Amino acid sequence of the human fibronectin receptor

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.     

Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.     

The alpha subunit of meprin A. Molecular cloning and sequencing, differential expression in inbred mouse strains, and evidence for divergent evolution of the alpha and beta subunits.

Meprin A, a membrane-bound oligomeric metalloendopeptidase, contains two different subunits, alpha and beta. We report here the cloning and sequencing of the alpha subunit cDNA. The translated polypeptide consists of 760 amino acids, including a preprosequence (77 amino acids) that precedes the NH2 terminus of the purified enzyme. The next 198 amino acids constitute the "astacin family" protease domain, which includes the astacin family signature sequence, HE(L,I)XHXXGFXHE(Q,H)XRXDRDX(Y,H)(V,I)X(I,V). An immunoglobulin/major histocompatibility complex protein signature was found at the end of the protease domain. At the COOH terminus of the alpha subunit, there is an epidermal growth factor-like domain, followed by a transmembrane domain, and six additional amino acids. Ten potential glycosylation sites have been identified, and at least three of those sites are glycosylated. Northern blot analyses of kidney tissue from C57BL/6 and C3H/He mice indicate that variations in meprin A activity in these strains reflect differences in the levels of the alpha subunit mRNA. Several internal peptide sequences obtained from the beta subunit indicate that it is approximately 50% identical to the alpha subunit. Furthermore, NH2-terminal sequence analyses (39 residues) indicate that rat and mouse alpha are 79% identical, rat and mouse beta are 74% identical, and that alpha and beta subunits for both species are 47% identical. These data indicate that alpha and beta are closely related products of divergent evolution.     

Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains

Macrophages express a cell surface receptor which mediates phagocytosis and pinocytosis of particles and solutes containing mannose (fucose and N-acetylglucosamine are also ligands for the receptor). An apparently identical protein has been isolated from human placenta. Proteolytic fragments of the placental receptor were sequenced so that oligonucleotide probes complementary to the receptor cDNA could be generated. These probes were used to isolate cDNA clones covering the entire coding portion of the mRNA for the receptor. Confirmation that these clones encode the mannose receptor was obtained by expression in rat fibroblasts. The expressed protein mediates uptake and degradation of mannose-conjugated serum albumin. The deduced amino acid sequence of the receptor reveals that it is most likely to be a type I transmembrane protein (COOH terminus on the cytoplasmic side of the membrane) since the mature polypeptide is preceded by a signal sequence and a hydrophobic stop transfer sequence is located 45 amino acids from the COOH terminus. The extracellular portion of the receptor polypeptide consists of three types of domains. The first 139 amino acids constitute a cysteine-rich segment which does not resemble other known sequences. There follows a domain which closely resembles fibronectin type II repeats. The remainder of the extracellular portion of the receptor is composed of eight segments homologous with the C-type carbohydrate-recognition domains of the asialoglycoprotein receptor, mannose binding proteins, and other Ca2(+)-dependent animal lectins. This structure suggests that the receptor may contain multiple ligand-binding domains thus accounting for its tight binding to highly multivalent ligands.     

Molecular cloning and nucleotide sequence of a cDNA clone coding for the cell attachment domain in human fibronectin

A cDNA clone coding for the cell attachment domain in human fibronectin has been isolated using synthetic oligonucleotides. Three sets of mixed tetradecamer oligonucleotides were synthesized based on amino acid sequences in the 108-amino acid cell attachment domain (Pierschbacher, M. D., Ruoslahti, E., Sundelin, J., Lind, P., and Peterson, P. A. (1982) J. Biol. Chem. 257, 9593-9597). One of these sets was made complementary to amino acids located near the COOH terminus of the cell attachment domain and synthesized as a mixture of 24 sequences. This oligonucleotide mixture was used to prime cDNA synthesis with mRNA prepared from a human fibrosarcoma as a template. A cDNA library was constructed with the oligonucleotide-primed sequences in the vector pBR322. Colonies that hybridized with the primer were isolated from the library and further identified by hybridization with oligonucleotides deduced from an amino acid sequence located 45 amino acid residues NH2-terminal of the primer sequence. One clone which hybridized to both probes was characterized in detail. The insert was 380 base pairs long and its nucleotide sequence agreed completely with the corresponding amino acid sequence of human plasma fibronectin, showing that the sequences for this region are identical in plasma fibronectin and fibronectin from a cell line. This clone should be useful for studies on the expression of fibronectins and may also allow for the production of the biologically active cell attachment domain of fibronectin in bacteria.     

Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor alpha-subunits and platelet glycoprotein IIb

The fibronectin receptor (FnR), the vitronectin receptor (VnR), and the platelet membrane glycoprotein (GP) IIb-IIIa complex are members of a family of cell adhesion receptors, which consist of noncovalently associated alpha- and beta-subunits. The present study was designed to compare the cDNA-derived protein sequences of the alpha-subunits of human FnR, VnR, and platelet GP IIb. cDNA clones for the alpha-subunit of the FnR (FnR alpha) were obtained from a human umbilical vein endothelial (HUVE) cell library by using an oligonucleotide probe designed from a peptide sequence of platelet GP IIb. cDNA clones for platelet GP IIb were isolated from a cDNA expression library of human erythroleukemia cells by using antibodies. cDNA clones of the VnR alpha-subunit (VnR alpha) were obtained from the HUVE cell library by using an oligonucleotide probe from the partial cDNA sequence for the VnR alpha. Translation of these sequences showed that the FNR alpha, the VnR alpha, and GP IIb are composed of disulfide-linked large (858-871 amino acids) and small (137-158 amino acids) chains that are posttranslationally processed from a single mRNA. A single hydrophobic segment located near the carboxyl terminus of each small chain appears to be a transmembrane domain. The large chains appear to be entirely extracellular, and each contains four repeated putative Ca2+-binding domains of about 30 amino acids that have sequence similarities to other Ca2+-binding proteins. The identity among the protein sequences of the three receptor alpha-subunits ranges from 36.1% to 44.5%, with the Ca2+-binding domains having the greatest homology. These proteins apparently evolved by a process of gene duplication.     

The sequence of a cloned cDNA for the beta subunit of bovine thyrotropin predicts a protein containing both NH2-and COOH-terminal extensions

Cloned cDNAs containing sequences coding for the beta subunit of bovine thyrotropin have been identified. The complete nucleotide sequence of the largest of the beta subunit cDNA inserts has been determined. This cDNA contains 35 nucleotides from the 5' untranslated region of thyrotropin beta subunit mRNA and 60 nucleotides coding for an NH2-terminal precursor segment. This is followed by 339 nucleotides which code for the published amino acid sequence of the thyrotropin beta subunit. Following the 339 nucleotide beta subunit coding sequence, no termination codon is encountered for another 15 nucleotides. Thus, the cDNA codes for a thyrotropin beta subunit containing an additional 5 amino acids at the COOH terminus. The cDNA also contains 82 nucleotides of 3' untranslated sequence followed by a short poly(A) segment. Comparison of the bovine cDNA sequence to the recently described mouse thyrotropin beta subunit cDNA sequence reveals considerable homology throughout the coding sequence, including the COOH-terminal extension. These findings suggest the possibility that a thyrotropin beta subunit precursor is processed at both the NH2 and COOH termini.     

Deletions in the large (beta) subunit of a hetero-oligomeric aminoacyl-tRNA synthetase

Glycyl-tRNA synthetase is one of two aminoacyl-tRNA synthetases in Escherichia coli that is comprised of heterologous subunits which are organized in an alpha 2 beta 2 quaternary structure. The two subunits are encoded by a single mRNA with the region for alpha (303 codons) subunit followed by that for beta (689 codons) subunit. Five COOH-terminal deletions in the beta subunit coding region have been created. Each deletion protein has been investigated for its synthesis and stability in vivo, adenylate synthesis activity in vitro, and aminoacylation activity in vivo and in vitro. This has been done in the presence of free alpha subunit and, additionally, with alpha subunit that is fused by its carboxyl terminus to the amino terminus of each of the beta subunit deletion proteins. With a fused or unfused alpha chain, over 100 amino acids can be deleted from the carboxyl terminus of the beta chain without loss of in vivo complementation of a delta glyS (deletion) strain. Further analysis shows that the alpha subunit and approximately the amino-terminal half of the beta subunit are sufficient for the adenylate synthesis activity. In particular, a deletion of 306 amino acids from the COOH terminus of the beta subunit has little effect on the Km parameter for ATP or glycine in the pyrophosphate exchange reaction. The tRNA-dependent step in aminoacylation requires additional beta subunit sequences on the COOH-terminal side of those needed for adenylate synthesis. In these respects, the functional organization of the beta chain parallels that of several aminoacyl-tRNA synthetases which have only homologous subunits. In the case of the glycine enzyme, however, the heterologous alpha subunit is required for the elucidation of activities encoded by functional determinants of the beta chain.     

