of patients and only in the late stage of disease, suggesting that early negative selection against HIV-1-<n>CXCR4</n> interaction may exist. Here, we report that the HIV-1 <n>Tat</n> protein, which is secreted from virus-infected cells, is a <n>CXCR4</n>-specific antagonist. Soluble <n>Tat</n> selectively inhibited the entry and replication of X4, but not R5, virus in peripheral blood mononuclear cells (PBMCs). We propose that one functional consequence of secreted <n>Tat</n> is to select against X4 viruses, thereby influencing the early in vivo course of HIV-1 disease.
11003645>SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA. SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human <n>scaffold attachment factor A</n> (<n>SAF-A</n>) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.
10982864>Interaction of the <n>U3-55k</n> protein with U3 snoRNA is mediated by the box B/C motif of U3 and the WD repeats of <n>U3-55k</n>. U3 small nucleolar RNA (snoRNA) is a member of the Box C/D family of snoRNAs which functions in ribosomal RNA processing. <n>U3-55k</n> is a protein that has been found to interact with U3 but not other members of the Box C/D snoRNA family. We have found that interaction of the <n>U3-55k</n> protein with U3 RNA in vivo is mediated by the conserved Box B/C motif which is unique to U3 snoRNA. Mutation of Box B and Box C, but not of other conserved sequence elements, disrupted interaction of <n>U3-55k</n> with U3 RNA. Furthermore, a fragment of U3 containing only these two conserved elements was bound by <n>U3-55k</n> in vivo. RNA binding assays performed in vitro indicate that Box C may be the primary determinant of the interaction. We have cloned the cDNA encoding the Xenopus laevis <n>U3-55k</n> protein and find strong homology to the human sequence, including six WD repeats. Deletion of WD repeats or sequences near the C-terminus of <n>U3-55k</n> resulted in loss of association with U3 RNA and also loss of localization of <n>U3-55k</n> to the nucleolus, suggesting that protein-protein interactions contribute to the localization and RNA binding of <n>U3-55k</n> in vivo.
10967329>Convergent evolution with combinatorial peptides. Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon 'convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein-protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein-protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis.
10956659>Interaction of the <n>factor XIII</n> activation peptide with <n>alpha-thrombin</n>. Crystal structure of its enzyme-substrate analog complex. The serine protease <n>thrombin</n> proteolytically activates <n>blood coagulation factor XIII</n> by cleavage at residue Arg(37); <n>factor XIII</n> in turn cross-links <n>fibrin</n> molecules and gives mechanical stability to the blood clot. The 2.0-A resolution x-ray crystal structure of human <n>alpha thrombin</n> bound to the factor XIII-(28-37) decapeptide has been determined. This structure reveals the detailed atomic level interactions between the <n>factor XIII</n> activation peptide and <n>thrombin</n> and provides the first high resolution view of this functionally important part of the <n>factor XIII</n> molecule. A comparison of this structure with the crystal structure of <n>fibrinopeptide A</n> complexed with <n>thrombin</n> highlights several important determinants of <n>thrombin</n> substrate interaction. First, the P1 and P2 residues must be compatible with the geometry and chemistry of the S1 and S2 specificity sites in <n>thrombin</n>. Second, a glycine in the P5 position is necessary for the conserved substrate conformation seen in both factor XIII-(28-37) and <n>fibrinopeptide A</n>. Finally, the hydrophobic residues, which occupy the aryl binding site of <n>thrombin</n> determine the substrate conformation further away from the catalytic residues. In the case of <n>factor XIII</n>-(28-37), the aryl binding site is shared by hydrophobic residues P4 (Val(34)) and P9 (Val(29)). A bulkier residue in either of these sites may alter the substrate peptide conformation.
10938104>Potentiation of <n>GATA-2</n> activity through interactions with the <n>promyelocytic leukemia protein</n> (<n>PML</n>) and the t(15;17)-generated <n>PML-retinoic acid receptor alpha oncoprotein</n>. The hematopoietically expressed GATA family of transcription factors function as key regulators of blood cell fate. Among these, <n>GATA-2</n> is implicated in the survival and growth of multipotential progenitors. Here we report that the <n>promyelocytic leukemia protein</n> (<n>PML</n>) can complex with <n>GATA-2</n> and potentiate its transactivation capacity. The binding is mediated through interaction of the zinc finger region of <n>GATA-2</n> and the B-box domain of <n>PML</n>. The B-box region of <n>PML</n> is retained in the <n>PML</n>-<n>RARalpha</n> (<n>retinoic acid receptor alpha</n>) fusion protein generated by the t(15;17) translocation characteristic of acute promyelocytic leukemia (APL). Consistent with this, we provide evidence that <n>GATA-2</n> can physically associate with <n>PML</n>-<n>RARalpha</n>. Functional experiments further demonstrated that this interaction has the capacity to render GATA-dependent transcription inducible by retinoic acid, raising the possibility that GATA target genes may be involved in the molecular pathogenesis of APL.
10921882>Negative regulation of <n>PI 3-kinase</n> by <n>Ruk</n>, a novel adaptor protein. <n>Class I(A) phosphatidylinositol 3-kinase</n> (<n>PI 3-kinase</n>) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, <n>Ruk(l)</n>, which forms complexes with the <n>PI 3-kinase</n> holoenzyme in vitro and in vivo. This interaction involves the proline-rich region of <n>Ruk</n> and the SH3 domain of the p85 alpha regulatory subunit of the <n>class I(A) PI 3-kinase</n>. In contrast to many other adaptor proteins that activate <n>PI 3-kinase</n>, interaction with <n>Ruk</n>(l) substantially inhibits the lipid kinase activity of the enzyme. Overexpression of <n>Ruk</n>(l) in cultured primary neurons induces apoptosis, an effect that could be reversed by co-expression of constitutively activated forms of the p110 alpha catalytic subunit of <n>PI 3-kinase</n> or its downstream effector <n>PKB</n>/<n>Akt</n>. Our data provide evidence for the existence of a negative regulator of the <n>PI 3-kinase</n> signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between <n>Ruk</n>, <n>CIN85</n> and <n>CD2AP</n>/<n>CMS</n> suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.
10908344>The human <n>RAD18 gene product</n> interacts with <n>HHR6A</n> and <n>HHR6B</n>. During DNA replication, lesion bypass is an important cellular response to unrepaired damage in the genome. In the yeast Saccharomyces cerevisiae, <n>Rad6</n> and <n>Rad18</n> are required for both the error-free and error-prone lesion bypass mechanisms. Furthermore, <n>Rad6</n>-<n>Rad18</n> interaction is thought to be critical at an early step during lesion bypass in yeast. Two closely related human homologs of yeast <n>Rad6</n> have been identified as <n>HHR6A</n> and <n>HHR6B</n>. Here, we report a full-length cDNA coding for the human homolog of yeast <n>Rad18</n>. The human RAD18 gene codes for a protein of 484 amino acid residues with a calculated molecular weight of 54 804 Da, and the gene is localized to chromosome 3 between reference intervals D3S3591 and D3S1283. Human <n>RAD18 protein</n> (<n>hRAD18</n>) was found to interact with <n>HHR6A</n> and <n>HHR6B</n>. When co-expressed in yeast cells, stable <n>hRAD18</n>-<n>HHR6A</n> and <n>hRAD18</n>-<n>HHR6B</n> protein complexes were identified and purified to near homogeneity. Thus, through interaction and complex formation with <n>HHR6A</n> and <n>HHR6B</n>, <n>RAD18 protein</n> may play an important role in lesion bypass mechanisms in humans. Consistent with its role as a fundamental lesion bypass protein, the RAD18 gene is ubiquitously expressed in various human tissues.
10884290><n>Apolipoprotein E</n>;-<n>low density lipoprotein receptor</n> interaction. Influences of basic residue and amphipathic alpha-helix organization in the ligand. Conserved lysines and arginines within amino acids 140-150 of <n>apolipoprotein (apo) E</n> are crucial for the interaction between <n>apoE</n> and the <n>low density lipoprotein receptor</n> (<n>LDLR</n>). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of <n>apoE</n> (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on <n>apoE</n> and an acidic residue on the <n>LDLR</n>. Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important. Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the <n>low density lipoprotein receptor</n>.
10862613><n>SUMO-1</n> conjugation to human <n>DNA topoisomerase II</n> isozymes. <n>Topoisomerase I</n>-mediated DNA damage induced by camptothecin has been shown to induce rapid small <n>ubiquitin</n>-related modifier <n>(SUMO)-1</n> conjugation to <n>topoisomerase I</n>. In the current study, we show that <n>topoisomerase II</n>-mediated DNA damage induced by teniposide (VM-26) results in the formation of high molecular weight conjugates of both <n>topoisomerase II alpha</n> and <n>II beta</n> isozymes in HeLa cells. Immunological characterization of these conjugates suggests that both <n>topoisomerase II alpha</n> and <n>II beta</n> isozymes are conjugated to <n>SUMO-1</n>. The involvement of <n>SUMO-1</n>/<n>UBC9</n> in the modification of <n>topoisomerase II</n> isozymes is also supported by the demonstration of physical interaction between <n>topoisomerase II</n> and <n>SUMO-1</n>/<n>UBC9</n>. Surprisingly, <n>ICRF-193</n>, which does not induce <n>topoisomerase II</n>-mediated DNA damage but traps <n>topoisomerase II</n> into a circular clamp conformation, is also shown to induce similar <n>SUMO-1</n> conjugation to <n>topoisomerase II</n> isozymes. In addition, we show that both oxidative and heat shock stresses, which can cause protein damage, rapidly increase nuclear <n>SUMO-1</n> conjugates. These studies raise the question on whether <n>SUMO-1</n> conjugation to topoisomerases is an indirect result of a DNA damage response or a direct result because of protein conformational changes.
10851237>Specific sequences of the <n>Sm</n> and <n>Sm-like</n> (<n>Lsm</n>) proteins mediate their interaction with the <n>spinal muscular atrophy disease gene product</n> (<n>SMN</n>). The <n>spinal muscular atrophy disease gene product</n> (<n>SMN</n>) is crucial for <n>small nuclear ribonuclear protein</n> (<n>snRNP</n>) biogenesis in the cytoplasm and plays a role in pre-mRNA splicing in the nucleus. <n>SMN</n> oligomers interact avidly with the <n>snRNP</n> core proteins <n>SmB</n>, <n>-D1</n>, and <n>-D3</n>. We have delineated the specific sequences in the <n>Sm</n> proteins that mediate their interaction with <n>SMN</n>. We show that unique carboxyl-terminal arginine- and glycine-rich domains comprising the last 29 amino acids of <n>SmD1</n> and the last 32 amino acids of <n>SmD3</n> are necessary and sufficient for <n>SMN</n> binding. Interestingly, <n>SMN</n> also interacts with at least two of the U6-associated <n>Sm-like</n> (<n>Lsm</n>) proteins, <n>Lsm4</n> and <n>Lsm6</n>. Furthermore, the carboxyl-terminal arginine- and glycine-rich domain of <n>Lsm4</n> directly interacts with <n>SMN</n>. This suggests that <n>SMN</n> also functions in the assembly of the U6 snRNP in the nucleus and in the assembly of other <n>Lsm</n>-containing complexes. These findings demonstrate that arginine- and glycine-rich domains are necessary and sufficient for <n>SMN</n> interaction, and they expand further the range of targets of the <n>SMN</n> protein.
10831590>Sites in the A2 subunit involved in the interfactor VIIIa interaction. <n>Factor VIIIa</n> is a trimer of the A1, A2, and A3-C1-C2 subunits. Regions in the A2 subunit that interact with the A1/A3-C1-C2 dimer were localized using synthetic peptides derived from A2 sequences showing high probability of being surface exposed. Peptides were restricted to residues 373-562 of A2 based on the earlier observation that this region of A2 reacts with A1 using a zero length cross-linker. Peptides were assessed for their capacity to inhibit the reconstitution of <n>factor VIIIa</n> from the isolated A1/A3-C1-C2 dimer and A2 subunit. Reconstitution was monitored using both regeneration of <n>factor VIIIa</n> activity and fluorescence quenching of an acrylodan-labeled A2 (Ac-A2) by fluorescein-labeled A1/A3-C1-C2. The activity assay identified four peptides as inhibitors, residues 373-395 (IC(50) = 65 micrometer), 418-428 (IC(50) = 25 micrometer), 482-493 (IC(50) = 325 micrometer), and 518-533 (IC(50) = 585 micrometer). The 373-395 and 518-533 peptides eliminated the fluorescence quenching of Ac-A2, whereas the 418-428 peptide reduced but did not eliminate Ac-A2 quenching. Peptide 482-493 had no effect on the fluorescence quenching of Ac-A2 suggesting that the peptide did not directly affect reassociation of the <n>factor VIIIa</n> subunits. These results identify three regions in the A2 subunit (373-395, 418-428, and 518-533) that interact with the A1/A3-C1-C2 dimer. Furthermore, comparison of results obtained using the two assays distinguish inhibition of the intersubunit interactions from intermolecular interactions.
