WO1992012236A1 - NOUVELLE SOUS-UNITE β D'INTEGRINE ET SES PROCEDES D'UTILISATION - Google Patents

NOUVELLE SOUS-UNITE β D'INTEGRINE ET SES PROCEDES D'UTILISATION Download PDF

Info

Publication number
WO1992012236A1
WO1992012236A1 PCT/US1991/000236 US9100236W WO9212236A1 WO 1992012236 A1 WO1992012236 A1 WO 1992012236A1 US 9100236 W US9100236 W US 9100236W WO 9212236 A1 WO9212236 A1 WO 9212236A1
Authority
WO
WIPO (PCT)
Prior art keywords
integrin
subunit
ligand
binding
amino acid
Prior art date
Application number
PCT/US1991/000236
Other languages
English (en)
Inventor
Dean Sheppard
Vito Quaranta
Robert Pytela
Original Assignee
The Regents Of The University Of California
Scripps Clinic And Research Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California, Scripps Clinic And Research Center filed Critical The Regents Of The University Of California
Priority to PCT/US1991/000236 priority Critical patent/WO1992012236A1/fr
Priority to CA002100352A priority patent/CA2100352A1/fr
Priority to JP3504047A priority patent/JPH07500721A/ja
Publication of WO1992012236A1 publication Critical patent/WO1992012236A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily

Definitions

  • This invention relates to receptors for adhesion peptides, and more specifically to a novel receptor subunit having affinity for extracellular matrix molecules.
  • Multicellular organisms such as man, have some 10 u cells which can be divided into a minimum of fifty different types, such as blood cells and nerve cells.
  • cell adhesion mechanisms appear to be of importance in mediating patterns of cellular growth, migration and differentiation, whereby cells develop specialized characteristics so as to function as, for example, muscle cells or liver cells. Cell adhesion mechanisms are also implicated in dedifferentiation and invasion, notably where cells lose their specialized forms and become metastasizing cancer cells.
  • the Arg-Gly-Asp sequence is a cell attachment site at least in fibronectin, vitronectin, fibrinogen von Willibrand, thrombopondin, osteopontin, and possibly various collagens, laminin and tenascin. Despite the similarity of their cell attachment sites, these proteins can be recognized individually by their interactions with specific receptors.
  • the integrins are a large family of cell surface glycoproteins that mediate cell-to-cell and cell- to-matrix adhesion as described, for example, in the Ruoslahti and Pierschbacher article cited above. All known members of this family of adhesion receptors are heterodimers consisting of an ⁇ and a ⁇ subunit noncovalently bound to each other. When the integrin family was first identified, integrins were grouped into three subfamilies based on the three ⁇ subunits that were initially recognized (&. , B 2 and ⁇ 3 ) . Over the past few years, the primary structures of three integrin ⁇ subunits from mammalian cells and one from Drosophila have been deduced from cDNA.
  • the present invention relates to a substantially purified ⁇ subunit of an integrin cell surface receptor designated as ⁇ 6 .
  • the amino acid sequence of ⁇ 6 is provided in Figure 3.
  • the present invention also relates to amino acid fragments specific to B 6 that have a variety of uses.
  • the invention further relates to vectors having a gene encoding such fragments. Host cells containing such vectors are also provided.
  • the nucleic acids encoding ⁇ 6 as well as nucleic acids that specifically hybridize with the nucleic acids encoding B 6 sequences are other aspects of the present invention.
  • the present invention relates to a substantially purified integrin comprising & 6 bound to an a subunit, particularly ⁇ v or ⁇ F .
  • Methods of blocking the attachment of the ⁇ 6 -containing integrins to its ligand and of detecting the binding of such integrins to its ligand are also provided.
  • the present invention also relates to methods of increasing or decreasing cell adhesion in cells expressing a ⁇ 6 -containing integrin by overexpressing the integrin or by binding the integrin with a ligand, such as vitronectin.
  • Figure 1 shows the design of PCR primers.
  • Figure 2 shows a map of sequencing strategy.
  • Figure 3 shows the nucleotide sequence and amino acid translation for human (H) and guinea pig (GP)
  • Figure 4 shows the alignment of ⁇ 6 with four previously reported integrin ⁇ subunits.
  • Figure 5 shows the alignment of partial nucleotide and amino acid sequences from human (H) and guinea pig (GP) £>. , B z , B 3 , and & 6 for the region just downstream from the B3F primer.
  • the present invention provides a composition of matter relating to a novel, substantially purified integrin ⁇ subunit, referred to herein as E> 6 .
  • E> 6 The amino acid sequence of ⁇ 6 is also provided and is shown in Figure 3.
  • substantially purified is meant substantially free of contaminants normally associated with a native or natural environment.
  • ⁇ 6 is meant a polypeptide having substantially the same amino acid sequence and binding functions of the polypeptides encoded by the sequences set forth in Figure 3 for human and guinea pig B 6 .
  • modified amino acid sequences that do not substantially destroy the functions and retain the essential sequence of ⁇ 6 are included within the definition of ⁇ 6 .
  • Amino acid sequences such as the sequence for ⁇ . , B 2 and ⁇ 3 , having less than 50% homology with the sequence of ⁇ 6 , are not substantially the same sequence and, therefore, do not fall within the definition of B 6 .
  • additions, deletions or substitutions can be made and tested to determine their effect on the function of B 6 .
  • certain amino acids such as the conserved cystines, for example, can be modified to alter a binding function of B 6 .
  • the ⁇ subunit of the present invention is clearly different from &. , ⁇ -, ⁇ 3 and other ⁇ subunits that have recently been discovered.
  • the 11-amino acid carboxyl- terminal extension on ⁇ 6 distinguishes it from ⁇ -,, ⁇ 2 , and ⁇ 3 .
  • the short cytoplasmic tails of ⁇ ,,, B 2 , and B 3 are thought to be sites of interaction with the cytoskeleton and regions for the transduction of signals initiated by interactions of the large extracellular domains with ligands. These cytoplasmic tails may also be targets for regulation of integrin function.