The human leukocyte adhesion glycoprotein Mac-1 (complement receptor type 3, CD11b) alpha subunit. Cloning, primary structure, and relation to the integrins, von Willebrand factor and factor B

Mac-1 (CD 11b/CD18) is a leukocyte adhesion heterodimeric glycoprotein which functions both as a receptor for iC3b (CR3) and in several cell-cell and cell-substrate adhesive interactions. We describe full-length cDNA clones for the alpha subunit of Mac-1. Mac-1 alpha subunit message was detected in blood monocytes and phorbol-12-myristate acetate-induced myeloid cell lines, but not in cells of the T or B lineages, correlating with Mac-1 protein surface expression. The alpha subunit of Mac-1 is a transmembrane protein of 1137 residues with a long extracellular domain (1092 residues) and a 19-amino acid cytoplasmic tail. The extracellular domain contains three putative divalent cation-binding sequences and 19 potential N-glycosylation sites. The amino acid sequence of Mac-1 alpha shows that it is a member of the integrin superfamily; Mac-1 alpha shows 63% identity to the alpha subunit of the leukocyte adhesion glycoprotein p150.95 and 25% to the alpha subunits of the extracellular matrix receptors platelet glycoprotein IIb/IIIa, the fibronectin receptor, and the vitronectin receptor. The Mac-1 alpha subunit putative divalent cation-binding sites and the flanking regions exhibit a high degree of identity both to the p150.95 alpha subunit (87% identity at the amino acid level) and to the rest of the integrin alpha subunits (38%). The alpha subunit of Mac-1, like the p150.95 alpha subunit, contains a domain of 187 amino acids in the extracellular region which is absent in other integrins. This leukocyte or "L" domain is homologous to the A domains of von Willebrand factor, which in turn are homologous to regions of the C3-binding proteins factor B and C2. These findings draw attention to this region of Mac-1 as a potential binding site for iC3b.     

Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen

alpha 3 beta 1 (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 alpha 3 subunit was selected from an endothelial cell lambda gt11 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 alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3- 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 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha 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 alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 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 alpha 3.     

Cloning, primary structure and properties of a novel human integrin beta subunit.

The originally described integrin beta subunits that define the three subfamilies of integrin heterodimers are beta 1, beta 2 and beta 3. In this paper, we describe the isolation of a cDNA coding for a novel human integrin beta subunit, designated as beta 5. The beta 5 cDNA was isolated from a human thymic epithelial cell library, using oligonucleotide probes that were designed from a region highly conserved among the known beta 1, beta 2 and beta 3 sequences. The beta 5 cDNA codes for 799 (or 796) amino acids, including a 23 amino acid leader sequence. There are 776 (or 773) amino acids in the mature protein, which includes a long extracellular domain of 696 amino acids, a transmembrane domain and an intracellular C-terminal domain of 57 amino acids. The beta 5 sequence resembled the known beta 3, beta 1 and beta 2 sequences by 55, 43 and 38%, respectively, including conservation of 56/56 cysteines. Rabbit antiserum was prepared against a 20 amino acid synthetic peptide predicted from the beta 5 C-terminal sequence. This serum immunoprecipitated a beta 5 protein that was 100,000 Mr (reduced) and 95,000 Mr (nonreduced). Only a single alpha subunit was detected in association with beta 5, and that alpha subunit was immunochemically indistinguishable from the alpha v subunit previously found as part of the vitronectin receptor complex. By immunoprecipitation, beta 5 was most prevalent on carcinoma cell lines, was also present on hepatoma and fibroblast cell lines, and was absent from lymphoblastoid cells and platelets.     

