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1.
Xenobiotica ; 38(3): 340-52, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18274961

ABSTRACT

Intercellular adhesion molecule (ICAM)-1988 is a small molecule lymphocyte function-associated antigen-1 (LFA-1) antagonist being considered for its anti-inflammatory properties. Following intravenous administration of ICAM1988, clearances in mice, rats, dogs, and monkeys were 17.8, 3.31, 15.4, and 6.85 ml min(-1) kg(-1), respectively. In mass balance studies using [(14)C]-ICAM1988 in rats dosed intravenously, unchanged ICAM1988 contributed to 25.1% of the dose. In rats, the systemic bioavailability of ICAM1988 was improved to 0.28 when the drug was administered orally as its isobutyl ester, ICAM2660. In rats, this was consistent with the complete in vitro conversion of ICAM2660 to ICAM1988 in plasma, and liver and intestinal S9. In dogs and monkeys, ICAM2660 did not improve the bioavailability of ICAM1988. This is consistent with limited in vitro conversion of ICAM2660 to ICAM1988 in plasma and liver S9. In human in vitro studies, ICAM2660 conversion to ICAM1988 in liver was similar to rats while no conversion in plasma and intestinal S9 fractions were observed. Based on the in vitro metabolism similarities of human and rat, it would be anticipated that in human oral administration of ICAM2660 would improve the systemic exposure of ICAM1988.


Subject(s)
Acrylamides/metabolism , Acrylamides/pharmacokinetics , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/pharmacokinetics , Lymphocyte Function-Associated Antigen-1/metabolism , Prodrugs/metabolism , Prodrugs/pharmacokinetics , beta-Alanine/analogs & derivatives , Absorption/drug effects , Acrylamides/chemistry , Acrylamides/pharmacology , Animals , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/pharmacology , Dogs , Drug Evaluation, Preclinical , Drug Stability , Haplorhini , Humans , Injections, Intravenous , Liver/drug effects , Liver/metabolism , Mice , Prodrugs/chemistry , Prodrugs/pharmacology , Rats , Tissue Distribution/drug effects , beta-Alanine/chemistry , beta-Alanine/metabolism , beta-Alanine/pharmacokinetics , beta-Alanine/pharmacology
2.
Science ; 295(5557): 1086-9, 2002 Feb 08.
Article in English | MEDLINE | ID: mdl-11834839

ABSTRACT

The protein-protein interaction between leukocyte functional antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) is critical to lymphocyte and immune system function. Here, we report on the transfer of the contiguous, nonlinear epitope of ICAM-1, responsible for its association with LFA-1, to a small-molecule framework. These LFA-1 antagonists bound LFA-1, blocked binding of ICAM-1, and inhibited a mixed lymphocyte reaction (MLR) with potency significantly greater than that of cyclosporine A. Furthermore, in comparison to an antibody to LFA-1, they exhibited significant anti-inflammatory effects in vivo. These results demonstrate the utility of small-molecule mimics of nonlinear protein epitopes and the protein epitopes themselves as leads in the identification of novel pharmaceutical agents.


Subject(s)
Immunosuppressive Agents/pharmacology , Intercellular Adhesion Molecule-1/immunology , Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Thiophenes/chemical synthesis , Thiophenes/pharmacology , beta-Alanine/chemical synthesis , beta-Alanine/pharmacology , Amino Acid Sequence , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cyclosporine/pharmacology , Dermatitis, Irritant/drug therapy , Dinitrofluorobenzene , Drug Design , Enzyme-Linked Immunosorbent Assay , Epitopes , Female , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/pharmacology , Immunosuppressive Agents/chemical synthesis , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/metabolism , Intercellular Adhesion Molecule-1/chemistry , Lymphocyte Culture Test, Mixed , Lymphocyte Function-Associated Antigen-1/immunology , Mice , Mice, Inbred BALB C , Molecular Mimicry , Mutagenesis , Protein Structure, Secondary , Structure-Activity Relationship , Thiophenes/chemistry , Thiophenes/metabolism , beta-Alanine/analogs & derivatives , beta-Alanine/chemistry , beta-Alanine/metabolism
3.
Mol Microbiol ; 34(5): 926-40, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10594819

