The Primary Structure of the VLA-2/Collagen Receptor .2 Subunit (Platelet GPla): Homology to Other Integrins and the Presence of a Possible Collagen-binding Domain

VLA-2 (also called gpla/IIa on platelets) is a collagen receptor with a unique ot subunit and a subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 o~ 2 subunit have been selected from a hgtll library by specific antibody screening. The 5,374bp 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 ot z protein confirmed the identity of the 15 NH2terminal amino acids. Overall, the ot 2 amino acid sequence was 18-25% similar to the sequences known for other integrin ot subunits. In particular, the a2 sequence matched other integrin c~ 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 ot 2 sequence has a 191-amino acid insert (called the 1-domain), previously found only in leukocyte integrins of the 82 integrin family. The oe 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 c~ 2 does not match the previously reported o~ 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. V L A 2 is an c~/~/-subunit cell surface heterodimer strongly implicated as receptor for collagen because (a) VLA-2 has been shown to be identical (61) to a 150,000/ll0,000-Mr structure recognized by the mAb P1H5, which specifically blocks human fibrosarcoma cell (66) and platelet (31) attachment to collagen; (b) patients deficient in platelet protein Ia (or subunit of VLA-2) also lacked responsiveness to collagen (27, 41); and (c) the mAb 12F1 was used to identify VLA-2 as a 160,000/130,000-Mr (nonreduced) platelet protein complex that mediates Mg2+-dependent adhesion to collagen (51, 52). Also in this regard, antigens that strongly resemble VLA-2 in size have been implicated in hepatocyte cell attachment to type I collagen (14). The cell surface heterodimer VLA-2 was initially characterized as a "very late antigen" appearing on activated T cells (19, 20). Later the mAb 12F1 was used to identify VLA-2 on platelets and on many other cell types, including most rapidly growing, adherent cell lines (43). Major portions of the platelet gpla and IIa are probably the same as the VLA-2 Y. Takada is currently at Biochemistry Division, National Cancer Center Research Institute, 1-1, Tsukiji, 5-chome, Chuo-ku Tokyo, 104 Japan. c~ and/3 subunits, respectively (31, 44). Thus the new platelet-specific alloantigens Br ~ and Br b, which reside on the platelet Ia-IIa complex (53), may be variable epitopes on VLA-2. In other studies, an antibody (called 5E8) that recognizes VLA-2 (Hemler, M., and C. Crouse, unpublished results) has been described on most primary human lung tumors (68), and ~25I-coupled 5E8 antibody has been used to inhibit the growth of lung tumor cell lines in vitro (57). The expression of VLA-2 can be up regulated on lymphocytes in response to mitogen or antigen stimulation (17) and on fibroblasts in response to serum (10). Conversely, VLA-2 expression slowly diminished when those stimuli were withdrawn and/or cells became quiescent (10, 17). Also, VLA-2 expression can be up regulated by TGF/~ (15). Like other cell surface receptors for extracellular matrix components, VLA-2 belongs to the integrin superfamily (26, 49). Three subfamilies of that superfamily that are defined for humans are (a) the six (or more) VLA proteins (16, 23); (b) the leukocyte adhesion molecules, LFA-1, Mac-l, and p150,95 (56); and (c) the cytoadhesins, which include the vitronectin receptor and platelet gplIb/IIIa (13). Within each © The Rockefeller University Press, 0021-9525/89/07/397/11 $2.00 The Journal of Cell Biology, Volume 109, July 1989 397-407 397 on July 9, 2017 jcb.rress.org D ow nladed fom family, the member heterodimers are each composed of a unique ot subunit associated with a common 13 subunit. Analyses of human eDNA clones for the/3 subunits of each subfamily (called 13, 135, and/33, respectively) have revealed that they are 44-47% similar (2, 12, 28, 33, 62), suggesting a common evolutionary origin. The evolutionary conservation of/3t is emphasized by the homology (82-86%) mainmined between/$~s from widely diverging species, such as human, chicken, and frog (9), and 45 % homology between human /3, and a /31-like structure from Drosophila (36). Also, complete sequencing of several integrin ot subunitsincluding those from a fibronectin receptor (or 5) (2, 11), the vitronectin receptor (58), platelet gplIb/IIIa (11, 45), Mac-1 (3, 8, 46), p150,95 (7), and the Drosophila PS2 antigen (6)-have revealed 20-60% similarity between any pair. Some of these integrin structures are receptors for ligands containing the amino acid signal sequence Arg-Gly-Asp (RGD) or a closely related sequence (49). Although NH2terminal