AN INTEGRIN HETERODIMER AND AN ALPHA SUBUNIT THEREOF

Abstract: A recombinant or isolated integrin heterodimer comprising a novel subunit alpha 11 in association with a subunit beta is described. The integrin or the subunit alpha 11 can be used as marker or target of all types of cells. The integrin or subunit alpha 11 thereof can be used as marker or target in different physiological or therapeutic methods. They can also be used as active ingredients in pharmaceutical compositions and vaccines.

Description: AN INTEGRIN HETERODIMER AND AN ALPHA SUBUNIT THEREOF FIELD OF THE INVENTION The present invention relates to a recombinant or isolated integrin heterodimer comprising a subunit all and a subunit β, the subunit all thereof, homologues and fragments of said integrin and of said subunit all, processes of producing the same, polynucleotides and oligonucleotides encoding the same, vectors and cells comprising the same, binding entities binding specifically to binding sites of the same, and the use of the same. BACKGROUND OF THE INVENTION Integrins are heterodimers composed of non-covalent- ly associated α- and β-chains which connect cells to the extracellular matrix or to other cells (1) . In addition to acting as mechanical links between the cytoskeleton and extracellular ligands, integrins are signal transducing receptors which influence processes such as cell proliferation, cell migration and cell differentiation (2-4) . Integrins can be grouped into subfamilies based on shared β-chains, shared ligand binding properties, or shared structural features of the α-chains. Currently 17 α-chains and 8 β-chains have been identified (5) . Of the subfamilies with shared β-chains, the βl subfamily has the most members. To date, 11 integrin α-chains asso- ciated with the βl-chain have been identified and characterized, αl-αlO and αv (5) . Several integrins bind the sequence RGD in their respective ligands (1) . Of those integrins identified so far, α4-, α5-, α8-, αllb- and αv-chains form heterodimers that mediate RGD-dependent interactions. The ligands containing RGD are generally found in the interstitial type of extracellular matrix. Major non-RGD dependent ligands include various collagen and laminin isoforms. Although both collagens and laminins contain the RGD sequence in their primary sequences, these RGD sequences are cryptic (6-9) and normally not accessible to cells in the native proteins, but they may be exposed during growth and reorganization events of the extracellular matrix. Another subdivision of integrins can be made based on structural similarities of the α-chains. A number of integrins contain an extracellular I -domain (10,11) which is homologous to collagen binding A-domains present in von Willebrand factor (12) . The I-domain constitutes an inserted domain of approximately 200 amino acids which is present in 8 known integrins (αl, α2 , αlO, αL, αM, αX, αD and αE) (5,10) . Structural analysis of integrin I-domains crystallized in the presence of Mg 2+ have revealed the presence of a characteristic "MIDAS" (metal ion dependent adhesion site) motif, shown to be critical for ligand binding (13) . Integrin α-chains containing the I-domain are not cleaved into heavy and light chains, although the rat αl chain possesses a proteolytic cleavage site near the membrane spanning region (14,15) . For I-domain integrins the principal ligand binding sites are found within the I-domain (10). Known ligands for I-domains found within the βl integrin subfamily include laminins and collagens (αlβl and α2βl integrins) (16-19) , and Echo- virus (α2βl integrin) (20) . Structure comparisons have suggested that integrins fold into a so-called 7-bladed β-propeller structure which forms one globular domain with the ligand binding region on the upper surface (21) . The I-domain is in- serted between blade 2 and 3 in this propeller and divalent cation binding sites are located on the lower surface in blades 4-7 (22,23). Studies of β2 integrins have revealed that proper folding of the β2 -chain is dependent on the presence of the αL-chain but that the I- domain folds independently of other structural elements in the α- and β-chains (24) . In integrin α-chains, a less conserved stalk region separates the predicted β-pro- peller from the short transmembrane region. This stalk region is possibly involved in transducing conformational changes between the extracellular and intracellular regions, as well as mediating protein-protein interactions. Although integrins take part in cell signalling events, the cytoplasmic tail is short and lacks enzymatic activity. The sequence GFFKR is conserved in a majority of integrin α-subunits cytoplasmic tails and has been shown to be important for calreticulin binding (25) . Cellular interactions with the extracellular matrix during muscle formation and in muscular dystrophy have received increased interest during the past years. In the early 1960 's a mutant was described in Drosophila which was characterized by the detachment of muscles from their attachment points at the time of the first embryonic muscle contraction, causing the embryos to assume a spheroid shape (26) . The mapping of the molecular defect in the lethal myospheroid mutant in 1988 to an integrin β-chain (27) , was the first evidence for a role of integrins in maintaining muscle integrity. More recently, refined analysis of Drosophila mutants have indicated distinct roles for integrins in muscle endpoint attachments and sarcomere structure (28) . The Drosophila integrins are all cleaved α-chains and share many fea- tures with vertebrate integrins such as the ability to cluster into focal contacts (29) . The finding that inactivation of the α7 integrin gene in mouse (30) , as well as mutations in the human ITGA7 gene (31) , both cause muscular dystrophy affecting mainly muscle attachment points, indicates a striking conservation of integrin function during evolution. Of the 11 members of the βl subfamily, α7 exists as a major integrin α-chain (32,33) associated with the βlD integrin chain in the adult skeletal muscle sarcolemma (34) . Intriguingly, mutations in the basement membrane protein laminin α2-chain (35-37) cause a more severe disease than that observed for the laminin receptor integrin α7βl (30) . This indicates that other receptors for laminins exist in muscle. A novel integrin has recently been identified on cultured human fetal muscle cells (38) . The present invention is related to, inter alia, the cloning and characterization of this novel I-domain containing, βl- associated integrin chain, which is expressed in muscle tissues . SUMMARY OF THE INVENTION The full-length cDNA for this integrin subunit, all, has now been isolated. The open reading frame of the cDNA encodes a precursor of 1188 amino acids. The predicted mature protein of 1166 amino acids contains 7 conserved FG-GAP repeats, an I-domain with a MIDAS motif, a short transmembrane region and a unique cytoplasmic domain of 24 amino acids containing the sequence GFFRS . all, like other I-domain integrins, lacks a dibasic cleavage site for generation of a heavy and a light chain, and contains three potentional divalent cation binding sites in re- peats 5-7. The presence of 22 inserted amino acids in the extracellular stalk portion (amino acids 804-826) distinguishes the all integrin sequence from other integrin a-chains. Amino acid sequence comparisons reveal the highest identity of 42% with αlO integrin chain. Immuno- precipitation with antibodies to all integrin captures a 145 kD protein, distinctly larger than the 140 kD α2 integrin chain when analyzed by SDS-PAGE under non- reducing conditions. Fluorescense in situ hybridization maps the integrin all gene to chromosome 15q23, in the vicinity of an identified locus for Bardet-Biedl syndrome. Based on Northern blotting integrin all mRNA levels are high in adult human uterus and in heart, and intermediate in skeletal muscle and some other tissues tested. During in vitro myogenic differentiation, all mRNA and protein are up-regulated. Studies of ligand binding properties show that αllβl binds collagen type I Sepharose and cultured muscle cells localize αllβl into focal contacts on collagen type I. DETAILED DESCRIPTION OF THE INVENTION The present invention relates in its different aspects to the following: A recombinant or isolated integrin subunit all comprising essentially the amino acid sequence shown in SEQ ID No. 1, or homologues or fragments thereof. The invention also encompasses integrin homologues of said integrin, isolated from other species, such as bovine integrin heterodimer comprising a subunit all in association with a subunit β, preferably βl, as well as homologues isolated from other types of human cells or from cells originating from other species. The term "homologues" in the context of the present invention is meant to imply proteins of a common evolutionary origin, having identical or similar functions, specifically requiring evidence based on gene structure and not merely a similarity of protein structure. The invention also encompasses a process of producing a recombinant integrin subunit all comprising essentially the amino acid sequence shown in SEQ ID No. 1, or homologues or fragments thereof, which process comprises the steps of a) isolating a polynucleotide comprising a nucleotide sequence coding for an integrin subunit all, or homologues