10821684>Interaction of <n>charybdotoxin S10A</n> with single <n>maxi-K channels</n>: kinetics of blockade depend on the presence of the beta 1 subunit. The <n>maxi-K channel</n> from bovine aortic smooth muscle consists of a pore-forming alpha subunit and a regulatory beta1 subunit that modifies the biophysical and pharmacological properties of the alpha subunit. In the present study, we examine <n>ChTX-S10A</n> blocking kinetics of single <n>maxi-K channels</n> in planar lipid bilayers from smooth muscle or from tsA-201 cells transiently transfected with either alpha or alpha+beta 1 subunits. Under low external ionic strength conditions, <n>maxi-K channels</n> from smooth muscle showed <n>ChTX-S10A</n> block times, 48 +/- 12 s, that were similar to those expressing alpha+beta 1 subunits, 51 +/- 16 s. In contrast, with the alpha subunit alone, <n>ChTX-S10A</n> block times were much shorter, 5 +/- 0.6 s, and were qualitatively similar to previously reported values for the skeletal muscle <n>maxi-K channel</n>. Increasing the external ionic strength caused a decrease in <n>ChTX-S10A</n> block times for <n>maxi-K channel</n> complexes of alpha+beta 1 subunits but not of alpha subunits alone. These findings indicate that it may be possible to predict the association of beta 1 subunits with native <n>maxi-K channels</n> by monitoring the kinetics of <n>ChTX</n> blockade of single channels, and they suggest that <n>maxi-K channels</n> in skeletal muscle do not contain a beta 1 subunit like the one present in smooth muscle. To further test this hypothesis, we examined the binding and cross-linking properties of [(125)I]-<n>IbTX</n>-D19Y/Y36F to both bovine smooth muscle and rabbit skeletal muscle membranes. [(125)I]-<n>IbTX</n>-D19Y/Y36F binds to rabbit skeletal muscle membranes with the same affinity as it does to smooth muscle membranes. However, specific cross-linking of [(125)I]-<n>IbTX</n>-D19Y/Y36F was observed into the beta 1 subunit of smooth muscle but not in skeletal muscle. Taken together, these data suggest that studies of <n>ChTX</n> block of single <n>maxi-K channels</n> provide an approach for characterizing structural and functional features of the alpha/beta 1 interaction.
10802067>The Krüppel-like transcriptional factors <n>Zf9</n> and <n>GKLF</n> coactivate the human <n>keratin 4</n> promoter and physically interact. <n>Zf9</n>/<n>CPBP</n>/<n>KLF6</n> is a widely expressed member of the Krüppel-like family of transcriptional factors which regulates gene expression in hepatic stellate cells. Because of its ubiquitous expression including in the esophagus, we have explored its function in the esophageal squamous epithelium, a model system to study cellular proliferation and differentiation. Reverse transcription-PCR (RT-PCR) and Western blot analyses revealed that <n>Zf9</n> was highly expressed in human esophageal squamous cancer cell lines. Additionally, <n>Zf9</n> localizes to the esophageal squamous epithelium by immunohistochemistry. Using transient transfection, <n>Zf9</n> transactivates the human <n>keratin 4</n> (K4) promoter reporter gene construct in a subset of the esophageal cancer cell lines through indirect mechanisms. Co-transfection of <n>Zf9</n> and <n>GKLF</n>/<n>KLF4</n>, which is also a member of the Krüppel-like factors and expressed in the esophageal squamous epithelium, leads to coactivation in an additive fashion. Furthermore, we demonstrate that there is a physical interaction between <n>GKLF</n> and <n>Zf9</n>, a novel finding for Krüppel-like family members.
10788327>Uncoupling <n>actin</n> filament fragmentation by <n>cofilin</n> from increased subunit turnover. The <n>actin depolymerizing factor</n> (<n>ADF</n>)/<n>cofilin</n> family of proteins interact with <n>actin</n> monomers and filaments in a pH-sensitive manner. When <n>ADF</n>/<n>cofilin</n> binds <n>F-actin</n> it induces a change in the helical twist and fragmentation; it also accelerates the dissociation of subunits from the pointed ends of filaments, thereby increasing treadmilling or depolymerization. Using site-directed mutagenesis we characterized the two <n>actin</n>-binding sites on human <n>cofilin</n>. One target site was chosen because we previously showed that the <n>villin</n> head piece competes with <n>ADF</n> for binding to <n>F-actin</n>. Limited sequence homology between <n>ADF</n>/<n>cofilin</n> and the part of the <n>villin</n> headpiece essential for actin binding suggested an actin-binding site on <n>cofilin</n> involving a structural loop at the opposite end of the molecule to the alpha-helix already implicated in <n>actin</n> binding. Binding through the alpha-helix is primarily to monomeric <n>actin</n>, whereas the loop region is specifically involved in filament association. We have characterized the <n>actin</n> binding properties of each site independently of the other. Mutation of a single lysine residue in the loop region abolishes binding to filaments, but not to monomers. Using the mutation analogous to the phosphorylated form of <n>cofilin</n> (<n>S3D</n>), we show that filament binding is inhibited at physiological ionic strength but not under low salt conditions. At low ionic strength, this mutant induces both the twist change and fragmentation characteristic of wild-type <n>cofilin</n>, but does not activate subunit dissociation. The results suggest a two-site binding to filaments, initiated by association through the loop site, followed by interaction with the adjacent subunit through the "helix" site at the opposite end of the molecule. Together, these interactions induce twist and fragmentation of filaments, but the twist change itself is not responsible for the enhanced rate of <n>actin</n> subunit release from filaments.
10772640>Stability and interactions of recombinant human <n>nerve growth factor</n> in different biological matrices: in vitro and in vivo studies. The purpose of this investigation was to characterize the stability, activity, and interactions of recombinant human <n>nerve growth factor</n> (<n>rhNGF</n>) in various biological matrices in vitro and in vivo. <n>rhNGF</n> (10 microg/ml) remained stable in human plasma for up to 4 days at 37 degrees C. There was a decrease in the recovery of <n>rhNGF</n> after incubation at lower concentrations (20 ng/ml) and for longer time periods (3 and 5 days at 37 degrees C). Size exclusion HPLC analysis indicated that <n>rhNGF</n> forms high molecular weight (HMW) complexes after long incubation periods. We confirmed that <n>alpha(2)-macroglobulin</n> (<n>alpha(2)M</n>) is the major plasma component that binds to <n>rhNGF</n>. Furthermore, this interaction was considerably increased by treatment of plasma with primary amines such as CH(3)NH(2). Changes in the pH environment did not affect the interaction of <n>rhNGF</n> with <n>alpha(2)M</n>. We also determined that the binding of <n>rhNGF</n> to CH(3)NH(2)-treated pure <n>alpha(2)M</n> or <n>alpha(2)M</n> present in human plasma substantially diminished its immunoreactivity and bioactivity detection. The interaction of <n>rhNGF</n> with activated <n>alpha(2)M</n> was reversed and inhibited by coincubation with dimethyl sulfoxide. Released <n>rhNGF</n> under these conditions was fully bioactive. (125)I-<n>rhNGF</n> also binds to <n>alpha(2)M</n> by forming similar (125)I-<n>rhNGF</n>/HMW complexes in plasma after i.v. administration in rats and mice. Sixty minutes after dosing in rats, most of the labeled material was in the form of a (125)I-<n>rhNGF</n>/HMW complex. These studies have provided a better understanding of the nature of the interactions of <n>rhNGF</n> with plasma components as well as methods to enhance, reverse, and inhibit these interactions.
10759550><n>Cadherin</n> interaction probed by atomic force microscopy. Single molecule atomic force microscopy was used to characterize structure, binding strength (unbinding force), and binding kinetics of a classical <n>cadherin</n>, <n>vascular endothelial (VE)-cadherin</n>, secreted by transfected Chinese hamster ovary cells as cis-dimerized full-length external domain fused to Fc-portion of human <n>IgG</n>. In physiological buffer, the external domain of <n>VE-cadherin</n> dimers is a approximately 20-nm-long rod-shaped molecule that collapses and dissociates into monomers (V-shaped structures) in the absence of Ca(2+). Trans-interaction of dimers is a low-affinity reaction (K(D) = 10(-3)-10(-5) M, k(off) = 1.8 s(-1), k(on) = 10(3)-10(5) M(-1) x s(-1)) with relatively low unbinding force (35-55 pN at retrace velocities of 200-4,000 nm x s(-1)). Higher order unbinding forces, that increase with interaction time, indicate association of <n>cadherins</n> into complexes with cumulative binding strength. These observations favor a model by which the inherently weak unit binding strength and affinity of <n>cadherin</n> trans-interaction requires clustering and cytoskeletal immobilization for amplification. Binding is regulated by low-affinity Ca(2+) binding sites (K(D) = 1.15 mM) with high cooperativity (Hill coefficient of 5.04). Local changes of free extracellular Ca(2+) in the narrow intercellular space may be of physiological importance to facilitate rapid remodeling of intercellular adhesion and communication.
10745073>Interaction of a novel cysteine and histidine-rich cytoplasmic protein with <n>galectin-3</n> in a carbohydrate-independent manner. We have used the yeast two-hybrid system to search for cytoplasmic proteins that might assist in the intracellular trafficking of the soluble <n>beta-galactoside-binding protein</n>, <n>galectin-3</n>. We utilised as bait murine full-length <n>galectin-3</n> to screen a murine 3T3 cDNA library. Several interacting clones were found to encode a partial open reading frame and a full-length clone was obtained by rapid amplification of cDNA ends methodology. In various assays in vitro the novel protein was shown to bind <n>galectin-3</n> in a carbohydrate-independent manner. The novel protein contains an unusually high content of cysteine and histidine residues and shows significant sequence homologies with several metal ion-binding motifs present in known proteins. Confocal immunofluorescence microscopy of permeabilised 3T3 cells shows a prominent perinuclear, as well as cytoplasmic, localisation of the novel protein.
10722602>Staphylococcus aureus <n>protein A</n> recognizes platelet <n>gC1qR</n>/<n>p33</n>: a novel mechanism for staphylococcal interactions with platelets. The adhesion of Staphylococcus aureus to platelets is a major determinant of virulence in the pathogenesis of endocarditis. Molecular mechanisms mediating S. aureus interactions with platelets, however, are incompletely understood. The present study describes the interaction between S. aureus <n>protein A</n> and <n>gC1qR</n>/<n>p33</n>, a multifunctional, ubiquitously distributed cellular protein, initially described as a binding site for the globular heads of <n>C1q</n>. Suspensions of fixed S. aureus or purified <n>protein A</n>, chemically cross-linked to agarose support beads, were found to capture native <n>gC1qR</n> from whole platelets. Moreover, biotinylated <n>protein A</n> bound specifically to fixed, adherent, human platelets. This interaction was inhibited by unlabeled <n>protein A</n>, soluble recombinant <n>gC1qR</n> (<n>rgC1qR</n>), or anti-<n>gC1qR</n> antibody F(ab')(2) fragments. The interaction between <n>protein A</n> and platelet <n>gC1qR</n> was underscored by studies illustrating preferential recognition of the <n>protein A</n>-bearing S. aureus Cowan I strain by <n>gC1qR</n> compared to recognition of the <n>protein A</n>-deficient Wood 46 strain, as well as inhibition of S. aureus Cowan I strain adhesion to immobilized platelets by soluble <n>protein A</n>. Further characterization of the <n>protein A</n>-<n>gC1qR</n> interaction by solid-phase enzyme-linked immunosorbent assay techniques measuring biotinylated <n>gC1qR</n> binding to immobilized <n>protein A</n> revealed specific binding that was inhibited by soluble <n>protein A</n> with a 50% inhibitory concentration of (3.3 +/- 0.7) x 10(-7) M (mean +/- standard deviation; n = 3). Rabbit <n>immunoglobulin G</n> (<n>IgG</n>) also prevented <n>gC1qR</n>-<n>protein A</n> interactions, and inactivation of <n>protein A</n> tyrosil residues by hyperiodination, previously reported to prevent the binding of <n>IgG</n> Fc, but not Fab, domains to <n>protein A</n>, abrogated <n>gC1qR</n> binding. These results suggest similar <n>protein A</n> structural requirements for <n>gC1qR</n> and <n>IgG</n> Fc binding. Further studies of structure and function using a truncated <n>gC1qR</n> mutant lacking amino acids 74 to 95 demonstrated that the <n>protein A</n> binding domain lies outside of the <n>gC1qR</n> amino-terminal alpha helix, which contains binding sites for the globular heads of <n>C1q</n>. In conclusion, the data implicate the platelet <n>gC1qR</n> as a novel cellular binding site for staphylococcal <n>protein A</n> and suggest an additional mechanism for bacterial cell adhesion to sites of vascular injury and thrombosis.
