Integrin alpha 2 cytoplasmic domain deletion effects: loss of adhesive activity parallels ligand-independent recruitment into focal adhesions.

Chinese hamster ovary (CHO) cells transfected with the integrin alpha 2 subunit formed a stable VLA-2 heterodimer that mediated cell adhesion to collagen. Within CHO cells spread on collagen, but not fibronectin, wild-type alpha 2 subunit localized into focal adhesion complexes (FACs). In contrast, alpha 2 with a deleted cytoplasmic domain was recruited into FACs whether CHO cells were spread on collagen or fibronectin. Thus, as previously seen for other integrins, the alpha 2 cytoplasmic domain acts as a negative regulator, preventing indiscriminate integrin recruitment into FACs. Notably, ligand-independent localization of the VLA-2 alpha 2 subunit into FACs was partially prevented if only one or two amino acids were present in the alpha 2 cytoplasmic domain (beyond the conserved GFFKR motif) and was completely prevented by four to seven amino acids. The addition of two alanine residues (added to GFFKR) also partially prevented ligand-independent localization. In a striking inverse correlation, the same mutants showing increased ligand-independent recruitment into FACs exhibited diminished alpha 2-dependent adhesion to collagen. Thus, control of VLA-2 localization may be closely related to the suppression of cell adhesion to collagen. In contrast to FAC localization and collagen adhesion results, VLA-2-dependent binding and infection by echovirus were unaffected by either alpha 2 cytoplasmic domain deletion or exchange with other cytoplasmic domains.     

VLA-2 (α2β1) Integrin Promotes Rotavirus Entry into Cells but Is Not Necessary for Rotavirus Attachment

In an attempt to identify the rotavirus receptor, we tested 46 cell lines of different species and tissue origins for susceptibility to infection by three N-acetyl-neuraminic (sialic) acid (SA)-dependent and five SA-independent rotavirus strains. Susceptibility to SA-dependent or SA-independent rotavirus infection varied depending on the cell line tested and the multiplicity of infection (MOI) used. Cells of renal or intestinal origin and transformed cell lines derived from breast, stomach, bone, or lung were all susceptible to rotavirus infection, indicating a wider host tissue range than previously appreciated. Chinese hamster ovary (CHO), baby hamster kidney (BHK-21), guinea pig colon (GPC-16), rat small intestine (Rie1), and mouse duodenum (MODE-K) cells were found to support only limited rotavirus replication even at MOIs of 100 or 500, but delivery of rotavirus particles into the cytoplasm by lipofection resulted in efficient rotavirus replication. The rotavirus cell attachment protein, the outer capsid spike protein VP4, contains the sequence GDE(A) recognized by the VLA-2 (alpha2beta1) integrin, and to test if VLA-2 is involved in rotavirus attachment and entry, we measured infection in CHO cells that lack VLA-2 and CHO cells transfected with the human alpha2 subunit (CHOalpha2) or with both the human alpha2 and beta1 subunits (CHOalpha2beta1) of VLA-2. Infection by SA-dependent or SA-independent rotavirus strains was 2- to 10-fold more productive in VLA-2-expressing CHO cells than in parental CHO cells, and the increased susceptibility to infection was blocked with anti-VLA-2 antibody. However, the levels of binding of rotavirus to CHO, CHOalpha2, and CHOalpha2beta1 cells were equivalent and were not increased over binding to susceptible monkey kidney (MA104) cells or human colonic adenocarcinoma (Caco-2, HT-29, and T-84) cells, and binding was not blocked by antibody to the human alpha2 subunit. Although the VLA-2 integrin promotes rotavirus infection in CHO cells, it is clear that the VLA-2 integrin alone is not responsible for rotavirus cell attachment and entry. Therefore, VLA-2 is not involved in the initial attachment of rotavirus to cells but may play a role at a postattachment level.     