or fragments thereof, b) constructing an expression vector comprising the isolated polynucleotide, c) transforming a host cell with said expression vector, d) culturing said transformed host cell in a culture medium under conditions suitable for expression of inte- grin subunit all, or homologues or fragments thereof, in said transformed host cell, and, optionally, e) isolating the integrin subunit all, or homologues or fragments thereof, from said transformed host cell or said culture medium. The transformation can be performed in vi tro, in si tu or in vivo . In further aspects, the invention encomppases : - A process of providing an integrin subunit all, or homologues or fragments thereof, whereby said subunit is isolated from a cell in which it is naturally present. - An isolated polynucleotide comprising a nucleotide coding for said integrin subunit all, or for homologues or fragments thereof, which polynucleotide comprises essentially the nucleotide sequence shown in SEQ ID No. 1 or suitable parts thereof. - An isolated polynucleotide or oligonucleotide which hybridises to a polynucleotide or oligonucleotide encoding said integrin subunit all or homologues or fragments thereof, wherein said isolated polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding an integrin subunit αlO. - A vector comprising a polynucleotide or oligonucleotide coding for said integrin subunit all, or homologues or fragments thereof, which polynucleotide or oligonucleotide comprises the nucleotide sequence shown in SEQ ID No. 1 or parts thereof. - A vector comprising a polynucleotide or oligonucleotide which hybridises to a DNA or RNA encoding an integrin subunit all or homologues or fragments thereof, wherein said polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding an integrin subunit αlO. - A cell containing the vector as defined above. - A cell generated during the process as defined above, in which a polynucleotide or oligonucleotide coding for said integrin subunit all, or homologues or fragments thereof, which polynucleotide or oligonucleotide comprises essentially the nucleotide sequence shown in SEQ ID No. 1 or parts thereof, has been stably integrated in the cell genome. - Binding sites of the amino acid sequence of the integrin subunit all, or of homologues or fragments thereof, said binding sites having the capability of binding specifically to entities chosen from the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, polyclonal and monoclonal antibodies, and fragments thereof. - Binding entities having the capability of binding specifically to integrin subunit all comprising the amino acid sequence of SEQ ID No . 1 or to homologues or fragments thereof, preferably chosen from the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, polyclonal and monoclonal antibodies, and fragments thereof. - A recombinant or isolated integrin heterodimer comprising a subunit all and a subunit β, in which the subunit all comprises essentially the amino acid sequence shown in SEQ ID No. 1, or homologues and fragments thereof. Said subunit β is preferably βl . - A process of producing a recombinant integrin heterodimer comprising a subunit all and a subunit β, in which the subunit all comprises essentially the amino acid sequence shown in SEQ ID No. 1, or homologues or fragments thereof, which process comprises the steps of a) isolating one polynucleotide comprising a nucleotide sequence coding for a subunit all of an integrin heterodimer and, optionally, another polynucleotide com- prising a nucleotide sequence coding for a subunit β of an integrin heterodimer, or polynucleotides or oligo- nucleotides coding for homologues or fragments thereof having similar biological activity, b) constructing an expression vector comprising said isolated polynucleotide coding for said subunit all optionally in combination with an expression vector com- prising said isolated nucleotide coding for said sub- unit β, c) transforming a host cell with said expression vector or vectors, which transformation may be performed in vi tro, in si tu or in vivo, d) culturing said transformed host cell in a culture medium under conditions suitable for expression of an integrin heterodimer comprising a subunit all and a sub- unit β, or homologues or fragments thereof, in said transformed host cell, and, optionally, e) isolating the integrin heterodimer comprising a subunit all and a subunit β, or homologues or fragments thereof, or the all subunit thereof from said transformed host cell or said culture medium. - A process of providing an integrin heterodimer comprising a subunit all and a subunit β, or homologues or fragments thereof having similar biological activity, whereby said integrin heterodimer is isolated from a cell in which it is naturally present. - A cell containing i) a first vector, said first vector comprising a polynucleotide or oligonucleotide coding for a subunit all of an integrin heterodimer, or for homologues or parts thereof, which polynucleotide or oligonucleotide comprises essentially the nucleotide sequence shown in SEQ ID No. 1 or parts thereof, and ii) a second vector, said second vector comprising a polynucleotide or oligonucleotide coding for a subunit β of an integrin heterodimer, or for homologues or frag- ments thereof. - Binding sites of an integrin heterodimer as defined above, or of homologues or fragments thereof, said binding sites having the capability of binding specifically to entities chosen among the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, polyclonal and monoclonal antibodies, and fragments thereof. - Binding entities having the capability of binding specifically to said integrin heterodimer, or to homologues or fragments thereof, or a subunit all thereof. Said subunit β is preferably βl.The binding entities are preferably chosen among the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, and fragments thereof. - A fragment of the integrin subunit all, which fragment is a peptide chosen from the group comprising peptides of the cytoplasmic domain, especially a peptide comprising essentially the ammo acid sequence KLGFFRSARRRREPGLDPTPKVLE, of the I -domain, especially a peptide comprising essentially the ammo acid sequence from about ammo acid No. 159 to about ammo acid No. 355 of SEQ ID No. 1, and the extracellular extension region, especially a peptide comprising esentially the ammo acid sequence from about ammo acid No. 804 to about ammo acid No. 826 of SEQ ID No. 1. - A method of producing a fragment of the integrin subunit all as defined above, which method comprises a sequential addition of ammo acids . This method comprises adding and removing protective groups a manner known by the man skilled in the art. - A polynucleotide or oligonucleotide coding for a fragment of the integrin subunit all as defined above. - Binding sites of a fragment as defined above, said binding sites having the capability of binding specifically to entities chosen from the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, and fragments thereof. - Binding entities havmg the capability of binding specifically to a fragment as defined of the human integrin subunit all as defined above. Preferably, said binding entities are chosen from the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, and fragments thereof. - A process of using an integrin subunit all comprising essentially the amino acid sequence shown in SEQ ID No. 1 or an integrin heterodimer comprising said subunit all and a subunit β, or a homologue or fragment of said integrin or subunit, as a marker or target molecule of cells or tissues expressing said integrin subunit all, which cells or tissues are of animal including human origin. Especially, said subunit β is βl . In embodiments of this process, said fragment is a peptide chosen from the above defined group. In one embodiment of said process, the cells are chosen from the group comprising fibroblasts, muscle cells, chondrocytes, osteoblasts, mesenchymally derived cells and stem cells. Especially, said process is used during pathological conditions involving said subunit all. Said pathological conditions comprise in one embodiment damage of muscles, muscle dystrophy, fibrosis or wound healing. In another embodiment, said pathological conditions comprise damage of cartilage and/or bone, or cartilage and/or bone diseases. In a still further embodiment, said pathological conditions comprise trauma, rheumatoid arthritis, osteoarthritis or osteoporosis. In a further embodiment, said process is a process for detecting the formation of cartilage during embryonic development, or for detecting physiological or therapeutic reparation of cartilage and/or muscle, or for selection and analysis, or for sorting, isolating or purification of chondrocytes and/or muscle cells, or for detecting regeneration of cartilage or chondrocytes during transplantation of cartilage or chondrocytes, respectively, or of muscle or muscle cells during transplantation of muscle or muscle cells, respectively, or for studies of differentiation of condrocytes or muscle cells. Said process may be and in vi tro, an in si tu or an in vivo process. - A process of using binding entities having the capability of binding specifically to binding sites of an integrin subunit all as defined above, or of an integrin heterodimer comprising said subunit all and a subunit β, or to homologues or