ABSTRACT

The spirochaetal agents of Lyme disease, Borrelia burgdorferi (sensu lato) bind to integrins alphaIIbbeta3, alphavbeta3 and alpha5beta1 in purified form and on the surfaces of human cells. Using a phage display library of B. burgdorferi (sensu stricto) DNA, a candidate ligand for beta3-chain integrins was identified. The native B. burgdorferi protein, termed p66, is known to be recognized by human Lyme disease patient sera and to be expressed on the surface of the spirochaete. We show here that recombinant p66 binds specifically to beta3-chain integrins and inhibits attachment of intact B. burgdorferi to the same integrins. When expressed on the surface of Escherichia coli, this protein increases the attachment of E. coli to a transfected cell line that expresses alphavbeta3, but not to the parental cell line, which expresses no beta3-chain integrins. Localization of p66 on the surface of B. burgdorferi, the ability of recombinant forms of the protein to bind to beta3-chain integrins and the fact that p66 and B. burgdorferi bind to beta3-chain integrins in a mutually exclusive manner make p66 an attractive candidate bacterial ligand for integrins alphaIIbbeta3 and alphavbeta3.


Subject(s)
ATP-Binding Cassette Transporters , Antigens, CD/metabolism , Bacterial Proteins , Borrelia burgdorferi Group/metabolism , Escherichia coli Proteins , Monosaccharide Transport Proteins , Platelet Membrane Glycoproteins/metabolism , Porins/genetics , Porins/metabolism , Antigens, CD/genetics , Bacterial Adhesion , Borrelia burgdorferi Group/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Integrin beta3 , Ligands , Maltose-Binding Proteins , Peptide Library , Platelet Membrane Glycoproteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
4.
Proc Natl Acad Sci U S A ; 96(1): 242-7, 1999 Jan 05.
Article in English | MEDLINE | ID: mdl-9874803

ABSTRACT

The human pathogenic bacterium group A Streptococcus produces an extracellular cysteine protease [streptococcal pyrogenic exotoxin B (SpeB)] that is a critical virulence factor for invasive disease episodes. Sequence analysis of the speB gene from 200 group A Streptococcus isolates collected worldwide identified three main mature SpeB (mSpeB) variants. One of these variants (mSpeB2) contains an Arg-Gly-Asp (RGD) sequence, a tripeptide motif that is commonly recognized by integrin receptors. mSpeB2 is made by all isolates of the unusually virulent serotype M1 and several other geographically widespread clones that frequently cause invasive infections. Only the mSpeB2 variant bound to transfected cells expressing integrin alphavbeta3 (also known as the vitronectin receptor) or alphaIIbbeta3 (platelet glycoprotein IIb-IIIa), and binding was blocked by a mAb that recognizes the streptococcal protease RGD motif region. In addition, mSpeB2 bound purified platelet integrin alphaIIbbeta3. Defined beta3 mutants that are altered for fibrinogen binding were defective for SpeB binding. Synthetic peptides with the mSpeB2 RGD motif, but not the RSD sequence present in other mSpeB variants, blocked binding of mSpeB2 to transfected cells expressing alphavbeta3 and caused detachment of cultured human umbilical vein endothelial cells. The results (i) identify a Gram-positive virulence factor that directly binds integrins, (ii) identify naturally occurring variants of a documented Gram-positive virulence factor with biomedically relevant differences in their interactions with host cells, and (iii) add to the theme that subtle natural variation in microbial virulence factor structure alters the character of host-pathogen interactions.


Subject(s)
Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Integrins/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Receptors, Vitronectin/metabolism , Streptococcus pyogenes/pathogenicity , Alleles , Animals , Bacterial Proteins , CHO Cells , Cell Adhesion/drug effects , Cricetinae , Endothelium, Vascular/cytology , Genetic Variation , Humans , Integrins/genetics , Oligopeptides/genetics , Oligopeptides/metabolism , Peptide Fragments/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Protein Binding , Receptors, Vitronectin/genetics , Recombinant Proteins , Streptococcus pyogenes/enzymology
5.
J Biol Chem ; 273(44): 28937-44, 1998 Oct 30.
Article in English | MEDLINE | ID: mdl-9786897

