Recombinant soluble human alpha 3 beta 1 integrin: purification, processing, regulation, and specific binding to laminin-5 and invasin in a mutually exclusive manner

Using insect cells, we expressed large quantities of soluble human integrin alpha 3 beta 1 ectodomain heterodimers, in which cytoplasmic and transmembrane domains were replaced by Fos and Jun dimerization motifs. In direct ligand binding assays, soluble alpha 3 beta 1 specifically bound to laminin-5 and laminin-10, but not to laminin-1, laminin-2, fibronectin, various collagens, nidogen, thrombospondin, or complement factors C3 and C3b. Soluble alpha 3 beta1 integrin also bound to invasin, a bacterial surface protein, that mediates entry of Yersinia species into the eukaryotic host cell. Invasin completely displaced laminin-5 from the alpha 3 beta 1 integrin, suggesting sterically overlapping or identical binding sites. In the presence of 2 mM Mg2+, alpha 3 beta 1's binding affinity for invasin (Kd = 3.1 nM) was substantially greater than its affinity for laminin-5 (Kd > 600 nM). Upon addition of 1 mM Mn2+, or activating antibody 9EG7, binding affinity for both laminin-5 and invasin increased by about 10-fold, whereas the affinity decreased upon addition of 2 mM Ca2+. Thus, functional regulation of the purified soluble integrin alpha 3 beta 1 ectodomain heterodimer resembles that of wild-type membrane-anchored beta 1 integrins. The integrin alpha 3 subunit was entirely cleaved into disulfide-linked heavy and light chains, at a newly defined cleavage site located C-terminal of a tetrabasic RRRR motif. Within the alpha 3 light chain, all potential N-glycosylation sites bear N-linked mannose-rich carbohydrate chains, suggesting an important structural role of these sugar residues in the stalk-like region of the integrin heterodimer. In conclusion, studies of our recombinant alpha 3 beta 1 integrin have provided new insights into alpha 3 beta1 structure, ligand binding function, specificity, and regulation.     

A cyclic hexapeptide is a potent antagonist of alpha 4 integrins

The alpha 4 integrins mediate leukocyte adhesion to specific counter-receptors, including vascular cell adhesion molecule-1 (VCAM-1), the fibronectin splice variant containing connecting segment 1 (CS1), and mucosal addressin cell adhesion molecule-1. A series of cyclized peptides based on the LDV sequence of CS1 were synthesized and assayed for inhibition of alpha 4 integrin binding. The most potent peptide, C*WLDVC* (where * indicates disulfide-linked residues), inhibited alpha 4 beta 1-dependent binding of lymphocytes to VCAM-1 and CS1 with half-maximal inhibition achieved at 1 to 3 microM of peptide. The peptide proved more potent when the lymphocytes were activated with 1 mM MnCl2; half-maximal inhibition was reached at 0.4 and 0.05 microM for VCAM-1 and CS1, respectively. This represents a 100- to 800-fold increase in potency over a linear CS1 peptide in these same assays. C*WLDVC* also inhibited alpha 4 beta 7-dependent lymphocyte binding to the ligands mucosal addressin cell adhesion molecule-1, VCAM-1 and CS1. Immunoprecipitation of radiolabeled integrin indicated that the peptide could bind alpha 4 beta 1 and alpha 4 beta 7 directly and elute alpha 4 beta 1 from a CS1-conjugated agarose resin. The peptide showed selectivity for alpha 4 integrins in that it effectively inhibited alpha 4 beta 1-dependent, but not alpha 5 beta 1-dependent, binding of cells to intact fibronectin. Due to its small size and potency, C*WLDVC* may serve as a useful tool for the study of alpha 4 integrin biology and the development of small molecule therapeutics.     