Biochemical characterization of VLA-1 and VLA-2. Cell surface heterodimers on activated T cells

The very late antigen complexes VLA-1 and VLA-2 which appear on long-term activated human T cells have been characterized with respect to 1) subunit arrangement, 2) location of monoclonal antibody (MAb) binding sites, 3) carbohydrate content, and 4) protein homology. Cross-linking experiments showed that the VLA-1 complex is a heterodimer composed of an Mr 210,000 subunit (alpha 1) in acid-labile association with an Mr 130,000 subunit (beta). The VLA-2 complex is a heterodimer with an Mr 165,000 subunit (alpha 2) in base-labile association with the Mr 130,000 beta subunit. The subunits of VLA-1 (alpha 1 beta) and VLA-2 (alpha 2 beta) each appear to be arranged with 1:1 stoichiometry. The MAb A-1A5 has been shown to bind to an epitope on the common beta subunit, consistent with its recognition of both the VLA-1 and VLA-2 heterodimers. On the other hand, MAb TS2/7 bound to an epitope of the alpha 1 subunit, thus explaining the specific recognition of the VLA-1 heterodimer by TS2/7. Digestion of the alpha 1, alpha 2, and beta subunits with neuraminidase and with endoglycosidase F revealed that each subunit contains substantial sialic acid and N-linked carbohydrate. By one-dimensional peptide mapping, the alpha 1, alpha 2, and beta subunits were shown to be highly nonhomologous with respect to each other, although each subunit from different T cell sources appeared highly homologous if not identical.     

Use of the monoclonal antibody 12F1 to characterize the differentiation antigen VLA-2

A monoclonal antibody, 12F1, has been produced that specifically immunoprecipitates the human cell surface structure VLA-2 from platelets and long-term activated T cells, as well as from fibroblast and neuroblastoma cell lines. Cross-linking studies indicate that the VLA-2 structure exists on the cell surface as a 165,000 Mr heavy chain (alpha 2) in noncovalent 1:1 association with a 130,000 Mr light chain (beta). The monoclonal antibody A-1A5, which reacts with the beta subunit common to all VLA structures, was able to completely preclear VLA-2, indicating that all of the alpha 2 subunit was associated with VLA beta-chain. The specificity of 12F1 for VLA-2 allowed independent immunoprecipitation and flow cytometry analysis of this alpha 2 beta structure separate from any other VLA structures that may have been present such as VLA-1 or free beta-subunit. Subunit dissociation studies were used to demonstrate that 12F1 recognizes an epitope on the alpha 2 chain on VLA-2, which is consistent with the 12F1 specificity for VLA-2 alone among the VLA proteins. Analysis of activated T cells indicated that VLA-2, like VLA-1, is another "very late" appearing T cell activation antigen that arises concurrently with VLA-1 starting at day 7 and increasing through 2 wk. VLA-2 was found on many of the same cells as VLA-1 (inactivated T cells, T cell leukemia cells, fibroblasts, SK-N-SH neuroblastoma cells), but VLA-1 and VLA-2 can be expressed independently, because VLA-2 was also present on VLA-1-negative cells such as HSB and platelets, and VLA-1 was present on VLA-2-negative C8215 cells.     

Echovirus 1 replication, not only virus binding to its receptor, VLA-2, is required for the induction of cellular immediate-early genes.

Induction of immediate-early genes c-jun, junB, and c-fos was demonstrated during echovirus 1 infection in a human osteogenic sarcoma (HOS) cell line. Tenfold induction was seen at 10 h postinfection, corresponding approximately to the end of the first replication cycle of the virus. Echovirus 1 uses VLA-2 integrin as its cellular receptor, and ligand binding by integrin is known to trigger signal transduction pathways ultimately activating immediate-early genes. In the present study, however, VLA-2 binding alone was not sufficient to induce their expression; viral replication was needed. This conclusion was based on the observations that no stimulation of the immediate-early genes occurred in the MG-63 cell line where the virus attached only to VLA-2 but was not able to replicate and that induction of these genes was observed when the HOS cells were infected with echovirus type 7, known to use a different cellular receptor. Induction was not seen in the presence of the antiviral compound WIN 54954, which evidently inhibits the uncoating but not receptor binding of echovirus 1, suggesting that viral replication triggers the activation of the immediate-early genes. The induction of these genes may have a role in viral replication and in the pathogenesis of infection.     