ABSTRACT

By extensive mutagenic analysis of the inserted domain (I-domain) of the alpha-chain (CD11a) of the leukocyte function-associated antigen-1 (LFA-1), we have defined a putative binding surface for intercellular adhesion molecules 1 and 2 (ICAM-1 and -2). This analysis showed that individually mutating Leu-205 or Glu-241 to alanine completely abolished LFA-1 binding to ICAM-1 or -2 without affecting I-domain structure, as assayed by antibody binding. Mutating Thr-243 to alanine also had a profound effect on LFA-1 binding to ICAM-1 and -2, as seen by complete loss of binding to ICAM-1 and a significant reduction (70% decrease) in binding to ICAM-2. Mutating Glu-146 to alanine reduced LFA-1 binding to ICAM-1 or -2 by 70%, and mutating His-264 or Glu-293 to alanine reduced binding to ICAM-1 or -2 by about 30-40%. Mutating Thr-175 to alanine reduced binding to ICAM-1 by about 30% and binding to ICAM-2 by about 70%. Interestingly, mutating Lys-263 to alanine preferentially abolished LFA-1 binding to ICAM-2. Using these data, we have generated a model of the interface between the LFA-1 I-domain and residues in the first domain of ICAM-1 that have been shown to be critical for this interaction. In addition, this model, together with the ICAM-2 crystal structure, has been used to map residues that are likely to mediate LFA-1 I-domain binding to ICAM-2.


Subject(s)
Antigens, CD/metabolism , Cell Adhesion Molecules/metabolism , Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Amino Acid Sequence , Antigens, CD/chemistry , Antigens, CD/genetics , Binding Sites , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Epitopes/metabolism , Intercellular Adhesion Molecule-1/chemistry , Intercellular Adhesion Molecule-1/genetics , Lymphocyte Function-Associated Antigen-1/chemistry , Lymphocyte Function-Associated Antigen-1/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis
6.
Infect Immun ; 66(5): 1946-52, 1998 May.
Article in English | MEDLINE | ID: mdl-9573074

ABSTRACT

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin alpha(IIb)beta3 was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins alpha(v)beta3 and alpha5beta1, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified alpha(v)beta3 and alpha5beta1. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified alpha(v)beta3 and alpha5beta1 was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to alpha(v)beta3 was also shown by using a transfected cell line that expresses this receptor but not alpha(IIb)beta3. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both alpha5beta1 and alpha(v)beta3. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.


Subject(s)
Bacterial Adhesion , Borrelia burgdorferi Group/physiology , Receptors, Fibronectin/physiology , Receptors, Vitronectin/physiology , Cell Line , Humans
7.
Mol Biol Cell ; 8(3): 501-15, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9188101

ABSTRACT

Intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the Ig superfamily and is a counterreceptor for the beta 2 integrins: lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), complement receptor 1 (MAC-1, CD11b/CD18), and p150,95 (CD11c/CD18). Binding of ICAM-1 to these receptors mediates leukocyte-adhesive functions in immune and inflammatory responses. In this report, we describe a cell-free assay using purified recombinant extracellular domains of LFA-1 and a dimeric immunoadhesin of ICAM-1. The binding of recombinant secreted LFA-1 to ICAM-1 is divalent cation dependent (Mg2+ and Mn2+ promote binding) and sensitive to inhibition by antibodies that block LFA-1-mediated cell adhesion, indicating that its conformation mimics that of LFA-1 on activated lymphocytes. We describe six novel anti-ICAM-1 monoclonal antibodies, two of which are function blocking. Thirty-five point mutants of the ICAM-1 immunoadhesin were generated and residues important for binding of monoclonal antibodies and purified LFA-1 were identified. Nineteen of these mutants bind recombinant LFA-1 equivalently to wild type. Sixteen mutants show a 66-2500-fold decrease in LFA-1 binding yet, with few exceptions, retain binding to the monoclonal antibodies. These mutants, along with modeling studies, define the LFA-1 binding site on ICAM-1 as residues E34, K39, M64, Y66, N68, and Q73, that are predicted to lie on the CDFG beta-sheet of the Ig fold. The mutant G32A also abrogates binding to LFA-1 while retaining binding to all of the antibodies, possibly indicating a direct interaction of this residue with LFA-1. These data have allowed the generation of a highly refined model of the LFA-1 binding site of ICAM-1.