  • the distinctive 11- amino acid cytoplasmic tail of B 6 indicates that its regulation or pathways for signal transduction may be different from those of ⁇ -,, ⁇ 2 and B 3 .
  • ⁇ subunit originally called ⁇ ⁇ was identified in epithelial-derived tumor cells in association with the integrin subunit ⁇ v as described, for example, in Cheresh et al., Cell 57:59-69 (1989).
  • This ⁇ subunit having a distinctive amino-terminal sequence, was recently renamed ⁇ 5 . Based on recent studies of purified preparations, ⁇ 5 clearly differs from the ⁇ subunit of the present invention. Because the ⁇ subunit described in the present report is distinct from each of the five ⁇ subunits for which sequence information is available, it has been designated as ⁇ 6 .
  • B s integrin ⁇ subunits
  • MG-63 human osteosarcoma cell line
  • AF1523 fibroblast cell line
  • human endothelial cells as described, for example, in Freed et al., EMBO J. 8:2955-2965 (1989).
  • This subunit is also different from B 6 since B s is expressed in MG-63 cells while B 6 is not expressed in these cells as shown in Table 1.
  • the other novel integrin ⁇ subunit identified by co-immunoprecipitation of known o subunits, ⁇ is a protein of about M r 95,000 that is found to be associated with ⁇ 4 , an subunit first found as part of the lymphocyte homing receptor VLA-4 as described, for example, in Holzmann et al.. Cell 45:37-46 (1989).
  • This subunit is also distinct from B 6 since ⁇ p is expressed in lymphocytes while B 6 is not expressed in lymphocytes as shown in Table 1.
  • Leukocytes The invention also provides an integrin comprising B 6 bound to an ⁇ subunit.
  • ⁇ 6 consistent with recent findings of other ⁇ subunits, can associate with a variety of ⁇ subunits to form a functional integrin.
  • ⁇ 6 associates with ⁇ v .
  • B 6 associates with another ⁇ subunit referred to herein as ⁇ F .
  • the ⁇ v ⁇ 6 integrin, as well as other integrins containing & b can bind molecules, for example extracellular matrix molecules. Such molecules are referred to herein as ligands.
  • certain B 6 -containing integrins can bind Arg-Gly-Asp- containing polypeptides such as vitronectin or fibronectin.
  • the binding of ⁇ 6 -containing integrins to various ligands can be determined according to procedures known in the art and as described for example, in
  • the invention also provides an amino acid fragment specific to B 6 . Since B 6 is a novel molecule, it contains many fragments which are specific for this ⁇ subunit. Fragments specific to B 6 contain sequences having less than 50% homology with sequences of other known integrin ⁇ subunit fragments. These fragments are necessarily of sufficient length to be distinguishable from known fragments and, therefore, are "specific for ⁇ 6 .” The amino acid sequence of such fragments can readily be determined by referring to the figures which identify the ⁇ 6 amino acid sequences. These fragments also retain the binding function of the ⁇ 6 subunit and can therefore be used, for example, as immunogens to prepare reagents specific for ⁇ 6 or as an indicator to detect the novel B 6 -containing integrin of the present invention. One skilled in the art would know of other uses for such fragments.
  • the invention also provides a reagent having specificity for an amino acid sequence specific for B 6 .
  • B 6 is a novel protein with at least 50% amino acid differences over related ⁇ subunits
  • reagents such as antibodies, which are specifically reactive with amino acid sequences specific for B 6 and thereby immunologically distinguish ⁇ 6 from other molecules.
  • Various methods of making such antibodies are well established and are described, for example, in Antibodies, A Laboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Laboratory 1988, pp. 139- 283 and Huse et al., Science 24:1275-1280 (1988).
  • the invention also provides nucleic acids which encode B 6 . Examples of such sequences are set forth in Figure 3. Following standard methods as described, for example, in Maniatis et al., Molecular Cloning, Cold Spring Harbor (1982) , nucleic acid sequences can be cloned into the appropriate expression vector. The vector can then be inserted into a host, which will then be capable of expressing recombinant proteins. Thus, the invention also relates to vectors containing nucleic acids encoding such sequences and to hosts containing these vectors.
  • nucleic acids that can be used as probes for diagnostic purposes. Such nucleic acids can hybridize with a nucleic acid having a nucleotide sequence specific for B 6 but do not hybridize with nucleic acids encoding non-B 6 proteins, particularly other cell surface receptors. These nucleic acids can readily be determined from the sequence of B 6 and synthesized using a standard nucleic acid synthesizer. Nucleic acids are also provided which specifically hybridize to either the coding pr non-coding DNA of ⁇ 6 .
  • Integrin cell surface receptors bind ligands, such as extracellular matrix molecules.
  • the binding of the integrin to the ligand can be blocked by various means.
