Chick integrin alpha V subunit molecular analysis reveals high conservation of structural domains and association with multiple beta subunits in embryo fibroblasts

Abstract

We have cloned and characterized a chick homologue of the human vitronectin receptor alpha subunit (alpha v) whose primary sequence is 83% identical with its human counterpart but less than 40% identical with any other known integrin alpha subunit. Comparison of the chick and human sequences reveals several highly conserved regions, including the cytoplasmic domain. The putative ligand binding domain contains alpha v-specific residues that may contribute to ligand binding specificity. These are concentrated in three regions that are located before and between the first three Ca2+ binding domains. Polyclonal antibodies raised against two peptides deduced from the putative cytoplasmic and extracellular domains of the chick alpha v sequence recognize specifically integrin heterodimers in chick embryo fibroblasts. At least three putative beta subunits coimmunoprecipitate with the chick alpha v subunit. In addition to a protein with the same molecular weight as beta 3 (94K), protein bands of Mr 84K and 110K are also coprecipitated. By successive immunodepletions, we demonstrate that this latter Mr 110K subunit is beta 1, which appears to be one of the alpha v-associated subunits in chick embryo fibroblasts.

FIGURE 1

Restriction map of the probes and purified cDNAs. (a) Map of the EcoRI (R) and PstI (P) restriction fragments and EcoRI–BglII (B) fragment in (b), from human α_v cDNAs used as probes for screening at low stringency. (c) Restriction map of the purified chick cDNAs. The box indicates the location of the main open reading frame. (EcoRI, R; BglII, B; PstI, P; HindIII, H). (d) Map of the six chick embryonic (E10) cDNAs (1.1, 1.3, 1.4, 1.5, 1.7, 1.8) purified with the probes mentioned in (a). (e) Map of six cDNAs (d10, d11, d12, d14, d15, and d17) purified from a chick brain (E13) cDNA library.

FIGURE 2

cDNA and deduced amino acid sequence of the chick α_v subunit and comparison with the human α_v subunit amino acid sequence. Amino acids are abbreviated with the single-letter code. The amino acids in the mature human α_v sequence that are different from the residues in the chick α_v sequence are mentioned below the corresponding chick residues. Nonconservative substitutions are indicated by an asterisk. Amino acid substitutions within the following groups were considered as conservative: A, S, T; M, I, L, V; D, E; F, Y, W; R, K; N, Q. The three amino acids residues in the human α_v sequence that do not have chick equivalents are listed with a (#). The four putative metal binding (I-IV) and transmembrane (TM) domains are underlined. The arrows pointing upward indicate the two potential cleavage sites: after the signal peptide (between residues −1 and 1) and within the extracellular domain (between residues 857 and 858). The 15 potential N-glycosylation sites are followed by a (@).

FIGURE 3

RNA hybridization analysis. Chick embryo fibroblast poly(A+) RNA was electrophoresed through a denaturing agarose gel, transferred to a membrane, and probed with the chick α_v subunit d10 cDNA. The numbers at left indicate RNA size standards in kilobases.

FIGURE 4

Immunoprecipitation of chick embryo fibroblasts (CEFs). Surface-labeled CEF extracts were immunoprecipitated with (1) preimmune and (2) immune sera raised against the C peptide of the chick α_v subunit, (3) preimmune and (4) immune sera raised against the L peptide of the chick α_v subunit, and (5) integrin β₁-specific serum and (6) α_vβ₃-specific monoclonal antibody LM609. After protein A–Sepharose precipitation, the samples were washed and electrophoresed in nonreducing (A) and reducing (B) conditions. The gels were then dried and autoradiographed. The numbers at left indicate molecular weight markers × 10⁻³.

FIGURE 5

Immunodepletion with anti-β₁ polyclonal antibodies. Surface-labeled CEF extracts were immunodepleted 7 times with successive doses of anti-β₁ subunit serum (lanes 1–7). The depleted extract was then mixed with the serum raised against the C peptide derived from the chick α_v subunit cytoplasmic domain (lane 8). An identical CEF extract sample was depleted 7 times with normal rabbit serum and finally was depleted 7 times with normal rabbit serum and finally immunoprecipitated (lane 9) with the same α_v subunit specific serum used in lane 8.

FIGURE 6

RGD binding domains comparison. Amino acid comparison of the RGD binding domain from the human α_v subunit (huVNR) and its chick counterpart (chVNR), with the homologous sequence from the α₅ subunit of the human fibronectin receptor (huFNR). Boxes indicate identical residues between chick and human vitronectin receptors (two upper row boxes) or between the three compared α subunits (three row boxes). Nonconservative substitutions between the two α_v and the α₅ sequences are indicated by an asterisk, using the rule defined in the Figure 2 legend. The three main regions, A, B, and C, containing vitronectin receptor specific sequences are underlined by dashed lines. Amino acid numbers correspond to the human sequence (Fitzgerald et al., 1987).