Specific overproduction of very late antigen 1 integrin in two human neuroblastoma cell lines selected for resistance to detachment by an Arg-Gly-Asp-containing synthetic peptide

Very late antigen (VLA) 1 is a member of the family of integral plasma-membrane glycoproteins known as integrins. It is a heterodimer composed of an alpha subunit of Mr 200,000, noncovalently associated with a beta subunit of Mr 110,000 which is shared by other VLA molecules (VLA-2-5). Unlike most of the other VLA proteins which have been shown to be receptors for various extracellular matrix proteins, the ligand for VLA-1 is unknown. Utilizing polyclonal antisera against the human fibronectin receptor as well as alpha subunit-specific monoclonal antibodies and cDNA probes, we have been able to demonstrate that in two human neuroblastoma cell lines, IMR-32 and SK-N-SH, the common beta subunit is associated with alpha 1, alpha 2, alpha 3, and alpha 5 subunits. By culturing these two cell lines in the presence of a synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, which contains the Arg-Gly-Asp cell attachment promotion tripeptide, we have isolated variant cell lines resistant to the detachment effects of this peptide. Peptide-resistant SK-N-SH and IMR-32 neuroblastoma cells exhibit weaker attachment to type I collagen and laminin, but a similar level of attachment to fibronectin as compared to the parental cells. Although the peptide-resistant variant cell lines proliferate at a rate similar to that of the parental cell lines, they stably overproduce (up to 20-fold) the alpha 1 subunit (VLA-1) specifically; and in the IMR-32 variant cells, the common beta 1 subunit is also overproduced. The level of expression of alpha 2 and alpha 3 subunits, however, is considerably reduced and that of the alpha 5 subunit is unchanged relative to the parental cells. These data suggest that the expression of integrin alpha subunits can be regulated differentially and independently of the beta subunit and that the VLA-1 heterodimer has an important function in mediating Arg-Gly-Asp-dependent cell adhesion or other phenotypic properties in human neuroblastoma cells.     

Characterization of the cell surface heterodimer VLA-4 and related peptides

A monoclonal antibody (B-5G10) was produced which specifically recognizes the Mr 150,000/130,000 VLA-4 complex on the surface of human cells. Cross-linking studies indicated that the Mr 150,000 alpha 4 subunit of VLA-4 is in noncovalent 1:1 association with the Mr 130,000 VLA beta subunit. In the absence of cross-linking, the VLA-4 alpha 4 beta subunit complex was easily dissociated, especially in Nonidet P-40 detergent, or at elevated pH (above 8.0). Studies of dissociated subunits showed that B-5G10 recognizes an epitope on the Mr 150,000 alpha 4 subunit of VLA-4, whereas the beta subunit is immunologically identical to the Mr 130,000 beta subunit common to all VLA heterodimers. VLA-4 is widely distributed on hematopoietic cells, including thymocytes, peripheral blood lymphocytes, monocytes, activated T cells, T and B lymphoblastoid cell lines, and myeloid cell lines. However, VLA-4 is only weakly expressed on most adherent cell lines tested. Immunoprecipitates of VLA-4 often contain additional proteins of Mr 80,000 and Mr 70,000. These are probably derived from the Mr 150,000 alpha 4 subunit because: 1) they are both recognized by anti-alpha 4 sera, but not anti-beta sera; 2) the sum of their sizes is equal to the size of alpha 4; 3) they are selectively coexpressed with alpha 4 and not other VLA alpha subunits; 4) the Mr 80,000 protein has an identical NH2-terminal sequence to alpha 4; 5) like alpha 4, the Mr 70,000 and 80,000 peptides can variably associate with the VLA beta subunit; and 6) trypsin appears to cleave the Mr 150,000 alpha 4 subunit into products of Mr 70,000 and 80,000.     

Multiple functional forms of the integrin VLA-2 can be derived from a single alpha 2 cDNA clone: interconversion of forms induced by an anti- beta 1 antibody

The integrin VLA-2 was previously found to bind to either collagen alone, or collagen plus laminin, but the mechanism for this cell- specific functional difference was unknown. Here we transfected VLA-2 alpha 2 subunit cDNA into K562 cells and obtained VLA-2 (called Form-O) which bound to neither collagen nor laminin. We then used a Matrigel selection procedure to enrich for a minor subpopulation of K562 cells stably expressing a form of VLA-2 (Form-C) that bound to collagen but not laminin. In contrast, the same alpha 2 cDNA transfected into RD cells yielded VLA-2 (Form-CL) which bound to both collagen and laminin. These Form-O, -C, and -CL activities were stably expressed during extended cell culture, and could not be qualitatively altered by adding phorbol esters or by exchaning the resident divalent cations. However, addition of stimulatory anti-beta 1 antibodies (TS2/16, A-1A5) rapidly converted VLA-2 Form-O and Form-C into Form-CL. Anti-beta 1 antibody stimulation of VLA-2 activity was observed not only on whole cells, but also with solubilized receptors. These results suggest (a) that the ligand binding specificity of VLA-2 can be determined by its cellular environment, rather than by variations in the primary sequence of the alpha 2 subunit, (b) that stably inactive or partly active VLA-2 can be rapidly converted to a fully active form through conformational changes initiated at a nonligand binding site on the beta 1 subunit, and (c) that the mechanisms for VLA-2 stimulation by phorbol ester and by antibody are quite distinct, because the latter does not require an intact cell.     