10706722>Chemokines fail to up-regulate <n>beta 1 integrin</n>-dependent adhesion in human Th2 T lymphocytes. Th1 and Th2 cells are functionally distinct subsets of CD4+ T lymphocytes whose tissue-specific homing to sites of inflammation is regulated in part by the differential expression of <n>P-</n> and <n>E-selectin</n> ligands and selected chemokine receptors. Here we investigated the expression and function of <n>beta 1 integrins</n> in Th1 and Th2 cells polarized in vitro. Th1 lymphocytes adhere transiently to the extracellular matrix ligands <n>laminin 1</n> and <n>fibronectin</n> in response to chemokines such as <n>RANTES</n> and <n>stromal cell-derived factor-1</n>, and this process is paralleled by the activation of the <n>Rac1 GTPase</n> and by a rapid burst of <n>actin</n> polymerization. Selective inhibitors of <n>phosphoinositide-3 kinase</n> prevent efficiently all of the above processes, whereas the <n>protein kinase C</n> inhibitor bisindolylmaleimide prevents chemokine-induced adhesion without affecting <n>Rac1</n> activation and <n>actin</n> polymerization. Notably, chemokine-induced adhesion to <n>beta 1 integrin</n> ligands is markedly reduced in Th2 cells. Such a defect cannot be explained by a reduced sensitivity to chemokine stimulation in this T cell subset, nor by a defective activation of the signaling cascade involving <n>phosphoinositide-3 kinase</n>, <n>Rac1</n>, and <n>actin</n> turnover, as all these processes are activated at comparable levels by chemokines in the two subsets. We propose that reduced <n>beta 1 integrin</n>-mediated adhesion in Th2 cells may restrain their ability to invade and/or reside in sites of chronic inflammation, which are characterized by thickening of basement membranes and extensive fibrosis, requiring efficient interaction with organized extracellular matrices.
10679202>Cloning and characterization of a novel adaptor protein, <n>CIN85</n>, that interacts with <n>c-Cbl</n>. The <n>c-Cbl</n> protooncogene product is a prominent substrate of protein tyrosine kinases and is rapidly tyrosine-phosphorylated upon stimulation of a wide variety of cell-surface receptors. We have identified a novel <n>c-Cbl</n>-interacting protein termed <n>CIN85</n> with a molecular mass of 85 kDa which shows similarity to adaptor proteins, <n>CMS</n> and <n>CD2AP</n>. <n>CIN85</n> mRNA is expressed ubiquitously in normal human tissues and cancer cell lines analyzed. <n>CIN85</n> was basally associated with <n>c-Cbl</n>. For interaction of <n>CIN85</n> with <n>c-Cbl</n>, the second SH3 domain of <n>CIN85</n> was shown to serve as a central player. The <n>CIN85</n>-<n>c-Cbl</n> association was enhanced shortly after stimulation of 293 cells with <n>epidermal growth factor</n> (<n>EGF</n>) and gradually diminished to a basal level, which correlated with a tyrosine phosphorylation level of <n>c-Cbl</n>. Our results suggest that <n>CIN85</n> may play a specific role in the <n>EGF</n> receptor-mediated signaling cascade via its interaction with <n>c-Cbl</n>.
10644998><n>EB3</n>, a novel member of the <n>EB1</n> family preferentially expressed in the central nervous system, binds to a CNS-specific <n>APC</n> homologue. <n>APCL</n>, a homologue of the <n>adenomatous polyposis coli (APC) tumor suppressor</n>, can deplete cytoplasmic <n>beta-catenin</n> like <n>APC</n>. However, as its biological function remains unclear, we have been using a yeast two-hybrid system to search for proteins that associate with its carboxyl region. Among several cDNA clones we isolated from a fetal-brain cDNA library as candidates, six included an identical sequence with significant homology to <n>EB1</n>, a protein known to bind to <n>APC</n>. The full-length cDNA of this novel homologue of <n>EB1</n>, named <n>EB3</n>, encoded a protein of 282 amino acids with 54% identity to <n>EB1</n>, and it was expressed preferentially in brain tissue on Northern blots. Confocal microscopy demonstrated that exogenous <n>EB3</n>, like <n>EB1</n>, is associated with the cytoplasmic microtubule network. Moreover, in these experiments <n>EB3</n> and <n>APCL</n> appeared together in the perinucleus and the cytoplasmic microtubule network. Since <n>APCL</n> is also expressed highly and specifically in the central nervous system, <n>APCL</n>-<n>EB3</n> interaction may be specific to the CNS, possibly involving stability and/or extension of microtubules during neuritogenesis.
10617208>Inhibition of msl-2 splicing by <n>Sex-lethal</n> reveals interaction between <n>U2AF35</n> and the 3' splice site AG. The protein <n>Sex-lethal</n> (<n>SXL</n>) controls dosage compensation in Drosophila by inhibiting the splicing and translation of male-specific-lethal-2 (msl-2) transcripts. Here we report that splicing inhibition of msl-2 requires a binding site for <n>SXL</n> at the polypyrimidine (poly(Y)) tract associated with the 3' splice site, and an unusually long distance between the poly(Y) tract and the conserved AG dinucleotide at the 3' end of the intron. Only this combination allows efficient blockage of U2 small nuclear ribonucleoprotein particle binding and displacement of the large subunit of the U2 auxiliary factor (<n>U2AF65</n>) from the poly(Y) tract by <n>SXL</n>. Crosslinking experiments with ultraviolet light indicate that the small subunit of <n>U2AF</n> (<n>U2AF35</n>) contacts the AG dinucleotide only when located in proximity to the poly(Y) tract. This interaction stabilizes <n>U2AF65</n> binding such that <n>SXL</n> can no longer displace it from the poly(Y) tract. Our results reveal a novel function for <n>U2AF35</n>, a critical role for the 3' splice site AG at the earliest steps of spliceosome assembly and the need for a weakened <n>U2AF35</n>-AG interaction to regulate intron removal.
10601333><n>Gemin3</n>: A novel DEAD box protein that interacts with <n>SMN</n>, the spinal muscular atrophy gene product, and is a component of gems. The survival of motor neurons (<n>SMN</n>) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The <n>SMN</n> protein is part of a complex containing several proteins, of which one, <n>SIP1</n> (<n>SMN interacting protein 1</n>), has been characterized so far. The <n>SMN</n> complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, <n>SMN</n> and <n>SIP1</n> interact with the <n>Sm</n> core proteins of spliceosomal small nuclear ribonucleoproteins (<n>snRNP</n>s), and they play a critical role in <n>snRNP</n> assembly. In the nucleus, <n>SMN</n> is required for pre-mRNA splicing, likely by serving in the regeneration of <n>snRNPs</n>. Here, we report the identification of another component of the <n>SMN</n> complex, a novel DEAD box putative RNA helicase, named <n>Gemin3</n>. <n>Gemin3</n> interacts directly with <n>SMN</n>, as well as with <n>SmB</n>, <n>SmD2</n>, and <n>SmD3</n>. Immunolocalization studies using mAbs to <n>Gemin3</n> show that it colocalizes with <n>SMN</n> in gems. <n>Gemin3</n> binds <n>SMN</n> via its unique COOH-terminal domain, and <n>SMN</n> mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in <n>Gemin3</n> suggests that it may provide the catalytic activity that plays a critical role in the function of the <n>SMN</n> complex on RNPs.
10572088>Kinetic resolution of two mechanisms for high-affinity <n>granulocyte-macrophage colony-stimulating factor</n> binding to its receptor. <n>Granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) is an important hematopoietic cytokine that exerts its effects by interaction with the <n>GM-CSF receptor</n> (<n>GMR</n>) on the surface of responsive cells. The <n>GM-CSF receptor</n> consists of two subunits: GMRalpha, which binds <n>GM-CSF</n> with low affinity, and <n>GMR</n>beta, which lacks intrinsic ligand-binding capability but complexes with GMRalpha to form a high-affinity receptor (<n>GMRalpha/beta</n>). We conducted dynamic kinetic analyses of <n>GM-CSF</n> receptors to define the role of GMRbeta in the interaction of ligand and receptor. Our data show that <n>GMRalpha/beta</n> exhibits a higher k(on) than GMRalpha, indicating that GMRbeta facilitates ligand acquisition to the binding pocket. Heterogeneity with regard to <n>GM-CSF</n> dissociation from <n>GMRalpha/beta</n> points to the presence of loose and tight ligand-receptor complexes in high-affinity binding. Although the loose complex has a k(off) similar to GMRalpha, the lower k(off) indicates that GMRbeta inhibits <n>GM-CSF</n> release from the tight receptor complex. The two rates of ligand dissociation may provide for discrete mechanisms of interaction between <n>GM-CSF</n> and its high-affinity receptor. These results show that the beta subunit functions to stabilize ligand binding as well as to facilitate ligand acquisition.
10551879><n>Cyclic AMP-dependent protein kinase</n> binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of <n>green fluorescent protein</n> fusion proteins. A-kinase anchoring proteins tether <n>cAMP-dependent protein kinase</n> (<n>PKA</n>) to specific subcellular locations. The purpose of this study was to use fluorescence resonance energy transfer to monitor binding events in living cells between the type II regulatory subunit of <n>PKA</n> (RII) and the RII-binding domain of the human <n>thyroid RII anchoring protein</n> (<n>Ht31</n>), a peptide containing the PKA-binding domain of an A-kinase anchoring protein. RII was linked to <n>enhanced yellow fluorescent protein</n> (<n>EYFP</n>), <n>Ht31</n> was linked to <n>enhanced cyan fluorescent protein</n> (<n>ECFP</n>), and these constructs were coexpressed in Chinese hamster ovary cells. Upon excitation of the donor fluorophore, <n>Ht31</n>.<n>ECFP</n>, an increase in emission of the acceptor fluorophore, RII.<n>EYFP</n>, and a decrease in emission from <n>Ht31</n>.<n>ECFP</n> were observed. The emission ratio (acceptor/donor) was increased 2-fold (p &#60; 0.05) in cells expressing <n>Ht31</n>.<n>ECFP</n> and RII.<n>EYFP</n> compared with cells expressing <n>Ht31P</n>.<n>ECFP</n>, the inactive form of <n>Ht31</n>, and RII.<n>EYFP</n>. These results provide the first in vivo demonstration of RII/<n>Ht31</n> interaction in living cells and confirm previous in vitro findings of RII/<n>Ht31</n> binding. Using surface plasmon resonance, we also showed that the <n>green fluorescent protein</n> tags did not significantly alter the binding of <n>Ht31</n> to RII. Thus, fluorescence resonance energy transfer can be used to directly monitor protein-protein interactions of the <n>PKA</n> signaling pathway in living cells.
10527805>Alzheimer's disease associated <n>presenilin 1</n> interacts with HC5 and ZETA, subunits of the catalytic 20S proteasome. Proteolytic processing and degradation tightly regulate the amount of stable, functional <n>presenilin 1</n> (<n>PSEN1</n>) in the cell. The approximately 46-kDa <n>PSEN1</n> holoprotein is cleaved into an approximately 30-kDa N-terminal fragment (NTF) and an approximately 20-kDa C-terminal fragment (CTF) by an unknown protease. The fragments are stabilized in a high molecular weight complex and nonincorporated fragments and excess holoprotein are degraded by the 26S proteasome. The tight balance between, on the one hand, processing and incorporation into the stable complex and, on the other hand, proteolytic degradation of excess <n>PSEN1</n>, indicates that minor changes in one of these two processes could be pathologically relevant. Here we demonstrate the direct physical interaction between <n>PSEN1</n> and two subunits, HC5 and ZETA, of the 20S proteasome. These interactions were identified using an interaction trap screening and were further established in an in vitro binding assay. Furthermore, we were able to coimmunoprecipitate the transfected binding partners, as well as the endogenous <n>PSEN1</n> and ZETA proteins from HEK 293T cells. Finally, degradation of ubiquitinated wild-type and mutant <n>PSEN1</n> by the 26S proteasome was demonstrated. In conclusion, we report a direct interaction between <n>PSEN1</n> and subunits of the 20S catalytic particle of the 26S proteasome, further establishing the involvement of proteasomal degradation in the regulation of <n>PSEN1</n> turnover.