Identification of ligand binding sites on integrin alpha4beta1 through chemical cross-linking

We have used chemical cross-linking to identify sequences in integrin alpha4beta1 that are involved in its interactions with ligands. A recently described leucine-aspartic acid-valine (LDV)-based small molecule inhibitor of alpha4beta1 (BIO-1494), that contained a single reactive amino group for targeting the cross-linking, was used for these studies. The specificity of the interaction was defined by (i) the ability to block the interaction with a competitive inhibitor lacking the reactive group, (ii) the absolute requirement of divalent cations for cross-linking, and (iii) the lack of cross-linking to the functionally related integrin alpha4beta7. With ANB-NOS as the cross-linker, only the beta1 chain was labeled with BIO-1494, while with the more flexible cross-linker DSS both the alpha4 and beta1 chains were modified. Similar results were obtained when cross-linking was performed on K562 cells expressing alpha4beta1 but not on K562 cells expressing alpha2beta1. The site of cross-linking on the beta1 chain was localized by CNBr peptide mapping within residues 130-146, a region that contains the putative metal binding site DXSXS and for which analogous data had been generated with RGD binding to integrin alphaIIbbeta3. The striking similarity between the data we generated for an LDV ligand and published data for the RGD family supports the notion of a common ligand binding pocket formed by both integrin chains. The cross-linking strategy developed here should serve as a useful tool for studying alpha4beta1 function.     

Potent alpha 4 beta 1 peptide antagonists as potential anti-inflammatory agents

The migration, adhesion, and subsequent extravasation of leukocytes into inflamed tissues contribute to the pathogenesis of a variety of inflammatory diseases including asthma, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis. The integrin adhesion receptor alpha 4 beta 1 expressed on leukocytes binds to the extracellular matrix protein fibronectin and to the cytokine inducible vascular cell adhesion molecule-1 (VCAM-1) at inflamed sites. Binding of alpha 4 beta 1 to VCAM-1 initiates firm adhesion of the leukocyte to the vascular endothelium followed by extravasation into the tissue. Monoclonal antibodies generated against either alpha 4 beta 1 or VCAM-1 can moderate this inflammatory response in a variety of animal models. Recently peptides containing a consensus LDV sequence based on the connecting segment-1 (CS-1) of fibronectin and cyclic peptides containing an RCD motif have shown promise in modulating leukocyte migration and inflammation presumably by blocking the interaction of alpha 4 beta 1 with VCAM-1. Here we describe novel, highly potent, cyclic peptides that competitively inhibit alpha 4 beta 1 binding to VCAM-1 and fibronectin at sub nanomolar concentrations. The structure of a representative analog was determined via NMR spectroscopy and used to facilitate optimization of peptide leads. The peptides discussed here utilize similar functional groups as the binding epitope of VCAM-1, inhibit lymphocyte migration in vivo, and are highly selective for alpha 4 beta 1. Furthermore the structure--activity relationships described here have provided a template for the structure-based design of small molecule antagonists of alpha 4 beta 1-mediated cell adhesion processes.     

Activation of the fibrinogen binding site on platelets isolated from a patient with the Strasbourg I variant of Glanzmann's thrombasthenia

One proposed ligand binding site on platelet integrin alpha IIb beta 3 is the region of the beta 3 subunit encompassing amino acids 211-221. However, we recently showed that synthetic peptides corresponding to amino acids 211-221 inhibit fibrinogen binding to alpha IIb beta 3 by binding to alpha IIb beta 3 and not to fibrinogen. In this study, we show that AP6, a monoclonal antibody (MoAb) directed against amino acids 214-221 of beta 3, bound to immobilized active alpha IIb beta 3 but did not inhibit fibrinogen binding to the complex. We then determined whether nonfunctional alpha IIb beta 3 on platelets with a beta 3 Arg-214-->Trp mutation (Strasbourg I variant of Glanzmann's thrombasthenia or GTV) could be induced to aggregate after treatment with dithiothreitol (DTT). DTT has been shown to expose the fibrinogen receptor on normal platelets. DTT treatment of GTV platelets did result in the formation of the fibrinogen binding site as indicated by the binding of pI-55, an MoAb that only binds to the activated form of alpha IIb beta 3. Furthermore, DTT-treated GTV platelets aggregated in the presence of fibrinogen and divalent cations. This aggregation was inhibited by EDTA, RGDS, and the selective alpha IIb beta 3 antagonist, Ro 43-5054. These data show that Arg-214 of beta 3 is not required for fibrinogen binding or for platelet aggregation. However, this amino acid appears to be critical for the formation and for the maintenance of the correct tertiary structure of the fibrinogen binding site on alpha IIb beta 3.     

