cDNA sequence of the human integrin beta 5 subunit

A novel integrin receptor involved in cell adhesion to the matrix protein vitronectin has recently been described from a human lung epithelial-derived cell line (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69). This receptor has an alpha subunit that appears identical to the alpha v of the vitronectin receptor alpha v beta 3 expressed in melanoma and endothelial cells, but is complexed with a distinct beta subunit, beta 5. cDNA clones coding for beta 5 have been isolated and used to determine the mRNA and amino acid sequence of this new subunit. A 3.3-kilobase mRNA was found to code for a mature protein of 775 amino acid residues with a hydrophobic leader sequence of 24 amino acids. A 56% identity was found between the beta 5 and beta 3 protein sequences, making them the most closely related of the integrin beta subunits. Polymerase chain reaction abundance analysis revealed that alpha v and beta 5 mRNAs were found in seven very different cell lines, compared with beta 3 mRNA which was found in only three of the them, indicating that this new integrin receptor may be widely distributed.     

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.     

Cloning and expression of a divergent integrin subunit beta 8

Rabbit and human cDNA clones have been identified that encode a novel integrin beta subunit. The sequences that encode this subunit, which has been designated as beta 8, were isolated initially from rabbit placental cDNA libraries using an oligonucleotide probe derived from a highly conserved region of integrin beta subunit sequences. The rabbit clone was used to isolate human beta 8 cDNA clones from human placental and MG-63 osteosarcoma cell libraries. The putative beta 8 polypeptides, which comprise 769 and 768 residues in human and rabbit, respectively, show a high degree of inter-species conservation (approximately 90% identity). In contrast, beta 8 is distinct from the other integrin beta subunits. At the amino acid level human beta 8 ranges from 31 to 37% identity with human beta 1-7. The domain structure of beta 8 is typical of the integrin beta subunits. Human beta 8 has a 42-residue N-terminal signal peptide, a large extracellular domain (approximately 639 residues) that contains four cysteine-rich repeats, a transmembrane domain (approximately 30 residues), and a C-terminal cytoplasmic domain (approximately 58 residues). There are several structural features that are unique to the beta 8 polypeptide, as compared with the other integrin beta subunits. Six of the 56 cysteine residues that are conserved within the extracellular domains of beta 1, beta 2, beta 3, beta 5, beta 6, and the beta subunit from Drosophila are absent in the beta 8 polypeptide. Also, the cytoplasmic domain of the beta 8 subunit shares no homology with the cytoplasmic regions of any of the other integrin beta subunits. Northern analysis demonstrated an approximately 8-kilobase beta 8 mRNA in rabbit placenta, kidney, brain, ovary, and uterus. PCR analysis revealed that beta 8 mRNA is also present in several transformed human cell lines. The beta 8 polypeptide has been transiently expressed in 293 human embryonic kidney cells. A polyclonal antipeptide antibody specific for beta 8 and a polyclonal antibody that recognizes alpha v epitopes were used to show that beta 8 can complex with the endogenous alpha v subunit in 293 cells and that the resulting integrin is expressed as a cell surface complex.     

Complete amino acid sequence of a novel integrin beta subunit (beta 6) identified in epithelial cells using the polymerase chain reaction

The integrin family of adhesion receptors consists of several heterodimeric glycoproteins, each composed of one alpha and one beta subunit. Three different mammalian beta subunits, beta 1, beta 2, and beta 3, have been sequenced, but recent evidence suggests the existence of several others. Amplification of guinea pig airway epithelial cell cDNA with oligonucleotide primers designed to recognize consensus integrin beta subunit sequences led to the identification of a novel partial cDNA sequence. Clones containing portions of this sequence were used to screen cDNA libraries constructed from the human pancreatic carcinoma cell line FG-2 and identified a series of overlapping clones encoding the full-length sequence of the human homologue of this protein. This sequence of 788 amino acids is 43, 38, and 47% identical to the sequences of beta 1, beta 2, and beta 3, respectively. Features shared between this novel protein and the previously sequenced beta subunits include the positions of all 56 cysteine residues in the extracellular domain, the single putative transmembrane domain, and the short putative cytoplasmic domain. However, a unique 11-amino acid extension at the carboxyl terminus, not present in any of the other beta subunits, is suggestive of distinctive interactions with cytoplasmic components. Comparison of the human and guinea pig sequences reveals a high degree (94%) of cross-species conservation. Because this protein is clearly distinct from the two other recently described integrins beta 4 and beta 5, we propose to designate it beta 6.     