10506185>A peptide representing the carboxyl-terminal tail of the <n>met receptor</n> inhibits kinase activity and invasive growth. Interaction of the <n>hepatocyte growth factor</n> (<n>HGF</n>) with its receptor, the <n>Met tyrosine kinase</n>, results in invasive growth, a genetic program essential to embryonic development and implicated in tumor metastasis. Met-mediated invasive growth requires autophosphorylation of the receptor on tyrosines located in the kinase activation loop (Tyr(1234)-Tyr(1235)) and in the carboxyl-terminal tail (Tyr(1349)-Tyr(1356)). We report that peptides derived from the <n>met receptor</n> tail, but not from the activation loop, bind the receptor and inhibit the kinase activity in vitro. Cell delivery of the tail receptor peptide impairs <n>HGF</n>-dependent <n>Met</n> phosphorylation and downstream signaling. In normal and transformed epithelial cells, the tail receptor peptide inhibits <n>HGF</n>-mediated invasive growth, as measured by cell migration, invasiveness, and branched morphogenesis. The <n>Met</n> tail peptide inhibits the closely related <n>Ron receptor</n> but does not significantly affect the <n>epidermal growth factor</n>, <n>platelet-derived growth factor</n>, or <n>vascular endothelial growth factor receptor</n> activities. These experiments show that carboxyl-terminal sequences impair the catalytic properties of the <n>met receptor</n>, thus suggesting that in the resting state the nonphosphorylated tail acts as an intramolecular modulator. Furthermore, they provide a strategy to selectively target the MET proto-oncogene by using small, cell-permeable, peptide derivatives.
10487747><n>GRASP55</n>, a second mammalian <n>GRASP</n> protein involved in the stacking of Golgi cisternae in a cell-free system. We have identified a 55 kDa protein, named <n>GRASP55</n> (<n>Golgi reassembly stacking protein of 55 kDa</n>), as a component of the Golgi stacking machinery. <n>GRASP55</n> is homologous to <n>GRASP65</n>, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system. <n>GRASP65</n> exists in a complex with the <n>vesicle docking protein receptor GM130</n> to which it binds directly, and the <n>membrane tethering protein p115</n>, which also functions in the stacking of Golgi cisternae. <n>GRASP55</n> binding to <n>GM130</n>, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system. Cryo-electron microscopy revealed that <n>GRASP65</n>, like <n>GM130</n>, is present on the cis-Golgi, while <n>GRASP55</n> is on the medial-Golgi. Recombinant <n>GRASP55</n> and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for <n>GRASP65</n>. These results demonstrate that <n>GRASP55</n> and <n>GRASP65</n> function in the stacking of Golgi cisternae.
10448035>Human <n>Rad51</n> amino acid residues required for <n>Rad52</n> binding. The <n>Rad51</n> protein, a homologue of the bacterial <n>RecA</n> protein, is an essential factor for both meiotic and mitotic recombination. The N-terminal domain of the human <n>Rad51</n> protein (<n>HsRad51</n>) directly interacts with DNA. Based on a yeast two-hybrid analysis, it has been reported that the N-terminal region of the Saccharomyces cerevisiae <n>Rad51</n> protein binds <n>Rad52</n>;S. cerevisiae <n>Rad51</n> and <n>Rad52</n> both activate the homologous pairing and strand exchange reactions. Here, we show that the <n>HsRad51</n> N-terminal region, which corresponds to the <n>Rad52</n>-binding region of <n>ScRad51</n>, does not exhibit strong binding to the human <n>Rad52</n> protein (<n>HsRad52</n>). To investigate its function, the C-terminal region of <n>HsRad51</n> was randomly mutagenized. Although this region includes the two segments corresponding to the putative DNA-binding sites of <n>RecA</n>, all seven of the mutants did not decrease, but instead slightly increased, the DNA binding. In contrast, we found that some of these <n>HsRad51</n> mutations significantly decreased the <n>HsRad52</n> binding. Therefore, we conclude that these amino acid residues are required for the <n>HsRad51</n>.<n>HsRad52</n> binding. <n>HsRad52</n>, as well as S. cerevisiae <n>Rad52</n>, promoted homologous pairing between ssDNA and dsDNA, and higher homologous pairing activity was observed in the presence of both <n>HsRad51</n> and <n>HsRad52</n> than with either <n>HsRad51</n> or <n>HsRad52</n> alone. The <n>HsRad51</n> F259V mutation, which strongly impaired the <n>HsRad52</n> binding, decreased the homologous pairing in the presence of both <n>HsRad51</n> and <n>HsRad52</n>, without affecting the homologous pairing by <n>HsRad51</n> alone. This result suggests the importance of the <n>HsRad51</n>.<n>HsRad52</n> interaction in homologous pairing.
10425523>Amino acids within the extracellular matrix (ECM) binding region (201-218) of rat <n>insulin-like growth factor binding protein (IGFBP)-5</n> are important determinants in binding <n>IGF-I</n>. The highly conserved N-and C-terminal domains of IGFBPs are believed to participate in <n>IGF</n> binding, but only recently have some of the critical residues in the <n>IGFBP</n> sequence involved in ligand binding been identified. Here we describe two highly conserved amino acids in the C-terminal domain of rat <n>IGFBP-5</n> that are involved in binding <n>IGF-I</n>. Site-directed mutagenesis was used to produce two mutants, G203K and Q209A, of <n>rIGFBP-5</n>. Relative to wild-type <n>rIGFBP-5</n>, an 8-fold reduction in affinity for human <n>IGF-I</n> was found for recombinant G203K protein in both <n>IGF-I</n> ligand blots and solution phase ligand binding assays, and a 7-and 6-fold reduction for Q209A respectively. This shows that Gly203 and Gln209 in <n>IGFBP-5</n> are important determinants in binding <n>IGF-I</n>, and due to their complete conservation in all <n>IGFBP</n> sequences, we suggest that they are likely to be involved in binding <n>IGF-I</n> in all six binding proteins. In addition, these two non-basic residues lie within the ECM binding region (201-218) of <n>IGFBP-5</n>, demonstrating that the C-terminus contains partially overlapping <n>IGF-I</n> and ECM binding sites. We therefore propose that <n>heparin</n> binding to basic amino acids in <n>IGFBP-5</n> between 201-218 may physically occlude subsequent interaction between <n>IGF-I</n> and Gly203/Gln209, and that this may explain previous work of others showing reduced affinity of ECM bound <n>IGFBP-5</n> for <n>IGF-I</n>.
10400685>Intact LIM 3 and LIM 4 domains of <n>paxillin</n> are required for the association to a novel polyproline region (Pro 2) of <n>protein-tyrosine phosphatase-PEST</n>. The <n>focal adhesion protein p130</n>(<n>Cas</n>) was identified as a substrate for the <n>protein-tyrosine phosphatase (PTP)-PEST</n>, and the specificity of this interaction is mediated by a dual mechanism involving a Src homology 3 domain-mediated binding and PTP domain recognition. Recently, <n>paxillin</n> was also demonstrated to interact with <n>PTP-PEST</n> (Shen, Y., Schneider, G., Cloutier, J. F., Veillette, A., and Schaller, M. D. (1998) J. Biol. Chem. 273, 6474-6481). In the present study, we show that amino acids 344-397 of <n>PTP-PEST</n> are sufficient for the binding to <n>paxillin</n>. We demonstrate that a proline-rich segment of <n>PTP-PEST</n> (Pro 2), 355PPEPHPVPPILTPSPPSAFP374, is essential for this interaction in vivo. Furthermore, mutation of proline residues within the Pro 2 motif reveal that proline 362 is critical for the binding of <n>paxillin</n>. Conversely, using deletion and point mutants of <n>paxillin</n>, LIM 3 and 4 domains were both found to be necessary for binding of <n>PTP-PEST</n>. Finally, using a "substrate trapping" approach, we demonstrate that, unlike <n>p130</n>(<n>Cas</n>), <n>paxillin</n> is not a substrate for <n>PTP-PEST</n>. In conclusion, we show that a novel proline-rich motif found in <n>PTP-PEST</n> serves as a ligand for the LIM domains of <n>paxillin</n>. Interestingly, the focal adhesion targeting of <n>paxillin</n> is mediated by LIM 3. Thus, we propose that <n>PTP-PEST</n>, by a competition with the ligand of <n>paxillin</n> in the focal adhesion complex, could contribute to the removal of <n>paxillin</n> from the adhesion sites and consequently promote focal adhesion turnover.
10380882><n>MBP1</n>: a novel mutant <n>p53</n>-specific protein partner with oncogenic properties. Using a yeast two-hybrid screening strategy with a common tumour-derived <n>p53</n> mutant as bait, we identified several mutant <n>p53</n>-interacting partners including the known proteins wild-type (wt) <n>p53</n>, <n>hUBC9</n> and <n>GBP</n>/<n>PIAS1</n>. In addition, a novel protein partner was identified which we have termed <n>MBP1</n>, for <n>Mutant p53-Binding Protein 1</n>. <n>MBP1</n> is a new member of the emerging fibulin gene family, which currently comprises <n>fibulin-1</n>, <n>fibulin-2</n> and <n>S1-5</n>. Expression of <n>MBP1</n> mRNA is differentially regulated both temporally during development of the mouse embryo and in a tissue-specific manner within the adult. Specific interaction between <n>MBP1</n> and mutant <n>p53</n> was illustrated by both two-hybrid analysis in yeast and co-immunoprecipitation in mammalian cells. <n>MBP1</n> displayed the following order of binding specificity towards different <n>p53</n> forms: H175 > G281 > H273 > or = W248>wt <n>p53</n>. Thus, <n>MBP1</n> appears to bind preferentially to <n>p53</n> mutants of the 'structural' rather than 'contact' class, reflecting a potential bias towards those mutants having a significant alteration in conformation from that assumed by wt <n>p53</n>. We propose that <n>MBP1</n> is the product of a candidate oncogene as rates of both neoplastic transformation and tumour cell growth were shown to be significantly enhanced when the protein is ectopically overexpressed. Furthermore, <n>MBP1</n> may play a role in determining if a 'gain of function' effect is seen with certain <n>p53</n> mutants.
10357818><n>Latent membrane protein 1</n> of Epstein-Barr virus interacts with <n>JAK3</n> and activates STAT proteins. <n>Latent membrane protein 1</n> (<n>LMP1</n>) acts like a permanently activated receptor of the tumor necrosis factor (TNF)-receptor superfamily and is absolutely required for B cell immortalization by Epstein-Barr virus. Molecular and biochemical approaches demonstrated that <n>LMP1</n> usurps cellular signaling pathways resulting in the induction of <n>NF-kappaB</n> and <n>AP-1</n> via two C-terminal activating regions. We demonstrate here that a third region encompassing a proline rich sequence within the 33 bp repetitive stretch of <n>LMP1's</n> C-terminus is required for the activation of <n>Janus kinase 3</n> (<n>JAK3</n>). The interaction of <n>LMP1</n> and <n>JAK3</n> leads to the enhanced tyrosine auto/transphosphorylation of <n>JAK3</n> within minutes after crosslinking of a conditional <n>NGF</n>-R:<n>LMP1</n> chimera and is a prerequisite for the activation of STAT transcription factors. These results reveal a novel activating region in the <n>LMP1</n> C-terminus and identify the JAK/STAT pathway as a target of this viral integral membrane protein in B cells.
10318834>Shared and unique determinants of the <n>erythropoietin (EPO) receptor</n> are important for binding <n>EPO</n> and <n>EPO mimetic peptide</n>. We have shown previously that Phe93 in the extracellular domain of the <n>erythropoietin (EPO) receptor</n> (<n>EPOR</n>) is crucial for binding <n>EPO</n>. Substitution of Phe93 with alanine resulted in a dramatic decrease in <n>EPO</n> binding to the Escherichia coli-expressed extracellular domain of the <n>EPOR</n> (<n>EPO-binding protein</n> or <n>EBP</n>) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe93 forms extensive contacts with a peptide ligand in the crystal structure of the <n>EBP</n> bound to an <n>EPO-mimetic peptide</n> (<n>EMP1</n>), suggesting that Phe93 is also important for <n>EMP1</n> binding. We used alanine substitution of <n>EBP</n> residues that contact <n>EMP1</n> in the crystal structure to investigate the function of these residues in both <n>EMP1</n> and <n>EPO</n> binding. The three largest hydrophobic contacts at Phe93, Met150, and Phe205 and a hydrogen bonding interaction at Thr151 were examined. Our results indicate that Phe93 and Phe205 are important for both <n>EPO</n> and <n>EMP1</n> binding, Met150 is not important for <n>EPO</n> binding but is critical for <n>EMP1</n> binding, and Thr151 is not important for binding either ligand. Thus, Phe93 and Phe205 are important binding determinants for both <n>EPO</n> and <n>EMP1</n>, even though these ligands share no sequence or structural homology, suggesting that these residues may represent a minimum epitope on the <n>EPOR</n> for productive ligand binding.