Effects of Asp-369 and Arg-372 mutations on heme environment and function in human endothelial nitric-oxide synthase

Eight polar amino acid residues in the putative substrate-binding region from Thr-360 to Val-379 in human endothelial nitric-oxide synthase (eNOS) (Thr-360, Arg-365, Cys-368, Asp-369, Arg-372, Tyr-373, Glu-377, and Asp-378) were individually mutated. Only two of these residues, Asp-369 and Arg-372, were found to be essential for enzyme activity. A further series of mutants was generated by replacing these two residues with various amino acids and the mutant proteins were expressed in a baculovirus system. Mutant eNOS had a very low L-citrulline formation activity with the exception of D369E and R372K, which retained 27% and 44% of the wild-type enzyme activity, respectively. Unlike the wild-type enzyme, all mutants except D369E, R372K, and R372M had a low spin heme (Soret peak at 416 nm). All the Asp-369 mutants had higher Kd values for L-arginine (1-10 mM) than wild-type eNOS (0.4 microM) and an unstable heme-CO complex, and except for D369E, had a very low (6R)-5,6,7, 8-tetrahydro-L-biopterin (BH4) content. In contrast, each of Arg-372 mutants retained a considerable amount of BH4, had a moderate reduction in L-arginine affinity, and had a more stable heme-CO complex. 1-Phenylimidazole did not bind to wild-type eNOS heme, but bound to all Asp-369 and Arg-372 mutants (Kd ranged from 10 to 65 microM) except R372K. Heme spin-state changes caused by binding of 3, 5-lutidine appeared to depend on both charge and size of the side chains of residues 369 and 372. Furthermore, all Asp-369 and Arg-372 mutants were defective in dimer formation. These results suggest that residues Asp-369 and Arg-372 in eNOS play a critical role in oxygenase domain active-site structure and activity.     

Asymmetrical increases in dopamine turn-over in the nucleus accumbens and lack of changes in locomotor responses following unilateral dopaminergic depletions in the entorhinal cortex

Purified integrin alpha v beta 3 was used in solid-phase binding studies with chimeric hepatitis B cores which carry the RGD-containing loop of VP1 protein of the foot-and-mouth disease virus (FMDV). High levels of specific binding between the integrin and the particles were detected by enzyme-linked immunosorbent assays. The binding was Mn2+ cation dependent and could be competed with fibronectin, vitronectin, and the peptide GRGDSPK. Particles in which the RGD motif had been mutated to RGE failed to bind, indicating that the chimeric cores bound specifically to the ligand binding site of integrin alpha v beta 3. Electron micrographs showed several individual alpha v beta 3 molecules bound to the surface of each chimeric particle. Collectively, these data constitute firm evidence that the RGD-containing loop of FMDV is critical for binding to alpha v beta 3 and provide support for identification of alpha v beta 3 as a potential cellular receptor for FMDV.     

Studies in vitro on the role of alpha v and beta 1 integrins in the adhesion of human dermal fibroblasts to provisional matrix proteins fibronectin, vitronectin, and fibrinogen

Fibroblasts that migrate into a wound during the early stages of repair use cell surface integrins to interact with extracellular molecules as they move away from the interstitial matrix of normal tissue and into the provisional matrix of the wound. Therefore, to understand a critical phase of wound healing, it is necessary to understand the details of integrin involvement. Normal adult human dermal fibroblasts in culture express many receptors for the provisional matrix proteins fibronectin, vitronectin, and fibrinogen, including the integrins alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, alpha v beta 1, alpha v beta 3, and alpha v beta 5. We used quantitative flow cytometry to estimate the relative numbers of these receptors and immunoprecipitation to confirm the expression of alpha v beta 1. Adult human dermal fibroblasts primarily use beta 1 integrins, alpha 4 beta 1, alpha 5 beta 1, and possibly alpha v beta 1, for attachment to fibronectin. alpha v beta 3 and perhaps other integrins containing the alpha v subunit serve fibroblasts as secondary or auxiliary receptors for fibronectin. In contrast, these cells use alpha v integrins but probably not beta 1 integrins for attachment to vitronectin. alpha v beta 3 and alpha v beta 5 apparently act in concert to mediate attachment to vitronectin, and these two integrins may perform different functions during wound repair. Fibroblast adhesion to certain preparations of fibrinogen occurs, at least partially, through the small amount of fibronectin present in the preparations. Fibroblast attachment to fibrinogen purified free of fibronectin also occurs, and that was demonstrated with a sensitive new assay called electrical cell-substrate impedance sensing. Fibroblast attachment to pure fibrinogen can be inhibited by RGD peptide, suggesting that integrins are involved.     

