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Amino Acid Sequence of the Human Fibronectin Receptor

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Amino Acid Sequence of the Human Fibronectin Receptor Amino Acid Sequence of the Human Fibronectin Receptor W. Scott Argraves, Shintaro Suzuki, Hiroharu Arai, Katie Thompson, Michael D. Pierschbacher, and Erkki Ruoslahti Cancer Research Center, La Jolla Cancer Research Foundation, La Jolla, California 92037 Abstract. The amino acid sequence deduced from repeat strikingly rich in cysteine. The r subtmit se- eDNA of the human placental fibronectin receptor is quence is 46% homologous to the a subunit of the reported. The receptor is composed of two subunits: vitronectin receptor. The 13 subunit is 44% homolo- an 0t subunit of 1,008 amino acids which is processed gous to the human platelet adhesion receptor subunit into two polypeptides disulfide bonded to one another, IIIa and 47% homologous to a leukocyte adhesion and a 13 subunit of 778 amino acids. Each subunit has receptor 13 subunit. The high degree of homology near its COOH terminus a hydrophobic segment. This (85%) of the 13 subunit with one of the polypeptides of and other sequence features suggest a structure for the a chicken adhesion receptor complex referred to as receptor in which the hydrophobic segments serve as integrin complex strongly suggests that the latter poly- transmembrane domains anchoring each subunit to the peptide is the chicken homologue of the fibronectin membrane and dividing each into a large ectodomain receptor 13 subunit. These receptor subunit homologies and a short cytoplasmic domain. The r subunit ec- define a superfamily of adhesion receptors. The avail- todomain has five sequence elements homologous to ability of the entire protein sequence for the fibronec- consensus Ca2§ sites of several calcium- tin receptor will facilitate studies on the functions of binding proteins, and the 13 subunit contains a fourfold these receptors. HE cell surface receptors that bind to the adhesive gly- receptor superfamily is that they are heteromeric complexes coproteins fibronectin, vitronectin, fibrinogen, and having noncovalently associated tz and 13 subunits. Details of T von Willebrand factor have been shown to be structur- the structure of the receptors have come from recombinant ally and functionally related (Pytela et al., 1986). Further- DNA cloning work. The 13 subunit of the chicken fibronec- more, they have been shown to be members of a superfamily tin-laminin receptor complex (Tamkun et al., 1986), the 13 of receptors that include the leukocyte glycoproteins (LFA-1, subunit of the human LFA-1 cell surface glycoprotein Mac-l, and p150,95 (Springer et al., 1986) and another group (Kishimoto et al., 1987; Law et al., 1987), and the glycopro- of cell surface proteins referred to as VLA antigens (Hemler tein ma of the platelet l/b/IIIa complex (Fitzgerald et al., et al., 1987). Homologous forms of these receptors have been 1987) have recently been cloned and sequenced. Comparison defined in various species by using antibodies that interfere of primary sequences of these 13 subunits indicates that they with cell adhesion (Knudsen et al., 1981; Greve and Gottlieb, are related, having a high overall homology, and that they 1982; Horwitz et al., 1984; Brown and Juliano, 1985; Gian- share common structural features such as short cytoplasmic cotti et al., 1985). A family of cell surface glycoproteins that domains, putative transmembrane segments, and homolo- appear to be related to the vertebrate receptors has also been gous cysteine-rich domains. Results from our laboratory found in Drosophila (Wilcox and Leptin, 1985) and sea ur- concerning the a subunits of the human fibronectin receptor chin (Noll et al., 1985). It seems, then, that this superfamily and vitronectin receptor have revealed that the partial amino of receptors is diversely represented in cells of vertebrate and acid sequences deduced from cDNAs for the two subunits invertebrate organisms. The receptors for fibronectin, vitro- are homologous and have common structural elements such nectin, fibrinogen, and von Willebrand factor all bind to Arg- as potential membrane-spanning regions and short cytoplas- Gly-Asp (RGD) sequences (Pierschbacher and Ruoslahti, mic domains (Argraves et al., 1986; Suzuki et al., 1986). 1984; Plow et al., 1985; Pytela et al., 1985a, b, 1986; Hor- We have continued our efforts to determine the structure witz et al., 1985; Akiyama and Yamada, 1985; Hynes, 1987) of the fibronectin receptor expecting that it will lead to an within their respective ligands. It is not yet known whether understanding of the function of this and other related recep- RGD binding is a common functional trait among all mem- tors. Here we report the complete amino acid sequences, bers of the receptor superfamily. deduced from cDNA, of both the a and 13 subunits of the hu- A general structure characteristic of members of this man fibronectin receptor. The Rockefeller University Press, 0021-9525/87/09/1183/8 $2.00 The Journal of Cell Biology, Volume 105, September 1987 1183-1190 1183 