  • the binding of a B 6 - containing integrin can be blocked by a reagent that binds the B 6 subunit or the ⁇ 6 -containing integrin.
  • reagents include, for example, Arg-Gly- Asp-containing peptides and polypeptides, ligand fragments containing the integrin binding site, as well as antibodies specifically reactive with B 6 or a B 6 - containing integrin.
  • the blocking can be carried out by binding the ligand or fragment thereof, recognized by a ⁇ 6 -containing integrin with a reagent specific for the ligand at a site that inhibits the ligand from binding with the integrin. Since the binding of a B 6 -containing integrin to its ligand can mediate cell adhesion to an extracellular matrix molecule, preventing this binding can prevent cell adhesion. Alternatively, cell adhesion can be promoted by increasing the expression of B 6 -containing integrins by a cell.
  • the invention provides a method of detecting ligands which bind a B 6 -containing integrin.
  • the method comprises contacting a B 6 -containing integrin with a solution containing ligands suspected of binding B 6 -containing integrins. The presence of ligands which bind a ⁇ 6 -containing integrin is then detected.
  • a substantially purified integrin cell surface receptor subunit comprising B 6 .
  • a substantially purified integrin comprising ⁇ 6 bound to an ⁇ subunit. 4. The integrin of claim 3, wherein the subunit is ⁇ v .
  • a vector comprising a gene encoding for the amino acid fragment of claim 6.
  • a reagent having specificity for an amino acid sequence specific for B 6 8.
  • a substantially purified nucleic acid which specifically hybridizes with a nucleotide sequence of the nucleic acid of claim 11.
  • the reagent is an RGD-containing peptide or polypeptide.
  • the reagent is a ligand fragment containing an integrin binding site.
  • a method of detecting a ligand that binds a B 6 -containing integrin comprising contacting the ⁇ 6 - containing integrin with a solution containing the ligand suspected of binding ⁇ 6 -containing integrins and detecting the presence of the ligand bound to the B 6 -containing integrin.
  • a method of increasing cell adhesion in cells expressing a ⁇ 6 -containing integrin comprising overexpressing the ⁇ 6 -containing integrin in a cell.
  • a method of decreasing cell adhesion in cells expressing a ⁇ 6 -containing integrin comprising binding the ⁇ 6 -containing integrin with a ligand.
  • Moloney murine leukemia virus reverse transcriptase Bethesda Research Laboratories, Gaithersburg, MD
  • PCR polvmerase chain reaction
  • PCR was carried out in a reaction volume of 25-200 ⁇ l.
  • each PCR reaction contained 50 mM KC1, 10 mM Tris-HCl (pH 9.0 at 25"C), 1.5 mM MgCl 2 , 0.01% gelatin, 0.1% Triton X-100, 0.2 mM each of dATP, dGTP, dCTP and dTTP, and 0.05 units/ ⁇ l Taq DNA polvmerase (obtained from either United States
  • each reaction mixture was overlaid with mineral oil, heated to 95°C for 4 in. in a thermal cycler (Ericomp, San Diego, CA) , and then subjected to 30 cycles of PCR. Each cycle consisted of 45 seconds at 95 " C, 45 seconds at 53°C, and 1 min. at 72°C. Immediately after the last cycle, the sample was maintained at 72 ⁇ C for 10 min.
  • consensus primer mixtures were designed to anneal with the nucleotides encoding the highly conserved sequence Asp-Leu-Tyr-Tyr-Leu-Met-Asp-Leu (primer B1F) and Glu-Gly-Gly-Asp-Ala-Ile-Met-Gln (primer B2R) that flank an approximately 300-nucleotide region beginning approximately 130 amino acids from the amino terminus of each of the integrin ⁇ subunits sequenced to date.
  • the sequences of the primers identified herein are depicted in Figure 1.
  • a specific forward primer was designed to anneal with the sequence encoding the amino acids Pro-Leu-Thr-Asn-Asp- Ala-Glu-Arg (primer BTE2F) ending approximately 49 nucleotides from the 3* end of the region we had sequenced.
  • B3F additional forward primer
  • B3R and B4R reverse primers
  • PCR as described above was performed with cDNA from guinea pig tracheal epithelial cells and the primer pairs BTE2F/B3R and B3F/B4R.
  • the primer pair BTE2F/B3R yielded 1095 additional base pairs of new sequence. Based on this sequence another specific primer (BTE3F) was designed to recognize the sequence Val-Ser-Glu-Asp-Gly-Val near the 3' end of this sequence, and PCR was performed with this primer in combination with primer B4R.
  • BTE3F another specific primer
  • Figure 1 shows the design of PCR primers, ⁇ subunit consensus primer mixtures were designed on the basis of alignment of published sequences of human ⁇ -,, ⁇ 2 , B 3 and chicken ⁇ ,.
  • forward primers B1F and B3F
  • the primer sequences included a single nucleotide whenever possible for each of the first two nucleotides of each codon and were usually either degenerate or included deoxyinosine for the third base in codons for amino acids other than methionine.
  • Reverse primers (B2R, B3R, and B4R) were designed in the same manner for the complementary DNA strand. Two specific forward primers were designed to recognize B 6 .