Heterodimerization preferences of thyroid hormone receptor alpha isoforms

Thyroid hormone receptors (TRs) are members of the steroid hormone receptor superfamily and are encoded by two different genes, alpha and beta. Three isoforms (alpha 1, alpha 2, and alpha 3) are created by alternative splicing of the TR alpha gene. In TR alpha 2 and alpha 3, the distal half of the putative dimerization domain is disrupted and the carboxy terminus of the protein is substituted with different amino acids. To evaluate the properties of these alterations in the dimerization region, DNA binding and dimerization of TR alpha isoforms were studied by electrophoretic mobility shift assays. TR alpha 1 formed a monomer or a homodimer on certain thyroid hormone responsive elements (TREs), whereas TR alpha 2 and alpha 3 did not bind effectively to any of the TREs studied. TR alpha 1 formed a heterodimer with 9-cis retinoic acid receptor alpha (RXR alpha) on all TREs studied. Although TR alpha 2 did not bind as a homodimer, it did bind as a heterodimer with RXR alpha to DR4 and MHC-TRE. TR alpha 3 bound as a heterodimer to a broader repertoire of TREs, including DR4, MHC, ME, and F2-TRE. These results indicate that the alterations in the dimerization region in TR alpha 2 and alpha 3 abrogated homodimer binding, but differentially affected heterodimerization with RXR alpha on various TREs.     

Distribution, prevalence, and drug binding profile of gamma-aminobutyric acid type A receptor subtypes differing in the beta-subunit variant

Native gamma-aminobutyric acid type A (GABAA) receptors containing different beta-subunit variants were identified immunobiochemically with antisera recognizing selectively the beta 1-, beta 2-, or beta 3-subunit. As determined by immunoprecipitation, the beta 2-subunit was present in 55-60% of GABAA receptors, while only minor receptor populations contained the beta 1-subunit (16-18%) or the beta 3-subunit (19-25%). Since the sum of these values amounts to about 100%, it is concluded that GABAA receptors largely contain only a single type of beta-subunit. Pharmacologically, receptors containing the beta 2-subunit differed from those containing the beta 1- or beta 3-subunit by their differential affinities for benzodiazepine receptor ligands. The subunit composition was analyzed biochemically in receptors immunoprecipitated by the beta 2-subunit antiserum. The beta 2-subunit was preferentially associated with the alpha 1-subunit (rarely with the alpha 2-subunit) and with the gamma 2-subunit; negligible or no immunoreactivity was detected for the alpha 3-, alpha 5-, or beta 1-subunit. A stringent co-expression of alpha 1- and beta 2-subunits was confirmed by double immunofluorescence staining on the cellular level. Neurons expressing the beta 3-subunit immunoreactivity were largely double labeled by the alpha 2-subunit antiserum. Thus, the subunit combinations alpha 1 beta 2 gamma 2 and alpha 2 beta 3 gamma 2 represent two main GABAA receptor subtypes, which together amount to 75-85% of the diazepam-sensitive GABAA receptors.     

Identification of a domain on the integrin alpha5 subunit implicated in cell spreading and signaling

The alpha5 beta1 integrin is a cell surface receptor for fibronectin implicated in several cellular activities including cell proliferation, differentiation, and migration. The primary site at which the alpha5 beta1 integrin interacts with fibronectin is the RGD (Arg-Gly-Asp) amino acid sequence. In general, the sites on the integrin alpha subunits involved in ligand binding are not well characterized. Based on previous cross-linking studies, sequence alignment, predicted conformation, and intron-exon boundaries, we identified a 144-residue region (positions 223-367) on the alpha5 subunit as a putative binding region and divided it into four subdomains named domains I, II, III, and IV. Chimeric receptors were prepared in which sequences on the alpha5 subunit were exchanged with the corresponding sequences on the alpha6 subunit, which is specific for laminin and does not bind via an RGD sequence. The mutated human alpha5 integrin gene was transfected into CHO B2 cells, which are deficient in alpha5 expression. Only chimeras of domain III or IV express on the cell surface. Both of these chimeras decreased the adhesion, spreading, focal adhesion assembly, and migration on fibronectin. The adhesion of the chimeric receptors to fibronectin remained sensitive to the RGD peptide, and antibodies that inhibit interaction with the fibronectin synergy site and RGD loop remain inhibitory for the chimeras, indicating that our chimeras do not inhibit binding to either the RGD or synergy sites. Finally, the affinity of soluble fibronectin to cells via the alpha5 beta1 receptor decreased only about 3-fold. This decrease is substantially less than the observed effects on migration and spreading, which were not altered by changes in substrate concentration. Thus, the alteration in binding sites does not easily account for the changes in cell spreading and focal adhesion assembly. The tyrosine phosphorylation and focal adhesion assembly that are seen when cells expressing the wild type alpha5 receptor adhere to fibronectin were inhibited in cells expressing the chimeric receptors. Therefore, our results suggest that the chimeras of these domains likely interrupt alpha5-mediated conformational signaling.     