10219086>Directional binding of <n>HMG-I(Y)</n> on four-way junction DNA and the molecular basis for competitive binding with <n>HMG-1</n> and <n>histone H1</n>. <n>Histone H1</n>, <n>HMG-1</n> and <n>HMG-I(Y)</n> are mammalian nuclear proteins possessing distinctive DNA-binding domain structures that share the common property of preferentially binding to four-way junction (4H) DNA, an in vitro mimic of the in vivo genetic recombination intermediate known as the Holliday junction. Nevertheless, these three proteins bind to 4H DNA in vitro with very different affinities and in a mutually exclusive manner. To investigate the molecular basis for these distinctive binding characteristics, we employed base pair resolution hydroxyl radical footprinting to determine the precise sites of nucleotide interactions of both <n>HMG-1</n> and <n>histone H1</n> on 4H DNA and compared these contacts with those previously described for <n>HMG-I(Y)</n> on the same substrate. Each of these proteins had a unique binding pattern on 4H DNA and yet shared certain common nucleotide contacts on the arms of the 4H DNA molecule near the branch point. Both the <n>HMG-I(Y)</n> and <n>HMG-1</n> proteins made specific contacts across the 4H DNA branch point, as well as interacting at discrete sites on the arms, whereas the globular domain of <n>histone H1</n> bound exclusively to the arms of the 4H DNA substrate without contacting nucleotides at the crossover region. Experiments employing the chemical cleavage reagent 1, 10-orthophenanthroline copper(II) attached to the C-terminal end of a site-specifically mutagenized <n>HMG-I(Y)</n> protein molecule demonstrated that this protein binds to 4H DNA in a distinctly polar, direction-specific manner. Together these results provide an attractive molecular explanation for the observed mutually exclusive 4H DNA-binding characteristics of these proteins and also allow for critical assessment of proposed models for their interaction with 4H DNA substrates. The results also have important implications concerning the possible in vivo roles of <n>HMG-I(Y)</n>, <n>histone H1</n> and <n>HMG-1</n> in biological processes such as genetic recombination and retroviral integration.
10198166>Isolation and functional characterization of the human <n>90K</n> promoter. <n>90K</n> is a secreted protein thought to be involved in the body's defense against pathogens and cancer. To elucidate its transcriptional regulation, the promoter of human <n>90K</n> (HGMW-approved symbol <n>LGAL S3BP</n>) was isolated and characterized. Analysis of the 3. 3-kb 5'-flanking region revealed that it is a TATA-less promoter, but neither GC-rich nor dependent on SP1 sites. <n>RNase</n> protection assays detected one major transcription start site (+1) and several minor transcription start sites upstream and downstream. Deletion studies defined a minimal promoter (-103 --> -49) and indirectly suggested positive synergism between different elements within it. Consistent with the proposed function of <n>90K</n>, its promoter activity could be stimulated by poly(I). poly(C), mimicking viral infection. Two regions mediating induction by poly(I). poly(C) (-171 --> -112, -32 --> 46) were identified by deletion mutants. A small region around the minimal promoter (-99 --> -12) was highly homologous between human and mouse. While both human and mouse minimal promoters contained an <n>interferon</n>-responsive element (IRF-E), the human minimal promoter was not inducible by poly(I). poly(C) in contrast to that of the mouse. Point mutations 30 bp upstream of the IRF-E, however, conferred inducibility to the human minimal promoter, suggesting interaction between different promoter elements.
10089499>Biochemical and crystallographic characterization of homologous non-peptidic <n>thrombin</n> inhibitors having alternate binding modes. The X-ray crystallographic structure of [N-(3-phenylpropionyl)-N-(phenethyl)]-Gly-boroLys-OH (HPBK, Ki = 0. 42 nM, crystallographic R factor to 1.8 A resolution, 19.6%) complexed with human alpha-<n>thrombin</n> shows that the boron adopts a tetrahedral geometry and is covalently bonded to the active serine, Ser195. The HPBK phenethyl aromatic ring forms an edge-to-face interaction with the indole side chain of Trp215. Four HPBK analogs containing either electron-withdrawing or electron-donating substitutents at the 3' position of the phenethyl ring were synthesized in an attempt to modulate ligand affinity by inductive stabilization of the edge-to-face interaction. Refined crystallographic structures of the trifluoromethyl (Ki = 0.37 nM, crystallographic R factor to 2.0 A resolution = 18.7%), fluoro (Ki = 0.60; R factor to 2.3 A resolution = 18.4%), methoxy (Ki = 0.91 nM, R factor to 2.2 A resolution = 19.8%) and methyl (Ki = 0.20 nM, R factor to 2.5 A resolution = 16.9%) HPBK analogs complexed with <n>thrombin</n> revealed two binding modes for the closely related compounds. A less than 1.5-fold variation in affinity was observed for analogs (trifluoromethyl-HPBK and fluoro-HPBK) binding with the edge-to-face interaction. The slight inductive modulation is consistent with the overall weak nature of the edge-to-face interaction. Owing to an unexpected rotation of the phenethyl aromatic ring, the 3' substituent of two analogs, methoxy-HPBK and methyl-HPBK, made direct contact with the Trp215 indole side chain. Increased affinity of the 3' methyl analog is attributed to favorable interactions between the methyl group and the Trp215 indole ring. Differences in inhibitor, <n>thrombin</n> and solvent structure are discussed in detail. These results demonstrate the subtle interplay of weak forces that determine the equilibrium binding orientation of inhibitor, solvent and protein.
10050856>The <n>Rab5</n> effector <n>EEA1</n> is a core component of endosome docking. Intracellular membrane docking and fusion requires the interplay between soluble factors and SNAREs. The SNARE hypothesis postulates that pairing between a vesicular <n>v-SNARE</n> and a target membrane <n>z-SNARE</n> is the primary molecular interaction underlying the specificity of vesicle targeting as well as lipid bilayer fusion. This proposal is supported by recent studies using a minimal artificial system. However, several observations demonstrate that SNAREs function at multiple transport steps and can pair promiscuously, questioning the role of SNAREs in conveying vesicle targeting. Moreover, other proteins have been shown to be important in membrane docking or tethering. Therefore, if the minimal machinery is defined as the set of proteins sufficient to reproduce in vitro the fidelity of vesicle targeting, docking and fusion as in vivo, then SNAREs are not sufficient to specify vesicle targeting. Endosome fusion also requires cytosolic factors and is regulated by the small GTPase <n>Rab5</n>. Here we show that <n>Rab5</n>-interacting soluble proteins can completely substitute for cytosol in an in vivo endosome-fusion assay, and that the <n>Rab5</n> effector <n>EEA1</n> is the only factor necessary to confer minimal fusion activity. <n>Rab5</n> and other associated proteins seem to act upstream of <n>EEA1</n>, implying that <n>Rab5</n> effectors comprise both regulatory molecules and mechanical components of the membrane transport machinery. We further show that <n>EEA1</n> mediates endosome docking and, together with SNAREs, leads to membrane fusion.
9973489>Identification of residues in the first domain of human <n>Fc alpha receptor</n> essential for interaction with <n>IgA</n>. The FcR family contains multiple receptors for Igs, of which the most distantly related ( approximately 20%) is the <n>IgA</n> receptor (human <n>Fc alpha R</n>), being more homologous ( approximately 35%) to another family of killer-inhibitory receptor-related immunoreceptors with a 19q13.4 chromosomal location in humans. This study of the <n>Fc alpha R</n> demonstrated that, like several <n>IgG</n> receptors, <n>Fc alpha R</n> is a low affinity receptor for Ab (Ka approximately 106 M-1). Rapid dissociation of the rs<n>Fc alpha R</n>:<n>IgA</n> complex (t1/2 approximately 25 s) suggests that monomer <n>IgA</n> would bind transiently to cellular <n>Fc alpha R</n>s, while <n>IgA</n> immune complexes could bind avidly. Mutagenesis of histidyl 85 and arginyl 82, in the FG loop of domain 1, demonstrated that these residues were essential for the <n>IgA</n>-binding activity of <n>Fc alpha R</n>, while arginyl 87 makes a minor contribution to the binding activity of the receptor. This site is unusual among the Fc receptors (<n>Fc gamma RII</n>, <n>Fc gamma RIII</n>, and <n>Fc epsilon RI</n>), in which the ligand binding site is in domain 2 rather than domain 1, but like <n>Fc alpha R</n>, the FG loop comprises part of the ligand binding site. The putative F and G strands flanking the <n>Fc alpha R</n> ligand binding site are highly homologous in the other killer-inhibitory receptor-related immunoreceptors, suggesting they comprise a conserved structural element on which divergent FG loops are presented and participate in the specific ligand interactions of each of these receptors.
9890995>Definition of the sites of interaction between the protein <n>tyrosine phosphatase SHP-1</n> and <n>CD22</n>. <n>CD22</n> phosphorylation is an early event of B cell antigen receptor engagement and results in the recruitment of the negative regulatory tyrosine phosphatase, <n>SHP-1</n>. Peptides representing the potential phosphorylation sites within the cytoplasmic domain of <n>CD22</n> have been used to stimulate <n>SHP-1</n> catalytic activity and to inhibit the binding of <n>SHP-1</n> to <n>CD22</n> (Doody, G., Justement, L., Delibrias, C., Matthews, R., Lin, J., Thomas, M., and Fearon, D. (1995) Science 269, 242-244). However, the sites of phosphorylation within the cytoplasmic domain of <n>CD22</n> and the importance of each for the recruitment and activation of <n>SHP-1</n> remain unknown. Here we demonstrate that there are multiple sites within the cytoplasmic domain of <n>CD22</n> that interact with the Src homology 2 domains of <n>SHP-1</n>. Nevertheless, a minimum of two tyrosines in <n>CD22</n> is required for the association with <n>SHP-1</n>. Furthermore, both Src homology 2 domains of <n>SHP-1</n> are necessary for efficient binding to <n>CD22</n>.
9869654>Lipid binding-induced conformational changes in the N-terminal domain of human <n>apolipoprotein E</n>. The N-terminal domain of human <n>apolipoprotein E3</n> (<n>apoE3</n>) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its <n>low density lipoprotein receptor</n> recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1-183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-<n>apoE3</n>(1-183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 A to 44 A.The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable <n>apolipoproteins</n> and are consistent with the hypothesis that, upon lipid binding, <n>apoE3</n>(1-183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.
9843378>SH3 binding domains in the <n>dopamine D4 receptor</n>. The <n>dopamine D4 receptor</n> is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit <n>adenylyl cyclase</n>. The <n>dopamine D4 receptor</n> as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the <n>D4 receptor</n> are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the <n>D4 receptor</n> can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins <n>Grb2</n> and <n>Nck</n>. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the <n>dopamine D4 receptor</n> resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to <n>adenylyl cyclase</n> and <n>MAPK</n>, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the <n>D4 receptor</n> contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.
9820523>A site for <n>CD4</n> binding in the beta 1 domain of the MHC class II protein <n>HLA-DR1</n>. Using a lymphocyte binding assay, we have previously demonstrated that the <n>CD4</n> protein can mediate cell adhesion by direct interaction with MHC class II molecules. In this report, we have used this assay to test whether synthetic peptides, corresponding to DR beta sequences, could inhibit <n>CD4</n>-class II adhesion. A peptide derived from sequences within the <n>beta1</n> domain (DR beta 41-55), as well as two peptides derived from sequences within the beta 2 domain (DR beta 121-135 and DR beta 141-155), were shown to inhibit <n>CD4</n>-class II adhesion. Inasmuch as a site for <n>CD4</n> binding in the beta 2 domain had been previously documented, these studies were designed to investigate the role of the beta 1 domain as an additional site of interaction with <n>CD4</n>. Sixteen site-specific mutations were engineered within the <n>beta1</n> domain of DR beta 1*0101. Several mutations were shown to disrupt <n>CD4</n>-dependent T cell activation. Based on these results, we propose a model for the molecular interaction of <n>CD4</n> with MHC class II proteins in which both the beta 1 and beta 2 domains of class II interact with the two amino-terminal Ig-like domains of <n>CD4</n>.