Cloning of the murine lymphocyte function-associated molecule-1 alpha-subunit and its expression in COS cells

The lymphocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is an integrin that mediates adhesion of immune cells by interaction with two members of the Ig superfamily, ICAM-1 and ICAM-2. LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report here the isolation and expression of the murine alpha subunit cDNA (GenBank accession no. M60778). The deduced sequence comprises a 1061 amino acid extracellular domain, a 29 amino acid transmembrane region, and a 50 amino acid cytoplasmic domain. It has a 72% amino acid identity with its human counterpart and 34% identity with the murine Mac-1 alpha subunit. The murine LFA-1 alpha subunit could be expressed on the cell surface of a fibroblastoid cell line, COS, by cotransfection with either the human or murine beta subunit cDNA.     

Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain

The alpha 6 beta 4 complex is a member of the integrin superfamily of adhesion receptors. A human keratinocyte lambda gt11 cDNA library was screened using a monoclonal antibody directed against the beta 4 subunit. Two cDNAs were selected and subsequently used to isolate a complete set of overlapping cDNA clones. The beta 4 subunit consists of 1778 amino acids with a 683 amino acid extracellular domain, a 23 amino acid transmembrane domain and an exceptionally long cytoplasmic domain of 1072 residues. The deduced amino-terminal sequence is in good agreement with the published amino-terminal sequence of purified beta 4. The extracellular domain contains five potential N-linked glycosylation sites and four cysteine-rich homologous repeat sequences. The extracellular part of the beta 4 subunit sequence shows 35% identify with other integrin beta subunits, but is the most different among this class of molecules. The transmembrane region is poorly conserved, whereas the cytoplasmic domain shows no substantial identity in any region to the cytoplasmic tails of the known beta sequences or to other protein sequences. The exceptionally long cytoplasmic domain suggests distinct interactions of the beta 4 subunit with cytoplasmic proteins.     

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.     

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.     

The primary structure of the alpha 4 subunit of VLA-4: homology to other integrins and a possible cell-cell adhesion function.

VLA-4 is a cell surface heterodimer in the integrin superfamily of adhesion receptors. Anti-VLA-4 antibodies inhibited cytolytic T cell activity, with inhibitory activity directed against the effector T cells rather than their targets. Thus, whereas other VLA receptors appear to mediate cell--matrix interactions, VLA-4 may have a cell--cell adhesion function. To facilitate comparative studies of VLA-4 and other integrins, cDNA clones for the human alpha 4 subunit of VLA-4 were selected and then sequenced. The 3805 bp sequence encoded for 999 amino acids, with an N-terminus identical to that previously obtained from direct sequencing of purified alpha 4 protein. The alpha 4 amino acid sequence was 17-24% similar to other integrin alpha chains with known sequences. Parts of the alpha 4 sequence most conserved in other alpha chains include (i) the positions of 19/24 cysteine residues, (ii) three potential divalent cation binding sites of the general structure DXDXDGXXD and (iii) the transmembrane region. However, alpha 4 stands apart from all other known integrin alpha subunit sequences because (i) alpha 4 has neither an inserted I-domain, nor a disulfide-linked C-terminal fragment, (ii) its sequence is the most unique and (iii) only alpha 4 has a potential protease cleavage site, near the middle of the coding region, which appears responsible for the characteristic 80,000 and 70,000 Mr fragments of alpha 4.     

Sequence and tissue distribution of the integrin alpha 9 subunit, a novel partner of beta 1 that is widely distributed in epithelia and muscle [published erratum appears in J Cell Biol 1994 Feb;124(3):395]

The integrin family of adhesion receptors consists of several heterodimeric glycoproteins, each composed of one alpha and one beta subunit. A novel integrin alpha subunit partial cDNA isolated from TGF- beta stimulated guinea pig airway epithelial cells has previously been reported (Erle, D.J., D. Sheppard, J. Bruess, C. Ruegg, and R. Pytela. 1991. Am. J. Respir. Cell Mol. Biol. 5:170-177). We have now determined cDNA and amino acid sequence for the human homolog of this subunit, named alpha 9, from a human lung cDNA library, a human small intestine cDNA library, and cDNA from the cell lines U937, HL-60 and Tera-2. This sequence is predicted to encode a 1006-amino acid mature protein that shares 39% identity with the previously identified integrin subunit alpha 4. By Northern blot analysis, alpha 9 mRNA was detected in the human carcinoma cell lines Tera-2 and Caco-2. Anti-peptide antibodies against the predicted COOH-terminal sequence of alpha 9 immunoprecipitated a heterodimer (140 kD/115 kD nonreduced; 150 kD/130 kD reduced) from Tera-2 lysates. Immunodepletion of beta 1-containing integrins with Tera-2 lysates removed alpha 9 immunoreactivity, suggesting that beta 1 is the principal beta subunit partner for alpha 9 in these cells. alpha 9 was detected by immunohistochemistry in airway epithelium, in the basal layer of squamous epithelium, and in smooth muscle, skeletal muscle, and hepatocytes.     