Subunit structure of a laminin-binding integrin and localization of its binding site on laminin

A laminin receptor was isolated from human MG-63 osteosarcoma cells by affinity chromatography on human laminin. The isolated receptor was defined as the alpha 3 beta 1 integrin by immunoprecipitation with subunit-specific antibodies. A previously unclassified laminin-binding integrin from rat cells was shown also to contain the alpha 3 subunit. Both receptors bound to human and mouse laminin in a radioreceptor assay. They also both bound to some extent to fibronectin in this assay, but only the MG-63 cell receptor showed binding to type IV collagen. The binding of the radiolabeled receptor to insoluble laminin was inhibited by unlabeled receptor, by soluble laminin, and by chymotryptic fragments of laminin that have previously been shown to contain neurite-promoting and cell attachment-promoting activities. Moreover, the receptor binding was also inhibited by monoclonal antibodies capable of inhibiting the neurite-promoting activity of laminin and known to bind to laminin near the junction of the long arm and its terminal globule. One of these antibodies was reactive with fusion proteins expressed from laminin cDNA clones. The immunoreactive clones corresponded to the COOH-terminal end of the B1 subunit. These results identify the integrin-type laminin receptor isolated from the osteosarcoma cells as the alpha 3 beta 1 integrin and localize its binding site in close proximity of the B1 subunit COOH terminus.     

Amino acid sequence of the alpha subunit of human leukocyte adhesion receptor Mo1 (complement receptor type 3)

Mo1 (complement receptor type 3, CR3; CD11b/CD18) is an adhesion-promoting human leukocyte surface membrane heterodimer (alpha subunit 155 kD [CD11b] noncovalently linked to a beta subunit of 95 kD [CD18]). The complete amino acid sequence deduced from cDNA of the human alpha subunit is reported. The protein consists of 1,136 amino acids with a long amino-terminal extracytoplasmic domain, a 26-amino acid hydrophobic transmembrane segment, and a 19-carboxyl-terminal cytoplasmic domain. The extracytoplasmic region has three putative Ca2+-binding domains with good homology and one with weak homology to the "lock washer" Ca2+-binding consensus sequence. These metal-binding domains explain the divalent cation-dependent functions mediated by Mo1. The alpha subunit is highly homologous to the alpha subunit of leukocyte p150,95 and to a lesser extent, to the alpha subunit of other "integrin" receptors such as fibronectin, vitronectin, and platelet IIb/IIIa receptors in humans and position-specific antigen-2 (PS2) in Drosophila. Mo1 alpha, like p150, contains a unique 187-amino acid stretch NH2-terminal to the metal-binding domains. This region could be involved in some of the specific functions mediated by these leukocyte glycoproteins.     

A mutation linked with Bartter's syndrome locks Kir 1.1a (ROMK1) channels in a closed state.

Mutations in the inward rectifying renal K(+) channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. One disease-causing mutation removes the last 60 amino acids (332-391), implicating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated channels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles of this domain were systematically evaluated. When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion construct was indistinguishable from the GFP-wild-type channel, demonstrating that mutant channels are expressed on the oocyte plasma membrane in a nonconductive or locked-closed conformation. Incremental reconstruction of the COOH terminus identified amino acids 332-351 as the critical residues for restoring channel activity and uncovered the nature of the functional defect. Mutant channels that are truncated at the extreme boundary of the required domain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies the extreme COOH terminus as an important subunit interaction domain, controlling the efficiency of oligomerization. Collectively, these observations provide a mechanistic basis for the loss of function in one particular Bartter's-causing mutation and identify a structural element that controls open-state occupancy and determines subunit oligomerization. Based on the overlapping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.     

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.     

Promotion of the GTP-liganded state of the Go alpha protein by deletion of the C terminus.

G proteins are active as long as GTP is bound to the alpha subunit. Activation ends when GTP is cleaved to GDP that then stays bound to the active site. Agonist-liganded receptors allow formation of the active state by decreasing the affinity of alpha subunits for GDP allowing exchange of GDP for GTP. Since receptors interact with the C terminus of the alpha subunits, we tested whether deletion of the C terminus could mimic activation by receptors. Three deletions and one point mutation at the C terminus of alpha o were engineered in alpha o cDNA by the polymerase chain reaction, transcribed into RNA, and translated in a rabbit reticulocyte lysate. The ability of in vitro synthesized protein to bind guanine nucleotide was inferred from analysis of native tryptic cleavage patterns, while the ability of the proteins to associate with beta gamma was measured by sucrose density gradient centrifugation. Deletion of 14 amino acids, alpha oD[341], from the C terminus causes a large decrease in GDP affinity, with little or no change in guanosine 5'-3-O-(thio)triphosphate affinity. When GTP is present, alpha oD[341] remains in the activated conformation because exchange of GTP for GDP is rapid. Deletion of 10 amino acids, alpha oD[345], lowers GDP affinity, but less dramatically than in alpha oD[341]. Deletion of 5 amino acids, alpha oD[350], or mutation of Arg-349 to proline alpha oR[349P] has no detectable effects on GDP affinity. Deletion of up to 10 amino acids from the C terminus does not prevent formation of alpha beta gamma heterotrimers. We propose that the C terminus of the alpha subunit is a mobile region that blocks dissociation of GDP. Agonist-liganded receptors may move it aside to allow release of GDP, exchange for GTP, and activation of the alpha subunit.     