9799109>Characterization of <n>brain PCTAIRE-1 kinase</n> immunoreactivity and its interactions with <n>p11</n> and <n>14-3-3</n> proteins. An antibody directed against the C-terminal part of <n>PCTAIRE-1</n> recognized three proteins in rodent brain. The high-molecular-mass band is most abundant in the cerebellum, hippocampus and cortex. It migrated at the same apparent molecular mass as recombinant <n>PCTAIRE-1</n> and interacted, like recombinant <n>PCTAIRE-1</n>, with <n>p11</n> and <n>14-3-3</n> proteins. Combination of <n>p11</n> or <n>14-3-3</n> affinity resins with immunoprecipitation and peptide elution allowed us to obtain a purified full-length <n>PCTAIRE-1</n> preparation having significant kinase activity. These results suggest that <n>PCTAIRE-1</n> is an active kinase in brain. The catalytic core region of <n>PCTAIRE-1</n> which is common for all cyclin-dependent kinases, does not interact with <n>p11</n> and <n>14-3-3</n> proteins in the two-hybrid assay. Full interaction with <n>p11</n> and <n>14-3-3</n> proteins requires both, the N-terminal and C-terminal ends of <n>PCTAIRE-1</n>, suggesting that complex three-dimensional arrangements are responsible for these interactions. A low-molecular-mass protein (migrating at about 30 kDa) that was also recognized by the antibody directed against the carboxy-terminal part of <n>PCTAIRE-1</n>, is abundant and almost homogeneously distributed in all brain areas investigated. Database searches starting with the amino acid sequences of two peptides obtained by tryptic digestion of this protein yielded cDNA and genomic (a gene of about 10 kb on human chromosome 1q24-1q25 and clone 262D12) sequences, allowing us to compose a DNA sequence coding for a putative 26 kDa protein containing both peptides. This protein has no important sequence similarity with any other known protein. But many DNA sequences are found in databases with an almost 100% identity with parts of the 26 kDa protein coding sequence. Our results allow us to attribute these widely distributed cDNA sequences to an existing 26-kDa protein and to localize a gene within two recently published genomic sequences.
9786917><n>CBP</n> alleviates the intramolecular inhibition of <n>ATF-2</n> function. The transcription factor <n>ATF-2</n> (also called <n>CRE-BP1</n>), whose DNA-binding domain consists of a basic amino acid cluster and a leucine zipper (b-ZIP) region, binds to the cAMP response element as a homodimer or as a heterodimer with <n>c-Jun</n>. The amino-terminal region of <n>ATF-2</n> containing the transcriptional activation domain is phosphorylated by stress-activated kinases, which leads to activation of <n>ATF-2</n>. We report here that <n>CBP</n>, which was originally identified as a co-activator of <n>CREB</n>, directly binds to the b-ZIP region of <n>ATF-2</n> via a Cys/His-rich region termed C/H2, and potentiates trans-activation by <n>ATF-2</n>. The b-ZIP region of <n>ATF-2</n> was previously shown to interact with the amino-terminal region intramolecularly and to inhibit trans-activating capacity. The binding of <n>CBP</n> to the b-ZIP region abrogates this intramolecular interaction. The <n>adenovirus 13S E1A protein</n> which binds to the b-ZIP region of <n>ATF-2</n> also inhibited this intramolecular interaction, suggesting that both <n>CBP</n> and <n>13S E1A</n> share a similar function as positive regulators of <n>ATF-2</n>. We found that the b-ZIP regions of <n>c-Jun</n> and <n>CREB</n> also interact with the C/H2 domain of <n>CBP</n>, suggesting that <n>CBP</n> acts as a regulator for a group of b-ZIP-containing proteins. These results shed light on a novel aspect of <n>CBP</n> function as a regulator for a group of b-ZIP-containing proteins.
9761476>Lysine-50 is a likely site for anchoring the <n>plasminogen</n> N-terminal peptide to lysine-binding kringles. Interactions between the kringle 4 (K4) domain of human <n>plasminogen</n> (<n>Pgn</n>) and segments of the N-terminal Glu1-Lys77 peptide (NTP) have been investigated via 1H-NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka &#60; 0.05 mM(-1)). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Nalpha-acetyl-L-lysine-methyl ester (Ka approximately 0.2 mM(-1), peptides encompassing Lys50O consistently show higher Ka values. Among the investigated linear peptides, Nalpha-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys5O-Glu-Gln-NH2 (AcAFYHSK5OEQ-NH2) exhibits the strongest interaction with K4 (Ka approximately 1.4 mM(-1)), followed by AcYHSK50EQ-NH2 (Ka approximately 0.9 mM(-1)). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 --> Ser mutation was introduced (==> AcYHSS50EQ-NH2), binding was abolished. The Ile27-lle56 construct (L-NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant <n>Pgn</n> K1 (rK1) and K2 (rK2) modules, and of <n>Pgn</n> fragments encompassing the intact K4 and K5 domains, for binding L-NTP, was investigated. We find that L-NTP interacts with rK1, rK2, K4, and K5-all lysine-binding kringles-in a fashion that closely mimics what has been observed for the Glul-HSer57 N-terminal fragment of <n>Pgn</n> (CB-NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L-NTP and magnitudes of kringle-L-NTP binding affinities (rK1, Ka approximately 4.3 mM(-1); rK2, Ka approximately 3.7 mM(-1; K4, Ka approximately 6.4 mM(1); and K5, Ka approximately 2.1 mM(-1)) are essentially the same as for the corresponding kringle-CB-NTP pairs. Molecular modeling studies suggest that the Glu39-Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent-exposed kringle LBS surface.
9740790>The <n>V protein</n> of the paramyxovirus SV5 interacts with <n>damage-specific DNA binding protein</n>. The simian parainfluenza virus 5 (SV5) V/P gene encodes two proteins: <n>V</n> and the <n>phosphoprotein P</n>. The <n>V</n> and <n>P</n> proteins are amino coterminal for 164 residues, but they have unique carboxyl termini. The unique carboxyl terminus of <n>V</n> contains seven cysteine residues, resembles a zinc finger, and binds two atoms of zinc. In a <n>glutathione-S-transferase</n> (<n>GST</n>)-fusion protein selection of cell lysate assay, the <n>GST</n>-<n>V protein</n> was found to interact with the 127-kDa subunit (DDB1) of the <n>damage-specific DNA binding protein</n> (<n>DDB</n>) [also known as <n>UV-damaged DNA binding protein</n> (<n>UV-DDB</n>), <n>xeroderma pigmentosum group E binding factor</n> (<n>XPE-BF</n>), and the hepatitis B virus <n>X-associated protein 1</n> (<n>AP-1</n>)]. A reciprocal <n>GST</n>-DDB1 fusion protein selection assay of SV5-infected cell lysates showed that DDB1 and <n>V</n> interact, and it was found that <n>V</n> and DDB1 could be coimmunoprecipitated from SV5-infected cells or from cells expressing <n>V</n> and DDB1 using the vaccinia virus T7 expression system. The interaction of <n>V</n> and DDB1 involves the carboxyl-terminal domain of <n>V</n> in that either deletion of the <n>V</n> carboxyl-terminal domain or substitution of the cysteine residues (C189, C193, C205, C207, C210, C214, and C217) in the zinc-binding domain with alanine was able to disrupt binding to DDB1. The <n>V protein</n>s of the mumps virus, human parainfluenza virus 2 (hPIV2), and measles virus have also been found to interact with DDB1 in <n>GST</n>-fusion protein selection assays using in vitro transcribed and translated DDB1.
9716151>Effects of radiographic contrast agents on <n>thrombin</n> formation and activity. Clinical trials suggest that the risk of thrombosis during coronary angioplasty is lower with ionic contrast agents than with nonionic contrast agents. However, the molecular mechanisms underlying this effect are unknown. This study examined the effects of contrast agents on <n>thrombin</n> formation and its interaction with substrates, inhibitors, and ligands to define potential mechanisms by which contrast agents affect thrombus formation. Two ionic agents, diatrizoate and ioxaglate, and one nonionic agent, ioversol, were studied. Ionic agents inhibited <n>factor X</n> activation by the <n>tissue factor</n>-<n>factor VIIa</n> complex more potently than ioversol (53 +/- 3.7, 43.0 +/- 1.9, and 26.5 +/- 2.4% inhibition by diatrizoate, ioxaglate, and ioversol, respectively, at concentrations of 5%). Ionic contrast agents were potent inhibitors of <n>prothrombinase</n> function, inhibiting <n>thrombin</n> formation by >75% at contrast concentrations of 0.6% (p &#60;0.005). Ioversol inhibited <n>prothrombinase</n> to a significantly lesser extent than ionic agents. Clotting assays suggested that ioxaglate was the most potent inhibitor of <n>thrombin</n> generation in plasma despite having the least effect on <n>fibrin</n> polymerization. Contrast agents inhibited binding of <n>thrombin</n> to <n>fibrin</n>, with ionic agents producing a more potent effect than ioversol (p &#60;0.02). However, contrast agents did not inhibit <n>thrombin</n>-mediated platelet activation, had only a minor effect on inhibition of <n>thrombin</n> by <n>antithrombin III</n>, and did not affect <n>thrombin</n>-<n>hirudin</n> interactions. In summary, these studies identify specific mechanisms by which radiographic contrast agents inhibit <n>thrombin</n> formation and function -- i.e. inhibition of <n>tissue factor</n>-dependent <n>factor Xa</n> generation, inhibition of the <n>prothrombinase</n> complex, and inhibition of <n>thrombin</n> binding to <n>fibrin</n>. These findings may help to explain the reduced risk of thrombosis during coronary angioplasty associated with ionic contrast agents.
9683573>A novel human DnaJ protein, <n>hTid-1</n>, a homolog of the Drosophila tumor suppressor protein <n>Tid56</n>, can interact with the human papillomavirus type 16 <n>E7 oncoprotein</n>. We have cloned <n>hTid-1</n>, a human homolog of the Drosophila tumor suppressor protein <n>Tid56</n>, by virtue of its ability to form complexes with the human papillomavirus <n>E7 oncoprotein</n>. The carboxyl terminal cysteine-rich metal binding domain of <n>E7</n> is the major determinant for interaction with <n>hTid-1</n>. The carboxyl terminus of <n>E7</n> is essential for the functional and structural integrity of <n>E7</n> and has previously been shown to function as a multimerization domain. The <n>hTid-1</n> protein is a member of the DnaJ-family of chaperones. Its mRNA is widely expressed in human tissues, including the HPV-18-positive cervical carcinoma cell line HeLa and human genital keratinocytes, the normal host cells of the HPVs. The <n>hTid-1</n> gene has been mapped to the short arm of chromosome 16. The large tumor antigens of polyomaviruses encode functional J-domains that are important for viral replication as well as cellular transformation. The ability of HPV <n>E7</n> to interact with a cellular DnaJ protein suggests that these two viral oncoproteins may target common regulatory pathways through J-domains.
9657960>Chaperonins. The molecular chaperones are a diverse set of protein families required for the correct folding, transport and degradation of other proteins in vivo. There has been great progress in understanding the structure and mechanism of action of the chaperonin family, exemplified by Escherichia coli <n>GroEL</n>. The chaperonins are large, double-ring oligomeric proteins that act as containers for the folding of other protein subunits. Together with its co-protein <n>GroES</n>, <n>GroEL</n> binds non-native polypeptides and facilitates their refolding in an ATP-dependent manner. The action of the <n>ATPase</n> cycle causes the substrate-binding surface of <n>GroEL</n> to alternate in character between hydrophobic (binding/unfolding) and hydrophilic (release/folding). ATP binding initiates a series of dramatic conformational changes that bury the substrate-binding sites, lowering the affinity for non-native polypeptide. In the presence of ATP, <n>GroES</n> binds to <n>GroEL</n>, forming a large chamber that encapsulates substrate proteins for folding. For proteins whose folding is absolutely dependent on the full <n>GroE</n> system, ATP binding (but not hydrolysis) in the encapsulating ring is needed to initiate protein folding. Similarly, ATP binding, but not hydrolysis, in the opposite <n>GroEL</n> ring is needed to release <n>GroES</n>, thus opening the chamber. If the released substrate protein is still not correctly folded, it will go through another round of interaction with <n>GroEL</n>.