Human smooth muscle VLA-1 integrin: purification, substrate specificity, localization in aorta, and expression during development

A membrane glycoprotein complex was isolated and purified from human smooth muscle by detergent solubilization and affinity chromatography on collagen-Sepharose. The complex was identified as VLA-1 integrin and consisted of two subunits of 195 and 130 kD in SDS-PAGE. Liposomes containing the VLA-1 integrin adhered to surfaces coated with type I, II, III, and IV collagens, Clq subcomponent of the first component of the complement, and laminin. The liposomes specifically adhered to these proteins in a Ca2+, Mg2(+)-dependent manner, but did not bind to gelatin, fibronectin, and thrombospondin substrates. The expression of VLA-1 integrin in different human tissues and cell types, and during aorta smooth muscle development was studied by SDS-PAGE, and subsequent quantitative immunoblotting was performed with antibodies recognizing alpha 1 and beta 1 subunits of the VLA-1 integrin. A high level of VLA- 1 integrin expression was an exceptional feature of smooth muscles. Fibroblasts, endothelial cells, keratinocytes, striated muscles, and platelets contained trace amounts of VLA-1 integrin. In the 10-wk-old human fetal aorta, VLA-1 integrin was found only in smooth muscle cells whereas mesenchymal cells, surrounding aortic smooth muscle cells, were VLA-1 integrin negative. By the 24th wk of gestation, the amount of VLA- 1 integrin was significantly reduced in the aortic media (4.3-fold for alpha 1 subunit and 2.5-fold for beta 1 subunit) compared with that in the 10-wk-old aortic smooth muscle cells. After birth, the expression of VLA-1 integrin increased and in the 1.5-yr-old child aorta the VLA-1 integrin level was almost the same as in adult aortic media. Smooth muscle cells from intimal thickening of adult aorta express five times less alpha 1 subunit of VLA integrin that smooth muscle cells from adult aortic media. In primary culture of aortic smooth muscle cells, the content of the VLA-1 integrin was dramatically reduced and subcultured cells did not contain VLA-1 integrin at all.     

Sequence analysis of the downstream 5' nontranslated region of seven echoviruses with different neurovirulence phenotypes.

The downstream 5' nontranslated regions of seven echoviruses with different neurovirulent phenotypes were amplified and sequenced. Neurovirulent echovirus serotypes 4, 6, 9, 11, and 30 were identical to the putative poliovirus in 18S rRNA binding sequence and the flanking conserved sequences. Less neurovirulent echoviruses, serotypes 2 and 12, exhibited variations within these regions.     

Echovirus 1 interaction with the isolated VLA-2 I domain.

The isolated I domain of the integrin VLA-2, produced as a bacterial fusion protein, specifically bound echovirus 1 and prevented virus attachment to cells. These results demonstrate that the receptor structures critical for virus attachment are contained solely within the VLA-2 I domain and that soluble receptor fragments are capable of preventing infection.     

Optimal conditions for plaque assay of echoviruses in human embryonic lung fibroblast cells

The present paper gives optimal conditions for plaque assays tn human embryonic lung fibroblast cells with 45 echoviruses including 11 fresh isolates, covering 31 serotypes. All the viruses tested except echovirus type 23 produced plaques at a high titer in the same cells by the methods described in this communication. Plaque formations by a number of strains were markedly enhanced in size or number or both by the addition of polycation.     