Subject(s)
Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Binding Sites , Binding Sites, Antibody , Humans , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/immunology , Intercellular Adhesion Molecule-1/isolation & purification , Lymphocyte Function-Associated Antigen-1/genetics , Lymphocyte Function-Associated Antigen-1/isolation & purification , Mice , Models, Molecular , Mutagenesis, Site-Directed , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolism
8.
Bone ; 19(4): 317-28, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8894137

ABSTRACT

The extracellular matrix of bone is composed mainly of type I collagen. In this report we studied the role and collagen-binding properties of osteoclast integrins (alpha v, alpha 2, beta 1, and beta 3). Cell adhesion assays with rat osteoclasts and affinity chromatography/SDS-PAGE analysis with purified human osteoclast membranes demonstrated adhesion of osteoclasts to native type I collagen in a divalent cation and Arg-Gly-Asp (RGD)-dependent way via alpha 2 beta 1 integrin, whereas osteoclast adhesion to denatured collagen predominantly involved alpha v beta 3. In receptor-binding assays, the involvement of human recombinant alpha v beta 3 in adhesion to denatured collagen was confirmed. Additionally, osteoclasts adhered to type I collagen fibers and to monomeric types II-V collagen with characteristics similar to those on native monomeric type I collagen. Osteoclastic bone resorption in vitro was inhibited (> 40%) in the presence of alpha 2 and beta 1 antibodies. Using scanning laser confocal microscopy, alpha v beta 3, alpha 2, and beta 1 integrin were detected within podosomes in nonresorbing osteoclasts and in the ruffled border area and basolateral membrane in resorbing osteoclasts, but not in the sealing zone of resorbing osteoclasts. These results demonstrate that alpha 2 beta 1, in addition to alpha v beta 3, has an important role in osteoclast function and acts as a receptor for native, but not denatured, collagen.


Subject(s)
Bone Resorption/physiopathology , Cell Adhesion/physiology , Integrins/metabolism , Osteoclasts/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/therapeutic use , Binding, Competitive , Bone Resorption/drug therapy , Bone Resorption/metabolism , Cations, Divalent/metabolism , Chromatography, Affinity , Collagen/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Immunohistochemistry , Integrins/genetics , Integrins/immunology , Integrins/physiology , Membrane Proteins/metabolism , Oligopeptides/metabolism , Osteoclasts/cytology , Peptide Fragments/metabolism , Rats , Receptors, Immunologic/metabolism
9.
Science ; 273(5281): 1551-5, 1996 Sep 13.
Article in English | MEDLINE | ID: mdl-8703217

ABSTRACT

Integrin function is central to inflammation, immunity, and tumor progression. The urokinase-type plasminogen activator receptor (uPAR) and integrins formed stable complexes that both inhibited native integrin adhesive function and promoted adhesion to vitronectin via a ligand binding site on uPAR. Interaction of soluble uPAR with the active conformer of integrins mimicked the inhibitory effects of membrane uPAR. Both uPAR-mediated adhesion and altered integrin function were blocked by a peptide that bound to uPAR and disrupted complexes. These data provide a paradigm for regulation of integrins in which a nonintegrin membrane receptor interacts with and modifies the function of activated integrins.


Subject(s)
Cell Adhesion , Integrins/physiology , Receptors, Cell Surface/metabolism , Receptors, Cytoadhesin/metabolism , Amino Acid Sequence , CD18 Antigens/metabolism , Cell Line , Fibronectins/metabolism , Glycosylphosphatidylinositols/metabolism , Humans , Integrin beta1/metabolism , Integrins/metabolism , Ligands , Molecular Sequence Data , Receptors, Urokinase Plasminogen Activator , Recombinant Fusion Proteins/metabolism , Transfection , Urokinase-Type Plasminogen Activator/metabolism , Vitronectin/metabolism
10.
J Biol Chem ; 271(17): 9962-8, 1996 Apr 26.
Article in English | MEDLINE | ID: mdl-8626634