9575161>Characterization of human hect domain family members and their interaction with <n>UbcH5</n> and <n>UbcH7</n>. The hect domain protein family was originally identified by sequence similarity of its members to the C-terminal region of <n>E6-AP</n>, an E3 <n>ubiquitin</n>-protein ligase. Since the C terminus of <n>E6-AP</n> mediates thioester complex formation with <n>ubiquitin</n>, a necessary intermediate step in <n>E6-AP</n>-dependent <n>ubiquitination</n>, it was proposed that members of the hect domain family in general have E3 activity. The hect domain is approximately 350 amino acids in length, and we show here that the hect domain of <n>E6-AP</n> is necessary and sufficient for <n>ubiquitin</n> thioester adduct formation. Furthermore, the human genome encodes at least 20 different hect domain proteins, and in further support of the hypothesis that hect domain proteins represent a family of E3s, several of these are shown to form thioester complexes with <n>ubiquitin</n>. In addition, some hect domain proteins interact preferentially with <n>UbcH5</n>, whereas others interact with <n>UbcH7</n>, indicating that human hect domain proteins can be grouped into at least two classes based on their E2 specificity. Since E3s are thought to play a major role in substrate recognition, the presence of a large family of E3s should contribute to ensure the specificity and selectivity of <n>ubiquitin</n>-dependent proteolytic pathways.
9556563><n>Ciao 1</n> is a novel WD40 protein that interacts with the tumor suppressor protein <n>WT1</n>. The <n>Wilms tumor suppressor protein</n>, <n>WT1</n>, is a transcription factor capable of activating or repressing transcription of various cellular genes. The mechanisms involved in regulating the transcriptional activities of <n>WT1</n> are beginning to be unraveled. It appears that physical interactions of other cellular proteins (<n>p53</n> and <n>par-4</n>) with <n>WT1</n> can modulate the function of <n>WT1</n>. Here, we report the identification and cloning of a novel <n>WT1</n>-interacting protein termed <n>Ciao 1</n>, a member of the WD40 family of proteins. <n>Ciao 1</n> specifically interacts with <n>WT1</n> both in vitro and in vivo. This interaction alters the mobility of a <n>WT1</n>.DNA complex in gel shift assays, and results in a decrease in transcriptional activation mediated by <n>WT1</n>. <n>Ciao 1</n> does not inhibit binding of <n>WT1</n> to its consensus nucleotide sequence and does not affect the repression activity of <n>WT1</n>. Thus, <n>Ciao 1</n> appears to specifically modulate the transactivation activity of <n>WT1</n> and may function to regulate the physiological functions of <n>WT1</n> in cell growth and differentiation.
9571026>Structural and functional analysis of the 1:1 growth hormone:receptor complex reveals the molecular basis for receptor affinity. The designed G120R mutant of human growth hormone (<n>hGH</n>) is an antagonist and can bind only one molecule of the growth hormone receptor. We have determined the crystal structure of the 1:1 complex between this mutant and the receptor extracellular domain (hGHbp) at 2.6 A resolution, and used it to guide a detailed survey of the structural and functional basis for hormone-receptor recognition. The overall structure of the complex is very similar to the equivalent portion of the 1:2 complex, showing that formation of the active complex does not involve major conformational changes. However, a segment involved in receptor-receptor interactions in the 1:2 complex is disordered in this structure, suggesting that its productive conformation is stabilized by receptor dimerization.The hormone binding site of the receptor comprises a central hydrophobic patch dominated by Trp104 and Trp169, surrounded by a hydrophilic periphery containing several well-ordered water molecules. Previous alanine scanning showed that the hydrophobic "hot spot" confers most of the binding energy. The new structural data, coupled with binding and kinetic analysis of further mutants, indicate that the hot spot is assembled cooperatively and that many residues contribute indirectly to binding. Several hydrophobic residues serve to orient the key tryptophan residues; kinetic analysis suggests that Pro106 locks the Trp104 main-chain into a required conformation. The electrostatic contacts of Arg43 to <n>hGH</n> are less important than the intramolecular packing of its alkyl chain with Trp169. The true functional epitope that directly contributes binding energy may therefore comprise as few as six side-chains, participating mostly in alkyl-aromatic stacking interactions. Outside the functional epitope, multiple mutation of residues to alanine resulted in non-additive increases in affinity: up to tenfold for a hepta-alanine mutant. Contacts in the epitope periphery can therefore attenuate the affinity of the central hot spot, perhaps reflecting a role in conferring specificity to the interaction.
9563526>NMR analyses of the interactions of human <n>annexin I</n> with ATP, Ca2+, and Mg2+. Human <n>annexin I</n> is a member of the annexin family of calcium-dependent phospholipid binding proteins. The structure of an N-terminally truncated human <n>annexin I</n> (<n>delta-annexin I</n>) and its interactions with Ca2+, Mg2+, and ATP were studied at the atomic level using nuclear magnetic resonance (NMR) spectroscopy. Since <n>delta-annexin I</n> is a large protein, with a molecular weight of 35 kDa, a site-specific (carbonyl-13C, amide-15N) labeling technique was used to determine the interaction sites of <n>delta-annexin I</n> with Ca2+, Mg2+, and ATP. The 13C NMR study focused on the carbonyl carbon resonances of the histidine residues of <n>delta-annexin I</n>. We found that ATP binds to <n>delta-annexin I</n>, and that the ATP binding site is located in the 1-domain of <n>annexin I</n>. We also found that histidine-52 is involved in that site, and that the binding ratio of ATP to <n>delta-annexin I</n> is 1:1.
9488727>Association of <n>activating transcription factor 2</n> (<n>ATF2</n>) with the <n>ubiquitin</n>-conjugating enzyme <n>hUBC9</n>. Implication of the <n>ubiquitin</n>/proteasome pathway in regulation of <n>ATF2</n> in T cells. <n>activating transcription factor 2</n> (<n>ATF2</n>) is regulated by phosphorylation via the <n>Jun N-terminal kinase</n>, and its binding activity is markedly induced at late stages of T and B lymphocyte activation (Feuerstein, N., Firestein, R., Aiyer, N., Xiao, H., Murasko, D., and Cristofalo, V. (1996) J. Immunol. 156, 4582-4593). To identify proteins that interact specifically with <n>ATF2</n> in lymphocytes, the yeast two-hybrid interaction system was employed using <n>ATF2</n> cDNA as a "bait." In two separate screenings, a clone was identified that revealed a novel sequence with homology to several members of the <n>ubiquitin</n>-conjugating enzyme family. An identical sequence was recently reported as the human homolog of the yeast <n>UBC9</n>, <n>hUBC9</n>. Northern blot analysis revealed a 1.3-kilobase RNA transcript, which showed differential levels of expression in various human tissues and a moderate induction after a 48-h stimulation of peripheral blood T lymphocytes. An antibody that was generated against the bacterially expressed <n>glutathione S-transferase</n>-<n>hUBC9</n> detected a approximately 19-kDa protein, which localizes predominantly in the nuclei of T cells. Further quantitative assays using the yeast two-hybrid system confirmed a high and specific level of interaction of <n>hUBC9</n> with <n>ATF2</n> and lack of interaction with <n>lamin</n> or control vectors. Two other cyclic AMP-responsive element-binding transcription factors, <n>CREB</n> and <n>ATF1</n>, also showed significant levels of interaction with <n>hUBC9</n>. However, this interaction was severalfold lower as compared with <n>ATF2</n>. Far Western blot analysis confirmed the specific binding of <n>ATF2</n> and <n>hUBC9</n> also in vitro. Evidence is presented that indicates a physiological significance for the interaction of <n>hUBC9</n> with <n>ATF2</n>. (a) We show that <n>ATF2</n> is ubiquitinated in vivo and in vitro, and (b) <n>ATF2</n> ubiquitination in vitro is facilitated by addition of purified <n>hUBC9</n>. (c) <n>ATF2</n> is shown to undergo a proteolytic process, which is rapidly regulated upon T cell activation concomitant with induction of <n>ATF2</n> phosphorylation. (d) A proteasome inhibitor delays the down-regulation of <n>ATF2</n> phophorylation after T cell activation. Taken collectively, these results implicate a role for <n>hUBC9</n> and the <n>ubiquitin</n>/proteasome pathway in regulation of <n>ATF2</n> in T cells.
9514571>Binding of complement subcomponent <n>C1q</n> to Streptococcus pyogenes: evidence for interactions with the <n>M5</n> and <n>FcRA76</n> proteins. Binding of <n>C1q</n>, the first component of the complement system, to some human pathogens has been earlier reported. In the present study, direct binding of <n>C1q</n> to group A streptococci (GAS) of various serotypes as well as some other Gram-positive and Gram-negative species was demonstrated. The interaction between <n>C1q</n> and GAS was investigated more in detail. In hot neutral extracts of a number of GAS strains two components of 64 and 52 kDa, respectively, bound <n>C1q</n>; alkaline and SDS extracts yielded the 52 kDa component as the main <n>C1q</n>-binding substance. <n>Trypsin</n> treatment of the SDS extracts of two GAS strains suggested the <n>C1q</n>-binding component(s) to be of protein nature. <n>C1q</n>-binding material purified from the SDS extract of an avirulent strain, type T27, was separated in 12% SDS-PAGE and probed in Western blot with human <n>C1q</n> and <n>fibrinogen</n>, conjugated to <n>horse radish peroxidase</n> (<n>HRP</n>) as well as rabbit <n>IgG</n> antibodies complexed to <n>HRP</n> (PAP system). The 52 kDa component was non-reactive with <n>fibrinogen</n> or rabbit <n>IgG</n>. However, <n>C1q</n>-binding components purified from the alkaline extracts of two M-positive strains revealed strong binding of either <n>fibrinogen</n> (type M5) or both <n>fibrinogen</n> and rabbit <n>IgG</n> (type M76); the molecular mass of these components. 55 kDa and 43-40 kDa, respectively, was in agreement with the reported molecular mass of the <n>M5</n> and <n>FcRA76</n> proteins. Our findings suggest that <n>C1q</n> may interact with GAS through certain M-family proteins as well as by a so far unidentified surface factor of protein nature occurring in most GAS strains. The involvement of M-family proteins, regarded as virulence factors of these organisms, may suggest the interaction of GAS with <n>C1q</n> as biologically important.
9466043>DNA-binding activity of wild-type <n>p53</n> protein is mediated by the central part of the molecule and controlled by its C terminus. The DNA binding activity of wild type <n>p53</n> is central to its activity. The "central" part of the molecule, where most mutations appear in primary human tumors, is the actual DNA binding domain. The C-terminal part was shown to exert a negative effect on the DNA binding activity. In the present study we show that while anti-p53 antibodies recognizing the C terminus of the wild type <n>p53</n> facilitate DNA binding activity, blocking of the wild type specific epitope by specific anti-p53 antibodies, inhibited the DNA binding activity of the wild type <n>p53</n> protein. An alternatively spliced <n>p53</n> protein exhibits an augmented DNA binding activity. The fact that most <n>p53</n> mutants have lost the wild type <n>p53</n> conformation specific epitope, coupled with the observation that blocking of this site by binding specific antibodies, prevents the interaction of wild type <n>p53</n> with DNA, suggests that maintaining the correct structural conformation of this site is central for DNA binding activity. Still, the internal structure of the <n>p53</n> target and particularly the length of the sequence between the two tandem inverted repeats, is critical for protein-DNA interaction behavior.
9398285>Participation of the N-terminal region of <n>Cepsilon3</n> in the binding of human <n>IgE</n> to its high-affinity receptor <n>FcepsilonRI</n>. The binding of <n>immunoglobulin E</n> (<n>IgE</n>) to its high-affinity receptor (<n>FcepsilonRI</n>) expressed on mast cells and basophils is central to the development of an allergic reaction. Previous studies have implicated the third constant domain of <n>IgE</n>-Fc (<n>Cepsilon3</n>) as the site of the interaction with <n>FcepsilonRI</n>. We have prepared a series of site-directed mutants of human <n>IgE</n>-Fc, particularly focusing on the N-terminal "linker" region and AB loop of <n>Cepsilon3</n>. The kinetics of binding <n>IgE</n> and its Fc fragments to the immobilized receptor were determined by surface plasmon resonance (SPR), and two phases of binding were observed. We identified one mutation in the N-terminal linker region, R334S, that has a dramatic effect on binding. R334S lowers the affinity of <n>IgE</n>-Fc for <n>FcepsilonRI</n> by 120-fold, principally through an increase in the dissociation rate of the slower phase of the interaction. This mutation has a similar effect in <n>Fcepsilon3-4</n>, a truncated form of <n>IgE</n>-Fc which lacks the <n>Cepsilon2</n> domain pair, and thus it does not exert its effect through altering the quaternary structure of <n>IgE</n>-Fc, firmly implicating Arg334 as a contact residue in the complex. However R334S has no effect on the binding of <n>FcepsilonRII</n> (<n>CD23</n>), the low-affinity receptor for <n>IgE</n>, demonstrating the structural integrity of the mutated <n>IgE</n>-Fc. Circular dichroism spectroscopy and thermal stability studies further indicate that the R334S mutation does not disorder or destabilize the structure of <n>IgE</n>-Fc or <n>Fcepsilon3-4</n>. These results demonstrate the importance of the N-terminal linker region of <n>Cepsilon3</n> in the interaction of <n>IgE</n> with <n>FcepsilonRI</n>.