The I Domain is Essential for Echovirus 1 Interaction with VLA-2

VLA-2, the α2β1 integrin, mediates cell adhesion to collagen and laminin, and is the receptor for the human pathogen echovirus 1. Because of its similarity to domains present in other proteins that interact with collagen, a 191 amino acid region within the α2 subunit (the I domain) has been proposed as a potential site for ligand interactions. Although the α2 subunits of human and murine VLA-2 are 84% identical, human α2 promotes virus binding whereas murine α2 does not. We used murine/human chimeric α2 molecules to identify regions of the human molecule essential for virus binding. Virus bound efficiently to a chimeric protein in which the human I domain was inserted into murine α2, indicating that the human I domain is responsible for specific virus interactions. Monoclonal antibodies that inhibited virus attachment all recognized epitopes within the human I do-main, further suggesting that virus interacts with this portion of the molecule. Similarly, antibodies that prevented VLA-2-mediated cell adhesion to collagen also mapped to the I domain. These results indicate that the I domain plays a role in VLA-2 interactions both with virus and with extracellular matrix ligands.     

The Mouse VLA-2 Homologue Supports Collagen and Laminin Adhesion but Not Virus Binding

Human VLA-2 (α₂β₁) mediates cellular adhesion to collagen and laminin and cell attachment by the human pathogen echovirus 1. We report here the cloning, sequencing and functional expression of the mouse VLA-2 α subunit homologue. This integrin subunit is closely related to its human counterpart, with 84% amino acid identity between the human and murine proteins. Conserved structural features include an identical number of amino acids, the presence of an I domain, and identity in the number and position of N-linked glycosylation sites and putative divalent cation binding regions. Murine and human α₂ show 30% amino acid divergence within the cytoplasmic tail, a difference that can be detected with antisera directed against the C-terminal peptides. Functionally, mouse α₂ was capable of mediating cell attachment to collagen and laminin, and responded to both intra- and extracellular signals with changes in its ligand affinity. In contrast, unlike its human homologue, mouse α₂ did not promote binding of echovirus 1. Comparison of the primary structure of the homologues leads us to predict that echovirus 1 may bind in the region of the first two thirds of the human α₂ I domain, where the sequences are most divergent, whereas more conserved flanking regions, and the conserved terminal one third of the I domain, may be involved in adhesion to collagen and laminin.     

The VLA protein family. Characterization of five distinct cell surface heterodimers each with a common 130,000 molecular weight beta subunit

The family of human cell surface heterodimers which includes VLA-1 and VLA-2 is now shown to include three additional heterodimers, here called VLA-3, VLA-4, and VLA-5. Each of these separate VLA structures is composed of a distinct alpha subunit (Mr 110,000-200,000 nonreduced) noncovalently associated with a common beta subunit (Mr 110,000 nonreduced). Chemical cross-linking experiments provided evidence that each VLA complex exists predominantly as a 1:1 alpha beta heterodimer. The VLA proteins are widely distributed, with one or more of the heterodimers present on nearly all cell types tested. Evidence for five distinct VLA alpha subunits was obtained from differences observed in antibody recognition, cell distribution patterns, two-dimensional gel analyses, and V8 protease cleavage patterns. On the other hand, the beta subunit present in each heterodimer was immunochemically and electrophoretically indistinguishable, and yielded identical V8 cleavage fragments. Immunoblotting experiments revealed that besides the Mr 110,000 beta normally seen, another beta protein was present that is smaller in size (Mr 90,000 nonreduced), altered or deficient in glycosylation, and not available for cell surface radiolabeling.     

Multiple very late antigen (VLA) heterodimers on platelets. Evidence for distinct VLA-2, VLA-5 (fibronectin receptor), and VLA-6 structures

After removal of very late antigen (VLA) 2 material from a radiolabeled detergent lysate of platelets, another VLA heterodimer was precipitated using antibody to the common VLA beta subunit. This structure was identified as VLA-5 because it contained VLA beta plus an alpha subunit that was (i) recognized by anti-alpha 5 antibodies and (ii) cleaved by V8 protease to yield a characteristic alpha 5-like pattern of peptide fragments. Besides VLA-2 and VLA-5, a third heterodimer, here named VLA-6, was also present on platelets. VLA-6 (an alpha 6 beta complex) was defined using the monoclonal antibody GoH3 (Sonnenberg, A., Janssen, H., Hogervorst, F., Calafat, J., and Hilgers, J. (1987) J. Biol. Chem. 262, 10376-10383). Although it resembled VLA-5 in size, VLA-6 was different from VLA-5 because (i) removal of the alpha 5 subunit did not remove alpha 6, (ii) removal of alpha 6 by the GoH3 antibody did not remove alpha 5, (iii) the alpha 5 and alpha 6 subunits had very distinct one-dimensional V8 peptide maps, and (iv) the alpha 6 and alpha 5 subunits had distinct migration patterns on two-dimensional O'Farrell gels. The beta subunit of VLA-6 was identified as the common VLA beta subunit because (i) it was recognized by anti-VLA beta antibody and (ii) it yielded a V8 protease cleavage map characteristic of beta. VLA-6 was not readily seen in anti-VLA beta immunoprecipitations, apparently because the alpha 6 subunit is only loosely or partially associated with the VLA beta subunit. Because VLA-5 and VLA-6 both closely resemble the previously defined Ic-IIa platelet protein complex, it is likely that there is more than one platelet "Ic" protein complexed with IIa.     