  • the first (BTE2F) was designed to work across species and was thus degenerate or included deoxyinosine in the third codon position.
  • the second, BTE3F was not degenerate and was designed to only recognize guinea pig ⁇ 6 .
  • PCR fragments generated with the primer pairs B1F/B2R and BTE3F/B4R were uniformly labeled with alpha- [ 32 P]dCTP and used as probes to screen a random-primed cDNA library and an oligo-dT-primed cDNA library both constructed in the plasmid pTZ18R-BstXI (Invitrogen) from mRNA obtained from the human pancreatic carcinoma cell line FG-2. Plasmid was purified from clones found to hybridize with either region, and inserts were sequenced. A portion of insert DNA from one clone was in turn labeled and used to screen the same libraries.
  • Figure 2 shows a map of the sequencing strategy. Shown are the location of clones used to obtain the partial cDNA sequence of guinea pig B 6 (clones IF, 3L, 3N and 3Y, top) and the complete sequence of human B & (clones T1-T19 bottom) . Also shown is the location of the translated region (Protein) . The location of the transmembrane domain is shown by the letters TM. Clones shown often represent one of several identical clones. Internal sequence of clones with long inserts was obtained by restriction endonuclease digestion and relegation and by ligation of internal fragments into pBluescript.
  • PCR using cDNA from guinea pig airway epithelial cells and the consensus primer mixtures B1F and B2R amplified DNA fragments with the expected size of approximately 350 nucleotides.
  • fragment DNA was sequenced after cloning into pBluescript, recombinant clones each contained inserts with one of two distinct sequences.
  • Both of the guinea pig sequences included the integrin B subunit consensus sequences Ser-X-Ser- Met-X-Asp-Asp-Leu and Gly-Phe-Gly-Ser-Phe-Val , and both contained the 2 cysteine residues found in this region in all known integrin ⁇ subunits. These data suggest that one of the two sequences we obtained encoded a new member of the integrin ⁇ subunit family.
  • Figure 3 shows Nucleotide sequence and amino acid translation for human (H) and guinea pig (GP) B 6 .
  • the amino acid translation is denoted by the single letter code beneath the second nucleotide of each codon from the translated region of human B 6 .
  • For the guinea pig sequence only amino acids that differ from the human sequence are shown.
  • the numbers along the right-hand margin denote the nucleotide or amino acid number of the last entry on each line. The numbering system used starts with the first nucleotide or amino acid available for each sequence shown.
  • the nine potential sites for N- glycosylation in the putative extracellular domain of human B 6 are underlined.
  • This map predicts an mRNA of approximately 5 kilobases including at least a 226-nucleotide untranslated region at the 5 1 end and, a 2364-nucleotide open reading frame, and a 3' untranslated region of approximately 2.5 kilobases.
  • This molecule has been termed integrin B 6 .
  • Fig. 3 shows the partial nucleotide and complete amino acid sequences for human B 6 (excluding most of the 3*-untranslated region) and the alignment of the 1732 nucleotides of sequence obtained from PCR of guinea pig airway epithelial cell cDNA.
  • the amino acid sequences are 94% identical.
  • the 1732 nucleotides sequenced in both species 91% are identical.
  • Nine potential glycosylation sites present in the putative extracellular domain of human B 6 are shown by underlining. All seven of these sites that lie within the 577 amino acids obtained for guinea pig B 6 are also present in the guinea pig protein. If all of the potential glycosylation sites are occupied with oligosaccharides having an average molecular weight of 2,500, the predicted molecular weight of human B 6 would be 106,000.
  • Figure 4 shows the alignment of B 6 with four previously reported integrin ⁇ subunits.
  • Previously published sequences for human B. , human ⁇ 2 , human B 3 , the myospheroid gene product ( ⁇ myo) of Drosophila, and the novel sequence described as (B 6 ) are shown using the single letter amino acid code.
  • the transmembrane domain is underlined.
  • the regions used for constructing the consensus ⁇ subunit primers B1F (1), B2R (B2) , B3F/R (B3) , and B4R (B4) are labeled below the alignment in bold type.
  • the numbers along the right-hand margin denote the number of the last amino acid in each line beginning from the first amino acid of each putative signal sequence.
  • the overall percentage of identical amino acids between B 6 and the other human ⁇ subunits is 47% for B 3 , 42% for ⁇ -, and 38% for B 2 .
  • Human B 6 is also 39% identical to the Drosophila ⁇ subunit.
  • Human ⁇ -,, B 2 and ⁇ 3 and the Drosophila ⁇ subunit all have cytoplasmic regions consisting of 41 amino acids (beginning after the putative transmembrane domain shown by the underline in Fig. 4) .
  • B 6 contains each of the 10 conserved amino acid residues in this cytoplasmic region it also contains an 11-amino acid extension at the carboxyl terminus. B 6 also contains two Arg-Gly-Asp sequences, one at amino acids 514-516 and the other at 594-596. These regions could serve as recognition sites for other ligands of the integrin family.