fragments thereof, as markers or target molecules of cells or tissues expressing said integrin subunit all, which cells or tissues are of animal including human origin. Especially, said subunit β is βl. In embodiments of this process, said fragment is as defined above. In one embodiment, said process is a process for detecting the presence of an integrin subunit all corn- prising the amino acid sequence shown in SEQ ID No. 1, or of an integrin heterodimer comprising said subunit all and a subunit β, or of homologues or fragments thereof. Furthermore, embodiments of this process encompass similar embodiments as defined above in connection with the process of using the integrin subunit all as a marker or target molecule. - A process for detecting the presence of an integrin subunit all, or of a homologue or fragment of said integrin subunit, as defined above, on cells, whereby a polynucleotide or oligonucleotide chosen from the group comprising essentially a polynucleotide or oligonucleotide as shown in SEQ ID No. 1 is used as a marker under hybridisation conditions, wherein said polynucleotide or oligonucleotide fails to hybridise to a DNA or RNA encoding an integrin subunit αlO. Said cells may be chosen from the group comprising muscle cells. In embodiments of this process, said fragment is as defined above . Furthermore, embodiments of this process encompass similar embodiments as defined above in connection with the process of using the integrin subunit all as a marker or target molecule. - A pharmaceutical composition comprising as an active ingredient a pharmaceutical agent or an antibody which is capable of using an integrin heterodimer comprising a subunit all and a subunit β, or the subunit all thereof, or a homologue or fragment of said integrin or subunit all, as a target molecule. - A pharmaceutical composition comprising as an active ingredient a pharmaceutical agent or an antibody which is capable of stimulating cell surface expression or activation of an integrin heterodimer comprising a subunit all and a subunit β, or the subunit all thereof, or homologues or fragmens of said integrin or subunit all. In one embodiment, said composition is for use in stimulating, inhibiting or blocking the formation of muscles, cartilage, bone or blood vessels. - A vaccine comprising as an active ingredient at least one member of the group comprising an integrin heterodimer, which heterodimer comprises a subunit all and a subunit β, or the subunit all thereof, and mologues or fragments of said integrin or subunit all, and a polynucleotide and a oligonucleotide coding for said integrin subunit all. - A method of gene therapy, whereby a vector comprising a polynucleotide or oligonucleotide coding for a subunit all of an integrin heterodimer, or for homologues or fragments thereof, which polynucleotide or oligonucleotide comprises essentially the nucleotide sequence shown in SEQ ID NO: 1 or parts thereof, and optionally a second vector comprising a polynucleotide or oligonucleotide coding for a sbunit β of said integrin heterodimer, is administered to a subject suffering from pathological conditions involving said subunit all . - A method of using binding entities having the capability of binding specifically to binding sites of a integrin subunit all comprising substantially the amino acid sequence shown in SEQ ID No . 1, or of an integrin heterodimer comprising said subunit all and a subunit β, or to homologues or fragments thereof, for promoting adhesion of cells. - A method of using an integrin heterodimer comprising an integrin subunit all and a subunit β, or the subunit all thereof, or homologues or fragments of said integrin or subunit all, as a target for anti- adhesive drugs or molecules in tissues where adhesion impairs the function of the tissue. - A method of in vitro detecting the presence of integrin binding entities, comprising interaction of an integrin heterodimer comprising a subunit all and a sub- unit β, or the subunit all thereof, or homologues or fragments of said integrin or subunit, with a sample, thereby causing said integrin, subunit all, or homologue or fragment thereof, to modulate the binding to its natural ligand or other integrin binding proteins present in said sample. - A method of in vitro studying consequences of the interaction of a human heterodimer integrin comprising a subunit all and a subunit β, or the subunit all thereof, or homologues or fragments of said integrin or subunit, with an integrin binding entity and thereby initiate a cellular reaction. In one embodiment of this method, the consequences of said interactions are measured as altera- tions in cellular functions. - A method of using a polynucleotide or oligonucleotide encoding an integrin subunit all or homologues or fragments thereof as a target molecule. One embodiment of this method comprises hybridising a polynucleotide or oligonucleotide to the DNA or RNA encoding the integrin subunit all or homologue or fragment thereof, which polynucleotide or oligonucleotide fails to hybridise to a polynucleotide or oligonucleotide encoding an integrin subunit αlO. - A method of using binding entities having the capability of binding specifically to an integrin subunit αlO comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No . 2, or an integrin heterodimer comprising said subunit αlO and a subunit β, or to homologues or fragments thereof having similar biological activity, for promoting adhesion of chondrocytes and/or osteoblasts to surfaces of implants to stimulate osseo- integration. - A method of using an integrin heterodimer comprising an integrin subunit all and a subunit β, or the subunit αlO thereof, or homologues or fragments of said integrin or subunit αlO, as a target for anti- adhesive drugs or molecules in tendon, ligament, skeletal muscle or other tissues where adhesion impairs the function of the tissue. - A method of stimulating, inhibiting or blocking the formation of cartilage or bone, comprising administration to a subject a suitable amount of a pharmaceutical agent or an antibody which is capable of using an integrin heterodimer comprising a subunit all and a sub- unit β, or the subunit all thereof, or homologues or fragments of said integrin or subunit all, as a target molecule . EXPERIMENTAL PROCEDURES Cell cultures The human fetal myoblast/myotube cultures were derived from clone G6 originating from a thigh muscle of a 73-day old aborted fetus ((39); referred to as G6 hereafter) . Cultures of G6 and 2.5 years postnatal human satellite cells XXVI, a gift from Dr. Helen Blau (Stanford University, CA) , were grown as reported earlier (39) . Human rhabdomyosarcoma cell lines RD (ATCC No. CCL- 136) and A204 (ATCC No. CRL-7900) were grown in DMEM (Swedish Agricultural University, Uppsala) supplemented with 10% fetal calf serum. RNA isolation and cDNA synthesis Total RNA from G6 and XXVI myoblasts, the same cells differentiated for 3 or 7 days, and RD and A204 cell lines, was isolated using the RNeasy Midi kit (Qiagen) according to the manufacturer's instructions. Poly-A RNA was extracted from total RNA of G6 and XXVI cells using Dynabeads mRNA DIRECT kit (DYNAL A.S., Norway) . PCR based cloning and generation of human all probes First strand cDNA was generated from 1 μg of G6 mRNA using a reverse transcription PCR-kit (Perkin-Elmer) . Advantage cDNA Polymerase Mix (Clontech) was used in PCR amplifications using two different pairs of primers: (1) 5' ACG GGA GAC GTG TAC AAG TG 3' (forward), 5 ' -AAA GTG CTG AAC CTC CAC CC-3' (reverse) and (2) 5'-CAC CAT CCA CCA GGC TAT GC -3' (forward) , 5'-TTA GCG TTC CGT TAT AAA CA -3' (reverse). The PCR conditions were: 94°C, 4 min. ("hot start"); 94°C, 30 s ; 55°C, 30 s; and 72°C, 1 min., for 25 cycles. Two products, named PCR1 and PCR2 , were obtained (figure 1) , subcloned into the plasmid vector TA (Invitrogen), and sequenced. A single product of 1,4 kb in size, named PCR 3 (figure 1), was amplified using primers 1 (forward) and 2 (reverse) , and human heart Marathon-Ready cDNA (Clontech) as template. Anneal - ing temperatures in the applied touch-down program were: 68°C, 1 min., 5 cycles; 65°C, 1 min., 5 cycles; 60°C, 1 min, 25 cycles. Other steps were as described above. After the final cycle the reactions were extended for additional 7 min. at 72 °C followed by a hold step at 4°C. To obtain a sequence covering the 5' end, Rapid Amplification of cDNA Ends (RACE) was employed acording to the manufacturer's instructions (Marathon cDNA Amplification kit, Clontech) using cDNA prepared from G6 mRNA and the gene specific antisense primer: 5'-CTT GGA GAA CCT GAA GTT GGA GTT GAC -3'. Amplification was carried out applying the "touch-down" program (see above) . To identify relevant products, 10 μl of each RACE product was resolved on 1% agarose gel and subjected to Southern blot analysis as described previously (40) . PCR2 (see above) was labeled with [α- 3 P]dCTP (Amersham Pharmacia Biotech, Sweden) using the RedyPrimell DNA labeling system (Amersham Pharmacia Biotech, UK) , and used as a hybridi- zation probe. One specific signal was detected. Corresponding cDNA was purified (Gel Extraction kit, Quagen) , cloned into the TA vector and sequenced (see figure 1) . Screening of cDNA libraries A λZAP custom made G6 cDNA library (Stratagene, USA) was screened with PCR2 (see above) as a probe. The screening procedure (carried out as