Molecular Cloning and Expression ofthe cDNA for a3 Subunit ofHuman a3aJVLA-3), an Integrin Receptor for Fibronectin, Laminin, and Collagen

a301 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen . A partial cDNA clone (2 .4 kb) for the human a3 subunit was selected from an endothelial cell Agtll cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries . The reconstructed a3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an a3-specific mAb after transfection, suggesting that the cDNA is authentic . Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 CIA (VLA-3), a cell surface heterodimer composed of a3 and 0 1 subunits, is an integrin (1, 6, 19, 23, 27) that has been implicated as a receptor for collagen, laminin, and fibronectin (13, 17, 18, 49, 54) . The role of a3a1 has been somewhat difficult to assess, partly because its adhesive functions are often obscured by other collagen, laminin, and fibronectin receptors such as a201, a6,Q1 and a501 (13) . The binding of a3a 1 to its different ligands appears to be accomplished by multiple binding mechanisms. For example, U3,Q1 binding to fibronectin differs substantially from binding to collagen and laminin with respect to both the divalent cation requirements and the influence of RGD peptides (13) . In addition to being a receptor for extracellular matrix ligands, aA could possibly also bind to cell surface ligands, as inferred from x301 localization to cell-cell contact sites (7, 31, 35) . In normal tissue, COO, expression is limited to a few cell types, including kidney glomeruli, and the basal cells of epidermis and other epithelia (7, 16, 34, 35) . In contrast, nearly all cultured cell lines express a3N, except for lymphoid cells (16) . Underscoring the likely importance of C01-mediated cell adhesion, its expression levels have been © The Rockefeller University Press, 0021-9525/91/10/257/10 $2 .00 The Journal of Cell Biology, Volume 115, Number 1, October 1991 257-266 residues) . Overall, the a3 amino acid sequence was 25-37% similar to the other integrin a subunits that are cleaved, with most similarity to the a6 sequence (37%), and less similarity to those a subunits that have I domains (15-20%, excluding the I domain sequence itself) . Features most like those in other a subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain . The mass of a3 calculated from its amino acid sequence is 113,505 . The human a3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human a3 " noted to vary on different cell types and with different growth conditions . For example, a30 1 levels diminished in response to TGF,Q in one case (22), but were increased in other examples (21) . Also, a,81 can be induced by attachment of some cultured cells to extracellular matrix (40) while a3a1 levels decrease upon shifting of fibroblasts from exponential growth to quiescence (14) . On rat (37) and human (34) transformed cells, x3/3 1 levels were elevated, suggesting that a 3(3 1 could possibly contribute to tumorigenicity and/or invasiveness . However, in several other studies, «3a1 levels were either unchanged or decreased on malignant cells (11, 35, 36), emphasizing that regulation of a301 expression on cancer cells is complex . In this paper we have cloned, sequenced, and expressed in eukaryotic cells the cDNA for the 013 subunit of C01. We have found that a3 resembles other integrin a subunits in having a signal peptide, a long extracellular domain (959 residues) and a putative transmembrane domain and a short cytoplasmic segment (32 residues) . The extracellular domain includes 18 conserved cysteine residues and three metal binding domains of the general structure DX(D/N)X(D/N)GXXD. The 0e3 subunit amino acid sequence was 257 on M arch 1, 2013 jcb.rress.org D ow nladed fom Published October 1, 1991