  • PCR using the primer pair B3F/B4R amplified fragments of the expected size of approximately 750 nucleotides. Cloning and sequencing of the fragments did not result in any additional clones containing the novel ⁇ subunit sequence but did result in several clones with inserts encoding an amino acid sequence that was 97% identical to the corresponding region of human B 3 and several others encoding an amino acid sequence that was 93% identical to human B. (Fig. 5) . These are presumably the guinea pig homologues of B. and ⁇ 3 , respectively. The nucleotide sequences of guinea pig and human ⁇ ., are 80% identical, and those of guinea pig and human ⁇ 3 are 91% identical.
  • Figure 5 shows the alignment of partial nucleotide and amino acid sequences from Human (H) and guinea pig (GP) ⁇ -,, ⁇ 2 , ⁇ 3 , and B 6 for the region just downstream from the B3F primer. Amino acid translations denoted by the one-letter code are shown below the second nucleotide of each codon. For the guinea pig sequences, only amino acids that differ from the human sequences are shown. The numbers shown along the right-hand margin denote the nucleotide number for human B 6 . The sequences for human ⁇ , and B 3 are from previously published reports.
  • antisera against peptides from the cytoplasmic domain sequence of B 6 were prepared.
  • the following amino acid peptides from the cytoplasmic sequence of B 6 were prepared and used to immunize rabbits: RGSTSTFKNVTYKHR (residues 763-777) and YKHREKQKVDLSTDC (residues 774-788) .
  • the antisera were raised in rabbits according to standard procedures known in the art.
  • peptides were chemically coupled to keyhole lympet hemocyanin, and were injected in rabbits in either complete (first injection only) or incomplete Freund's adjuvant as described, for example, in Antibodies: A Laboratory Manual. E. Harlow and D. Lowe, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724. Antisera were termed 6830 (to peptides corresponding to residues 763-777) and 6341 (to peptides corresponding to residues 774-788) .
  • the resulting polyclonal antibodies were used to immunoprecipitate detergent lysates from the pancreatic carcinoma cell line FG-2 that had been surface radioiodinated according to procedures well known in the art such as described, for example, in Kajiji et al., EMBO J 3:673-680 (1989).
  • a complex of two bands was precipitated of respectively 150 kilodaltons (Kd) and 97 Kd in SDS-PAGE under non-reducing conditions. Under reducing conditions, the two bands migrated as a diffused band, extending from 130 Kd to 116 Kd. These bands were specific since pre-immune serum did not precipitate any of them and they were not present when the immunoprecipitation was carried out in the presence of the corresponding immunogenic peptide.
  • the same complex of two bands was precipitated by both the 6830 and 6841 antibodies, which were raised against independent peptides from the cytoplasmic sequence deduced from B 6 cDNA clones.
  • the other band, of 150 Kd or 130 Kd under non- reducing or reducing conditions, respectively, is likely to be an subunit since it dissociates after SDS-heat denaturation of the lysate, indicating that it is non- covalently associated with the B 6 polypeptide. Furthermore, similar to certain other integrin ⁇ chains, its molecular weight decreases under reducing conditions by about 20 Kd (130 Kd versus 150 Kd under non-reducing conditions) probably due to a disulfide linked small peptide that dissociates upon reduction.
  • the ⁇ 6 integrin complex was purified by immuno-affinity chro atography on a 6841-protein A sepharose matrix according to procedures well known in the art such as described, for example, in Kajiji et al., EMBO J 3:673- 680 (1989) .
  • the eluted material was immunoprecipitated with antibodies specific for ⁇ -,, ⁇ 2 , ⁇ 3 , ⁇ 5 , ⁇ 6 and ⁇ v , which are known to be expressed in FG-2 cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne des intégrines pratiquement pures renfermant une nouvelle sous-unité β appelée β6. La nouvelle sous-unité β6 forme des hétérodimères avec αV et αF. Cette invention concerne également des procédés permettant de contrôler l'adhérence cellulaire à l'aide d'intégrines renfermant la sous-unité β6.
PCT/US1991/000236 1991-01-11 1991-01-11 NOUVELLE SOUS-UNITE β D'INTEGRINE ET SES PROCEDES D'UTILISATION WO1992012236A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/US1991/000236 WO1992012236A1 (fr) 1991-01-11 1991-01-11 NOUVELLE SOUS-UNITE β D'INTEGRINE ET SES PROCEDES D'UTILISATION
CA002100352A CA2100352A1 (fr) 1991-01-11 1991-01-11 Nouvelle sous-unite .beta. d'integrine et applications
JP3504047A JPH07500721A (ja) 1991-01-11 1991-01-11 新規なインテグリンβサブユニット及びその使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/US1991/000236 WO1992012236A1 (fr) 1991-01-11 1991-01-11 NOUVELLE SOUS-UNITE β D'INTEGRINE ET SES PROCEDES D'UTILISATION
CA002100352A CA2100352A1 (fr) 1991-01-11 1991-01-11 Nouvelle sous-unite .beta. d'integrine et applications