described in (40) ) resulted in two clones representing the 5' non-coding region and the beginning of the coding part of integrin all (figure 1) . To obtain an additional sequence, a human uterus 5'- stretch λgtll cDNA library (Clontech) was screened with a mixture of PCRl and PCR2 as probes. The probes were labeled with [α- 32 P]dCTP using the Ready-To-Go DNA labeling beads (Amersham Pharmacia Biotech, Sweden) . Three clones (1.1-1.3 in figure 1) representing parts of all cDNA, were obtained. Rescreening of the human uterus 5' -stretch λgtll cDNA library with the probe λ290 (corresponding to 2183- 2473 in Fig. 1) yielded three more clones (2.1-2.3, figure 1) covering the rest of all cDNA. Positive clones were plaque purified, the phage DNA isolated using the Lambda Midi kit (Qiagen) and then sub-cloned into the Bluescript SK or pUC19 plasmid vectors before sequencing. Northern hybridization A filter containing 6 μg of the poly-A RNA from G6 and XXVI cells and 10 μg of the total RNA from RD and A204 cell lines, and a Human Multiple Tissue Northern Blot containing poly-A RNA from adult human tissues (Clontech) , were hybridized at 68°C in ExpressHyb solution (Clontech) with probes labeled as described above. The probes used were PCRl, PCR2 , cDNA clone 1.3 (figure 1), 3RA (1.8 kb cDNA specific for human integrin αl mRNA, a generous gift from E.E. Marcantonio (Columbia Univer ¬ sity, New York), a 1.1 kb cDNA clone recognizing human G3PHD mRNA and a 1.8 kb cDNA clone recognizing human β- actin (both from Clontech) . cDNA sequencing and sequence analysis All PCR fragments and cDNA clones were sequenced on both strands either manually (29) or using ABI 310 Genetic Analyzer automatic sequencer. Sequences were analyzed with the aid of MacVector™ 6.0, DNA Star, Faktura™NE 1.2.0, and Sequence Navigator 1.0.1 software programs. A distance tree of all I-domain containing integrin α subunits was assembled using SEAVIE and PHYLO-WIN softwares (41) . Percent similarity between every two members in the I-domain integrin subfamily was calculated by a formula I=(l-D)xl00, where "I" is identity and "D" is distance . Antibodies A polyclonal antiserum (all cyt) was produced against the peptide CRREPGLDPTPKVLE from the integrin all cytoplasmic domain. Peptide synthesis and conjugation to Keyhole limpet hemocyanin, immunization of rabbits and affinity purification was performed at Innovagen AB (Lund, Sweden) . The monoclonal antibody Mab 13 against integrin βl was obtained from S.K. Akiyama (NIEHS, NIH) . Monoclonal antibodies to integrin αl (clone FB12 , sold as MAB 1973) and integrin α2 (clone BHA2.1 sold as Mab 1998) were both obtained from Chemicon, Temecula, CA. The monoclonal antibody to vinculin (clone hVIN-1) was from Sigma (Saint Louis, MO, USA) . Secondary fluorescent antibodies (CY3™-coupled goat-anti rabbit IgG and FITC- coupled goat anti-mouse IgG of multiple labeling grade) were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA) . Immunoprecipitation and SDS-PAGE G6 and XXVI cells were labeled with [ 35 S] cysteine/ methionine and subjected to immunoprecipitation and SDS- PAGE as reported previously (38) . The two-step procedure used to dissociate integrin heterodimers was carried out as follows. After incubation of samples with βl antibody and capture with GammaBind G Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) , 100 μl of 1% SDS was added to the washed beads which were then boiled for 5 minutes. 10 mM Tris-HCl, pH 7.4 , 0.15 M NaCl and 1% Triton X-100 was added to a final volume of 1ml and the lysate was incubated with GammaBind G Sepharose for 1 hour. The incubation with GammaBind G was performed in order to ensure that no reactive βl antibodies remained. After removal of GammaBind G Sepharose, all integrin antibody was added for additional 2 hours, followed by capture with protein A Sepharose (Amersham Pharmacia Biotech) and boiling in SDS-PAGE sample buffer. Chromosomal localization Chromosomal localization of the human integrin all was performed by using a combination of FISH (Fluorescent In Situ Hybridization) technique and DAPI (4 ' , 6-diamidino-2-phenylindole) banding essentially as described earlier (42) . As a hybridization probe, the 1.4 kb RT-PCR product PCR3 was used. Surface iodination and affinity chromatographv Cultured XXVI cells were surface iodinated as described (38) . Labeled cells were solubilized in 1 ml of solubilization buffer (10 mM Tris-HCl pH 7.4 , 15 mM NaCl, 1% Triton X-100, ImM MgCl 2 , 1 mM CaCl 2 , ImM MnCl 2 ) , centrifuged at 14000 g for 20 min., and soluble membrane proteins were applied to a collagen type I Sepharose (bovine collagen type I from Vitrogen (Collagen Corp., Palo Alto) coupled to CNBr-activated Sepharose CL-4B at 3 mg/ml gel as described (14) ) , equilibrated in solubilization buffer. Following a one hour incubation the column was washed extensively with buffer A (10 mM Tris-HCl pH 7.4 , 50 mM NaCl, 1 mM MnCl 2 , 0.1% Triton X-100) and by 10 column volumes of buffer A without NaCl. Bound proteins were eluted with 20 mM EDTA, 10 mM Tris- HCl pH 7.4, 0.1% Triton X-100. Peak fractions were pooled and concentrated by immunoprecipitation with βl integrin and all integrin antibodies as described under Immunoprecipitation and SDS-PAGE. Eluted frations and captured proteins were analyzed on 7.5% SDS-PAGE gels followed by autoradiography . Indirect immunofluorescence Cells cultured on coverslips were washed in serum- free medium and fixed for 8 min. in acetone at -20 °C. Non-specific binding sites were blocked by incubating with 10% goat serum diluted in phosphate buffered saline. In the double immunofluorescence staining protocol, primary antibodies (anti-αll cyt (rabbit antibody) and anti- vinculin (mouse antibody) ) were simultaneously incubated with fixed cells for 1.5 hours at +37°C. Specifically bound antibodies were detected using anti -rabbit Cy3 IgG and anti-mouse FITC IgG. Stained cells were mounted in Vectashield™ mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA) and visualized and photographed under a Zeiss light microscipe equipped with optics for observing fluorescence. RESULTS AND DISCUSSION cDNA cloning of a novel integrin α-chain In order to determine the nature of the integrin chain that we had previously characterized on human fetal muscle cells and named αmt (38) , a number of approaches were used. Applying PCR with mRNA from fetal muscle cells as template together with degenerate primers to conserved regions of integrin α subunits (43) we amplified cDNA for αl, α4 , α5, α6 and αv integrin chains (data not shown), but failed to amplify the novel integrin. However, while searching through the literature we came across two inte- grin sequences obtained in a subtractive hybridization protocol comparing human primary myoblasts with the rhab- domyosarcoma cell line RD (44) . After having confirmed that these sequences could be amplified by PCR from human fetal G6 myoblast cDNA, PCR was performed assuming that these sequences were derived from the same transcript. In this manner a 1.4 kb cDNA fragment with integrin-like sequence was obtained. Screening of a human fetal myo- blast cDNA library and 5' RACE yielded additional 5' sequence. We determined the mRNA expression pattern in a number of human tissues (see below) and observed a high mRNA expression in the uterus. Screening of a uterus cDNA library resulted in the identification of the complete open reading frame. A schematic illustration of the cloning strategy is shown in figure 1. cDNA sequence and predicted amino acid sequence of all integrin chain By sequence analysis of cDNA clones and 5' RACE products we obtained a continuous sequence of 3983 nucleotides (nt) composed of 90 nt 5' non-coding sequence, 3564 nt open reading frame, and 326 nt 3' non- coding sequence. Translation of the sequence predicts an integrin α-chain like precursor of 1188 amino acids including a 22 amino acid long signal peptide (fig. 2, GenBank accession No. AF137378) . The mature 1166 amino acid long peptide is larger than any other currently identified integrin α-chain (the closest being αE, com- posed of 1160 amino acids (45) . The 1115 amino acid long predicted extracellular domain contains 7 FG-GAP repeats in the amino-terminal end with an inserted I-domain between repeats 2 and 3. The I-domain consists of 195 amino acids and includes a conserved MIDAS motif. In addition to the metal chelating site in the I-domain, three additional potential divalent cation binding motifs with the consensus sequence DXD/NXDXXXD are present in repeats 5-7. A total of 20 cysteines are located in the extracellular domain. Of these, 16 are conserved in the most closely related integrin αlO and αl chains and they may contribute to intramolecular disulphide bonds. The two non-conserved cysteines found at positions Cys 606 and Cys 988 most likely represent free unpaired cysteines while the two non-conserved cysteines Cys 806 and Cys 817 may pair to form a disulphide bond. Mapping of the cysteines in the suggested β-propeller structure shows that the first three disulphide bonds are likely to stabilize blades one and two of the β-propeller whereas the remaining bonds are found outside the propeller region, in the stalk region towards the transmembrane domain. 