9420225>Amino-terminal substitutions in the <n>CCR5</n> coreceptor impair <n>gp120</n> binding and human immunodeficiency virus type 1 entry. The <n>CC-chemokine receptor</n> <n>CCR5</n> is required for the efficient fusion of macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains with the plasma membrane of <n>CD4</n>+ cells and interacts directly with the viral surface glycoprotein <n>gp120</n>. Although receptor chimera studies have provided useful information, the domains of <n>CCR5</n> that function for HIV-1 entry, including the site of <n>gp120</n> interaction, have not been unambiguously identified. Here, we use site-directed, alanine-scanning mutagenesis of <n>CCR5</n> to show that substitutions of the negatively charged aspartic acid residues at positions 2 and 11 (D2A and D11A) and a glutamic acid residue at position 18 (E18A), individually or in combination, impair or abolish <n>CCR5</n>-mediated HIV-1 entry for the ADA and JR-FL M-tropic strains and the DH123 dual-tropic strain. These mutations also impair <n>Env</n>-mediated membrane fusion and the <n>gp120</n>-<n>CCR5</n> interaction. Of these three residues, only D11 is necessary for <n>CC-chemokine</n>-mediated inhibition of HIV-1 entry, which is, however, also dependent on other extracellular <n>CCR5</n> residues. Thus, the <n>gp120</n> and <n>CC-chemokine</n> binding sites on <n>CCR5</n> are only partially overlapping, and the former site requires negatively charged residues in the amino-terminal <n>CCR5</n> domain.
9352222>Increased expression of <n>CD44</n> on astrocytoma cells induced by binding <n>myelin basic protein</n>. An astrocytoma cell line (HTB-14), expressing high amounts of a <n>CD44</n> variant compared to other astrocytoma lines was shown to bind <n>myelin basic protein</n> to a greater extent than low expressing lines in a concentration-dependent manner. The <n>CD44</n> variant expressed by HTB-14 cells was determined to migrate in sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular mass of 100 kDa compared to that from white matter which had a molecular mass of 80 kDa. The most cationic component of <n>myelin basic protein</n> (<n>MBP</n>), (component 1) bound more avidly than the least cationic isomer (component 8). Internalization of <n>MBP</n> was demonstrated by immunogold electron microscopy and was localized to the perinuclear area with some gold particles in the cytoplasm but not near the plasma membrane. Colocalization with <n>glial fibrillary acid protein</n> suggested an interaction between these two molecules. Binding and internalization of <n>MBP</n> was accompanied by an increase in <n>CD44</n> as determined by quantitation of gold particles and the measurement of <n>CD44</n> by sandwich enzyme-linked immunosorbent assay. The implication of these studies for the mechanism of demyelination is discussed.
9334241>The <n>Groucho</n>/<n>transducin</n>-like enhancer of split transcriptional repressors interact with the genetically defined amino-terminal silencing domain of <n>histone H3</n>. <n>Groucho</n> is a transcriptional repressor implicated in Notch signaling and involved in neural development and segmentation in Drosophila. We are investigating the molecular mechanisms underlying the functions of <n>Groucho</n> and its mammalian homologs, the <n>transducin-like Enhancer of split</n> (<n>TLE</n>) proteins. We report that <n>Groucho</n>/<n>TLEs</n> are associated with chromatin in live cells and that they co-purify with isolated histones. Affinity chromatography and far Western blotting studies show further that native <n>Groucho</n>/<n>TLE</n> proteins interact specifically with <n>histone H3</n> and not with other core histones. This interaction is mediated by the <n>H3</n> amino-terminal domain previously shown by genetic analysis in yeast to be essential for the role of <n>H3</n> in transcriptional silencing. We also demonstrate that <n>Groucho</n>/<n>TLEs</n> form oligomeric structures in vivo. These combined findings suggest that transcription complexes containing <n>Groucho</n>/<n>TLEs</n> may associate with chromatin through interactions with the amino terminus of <n>histone H3</n> and that these interactions may be propagated along the chromosome due to the ability of <n>Groucho</n>/<n>TLE</n>s to participate in higher order structures.
9311810>Herpes simplex virus 1 alpha regulatory protein <n>ICP0</n> interacts with and stabilizes the cell cycle regulator <n>cyclin D3</n>. The herpes simplex virus 1 (HSV-1) <n>infected-cell protein 0</n> (<n>ICP0</n>) has the characteristics of a promiscuous transactivator of genes introduced into cells by infection or transfection. To identify cellular proteins interacting with <n>ICP0</n>, we used a domain of exon II of <n>ICP0</n> that is known to be crucial for regulatory function of the protein as bait in the yeast two-hybrid screen. Our results were as follows. (i) A cDNA in a positive yeast colony was found to encode <n>cyclin D3</n>, a cell cycle regulator of G1 phase. (ii) A purified chimeric protein consisting of <n>glutathione S-transferase</n> (<n>GST</n>) fused to <n>cyclin D3</n> specifically formed complexes with <n>ICP0</n> contained in HSV-1-infected cell lysate. (iii) To enhance the expression of <n>cyclin D3</n>, the gene was inserted into the viral genome and overexpressed in infected cells. The overexpressed <n>cyclin D3</n> colocalized with <n>ICP0</n> in nuclear structures characteristic of ND10 and which earlier have been reported to contain <n>ICP0</n>. (iv) The accumulation of <n>cyclin D3</n> protein in Vero cells infected with an <n>alpha0</n> deletion mutant was reduced relative to that of cells infected with wild-type virus or a recombinant virus in which the deleted <n>alpha0</n> sequences were restored. (v) Lysates of Spodoptera frugiperda Sf9 cells doubly infected with baculoviruses genetically engineered to express <n>cyclin D3</n> and <n>cyclin-dependent kinase 4</n> (<n>CDK4</n>) phosphorylated <n>GST</n> fused to <n>retinoblastoma protein</n> (<n>GST</n>-<n>pRb</n>) but did not phosphorylate the <n>GST</n>-<n>alpha0</n>(20-241) or <n>GST</n>-<n>alpha0</n>(543-768) fusion protein or immunoprecipitated <n>ICP0</n> proteins. Moreover, the chimeric <n>GST</n>-<n>ICP0</n>(exon II) protein shown to bind <n>cyclin D3</n> had no effect on the activity of the kinase on <n>GST</n>-<n>pRb</n> when added to mixtures of lysates of Sf9 cells which coexpressed <n>cyclin D3</n> and <n>CDK4</n>. These results indicate that <n>ICP0</n> interacts with, colocalizes with, and stabilizes the <n>cyclin D3</n> cell cycle regulator and does not affect its interaction with the cyclin-dependent kinase.
9261175>Association of the <n>T-cell protein tyrosine phosphatase</n> with <n>nuclear import factor p97</n>. Alternative splicing of the <n>T-cell protein tyrosine phosphatase</n> (<n>TCPTP</n>) transcript generates two forms of the enzyme that differ at their extreme C termini: a 48-kDa endoplasmic reticulum-associated form and a 45-kDa nuclear form. By affinity chromatography, using <n>GST</n>-<n>TCPTP</n> fusion proteins, we have isolated three cytoplasmic proteins of 120, 116, and 97 kDa that interact with <n>TCPTP</n>. The <n>p120</n> protein associated with residues 377-415 from the C terminus of the 48-kDa form of <n>TCPTP</n>, whereas the recognition site for <n>p97</n> and <n>p116</n> was mapped to residues 350-381 encompassing the <n>TCPTP</n> nuclear localization sequence (NLS). The <n>TCPTP</n> NLS was shown to be bipartite, requiring basic residues 350-358 (basic cluster I) and 377-381 (basic cluster II), the sites of interaction with <n>p97</n> and <n>p116</n>, for efficient nuclear translocation. The interaction between <n>p97</n>, <n>p116</n>, and the <n>TCPTP</n> NLS appeared unique in that these proteins did not form a stable interaction with the classical NLS of <n>SV40 large T antigen</n> or the standard bipartite NLS of <n>nucleoplasmin</n>. Sequence analysis of <n>p97</n> identified it as the <n>nuclear import factor p97</n> (<n>importin</n>-beta), which is an essential component of the nuclear import machinery. In assays in vitro in permeabilized cells, <n>p97</n> was necessary but not sufficient for optimal nuclear import of <n>TCPTP</n>. We found that <n>TCPTP</n> co-immunoprecipitated with the <n>nuclear import factor p97</n> from cell lysates and that purified recombinant <n>p97</n> and <n>TCPTP</n> interacted directly in vitro. These results indicate selectivity in the binding of <n>p97</n> and <n>p116</n> to the <n>TCPTP</n> NLS and suggest that <n>p97</n> may mediate events that are distinct from the classical nuclear import process. Moreover, these results demonstrate that the C-terminal segment of <n>TCPTP</n> contains docking sites for interaction with proteins that may function to target the enzyme to defined intracellular locations and in the process regulate <n>TCPTP</n> function.
9202023>Characterization of the WW domain of human <n>yes-associated protein</n> and its polyproline-containing ligands. We had previously identified the WW domain as a novel globular domain that is composed of 38-40 semiconserved amino acids and is involved in mediating protein-protein interaction. The WW domain is shared by proteins of diverse functions including structural, regulatory, and signaling proteins in yeast, nematode, and mammals. Functionally it is similar to the Src homology 3 domain in that it binds polyproline ligands. By screening a 16-day mouse embryo expression library, we identified two putative ligands of the WW domain of <n>Yes kinase-associated protein</n> which we named <n>WW domain-binding proteins 1</n> and <n>2</n>. These proteins interacted with the WW domain via a short proline-rich motif with the consensus sequence of four consecutive prolines followed by a tyrosine. Herein, we report the cDNA cloning and characterization of the human orthologs of <n>WW domain-binding proteins 1</n> and <n>2</n>. The products encoded by these cDNA clones represent novel proteins with no known function. Furthermore, these proteins show no homology to each other except for a proline-rich motif. By fluorescence in situ hybridization on human metaphase chromosomes, we mapped the human genes for <n>WW domain-binding proteins 1</n> and <n>2</n> to chromosomes 2p12 and 17q25, respectively. In addition, using site-directed mutagenesis, we determined which residues in the WW domain of <n>Yes kinase-associated protein</n> are critical for binding. Finally, by synthesizing peptides in which the various positions of the four consecutive proline-tyrosine motif and the five surrounding residues were replaced by all possible amino acid residues, we further elucidated the binding requirements of this motif.
9209500>Characterization of mouse <n>ALCAM</n> (<n>CD166</n>): the <n>CD6</n>-binding domain is conserved in different homologs and mediates cross-species binding. Activated leukocyte cell adhesion molecule (<n>ALCAM</n>; <n>CD166</n>) is a member of the immunoglobulin gene superfamily (IgSF) which is expressed by activated leukocytes and thymic epithelial cells and is a ligand for the lymphocyte antigen <n>CD6</n>. Herein, we report on the isolation and characterization of cDNA clones encoding mouse <n>ALCAM</n> (<n>mALCAM</n>). Comparison of the predicted amino acid sequence of <n>mALCAM</n> and human <n>ALCAM</n> (<n>hALCAM</n>) showed an overall identity of 93%. Binding studies with truncated forms of the extracellular region of <n>mALCAM</n> showed that the <n>CD6</n> binding site is located in the N-terminal Ig-like domain and that <n>mALCAM</n> is capable of binding both human and mouse <n>CD6</n>. Mutagenesis studies on <n>hALCAM</n> suggested that residues critical for <n>CD6</n> binding map to the predicted A'GFCC'C beta-sheet of <n>ALCAM's</n> N-terminal binding domain. Residue differences in the N-terminal domains of <n>mALCAM</n> and <n>hALCAM</n> were analyzed with the aid of a molecular model of <n>ALCAM</n>. All residues critical for <n>CD6</n> binding are conserved in both <n>mALCAM</n> and <n>hALCAM</n>, whereas residue differences map to the predicted BED face which is opposite the <n>CD6</n> binding site on <n>hALCAM</n>. These findings provide a molecular rationale for the observed cross-species <n>CD6</n>/<n>ALCAM</n> interaction and the apparent inability to generate monoclonal antibodies (mAb) against the <n>CD6</n> binding site. RNA blot analysis showed that mRNA transcripts encoding <n>mALCAM</n> are expressed in the brain, lung, liver, and the kidney, as well as by activated leukocytes and a number of cell lines. A rat mAb specific for <n>mALCAM</n> was produced and by two-color immunofluorescence studies was shown to bind to both activated <n>CD4</n>+ and CD8+ T cells.
9144171>Protein bind