Amino acid sequence of a novel integrin beta 4 subunit and primary expression of the mRNA in epithelial cells.

Using the polymerase chain reaction, we have isolated cDNA clones that encode a new integrin beta subunit--beta 4. Its cDNA, which is 5676 bp in length, has one long coding sequence (5256 bp), a polyadenylation signal and a poly(A) tail. The deduced sequence of 1752 amino acids is unique among the integrin beta subunits. It contains a putative signal sequence as well as a transmembrane domain that divides the molecule into an extracellular domain at the N-terminal side and a cytoplasmic domain at the C-terminal side. The extracellular domain exhibits a 4-fold repeat of cysteine-rich motif similar to those of other integrin beta subunits. Certain features of the extracellular domain, however, are unique to the beta 4 subunit sequence. Of the 56 conserved cysteine residues found within the extracellular domain of other mature beta subunits, eight such residues are deleted from the beta 4 subunit sequence. The cytoplasmic domain is much larger (approximately 1000 amino acids) than those of other beta subunits (approximately 50 amino acids) and has no significant homology with them. A protein homology search revealed that the beta 4 subunit cytoplasmic domain has four repeating units that are homologous to the type III repetition exhibited by fibronectin. The beta 4 subunit mRNA was expressed primarily in epithelial cells. The restricted expression and the new structural features distinguish the integrin beta 4 subunit from other integrin beta subunits.     

cDNA cloning and complete primary structure of the alpha subunit of a leukocyte adhesion glycoprotein, p150,95.

The leukocyte adhesion receptors, p150,95, Mac-1 and LFA-1 are integral membrane glycoproteins which contain distinct alpha subunits of 180,000-150,000 Mr associated with identical beta subunits of 95,000 Mr in alpha beta complexes. p150,95 alpha subunit tryptic peptides were used to specify oligonucleotide probes and a cDNA clone of 4.7 kb containing the entire coding sequence was isolated from a size-selected myeloid cell cDNA library. The 4.7-kb cDNA clone encodes a signal sequence, an extracellular domain of 1081 amino acids containing 10 potential glycosylation sites, a transmembrane domain of 26 amino acids, and a C-terminal cytoplasmic tail of 29 residues. The extracellular domain contains three tandem homologous repeats of approximately 60 amino acids with putative divalent cation-binding sites, and four weaker repeats which lack such binding sites. The cDNA clone hybridizes with a mRNA of 4.7 kb which is induced during in vitro differentiation of myeloid cell lines. The p150,95 alpha subunit is homologous to the alpha subunits of receptors which recognize the RGD sequence in extracellular matrix components, as has previously been shown for the beta subunits, supporting the concept that receptors involved in both cell-cell and cell-matrix interactions belong to a single gene superfamily termed the integrins. Distinctive features of the p150,95 alpha subunit include an insertion of 126 residues N-terminal to the putative metal binding region and a deletion of the region in which the matrix receptors are proteolytically cleaved during processing.     

The cDNA sequence and chromosomal location of the murine GABAA alpha 1 receptor gene.

The murine GABAA/benzodiazepine (GABAA/BZ) receptor alpha 1 subunit cDNA has been isolated from a BALB/c mouse brain library and sequenced. The cDNA is 2665 nucleotides long with an open reading frame of 455 amino acids. It shows significant homology to the GABAA receptor alpha 1 subunit cDNA sequences of other species. Excluding deletions, the murine GABAA alpha 1 receptor exhibits 96% nucleotide and 100% amino acid sequence homology to the rat alpha 1 receptor cDNA and over 91% nucleotide and 98% amino acid sequence homology to the bovine and human alpha 1 receptor cDNAs in the protein coding region. This murine cDNA was used to locate the alpha 1 receptor subunit gene, Gabra-1, to murine Chromosome 11 between Il-3 and Rel. This assignment extends proximally the segment of mouse Chromosome 11 with known homology to human chromosome 5.