16 potentional N-glycosylation sites are present in all. A search for sequence motifs reveals the presence of a 22 amino acid leucine zipper motif starting at position 951, and a 17 amino acid sequence starting at position 1082, which is similar to sequences found in G-protein coupled receptors . These sequences might represent funtional domains of importance for protein-protein interactions. The transmembrane region (amino acids 1142-1164) is 23 amino acids long and is followed by a cytoplasmic tail of 24 amino acids. The cytoplasmic tail contains the sequence GFFRS instead of the conserved GFFKR sequence, found in all other α-chains except α8-αl0. It will be interesting to determine the importance of this sequence in defining the cytoplasmic domain as well as its possible ability to bind calreticulin and other intracellular components. Comparison of integrin all chain with other integrin α chains Alignment of the predicted all integrin amino acid sequence with other integrin sequences shows the highest overall identity with αlO (42% identity) , αl (37% iden- tity) , and α2 (35% identity) , followed by the remaining I-domain containing integrin subunits. Of the non I- domain containing integrins, α4 and α9 are the most similar to all. A distance tree shows that αlO and all form a separate branch from the most closely related αl and α2 integrin chains (fig. 3) . The similarity with other integrins is particularly high in the N-terminal β-propeller part but lower in the stalk region. Comparison of αl integrin with α2 integrin has pointed to the presence of a 38-residue insert in the β-propeller region of αl integrin chain (15) . Like αl chain, all also contains inserted amino acids not present in the other I-domain containing integrin chains, however, in the all chain these are found within the stalk region at amino acids 804-826. The exact border of the predicted insertion varies depending on the alignment method and the parameters chosen, but is predicted to span at least 22 amino acids. The insert shows no significant similarity to other integrin sequences and contains two cysteines likely to form a disulphide bond (see fig. 2) . We do not believe that the predicted inserted sequence represents a cloning artifact since it is present in three inde- pendently analysed clones. Other examples of non I-domain inserted sequences are found in the Drosophila αPS2 chain, where developmentally regulated splicing in the ligand binding region modulates ligand affinity (46) . In α7 integrin chain, splicing in the extracellular domain between predicted blades 2 and 3 in the β-propeller generates XI and X2 variants, affecting the binding to laminin-1 in a cell-specific manner (47) . In the more closely related αl integrin chain the 38 extra amino acids are present in a position that is predicted to be in the beginning of the sixth blade of the 7-bladed propeller. So far there is no evidence that the extra amino acids in either αl or all arise by alternative splicing. In all the predicted inserted region is outside the β-propeller and most likely does not directly affect ligand binding, but might instead be involved in modifying protein-protein interactions or be important for outside-in or inside-out signalling. In this regard it is interesting to note that tetraspan proteins by binding to the stalk region of certain integrin α-chains can recruit PI -4 kinase and protein kinase C to integrin complexes (48) . Likewise the extracellular membrane-proximal parts of certain integrin α-chains have been shown to be involved in She-mediated integrin signalling (49) . Analysis of sequences identified during screening for genes upregulated during tadpole regression revealed a partial sequence, which at the time was reported to show the highest similarity to integrin αl (41% identity) (50) . This sequence, when translated (amino acids 1-116), shows 71% identity to human all and thus most likely represents the Xenopus orthologue of all rather than that of the αl . These data suggest that all is well conserved during evolution. Chromosomal localization of the integrin all gene A fluorescent cDNA probe was used for in situ hybridization on metaphase chromosome spreads. The analysis shows that the integrin all gene (ITGA11) is located on chromosome 15q23 (fig. 4) . The genes for I-domain containing integrins αl and α2 are both present on chromosome 5 (51,52), just as the genes for the closely related β2 integrin associated α-chains all map to chromosome 16 (53) . Interestingly, the all gene and the closely related αl and α2 genes, map to different chromosomes. It will be of evolutionary interest to determine the chromosomal localization of the integrin αlO gene. Curiously, a form of Bardet-Biedl syndrome characterized by retinitis pigmentosa, polydactyly, obesity, hypogenitalism, mental retardation, and renal anomalies maps to 15q22-23 (54) . Future studies will clarify a possible linkage of ITGA11 to Bardet-Biedl syndrome. Expression pattern of all mRNA in adult tissues Northern blot analysis of mRNA from various adult human tissues shows the highest level of expression of all in adult human uterus. A strong signal is also noted in heart, while intermediate levels of all mRNA are present in skeletal muscle and intermediate to low levels in other adult tissues tested (fig. 5 and data not shown) . For a comparison, the same blot was probed for the closely related αl integrin mRNA (fig. 5) . A striking difference in the expression levels of αl and all was observed in the smooth muscle rich uterus, which appears to lack αl . Immunohistochemical analysis and in situ hybridiza- tions will elucidate the detailed distribution of all protein and mRNA in muscle and other tissues. Neither αl (33) nor α2 (55) are present in muscle fibers, and the distribution of αlO in skeletal muscle tissues is not known (5) . Hence, no I-domain containing integrin has so far been reported to be expressed in the skeletal muscle sarcolemma. Recently the gene for αl integrin was inacti- vated in mice, resulting in mice with an apparently normal phenotype (56) . More careful analysis revealed a phenotype characterized by a hypocellular skin (57) and aberrant regulation of collagen synthesis (58) . It will be interesting to compare sites of overlapping expression between αl , α2 and αlO integrins, and use reagents to αlO and all to examine possible funcional compensatory mechanisms in αl integrin-deficient mice. Biochemical characterization of all protein Following the cloning of the full-length all inte- grin cDNA it was essential to determine if the predicted amino acid sequence was identical to the novel uncleaved βl integrin-associated α-chain that we had previously noted to be upregulated during in vitro differentiation of human myoblasts (38) . To answer this question we raised antibodies to the cytoplasmic tail of the integrin all chain. Immunoprecipitation from the human satellite cells showed that the antibodies precipitated a 145 kDa all band associated with a 115 kDa βl band (fig. 6, panel A) in SDS-PAGE under non-reducing conditions. Under re- ducing conditions the all band migrated as 155 kDa (see fig. 6, panel B) . From the translated amino acid sequence an Mr of 133 400 is predicted for the all chain. Taking the 16 potential glycosylation sites into account this fits well with the observed 155 kDa band in SDS-PAGE. Under non-reducing conditions the 145 kDa band is distinctly larger than α2 (fig. 6, panel A) and αlO integrin chains which co-migrate as 140 kDa bands and all migrates well below the 180 kDa integrin αl band. The α2 (59) and αlO (5) chains both contain 10 potentional glycosylation sites whereas αl contains 26 glycosylation sites (60) . The intermediate size of all in SDS-PAGE compared with αl and α2/αl0 is thus most likely a result of differential glycosylation . To show that all is associated with the βl subunit a two-step immunoprecipitation procedure was performed. Integrins were first precipitated with a monoclonal anti- βl integrin antibody and GammaBind G captured integrins were then dissociated by boiling in 1% SDS . In the second step, SDS was diluted tenfold and antibodies to all were added. As shown in fig. 6 panel A antibodies to all immu- noprecipitate only the 145 kDa band from the dissociated precipitate initially captured with βl antibodies. Induction of all mRNA and protein during myogenic differentiation in vitro It has previously been determined that αmt is the major integrin α-chain that is up-regulated during myogenic differentiation on human fetal myoblasts in vitro (38) . To compare all levels in myoblasts and myotubes, immuno-precipitates were analyzed from myoblast cultures in pro-liferation medium, and from parallel cultures allowed to differentiate and form myotubes in differentiation medium for 7 days. Immunoprecipitation with both βl and all antibodies showed that all, like amt, is strongly up-regulated at the protein level in differentiation cultures of human fetal muscle cells and satellite cells (fig. 6, panel B) . To determine if the up-regulation occurs at the mRNA or protein level we analyzed all mRNA from different differentiation stages (day 1, day 3 and day 7) (fig. 6, panel C) . Already at day 3 in differentiation medium a strong up-regulation of all mRNA was noted, establishing that the up-regulation of all integrin protein occurs as a result of increased transcription or mRNA stability. Based on similar SDS- PAGE migration patterns, similar behavior under reducing conditions, association