amino acid sequencing has suggested ,o40% shared residues among six different VLA ot subunits (24, 60), complete sequence information is available for only one VLA ot subunit (or 5) (2, 11). Thus, to further establish patterns of similarity and differences among VLA/integrin sequences and to gain basic information for future studies of structure, function, and regulation, the complete sequence of the VLA ot 2 subunit was obtained. Also, because the ot2-subunit NH2terminal sequence predicted from cDNA did not match the previously published "or 2" NH2-terminal sequence, experiments were carried out to resolve this discrepancy. Materials and Methods Purification of VLA-2 from Platelets and NHz-terminal Sequencing VLA-2 protein was purified from outdated platelets (from the Dana-Farber Cancer Institute [Boston, MA] blood bank or from the American Red Cross [Dedham, MAD by lectin-Sepharose and then by 12FI or A-1A5 immunoaffinity chromatography (60). The anti-~ 2 mAb 12F1 (43) and the ant i-~ mAb A-IA5 (18) were obtained as described. For NH2-terminal amino acid mierosequencing of the 62 subunit, the ~2 subunit was first separated from the/ff subunit by SDS-gel electrophoresis, transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore Continental Water Systems, Bedford, MA), stained with Coomassie blue, and destained as described (39). Excised membrane pieces containing 62 material were then sequenced using a gas-phase sequenator (470A; Applied Biosystem, Inc., Foster City, CA) equipped with a phenylhydantoin amino acid analyzer (120A; Harvard Microsequeneing Facility, Cambridge, MA). Preparation of aZ-Subunit-specific Antiserum Subunits migrating in the o~ position (,050 #g from A-IAS-Sepharose or 12FI-Sepharose) were reduced and separated using preparative 5% SDSPAGE, stained with Coomasie blue, and destained in 7 % acetic acid and 30% methanol. The band corresponding to a2 peptide was cut out and electroeluted into Tris/glycine/SDS buffer using an Elutrap (Schleicher & SehueU, Inc., Keene, NH). The eluted peptide was used for rabbit immunization as previously described (23). Also, 62 protein purified from 12F1Sepharose was used for immobilization onto CNBr-Sepharose (Pharmacia Fine Chemicals, Piseataway, N J) according to the manufacturer's instructions, except that 0.1% of SDS was included in the coupling buffer. Antibodies from rabbit anti-a2-subunit antiserum were then purified using the 62Sepharose affinity column. Briefly, the antiserum was passed through the column several times and the column was washed successively with 3 column volumes each of PBST (0.14 M NaCI, 10 mM sodium phosphate, 0.2% Tween 20), 0.2 M KSCN, and then PBST again. For elution of 62subunit-specific IgG, 3 M KSCN was added, and the eluate was desalted by Sephadex G-25 chromatography into PBST in the presence of 0.1% hemoglobin as carrier. Immunopurified IgG was further incubated with denatured Escherichia coil protein coupled to Sepharose, and with glycoprote, ins from Molt-4 cells coupled to Sepharose to remove additional nonspecific reactivity. The Molt-4 leukemic T cell line does not usually express VLA-2 (22). Production of Antibodies to an ~2 COOH-terminal Synthetic Peptide A 22-mer consisting of the COOH-terminal 21 amino acids predicted from the c~ 2 sequence, with an added cysteine at the NH2-terminal end of the peptide, was synthesized by Multiple Peptide Systems (San Diego, CA). This peptide was coupled to carrier protein (keyhole limpet hemocyanin) using m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Pierce Chemical Co., Rockford, IL) as previously described (29), and then rabbit antibodies were generated using standard techniques (38). Isolation of the Gene Encoding the ~2 Subunit A phage ),gill expression eDNA library of human lung fibroblast IMR-90 (CIontech Laboratories, Inc., Palo Alto, CA) was screened by using afffinitypurified rabbit antibodies against 62 subunit according to Young and Davis (67), except that alkaline phosphatase-conjugated anti-rabbit IgG was used as a second antibody. Alkaline phosphatase activity was detected by using an immunoscreening system (ProtoBlot; Promega Biotec, Madison, WI) with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates. Positive clones from the screening were plaque purified, and then phage DNAs were purified by the plate lysate method (37). After the insert was excised from the phage by Eco RI restriction enzyme digestion, the inserts were separated On agarose gels, electroeluted, and subcloned into pBluescript plasmid (Stratagene, La Jolla, CA). A eDNA library made in kgtll from endothelial cells was the kind gift from Dr. Tucker Collins (Brigham and Women's Hospital, Boston, MA).