Publications (1)

Publication Number Publication Date
WO1992012236A1 true WO1992012236A1 (fr) 1992-07-23

Family

ID=25676369

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/000236 WO1992012236A1 (fr) 1991-01-11 1991-01-11 NOUVELLE SOUS-UNITE β D'INTEGRINE ET SES PROCEDES D'UTILISATION

Country Status (2)

Country Link
CA (1) CA2100352A1 (fr)
WO (1) WO1992012236A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001000677A1 (fr) * 1999-06-28 2001-01-04 The University Of Newcastle Research Associates Limited Procede servant a moduler une activite cellulaire provoquee par integrine et agents de modulation utiles
WO2002076489A1 (fr) * 2001-03-09 2002-10-03 Dyax Corp. Groupes de liaison d'albumine serique
EP1492870A2 (fr) * 2002-04-12 2005-01-05 Raven Biotechnologies, Inc. Anticorps se liant a l'integrine alpha-v-beta-6 et leurs methodes d'utilisation
EP1565581A2 (fr) * 2002-08-20 2005-08-24 Millenium Pharmaceuticals, Inc. Compositions, kits, et procedes d'identification, d'evaluation, de prevention et de therapie de cancer du col
US7422883B2 (en) 2000-12-22 2008-09-09 Inter-K Pty Limited Map kinase integrin-binding domain
US9765113B2 (en) 2003-10-17 2017-09-19 Interk Peptide Therapeutics Limited Methods and agents for the treatment of cancer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CELL, Vol. 44, issued 28 February 1986, RUOSLAHTI et al., "Arg-Gly-Asp: A Versatile Cell Recognition Signal", pages 517-518. *
THE EMBO JOURNAL, Vol. 8, No. 10, issued 1989, FREED et al., "A Novel Integrin Beta Subunit is Associated with the Vitronectin Receptor Alpha Subunit (alpha) is a Human Osteosarcoma Cell Line and is a Substrate for Protein Kinase C", pages 2955-2965. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 265, No. 20, issued 15 July 1990, SHEPPARD et al., "Complete Amino Acid Sequence of a Novel Integrin B Subunit (B6) Identified in Epithelial Cells Using the Polymerase Chain Reaction", pages 11502-11507. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8618062B2 (en) 1999-06-26 2013-12-31 Inter-K Pty Limited Method of modulating integrin mediated cellular activity and agents useful for same
WO2001000677A1 (fr) * 1999-06-28 2001-01-04 The University Of Newcastle Research Associates Limited Procede servant a moduler une activite cellulaire provoquee par integrine et agents de modulation utiles
US8119594B1 (en) 1999-06-28 2012-02-21 Inter-K Pty Limited Method of modulating integrin mediated cellular activity and agents useful for same
US7422883B2 (en) 2000-12-22 2008-09-09 Inter-K Pty Limited Map kinase integrin-binding domain
WO2002076489A1 (fr) * 2001-03-09 2002-10-03 Dyax Corp. Groupes de liaison d'albumine serique
US7211395B2 (en) 2001-03-09 2007-05-01 Dyax Corp. Serum albumin binding moieties
EP1492870A2 (fr) * 2002-04-12 2005-01-05 Raven Biotechnologies, Inc. Anticorps se liant a l'integrine alpha-v-beta-6 et leurs methodes d'utilisation
EP1492870A4 (fr) * 2002-04-12 2005-08-03 Raven Biotechnologies Inc Anticorps se liant a l'integrine alpha-v-beta-6 et leurs methodes d'utilisation
EP1565581A2 (fr) * 2002-08-20 2005-08-24 Millenium Pharmaceuticals, Inc. Compositions, kits, et procedes d'identification, d'evaluation, de prevention et de therapie de cancer du col
EP1565581A4 (fr) * 2002-08-20 2008-01-02 Millennium Pharm Inc Compositions, kits, et procedes d'identification, d'evaluation, de prevention et de therapie de cancer du col
EP2305836A1 (fr) * 2002-08-20 2011-04-06 Millennium Pharmaceuticals, Inc. Compositions, kits et procédés d'identification, évaluation, prévention et thérapie du cancer du col de l'utérus
US9765113B2 (en) 2003-10-17 2017-09-19 Interk Peptide Therapeutics Limited Methods and agents for the treatment of cancer