with βl integrin chain, and up- regulation during in vitro differentiation of human fetal myoblasts, the present data show that all integrin is identical with αmt. Analysis of mRNA from the two rhabdomyosarcoma cell lines RD and A204 (fig. 6, panel C) did not provide evidence for the presence of all in either cell line. Based on the observed up-regulation of αllβl in human fetal muscle cells and the presence of all message in adult muscle we suggest that all integrin might be involved in early steps of muscle formation and that it in adult muscle tissues may fulfill a stabilizing role. The α7 integrin subunit is a major βl-associated integrin chain in muscle, but genetic deletion of α7 leads to a fairly mild muscular dystrophy (30) . Ligand binding specificity of αllβl integrin So far identified I-domain containing integrins of the βl integrin subfamily all bind collagens (5,15,59). For αl and α2 this binding capacity has been shown to reside within the I-domain (17,18). To determine if αllβl also binds collagen we performed collagen type I Sepharose chromatography of membrane proteins from surface- iodinated XXVI satellite cells. Direct analysis of the EDTA eluate revealed weak bands corresponding to the positions of αl , α2 , all and βl in parallel immunopre- cipitations (figur 7, panel 1) . The EDTA eluate was concentrated by immunoprecipitation with βl and all antibodies. As shown in figure 7, a prominent all band is present in the collagen I Sepharose eluate. The relatively weak βl band in the proteins captured with all antibodies indicates that the αllβl heterodimer partly dissociates in the presence of EDTA. To visualize the interaction of αllβl integrin with collagen I in intact cells, myogenic cells expressing αllβl were trypsinized and plated on collagen and fibronectin for 1 hour. The ability to form focal contacts was investigated by double immunofluorescence staining for all-chain and vinculin. As seen in panel 2 of figure 7 all localizes to vinculin positive focal contacts on collagen but not on fibronectin. Binding studies with all I-domain expressed as a bacterial GST-fusion protein also confirmed a specific affinity for collagen I (unpublished M. Hook, R. Rich, R. Owens) . Stable transfections of all cDNA into cells with various integrin backgrounds will allow a more detailed study of αllβl interactions with different collagen, and possibly also laminin, isoforms. Combined with in vivo distribution studies of αllβl this is likely to yield valuble information regarding the in vivo ligands for αllβl in different tissues. all integrin protein distribution in human embryo Morphologically normal human embryos (aged from 4 to 8 post-ovulatory weeks) were obtained from legal abortions induced by Mifepristone (RU486) at Hopital Broussais in Paris. All procedures were approved by the Ethical Committee of Saint-Vincent de Paul Hospital in Paris. Each sample was first examined macroscopically during dissection under a stereo-microscope. The development stage of the embryos was determined using established criteria. Tissues were collected shortly after delivery and frozen within the first 24 h post mortem on dry ice and stored at -80°C until used. Seven micron-thick cryostat sections were mounted on slides previously coated with a 2% 3-aminopropyl-triethoxysilane solution in acetone. The cryosection was left unfixed prior to blocking of non-specific binding sites with 10% goat serum diluted in phosphate buffered saline. For immunofluorscence, the section was incubated with all antibodies 1.5 h at +37°C. Specifically bound antibodies were detected using goat anti -rabbit Cy3 IgG (Jackson Immunoresearch) . The stained tissue section was mounted in Vectashield™ mounting medium (Vector Laboratories Inc.) and visualized and photographed under a Zeiss ligth microscope equipped with optics for observing fluorescence . The results obtained are shown in figure 8. 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Clones 1.1-1.3 and 2.1-2.3 are from the first and the second round of screening, respectively. Fragment 0.0 represents a 5' RACE product as well as a clone obtained from screening of the G6 library. PCR fragments 1-3 and a Sad fragment of a clone 1.3, λ290, are marked with thick lines. Names and positions of all the clones on a scheme are shown in tabulated form in B. B. Names of the PCR-amplified fragments and cDNA clones shown in A are in the left column, and their positions in the full length cDNA of integrin all in the right column. Figure 2. Nucleotide and dedued amino acid sequence of the human integrin all chain The putative signal peptide is underlined in bold, I-domain is boxed, potential N-linked glycosylation sites are marked with asterisks, cysteines are underlined, potential divalent cation binding motifs are double underlined and the transmembrane domain is underlined with dashes. A 22 amino acid insert is boxed in bold. Figure 3. A distance tree of the I-domain containing α- integrin subfamily members A tree was assembled based using ClustalW multiple alignment - based SEAVIEW and PHYLOWIN softwares. A scale at the bottom shows percent identity. Figure 4. Chromosome mapping of ITGA11 gene by fluores- cent in situ hybridization (FISH) A. Left panel shows the FISH signals on human chromosome 15; right panel shows the same mitotic figure stained with 4 ' , 6-diamino-2-phenylindole to identify human chromosome 15. B. Diagram of FISH mapping result for the probe PCR3 based on a detailed analyses of 10 different images. Each dot represents the double FISH signals detected on human chromosome 15. Figure 5. Expression of integrin all and αl subunit mRNAs in adult human tissues Integrin all mRNA and integrin αl mRNA were analyzed on a membrane with RNA from various adult human tissues where mRNA loading was normalized with respect to β-actin. Probes used for hybridizations are marked on the left. Size of molecular weight standard is marked to the right. Note that the β-actin probe reacts with 2 kb β/γ actin transcripts and the muscle specific 1.8 kb α-actin message . Figure 6. Biochemical characterization of integrin all chain and upregulation of corresponding protein and mRNA in myogenic cells A. all associates with βl integrin chain. Human XXVI and G6 muscle cells were metabolically labeled with [ 35 S] cysteine/methionine and integrins were immunoprecipitated with the indicated antibodies (βl, α2 and all) . Evidence for the association of integrin all with the βl subunit obtained by treating proteins precipitated with anti-βl antibodies with SDS followed by a second precipitation with all antibodies (ant-αll+SDS) . Precipitated proteins were resolved on 7.5% SDS-PAGE gels in the absence of reducing agents, followed by fluorography . B . Induction of integrin all upon myogenic differentiation in vitro. G6 muscle cells were metabolically labeled with [ 35 S] cysteine/methionine when growing in proliferation medium (mb-proliferating myoblasts) and after 7 days in differentiation medium) (mt-myotubes) . Integrins were precipitated with antibodies to βl and all and the precipitates were resolved on 7.5% SDS-PAGE gels both under non- reducing (UNREDUCED) and reducing (REDUCED) conditions. Lanes 1, 3, 5 and 7 are immunoprecipitations with the antibody to integrin βl, and lanes 2, 4, 6 and 8 with the antibody to integrin all. C Upregulation of integrin all mRNA in differentiated myogenic cells. mRNA was extracted from G6 and XXVI cells growing under proliferating (p) or differentiating (d) conditions for 3 days (d3) or 7 days (d7) . Total RNA was isolated from RD and A204 cells. Following separation of RNA on agarose gel and transfer to the membrane, the filter was hybridized with probes to all integrin (all) and glyce- raldehyde-3 -phosphate dehydrogenase (GAPDH) . Size of bands in RNA standard (in kb) are marked to the right. Figure 7. Ligand binding properties of αllβl integrin panel 1: Collagen binding integrins on XXVI cells. XXVI cells were surface iodinated and integrins were analyzed by immunoprecipitation and collagen I Sepharose affinity chromatography . Immunoprecipitation reveals the presence of βl integrins (lane 1) , αlβl (lane 2) , αllβl (lane 3) and α2βl (lane 4) at the surface of XXVI cells. EDTA eluted proteins bound to collagen I Sepharose con- tain weak band in the position of αl, all, a2 and βl integrin chains (lane 5) . Immunoprecipitations with βl integrin antibodies (lane 6) and all integrin antibodies (lane 7) confirm the presence of all and βl in the EDTA eluate . panel 2: αllβl localizes to focal contacts on collagen. Indirect immunofluorescent visualization of vinculin (A, B) and all integrin chain (C, D) in human XXVI satellite cells seeded on collagen type I (A and C) and fibronectin (B and D) . Note the localization of integrin all chain to focal contacts of cells allowed to attach to collagen and its complete absence on cells seeded on fibronectin. Vinculin is found in focal contacts on both substrates. A and C show the same cell double stained for both antigens. Scale bar is 20μm. Figur 8. all integrin protein distribution at 8 weeks of gestation. Composite of immunohistochemical staining of sagital section of human embryo at 8 weeks of gestation. Note high levels of all protein around vetrebrae (arrows) , in intervertebral disc (asterisks) , around ribs (thin arrows) and around forming cartilage in the forelimb (arrowhead) .