A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA- dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components

The leukocyte beta 1 integrin receptor very late activation antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium. A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes beta 1-subunit (CD29) of integrin receptors. In contrast to mAbs directed to VLA-4 alpha-subunit (alpha 4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL. Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin. This binding was blocked by mAbs to the VLA alpha-subunits alpha 6 (CD49f), or alpha 5 (CD49e) and alpha 4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte beta 1 integrins, and should prove useful in studying the regulation of beta 1 integrin function.     

Alpha 1 beta 1 integrin heterodimer functions as a dual laminin/collagen receptor in neural cells

A monoclonal antibody (3A3) raised against a rat neural cell line (PC12) was shown previously to bind to the surfaces of these cells, inhibiting substratum adhesion. Immunochemical and other data indicated that the heterodimer recognized by 3A3 was a member of the integrin family of adhesive receptors and had a beta 1 subunit. The relationship of the alpha subunit to other integrins was unknown. Here we show that 3A3 recognizes in rat tissues a heterodimer (approximately 185 kDa, approximately 110 kDa; unreduced) that is electrophoretically and immunochemically indistinguishable from the antigen in PC12 cells. Immunoaffinity purification of the heterodimer from neonatal rats and protein microsequencing indicate that the alpha subunit is identical at 11 or 13 N-terminal residues with VLA-1, an integrin on human hematopoietic cells. Monoclonal antibody 3A3 inhibits the attachment of rat astrocytes to laminin or collagen but not to fibronectin or polylysine. These data suggest strongly that the integrin recognized by 3A3 is the rat homologue of VLA-1, i.e., alpha 1 beta 1, and that alpha 1 beta 1 is a dual laminin/collagen receptor.     

Functional and structural analysis of VLA-4 integrin alpha 4 subunit cleavage.

The cell surface heterodimer VLA-4 (alpha 4 beta 1), a member of the integrin family of adhesion receptors, is involved in both cell-extracellular matrix and cell-cell adhesion. Unlike any other integrin alpha subunit, the intact (150 kDa) alpha 4 subunit of VLA-4 can sometimes be cleaved into two noncovalently associated fragments (80 and 70 kDa). Using biosynthetic and mixing experiments, we found that human alpha 4 cleavage is a regulated, compartmentalized event, occurring soon after maturation of the beta 1-associated alpha 4 subunit. Cleavage of alpha 4, which is increased following T cell activation, has been suggested to correlate with altered VLA-4 functions. To address directly the functional importance of alpha 4 cleavage, we have studied VLA-4-mediated adhesion functions in cells expressing intact alpha 4 in comparison with cells expressing cleaved alpha 4. For this purpose, we first sequenced the N terminus of the endogenously produced 70-kDa alpha 4 fragment and identified the alpha 4 cleavage site between Lys557-Arg558 and Ser559. To abolish cleavage, we converted Arg558 to Leu or Lys557 to Gln by site-directed mutagenesis of the alpha 4 cDNA and then transfected both mutant and wild type alpha 4 cDNAs into VLA-4-negative K562 cells. Whereas transfection with wild type alpha 4 cDNA yielded predominantly cleaved alpha 4 subunit, the Leu558-alpha 4 yielded only intact alpha 4 subunit, and Gln557-alpha 4 yielded mostly intact alpha 4 subunit. Transfectants with the intact or the cleaved alpha 4 were equally capable of engaging in VLA-4-dependent adhesion to vascular cell adhesion molecule-1 and to the Hep II fragment of fibronectin (40 kDa) and aggregated equally well in response to anti-alpha 4 antibodies. Thus, cleavage of the alpha 4 subunit in these transfectants did not alter any of the known VLA-4-mediated adhesion functions.     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.