Identification of N,N'-dicyclohexylcarbodiimide-reactive glutamic and aspartic acid residues in Escherichia coli transhydrogenase and the exchange of these by site-specific mutagenesis

Pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was investigated with respect to the role of glutamic and aspartic acid residues reactive to N,N'-dicyclohexylcarbodiimide (DCCD) and potentially involved in the proton-pumping mechanism of the enzyme. The E. coli transhydrogenase consists of an alpha (510 residues) and a beta (462 residues) subunit. DCCD reacts with the enzyme to inhibit catalytic activity and proton pumping. This reagent modifies Asp alpha 232, Glu alpha 238, and Glu alpha 240 as well as amino acid residue(s) in the beta subunit. Using the cloned and overexpressed E. coli transhydrogenase genes (Clarke, D. M., and Bragg, P. D. (1985) J. Bacteriol. 162, 367-373), Asp alpha 232 and Glu alpha 238 were replaced independently by site-specific mutagenesis. In addition, Asp alpha 232, Glu alpha 238, and Glu alpha 240 were replaced to generate triple mutants. The specific catalytic activities of the mutant transhydrogenases alpha D232N, alpha D232E, alpha D232K, alpha D232H, alpha E238K, and alpha E238Q as well as of the triple mutants alpha D232N, alpha E238Q, alpha E240Q and alpha D232H, alpha E238Q, alpha E240Q were in the range of 40-90% of the wild-type activity. Proton-pumping activity was present in all mutants. Examination of the extent of subunit modification by [14C]DCCD revealed that the label was still incorporated into both alpha and beta subunits in the Asp alpha 232 mutants, but that the alpha subunit was not labeled in the triple mutants. Catalytic and proton-pumping activities were nearly insensitive to DCCD in the triple mutants. This suggests that loss of catalytic and proton-pumping activities is associated with modification of the aspartic and glutamic acid residues of the alpha subunit. In the presence of the substrate NADPH, the rate of modification of the beta subunit by [14C]DCCD was increased, and there was a greater extent of enzyme inactivation. By contrast, NADH and 3-acetylpyridine-NAD+ protected the catalytic activity of the transhydrogenase from inhibition by DCCD. The protection was particularly marked in the E238Q and E238K mutants. It is concluded that the Asp alpha 232, Glu alpha 238, and Glu alpha 240 residues are not essential for catalytic activity or proton pumping. The inactivation by DCCD is likely due to the introduction of a sterically hindering group that reacts with the identified acidic residues close to the NAD(H)-binding site.     

Echistatin inhibits the migration of murine prefusion osteoclasts and the formation of multinucleated osteoclast-like cells

The vitronectin receptor alpha(v)beta3 is highly expressed in osteoclasts and was shown to play a critical role in osteoclast function in vivo. The objective of this study was to examine the role of alpha(v)beta3 integrin in osteoclast formation in vitro using the inhibitory disintegrin echistatin, an RGD-containing snake venom. We documented by immunocytochemistry and Northern blot analysis that during murine osteoclast-like cell (OCL) formation in a coculture of mouse osteoblastic MB1.8 cells and bone marrow cells there is increased expression of the alpha(v) and beta3 integrin subunits. Echistatin binds preferentially to the membrane fraction of isolated enriched OCLs (IC50 = 0.6 nM), and this binding is inhibited by vitronectin receptor-blocking polyclonal antibodies. Additionally, cross-linking of radiolabeled echistatin to OCLs, followed by immunoprecipitation with antibodies to vitronectin or fibronectin receptors, shows that alpha(v)beta3 integrin is the predominant receptor for echistatin in this system. In this coculture, echistatin completely inhibits the formation of multinucleated OCLs, but not that of mononuclear prefusion OCLs (pOCs). This inhibition is RGD and dose dependent (IC50 = 0.7 nM). We tested the hypothesis that inhibition of OCL formation may be due to interference with pOC migration and found that echistatin inhibited macrophage colony-stimulating factor-induced migration and fusion of pOCs (IC50 = 1 and 0.6 nM, respectively). Echistatin inhibition of pOCs migration and fusion is also RGD dependent. These results suggest that the integrin alpha(v)beta3 plays a role in pOC migration, which can explain the inhibitory effect of echistatin on multinucleated osteoclast formation in vitro.     