Claims: CLAIMS 1. A recombinant or isolated integrin subunit all comprising essentially the amino acid sequence shown in SEQ ID No. 1, and homologues and fragments thereof. 2. A process of producing a recombinant integrin subunit all comprising essentially the amino acid sequence shown in SEQ ID No. 1, and homologues and fragments thereof, which process comprises the steps of a) isolating a polynucleotide comprising a nucleotide sequence coding for a integrin subunit all, of for homologues and fragments thereof, b) constructing an expression vector comprising the isolated polynucleotide, c) transforming a host cell with said expression vector, d) culturing said transformed host cell in a culture medium under conditions suitable for expression of said integrin subunit all, of said homologues and fragments, in said transformed host cell, and, optionally, e) isolating the integrin subunit all, or homologues and fragments thereof, from said transformed host cell or said culture medium. 3. A process according to claim 2, step c, said transforming being an in vi tro or in si tu process. 4. A process according to claim 2, step c, said transforming being an in vivo process. 5. A process of providing an integrin subunit all, of homolouges or fragments thereof, whereby said subunit is isolated from a cell in which it is naturally present. 6. An isolated polynucleotide or oligonucleotide comprising a nucleotide coding for an integrin subunit all, or for homologues or fragments thereof, which polynucleotide or oligonucleotide comprises essentially the nucleotide sequence shown in SEQ ID No. 1 or suitable parts thereof. 7. An isolated polynucleotide or oligonucleotide which hybridises to a polynucleotide or oligonucleotide as defined in claim 4, whereby said isolated polynucleotide or oligonucleotide fails to hybridise to a polynucletide or oligonucleotide encoding an integrin subunit αlO. 8. A vector comprising a polynucleotide or oligonucleotide as defined in claim 6 or 7. 9. A cell containing the vector as defined in claim 8. 10. A cell, as generated by the process in steps a) to c) of claim 2, in which a polynucleotide or oligonucleotide coding for an integrin subunit all, or for homologues and fragments thereof, said polynucleotide or oligonucleotide comprising essentially the nucleotide sequence shown in SEQ ID No. 1 or parts thereof, has been stably integrated in the cell genome. 11. Binding sites of the amino acid sequence of the integrin subunit all, or of homologues and fragments thereof, as defined in claim 1, said binding sites having the capability of binding specifically to entities chosen from the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, polyclonal and monoclonal antibodies, and fragments thereof. 12. Binding entities having the capability of binding specifically to integrin subunit all, or to homologues or fragments thereof, as defined in claim 1, which entities are chosen from the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, polyclonal and monoclonal antibodies, and fragments thereof. 13. A recombinant or isolated integrin heterodimer comprising a subunit all and a subunit β, in which the subunit all comprises essentially the amino acid sequence shown in SEQ ID No. 1 or homologues or fragments thereof. 14. A recombinant or isolated integrin heterodimer according to claim 11, wherein the subunit β is βl . 15. A process of producing a recombinant integrin heterodimer comprising a subunit all and a subunit β, in which the subunit all comprises essentially the amino acid sequence shown in SEQ ID No. 1, or homologues or fragments thereof, which process comprises the steps of a) isolating one polynucleotide or oligonucleotide comprising a nucleotide sequence coding for said subunit all of said integrin heterodimer, or for said homologues or fragments thereof, and, optionally, another polynucleotide comprising a nucleotide sequence coding for said subunit β of an integrin heterodimer, or for homologues or fragments thereof, b) constructing an expression vector comprising said isolated polynucleotides or oligonucleotides c) transforming a host cell with said expression vector or vectors, d) culturing said transformed host cell in a culture medium under conditions suitable for expression of said integrin heterodimer, or said homologues or fragments thereof, in said transformed host cell, and, optionally, e) isolating said integrin heterodimer, or said homologues or fragments thereof, from said transformed host cell or said culture medium. 16. A process according to claim 15, step c, said transforming being an in vi tro process. 17. A process according to claim 15, step c, said transforming being an in vivo process. 18. A process of providing an integrin heterodimer comprising a subunit all and a subunit β, as defined in claim 13 or 14, or homologues or fragments thereof, whereby said integrin heterodimer is isolated from a cell in which it is naturally present. 19. A cell containing i) a first vector, said first vector comprising a polynucleotide or oligonucleotide coding for a subunit all of an integrin heterodimer, or for homologues or fragments thereof, which polynucleotide or oligonucleotide comprises essentially the nucleotide sequence shown in SEQ ID No. 1 or parts thereof, and ii) a second vector, said second vector comprising a polynucleotide or oligonucleotide coding for a subunit β of said integrin heterodimer. 20. Binding sites of an integrin heterodimer as defined in claim 13 or 14, or of homologues or fragments thereof, said binding sites having the capability of binding specifically to entities chosen among the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, polyclonal and monoclonal antibodies, and fragments thereof. 21. Binding entities having the capability of binding specifically to an integrin heterodimer as defined in claim 13 or 14, or to homologues or fragments thereof, said binding entities being chosen among the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, polyclonal and monoclonal antibodies, and fragments thereof. 22. A fragment of an integrin subunit all, which integrin subunit all comprises essentially the amino acid sequence shown in SEQ ID NO: 1, said fragment being a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the extracellular extension region. 23. A fragment according to claim 22, said fragment being a peptide from the cytoplasmic domain comprising essentially the amino acid sequence KLGFFRSARRRREPGLDPTPKVLE . 24. A fragment according to claim 22, which is a peptide comprising essentially the amino acid sequence of the extracellular domain, from about amino acid No. 804 to about amino acid no. 826 of SEQ ID No. 1. 25. A fragment according to claim 22, which is a peptide comprising essentially the amino acid sequence of the I-domain, from about amino acid No. 159 to about amino acid no. 355 of SEQ ID No. 1. 26. A method of producing a fragment of the integrin subunit all as defined in any one of claims 22-25, which method comprises a sequential addition of amino acids. 27. A polynucleotide or oligonucleotide coding for a fragment of the integrin subunit all as ' defined in any one of claims 22-25. 28. Binding sites of an integrin subunit all fragment as defined in any one of claims 22-25, said binding sites having the capability of binding specifically to entities chosen from the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, monoclonal and polyclonal antibodies, and fragments thereof. 29. Binding entities having the capability of binding specifically to an integrin subunit all fragment as defined in any one of claims 22-25, which binding entities are chosen from the group comprising proteins, peptides, carbohydrates, lipids, natural integrin binding ligands, monoclonal and polyclonal antibodies, and fragments thereof. 30. A process of using an integrin subunit all comprising essentially the amino acid sequence shown in SEQ ID No .1 or an integrin heterodimer comprising said subunit all and a subunit β, or homologues or fragments thereof, as a marker or target molecule of cells or tissues expressing said integrin subunit all, which cells or tissues are of animal including human origin. 31. A process according to claim 30, which is a process for determining the differentiation-state of cells during differentiation, development, in pathological conditions, in tissue regeneration, in transplantation, or in therapeutic and physiological reparation of tissues. 32. A process according to claim 31, which process is used during pathological conditions involving said subunit all. 33. A process according to claim 31, which patho- logical conditions are comprised within the group comprising damage of muscles, muscle dystrophy, fibrosis and wound healing. 34. A process according to claim 31, which pathological conditions are comprised within the group comprising damage of cartilage and/or bone, and cartilage and/or bone diseases. 35. A process according to claim 31, which pathological conditions are comprised within the group comprising trauma, rheumatoid arthritis, osteoarthritis and osteoporosis. 36. A process according to claim 30, which is a process for detecting the formation of cartilage during embryonic development . 37. A process according to claim 30, which is a process for detecting physiological or therapeutic repair of cartilage and/or muscle. 38. A process according to claim 30, which is a process for selection and analysis, or for sorting, isolating or purification of chondrocytes and/or muscle cells. 39. A process according to claim 30, which is a process for detecting regeneration of cartilage or chondrocytes during transplantation of cartilage or chondrocytes, respectively, or of muscle or muscle cells during transplantation of muscle or muscle cells, respec ively. 40. A process according to claim 30, which is a process for studies of differentiation of chondrocytes or muscle cells. 41. A process according to any one of claims 30-40, which is an in vi tro process. 42. A process according to any one of claims 30-40, which is an in si tu process. 43. A process according to any one of claims 30-40, which is an in vivo process. 44. A process according to any one of claims 30-43, whereby a fragment of said integrin subunit all is a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the extracellular extension region. 45. A process according to claim 44, whereby said fragment is a peptide comprising essentially the amino acid sequence KLGFFRSARRRREPGLDPTPKVLE from the cytoplasmic domain. 46. A process according to claim 44, whereby said fragment is a peptide comprising essentially the amino acid sequence of the extracellular domain, from about amino acid No. 804 to about amino acid no. 826 of SEQ ID No. 1. 47. A process according to claim 44, whereby said fragment is a peptide comprising essentially the amino acid sequence of the I-domain, from about amino acid No. 159 to about amino acid no. 355 of SEQ ID No. 1. 48. A process according to any one of claims 30-47, whereby a subunit β of the integrin heterodimer is βl . 49. A process according to claim 30, whereby said cells are chosen from the group comprising fibroblasts, muscle cells, chondrocytes, osteoblasts, mesenchymally derived cells and stem cells. 50. A process of using binding entities having the capability of binding specifically to binding sites of an integrin subunit all comprising essentially the amino acid sequence shown in SEQ ID No. 1, or an integrin heterodimer comprising said subunit all and a subunit β, or to homologues or fragments thereof, as markers or target molecules of cells or tissues expressing said integrin subunit all, which cells or tissues are of animal including human origin. 51. A process according to claim 50, which is a process for detecting the presence of an integrin subunit all comprising essentially the amino acid sequence shown in SEQ ID No. 1, or of an integrin heterodimer comprising said subunit all and a subunit β, or of homologues or fragments thereof. 