Materials and Methods sorbed antibodies that reacted specifically with the I~ subunit of the fibronectin receptor (Fig. 1). LP32 was found to con- cDNA Libraries and Screening tain a 2.5-kb cDNA insert that did not cross hybridize with ~,P7 or ~,P34. Nucleotide sequence analysis of the 2,485-bp A placental cDNA ~,gtll library (Millan, 1986) was immunologically screened according to the procedure of Young and Davis (1983). A rabbit insert of LP32 showed that it had a single open reading frame antiserum prepared against purified human placental fibronectin receptor of 1368 bp followed by a 3' untranslated region of 1117 bp that (Pytela et al., 1986) served as a source of antibodies for the screening. The lacked a poly (A) tail. This sequence, presumably represent- antibodies were purified from this antiserum by affinity chromatography on ing the I~ subunit, was not homologous to the ~t subunit se- a column of fibronectin receptor coupled to Sepharose (Pytela et al., 1987). These antibodies immunoblot both subunits of the receptor (Argraves et al., quence. The cDNAs for both the ~t and 13 subunits of the 1986) and immunoprecipitate both of the receptor subunits from cells sur- fibronectin receptor were used as probes for genomic DNA face labeled with 12~I (Dedhar et al., 1987). Clones that expressed insert- Southern hybridization analysis (Southern, 1975) and the encoded protein reactive with these antibodies were isolated and purified resulting hybridization patterns indicated that both subunits to homogeneity through successive screenings. To identify whether the are encoded by distinct, single copy genes (data not shown). selected clones corresponded to the r or I~ subunit of the fibronectin recep- tor, their insert-encoded proteins were used to affinity select antibodies from RNA hybridization analysis showed that the LP32 13 subunit antifibronectin receptor serum, as previously described (Argraves et al., cDNA insert hybridized to an ~4.2-kb placental transcript, 1986). Additional screenings were done with radiolabeled DNA fragments while the a subunit mRNA is ~4.9 kb in size (Fig. 2). from the 5' portions of the previously isolated cDNA inserts as probes. To isolate cDNAs that would correspond to additional DNA fragments were 32p labeled by random oligonucleotide priming (Feinberg and Vogelstein, 1984) and used to screen either the placental Lgtll protein-encoding sequence in the mRNAs of the fibronectin cDNA library or a primer extension cDNA library by methods described receptor ct and 13 subunits, the placental cDNA library was elsewhere (Maniatis et al., 1982). rescreened with DNA fragments from the 5' portions of LP7 The primer extension cDNA library was made based on the procedure and LP32. In both cases, sets of overlapping cDNA clones of Krawinkel and Zoebelein (1986). Placental poly (A) RNA was specif- were isolated and sequenced. After an additional round of ically primed with an oligonucleotide sequence which was derived from the 5' region of the fibronectin receptor r subunit cDNA ~,P7 (Argraves et al., screening using 5' fragments from the new clones as probes, 1986). First strand cDNA was made from 15 I~g of placental poly (A) RNA overlapping cDNA clones encoding the complete 13 subunit primed with 0.6 p.g of a 28-bp oligonucleotide (5' TGAATTCGAACCCTC- were obtained. However, cDNAs encoding the amino-ter- GGGATCCAACTCC 3'). The first strand cDNA was G-tailed and the sec- minal region of the a subunit were still lacking. A primer ond strand synthesis primed with the oligonucleotide TGAATTCGGATCI3. After Eco RI digestion, the double-stranded cDNA was size fractionated on extension cDNA library was prepared from placental poly Sephacryl S-1000 (Pharmacia Fine Chemicals, Piscataway, NJ) and cDNAs (A) RNA primed with an oligonucleotide the sequence of greater than 500 bp were selected and ligated to Eco RI cut Lgtll DNA which was derived from the 5' portion of LP7. The cDNA (Promega Biotec, Madison, WI). Phage DNA was packaged using Gigapack library that was obtained was screened with a cDNA frag- (Vector Cloning Systems, San Diego, CA). Greater than 90% of the result- ment from the 5' region of a previously selected ct subunit ing phage were recombinants. cDNA that extended the farthest toward the 5' direction. As DNA Sequencing a result, several clones were isolated that completed the amino acid sequence of the a subunit polypeptide. cDNA inserts from selected ~,gtll clones were subcloned into the phage vec- tor MI3 mpl9. The cDNA inserts were sequenced by the dideoxy chain ter- mination method (Sanger et al., 1977) by using deoxyadenosine 5'-r The a Subunit Sequence thiotriphosphate and oligonucleotide primers that were synthesized on the basis of preceding sequences. All sequences reported here are the result of The sequence of the cDNA encoding the human fibronectin sequencing both strands of the cDNA inserts. receptor et subunit and the deduced amino acid sequence of the ct subunit polypeptide is shown in Fig. 3. The cDNA RNA Hybridization Analysis sequence was derived from eight overlapping cDNA clones.
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