The thrombospondin receptor CD47 (IAP) modulates and associates with alpha2 beta1 integrin in vascular smooth muscle cells

The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a beta1 integrin, rather than alphav beta3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to alpha2 and beta1 integrin subunits and to IAP. A combination of antialpha2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antialphav beta3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between alpha2 beta1 and IAP was detected by the coimmunoprecipitation of both alpha2 and beta1 integrin subunits with IAP. These data suggest that IAP can associate with alpha2 beta1 integrin and modulate its function.     

Post-translational modifications of alpha5beta1 integrin by glycosaminoglycan chains. The alpha5beta1 integrin is a facultative proteoglycan

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.     

Orientation of the Ig domains of CD8 alpha beta relative to MHC class I

The cell surface glycoprotein CD8 functions as a coreceptor with the TCR for interaction with MHC class I. The cocrystal structure of the CD8 alpha alpha-MHC complex showed that one CD8 Ig domain provided the majority of the contact with MHC class I and that residue R4 of that domain contacted the alpha2 domain of MHC class I. We previously showed by mutational analysis that this residue was critical for binding to MHC class I. To determine which of the Ig domains for the CD8 alpha beta heterodimer would make the most contact with class I MHC, we expressed single-chain or dimeric forms of CD8 on COS-7 cells and measured the adhesion of MHC class I positive cells. We found that when one of the R4 residues was mutated in a CD8 alpha alpha homodimer binding comparable to that of wild type was observed, whereas a double R4 mutant severely impaired binding. However, when mutant CD8 alpha (R4K) was coexpressed with wild-type CD8 beta, binding was not observed. These results support the model in which it is CD8 alpha, not CD8 beta, that is making the most of the contact with MHC class I, including the alpha 2 domain. In addition, they demonstrate that a single-chain form of CD8 alpha alpha can bind to MHC class I.     

Identification of amino acid residues that form part of the ligand-binding pocket of integrin alpha5 beta1

Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel ligand peptide for integrin alpha5 beta1, which blocks alpha5 beta1-mediated cell adhesion to fibronectin (Koivunen, E., Wang, B., and Ruoslahti, E. (1994) J. Cell Biol. 124, 373-380). Here we have localized the binding site for RRETAWA on alpha5 beta1 using inhibitory monoclonal antibodies (mAbs) and site-directed mutagenesis. A cyclic peptide containing this sequence (*CRRETAWAC*) had little effect on the binding of most anti-alpha5 and anti-beta1 mAbs to alpha5 beta1 but completely blocked binding of the anti-alpha5 mAb 16 in a directly competitive manner. Hence, the binding site of RRETAWA appears to closely overlap with the epitope of mAb 16. *CRRETAWAC* also acted as a direct competitive inhibitor of the binding of Arg-Gly-Asp (RGD)-containing fibronectin fragments to alpha5 beta1, suggesting that the binding site for RRETAWA is also closely overlapping with that for RGD. However, differences between the binding sites of RRETAWA and RGD were apparent in that (i) RGD peptides allosterically inhibited the binding of mAb 16 to alpha5 beta1, and (ii) several mAbs that perturbed binding of alpha5 beta1 to RGD had little effect on binding of alpha5 beta1 to RRETAWA. A double mutation in alpha5 (S156G/W157S) blocked the interaction of both RRETAWA and mAb 16 with alpha5 beta1 but had no effect on fibronectin binding or on the binding of other anti-alpha5 mAbs. Ser156-Trp157 is located near the apex of a putative loop region on the upper surface of a predicted beta-propeller structure formed by the NH2-terminal repeats of alpha5. Our findings suggest that this sequence forms part of the ligand-binding pocket of alpha5 beta1. Furthermore, as Ser156-Trp157 is unique to the alpha5 subunit, it may be responsible for the specific recognition of RRETAWA by alpha5 beta1.