52. A process according to claim 50, which is a process for determining the differentiation-state of cells during differentiation, development, in pathological conditions, in tissue regeneration, in transplantation, or in therapeutic and physiological repair of tissues. 53. A process according to claim 52, which process is used during pathological conditions involving said subunit all. 54. A process according to claim 52, which pathological conditions are comprised within the group comprising damage of muscles, muscle dystrophy, fibrosis and wound healing. 55. A process according to claim 52, which pathological conditions are comprised within the group comprising damage of cartilage and/or bone, and cartilage and/or bone diseases. 56. A process according to claim 52, which patho- logical conditions are comprised within the group comprising trauma, rheumatoid arthritis, osteoarthritis and osteoporosis. 57. A process according to claim 52 which is a process for detecting the formation of cartilage during embryonic development. 58. A process according to claim 52, which is a process for detecting physiological or therapeutic reparation of cartilage and/or muscle. 59. A process according to claim 52, which is a process for selection and analysis, or for sorting, isolating or purification of chondrocytes and/or muscle cells . 60. A process according to claim 52, which is a process for detecting regeneration of cartilage or chondrocytes during transplantation of cartilage or chondrocytes, respectively, or of muscle or muscle cells during transplantation of muscle or muscle cells, respectively . 61. A process according to claim 52, which is a process for studies of differentiation of chondrocytes or muscle cells. 62. A process according to any one of claims 50-61, which is an in vi tro process. 63. A process according to any one of claims 50-61, which is an in si tu process. 64. A process according to any one of claims 50-61, which is an in vivo process. 65. A process according to any one of claims 50-61, whereby a fragment of said integrin subunit all is a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the extracellular extension region. 66. A process according to claim 65, whereby said fragment is a peptide comprising essentially the amino acid sequence KLGFFRSARRRREPGLDPTPKVLE from the cytoplasmic domain. 67. A process according to claim 65, whereby said fragment is a peptide comprising essentially the amino acid sequence of the extracellular domain, from about amino acid No. 804 to about amino acid no. 826 of SEQ ID No. 1. 68. A process according to claim 65, whereby said fragment is a peptide comprising essentially the amino acid sequence of the I-domain, from about amino acid No. 159 to about amino acid no. 355 of SEQ ID No. 1. 69. A process according to any one of claims 50-68, whereby a subunit β of the integrin heterodimer is βl . 70. A process according to claim 50, whereby said cells are chosen from the group comprising fibroblasts, muscle cells, chondrocytes, osteoblasts, mesenchymally derived cells and stem cells. 71. A process for detecting the presence of an inte ¬ grin subunit all, or of homologues or fragments of said integrin subunit, on cells, whereby a polynucleotide or oligonucleotide chosen from the group comprising a polynucleotide or oligonucleotide having essentially the nucleotide sequence as shown in SEQ ID No. 1, or homologues or fragments thereof, is used as a marker under hybridisation conditions, wherein said polynucleotide or oligonucleotide fails to hybridise to a polynucleotide or oligonucleotide encoding an integrin subunit αlO . 72. A process according to claim 71, which is a process for determining the differentiation-state of cells during differentiation, development, in pathological conditions, in tissue regeneration, in transplantation, or in therapeutic and physiological reparation of tissues. 73. A process according to claim 72, which process is used during pathological conditions involving said subunit all. 74. A process according to claim 72, which pathological conditions are comprised within the group comprising damage of muscles, muscle dystrophy, fibrosis and wound healing. 75. A process according to claim 72, which pathological conditions are comprised within the group comprising damage of cartilage and/or bone, and cartilage and/or bone diseases. 76. A process according to claim 72, which pathological conditions are comprised within the group comprising trauma, rheumatoid arthritis, osteoarthritis and osteoporosis. 77. A process according to claim 72, which is a process for detecting the formation of cartilage during embryonic development . 78. A process according to claim 72, which is a process for detecting physiological or therapeutic reparation of cartilage and/or muscle. 79. A process according to claim 72, which is a process for selection and analysis, or for sorting, isolating or purification of chondrocytes and/or muscle cells . 80. A process according to claim 72, which is a process for detecting regeneration of cartilage or chondrocytes during transplantation of cartilage or chondrocytes, respectively, or of muscle or muscle cells during transplantation of muscle or muscle cells, respectively. 81. A process according to claim 72, which is a process for studies of differentiation of chondrocytes or muscle cells. 82. A process according to any one of claims 71-81, which is an in vi tro process. 83. A process according to any one of claims 71-81, which is an in si tu process. 84. A process according to any one of claims 71-81, which is an in vivo process. 85. A process according to any one of claims 71-84, whereby said polynucleotide or oligonucleotide is a polynucleotide or oligonucleotide coding for a peptide chosen from the group comprising peptides of the cytoplasmic domain, the I-domain and the extracellular extension region. 86. A process according to claim 85, whereby said peptide is a peptide comprising essentially the amino acid sequence KLGFFRSARRRREPGLDPTPKVLE from the cytoplasmic domain. 87. A process according to claim 85, whereby said peptide is a peptide comprising essentially the amino acid sequence of the extracellular domain, from about amino acid No. 804 to about amino acid no. 826 of SEQ ID No. l. 88. A process according to claim 85, whereby said peptide is a peptide comprising essentially the amino acid sequence of the I-domain, from about amino acid No. 159 to about amino acid no. 355 of SEQ ID No. 1. 89. A process according to any one of claims 71-88, whereby a subunit β of the integrin heterodimer is βl . 90. A process according to claim 71, whereby said cells are chosen from the group comprising fibroblasts, muscle cells, chondrocytes, osteoblasts, mesenchymally derived cells and stem cells. 91. A pharmaceutical composition comprising as an active ingredient a pharmaceutical agent or an antibody which is capable of using an integrin heterodimer comprising a subunit all and a subunit β, or the subunit all thereof, or homologues or fragment of said integrin or subunit all, as a target molecule. 92. A pharmaceutical composition comprising as an active ingredient a pharmaceutical agent or an antibody which is capable of stimulating cell surface expression or activation of an integrin heterodimer comprising a subunit all and a subunit β, or the subunit all thereof, or homologues or fragmens of said integrin or subunit all. 93. A pharmaceutical composition according to claim 92, for use in stimulating, inhibiting or blocking the formation of muscles, cartilage, bone or blood vessels. 94. A vaccine comprising as an active ingredient at least one member of the group comprising an integrin heterodimer, which heterodimer comprises a subunit all and a subunit β, or the subunit all thereof, and mologues or fragments of said integrin or subunit all, and a polynucleotide and a oligonucleotide coding for said integrin subunit all. 95. A method of gene therapy, whereby a vector comprising a polynucleotide or oligonucleotide coding for a subunit all of an integrin heterodimer, or for homologues or fragments thereof, which polynucleotide or oligonucleotide comprises essentially the nucleotide sequence shown in SEQ ID NO: 1 or parts thereof, and optionally a second vector comprising a polynucleotide or oligonucleotide coding for a sbunit β of said integrin heterodimer, is administered to a subject suffering from pathological conditions involving said subunit all. 96. A method of using binding entities having the capability of binding specifically to binding sites of a integrin subunit all comprising substantially the amino acid sequence shown in SEQ ID No. 1, or of an integrin heterodimer comprising said subunit all and a subunit β, or to homologues or fragments thereof, for promoting adhesion of cells. 97. A method of using an integrin heterodimer comprising an integrin subunit all and a subunit β, or the subunit all thereof, or homologues or fragments of said integrin or subunit all, as a target for anti- adhesive drugs or molecules in tissues where adhesion impairs the function of the tissue. 98. A method of in vitro detecting the presence of integrin binding entities, comprising interaction of an integrin heterodimer comprising a subunit all and a sub- unit β, or the subunit all thereof, or homologues or fragments of said integrin or subunit, with a sample, thereby causing said integrin, subunit all, or homologue or fragment thereof, to modulate the binding to its natural ligand or other integrin binding proteins present in said sample. 99. A method of in vitro studying consequences of the interaction of a human heterodimer integrin comprising a subunit all and a subunit β, or the subunit all thereof, or homologues or fragments of said integrin or subunit, with an integrin binding entity and thereby initiate a cellular reaction. 100. A method according to claim 99, whereby the consequences of said interactions are measured as alterations in cellular functions. 101. A method of using a polynucleotide or oligonucleotide encoding an mtegrm subunit all or homologues or fragments thereof as a target molecule. 102. A method according to claim 101, comprising hybridising a polynucleotide or oligonucleotide to the DNA or RNA encoding the mtegrm subunit all or homologue or fragment thereof, which polynucleotide or oligonucleotide fails to hybridise to a polynucleotide or oligonucleotide encoding an mtegrm subunit αlO . 103. A method of using binding entities having the capability of binding specifically to an mtegrm subunit αlO comprising the ammo acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2, or an mtegrm heterodimer comprising said subunit αlO and a subunit β, or to homo- logues or fragments thereof having similar biological activity, for promoting adhesion of chondrocytes and/or osteoblasts to surfaces of implants to stimulate osseo- mtegration. 104. A method of using an mtegrm heterodimer comprising an mtegrm subunit all and a subunit β, or the subunit αlO thereof, or homologues or fragments of said mtegrm or subunit αlO, as a target for anti- adhesive drugs or molecules in tendon, ligament, skeletal muscle or other tissues where adhesion impairs the function of the tissue. 105. A method of stimulating, inhibiting or blocking the formation of cartilage or bone, comprising administration to a subject a suitable amount of a pharmaceutical agent or an antibody which is capable of using an mtegrm heterodimer comprising a subunit all and a sub- unit β, or the subunit all thereof, or homologues or fragments of said mtegrm or subunit all, as a target molecule .

Inventors: GULLBERG DONALD, GULLBERG, DONALD
Applicants: ACTIVE BIOTECH AB, GULLBERG DONALD, GULLBERG, DONALD
Application year: 2000
Main classification: C07K14
IPC codes: A61K35, A61K38, A61K39, A61K45, A61K48, A61P9, A61P19, A61P21, A61P43, C07K14, C07K16, C07K19, C12N1, C12N5, C12N15, C12P21, C12Q1