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.     

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD- containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.     

Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2)

Clusterin, a glycoprotein originally isolated from ram rete testis fluid, is a dimer composed of monomers with non-identical NH2-terminal amino acid sequences. In view of its possible role in cell-cell interactions in the seminiferous epithelium, we sought to identify such a protein in the rat. Using the bioassay developed for the ovine protein, rat clusterin was purified to apparent homogeneity by HPLC from primary Sertoli cell-enriched culture media. This protein is also a heterodimer consisting of monomers of Mr 43,000 (alpha) and Mr 35,000 (beta). NH2-Terminal amino acid sequence analysis indicated that the alpha subunit has a sequence of NH2-SLMPLSHYGPLSFHNMFQPFFDMIHQAQQA and the beta subunit, NH2-EQEFSDNELQELSTQGSRYVNKEIQNAVQG. These two subunits show marked similarity with the corresponding subunits of ram clusterin isolated from rete testis fluid. Using an antibody against the alpha subunit of rat clusterin, a cDNA clone was isolated from a rat testicular lambda gt11 cDNA library. Analyses of the amino acid sequence derived from the isolated rat clusterin cDNA and of the NH2-terminal amino acid sequences indicate that rat clusterin is identical to a Sertoli cell glycoprotein previously designated sulfated glycoprotein-2.     

cDNA cloning and chromosomal localization of human alpha(11) integrin. A collagen-binding, I domain-containing, beta(1)-associated integrin alpha-chain present in muscle tissues.

We previously identified a novel integrin alpha-chain in human fetal muscle cells (Gullberg, D., Velling, T., Sj?berg, G., and Sejersen, T. (1995) Dev. Dyn. 204, 57-65). We have now isolated the full-length cDNA for this integrin subunit, alpha(11). The open reading frame of the cDNA encodes a precursor of 1188 amino acids. The predicted mature protein of 1166 amino acids contains seven conserved FG-GAP repeats, an I domain with a metal ion-dependent adhesion site motif, a short transmembrane region, and a unique cytoplasmic domain of 24 amino acids containing the sequence GFFRS. alpha(11), like other I domain integrins, lacks a dibasic cleavage site for generation of a heavy chain and a light chain, and it contains three potential divalent cation binding sites in repeats 5-7. The presence of 22 inserted amino acids in the extracellular stalk portion (amino acids 804-826) distinguishes the alpha(11) integrin sequence from other integrin alpha-chains. Amino acid sequence comparisons reveal the highest identity of 42% with the alpha(10) integrin chain. Immunoprecipitation with antibodies to alpha(11) integrin captures a 145-kDa protein distinctly larger than the 140-kDa alpha(2) integrin chain when analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Fluorescence in situ hybridization maps the integrin alpha(11) gene to chromosome 15q23, in the vicinity of an identified locus for Bardet-Biedl syndrome. Based on Northern blotting, integrin alpha(11) mRNA levels are high in the adult human uterus and in the heart and intermediate in skeletal muscle and some other tissues tested. During in vitro myogenic differentiation, alpha(11) mRNA and protein are up-regulated. Studies of ligand binding properties show that alpha(11)beta(1) binds collagen type I-Sepharose, and cultured muscle cells localize alpha(11)beta(1) into focal contacts on collagen type I. Future studies will reveal the importance of alpha(11)beta(1) for muscle development and integrity in adult muscle and other tissues.     

The role of the carboxyl-terminal 6 amino acid extension of human TSH beta subunit

The nucleotide sequence of human thyroid stimulating hormone (hTSH) gene can encode a protein of 138 amino acids. However, the mature polypeptide is lacking 6 amino acids of the carboxyl-terminus (C-terminus), suggesting posttranslational cleavage of these residues. To analyze a possible function of these 6 amino acids, we expressed two hTSH beta cDNAs with or without the 6 codons for C-terminal extension, together with alpha subunit cDNA in CHO cells, and determined the amino acid sequence of C-terminus of hTSH beta. hTSH beta propeptides without C-terminal extension were glycosylated, associated with alpha subunit and secreted, as normal propeptides were, and its heterodimer with alpha subunit showed normal TSH bioactivity in FRTL-5 bioassay. These data indicate that the 6 amino acid C-terminal extension is not necessary for the hTSH maturation in the process of the biosynthesis and for its bioactivity.     

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.     

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

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.