Also Published As

Publication number Publication date
CA2100352A1 (fr) 1992-07-12

Similar Documents

Publication Publication Date Title
US20090253146A1 (en) Methods of expressing integrin beta6 subunits
Marcantonio et al. Antibodies to the conserved cytoplasmic domain of the integrin beta 1 subunit react with proteins in vertebrates, invertebrates, and fungi.
Takada et al. 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.
Sheppard et al. Complete amino acid sequence of a novel integrin beta subunit (beta 6) identified in epithelial cells using the polymerase chain reaction.
Ignatius et al. Identification of a neuronal laminin receptor: an Mr 200K/120K integrin heterodimer that binds laminin in a divalent cation-dependent manner
PAN et al. Sequence of extracellular mouse protein BM‐90/fibulin and its calcium‐dependent binding to other basement‐membrane ligands
Murdoch et al. Primary structure of the human heparan sulfate proteoglycan from basement membrane (HSPG2/perlecan). A chimeric molecule with multiple domains homologous to the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor.
Jin et al. Molecular cloning of the CD3 eta subunit identifies a CD3 zeta-related product in thymus-derived cells.
US5459061A (en) Hybridomas producing monoclonal antibodies which specifically bind to continuous epitope on the human EGF receptor and compete with EGF for binding to the EGF receptor
EP1464706A2 (fr) HEK5, HEK7, HEK8 et HEK11: des récepteurs protéine tyrosine kinase d'analogues de l'EPH
EP0491675A1 (fr) Améliorations relatives aux facteurs de croissance
EP0379904A1 (fr) Molécule souple apparentée à mais différente d'ICAM-1
JP2003521915A (ja) 反芻動物ＭＨＣクラスＩ−様Ｆｃレセプター
WO1994011502A2 (fr) Kinases du type recepteurs activine, proteines possedant des domaines de kinase de serine/threonine et leur utilisation
WO1992012236A1 (fr) NOUVELLE SOUS-UNITE β D'INTEGRINE ET SES PROCEDES D'UTILISATION
EP0580672B1 (fr) ANTIGENE SYNTHETIQUE DE PEPTIDE CDw52(CAMPATH-1)
Cone et al. Effects of β subunit cytoplasmic domain deletions on the recruitment of the integrin αvβ6 to focal contacts
JPH03504439A (ja) 免疫グロブリンＥのための高アフィニティー受容体のγサブユニットをコードするｃＤＮＡ
Prince et al. Characterization of a partial cDNA clone for the NILE glycoprotein and identification of the encoded polypeptide domain
AU602487B2 (en) Molecular cloning of cDNA for human factor VIIIR (von Willebrand factor)
WO2001079144A2 (fr) Fyn kinase comme cible de modulation de la transduction des signaux medies par l'integrine
AU6299390A (en) Fibulin
Wennogle et al. Stabilization of C5a receptor–G‐protein interactions through ligand binding
Fawcett et al. Molecular tools for the analysis of cell adhesion
JPH07500721A (ja) 新規なインテグリンβサブユニット及びその使用

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2100352

Country of ref document: CA