ABSTRACT

Several studies indicate that the I domain located in the alpha chain (CD11a) of leukocyte function-associated antigen-1 (LFA-1; CD11a/CD18) plays an essential role in ligand recognition. We recently identified three distinct epitopes (IdeA, IdeB, and IdeC) within the CD11a I domain, recognized by antibodies that block binding of LFA-1 to intercellular adhesion molecules (ICAM) 1, 2, and 3. In the present study, we used a series of human/murine CD11a I domain chimeras, to localize a fourth I domain epitope (IdeD), recognized by three independently derived anti-CD11a antibodies that selectively block the binding of LFA-1 to ICAM-3, but not to ICAM-1. The IdeD epitope depended on human CD11a residues Asp182 and Ser184 and was not present in CD11b or CD11c. Although mutation of Asp182 and Ser184 failed to abolish ICAM-3 adhesion of LFA-1 transfectants, alignment of these residues with the crystal structure of the CD11a I domain suggested that the IdeD epitope is located in close proximity to residues (Ile126 and Asn129) recently implicated in the ICAM-3 binding site. Interestingly, the IdeB and IdeC epitopes appeared to be in close proximity of a divalent cation binding pocket within the CD11a I domain that regulates both ICAM-1 and ICAM-3 adhesion. Taken together, these data indicate that distinct regions of the CD11a I domain contain epitopes for antibodies that either selectively inhibit binding of LFA-1 to ICAM-3, or interfere with both ICAM-1 and ICAM-3 binding of LFA-1.


Subject(s)
Antigens, CD , Antigens, Differentiation , Cell Adhesion Molecules/immunology , Lymphocyte Function-Associated Antigen-1/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Antigen-Antibody Reactions , Cell Adhesion , Enzyme Activation , Epitope Mapping , Humans , Mice , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary
11.
J Exp Med ; 183(3): 1247-52, 1996 Mar 01.
Article in English | MEDLINE | ID: mdl-8642267

ABSTRACT

We have identified amino acid residues within the evolutionarily conserved I domain of the alpha-chain (CD11a) of the leukocyte integrin leukocyte function-associated antigen (LFA) 1 that are critical for intercellular adhesion molecule (ICAM) 3 (CD50) binding. ICAM-3, a ligand of LFA-1, is thought to mediate intercellular adhesion essential for the initiation of immune responses. Using a panel of human/murine I domain chimeras and point mutants, we observed that the Ile-Lys-Gly-Asn motif, located in the NH2-terminal part of the CD11a I domain, is required for ICAM-3 but not ICAM-1 binding. These findings demonstrate that the I domain of CD11a contains distinct functional subdomains for ligand specific binding. An aspartic acid located at position 137, which is essential to ICAM-1/LFA-1 interactions (Edwards, C.P., M. Champe, T. Gonzalez, M.E. Wessinger, S.A. Spencer, L.G. Presta, P.W. Berman, and S.C. Bodary. 1995. J. Biol. Chem. 270:12635-12640), was also critical for ICAM-3 binding, whereas Ser at position 139 did not effect ICAM-1 or ICAM-3 binding. A synthetic peptide containing the Ile-Lys-Gly-Asn motif inhibited ICAM-3-dependent adhesion and proliferation of T cells at micromolar concentrations, suggesting that this peptide interferes with immune recognition. These observations underscore the importance of ICAM-3 in leukocyte function, and may lead to development of a new category of immunosuppressive agents.


Subject(s)
Antigens, CD , Antigens, Differentiation , Cell Adhesion Molecules/metabolism , Cell Adhesion , Lymphocyte Function-Associated Antigen-1/chemistry , Lymphocyte Function-Associated Antigen-1/physiology , Amino Acid Sequence , Animals , Binding Sites , Biological Evolution , Cell Adhesion/drug effects , Cell Line , Conserved Sequence , Gene Expression , Humans , Kinetics , Lymphocyte Function-Associated Antigen-1/immunology , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Peptides/chemistry , Peptides/pharmacology , Point Mutation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Transfection
12.
J Biol Chem ; 270(21): 12635-40, 1995 May 26.
Article in English | MEDLINE | ID: mdl-7539005

ABSTRACT

Leukocyte function-associated antigen-1 (LFA-1) is a cell surface adhesion receptor for intercellular adhesion molecule-1, -2, and -3 (ICAM-1, -2, -3). Using human/murine chimeras of the I-domain of the LFA-1 alpha subunit (CD11a), we recently identified the epitopes recognized by eight monoclonal antibodies against CD11a that inhibit LFA-1 binding to ICAM-1. In this report, we determined that replacement of the entire human I-domain with the entire murine I-domain in CD11a completely abrogated LFA-1 binding to human ICAM-1 without affecting the gross conformation or heterodimer formation of LFA-1, as assayed by antibody binding. In order to assess which residues of the I-domain are responsible for binding to ICAM-1, we tested the ability of a panel of human/murine I-domain chimeras to bind to human ICAM-1. When complexed with CD18, all CD11a chimeras bound ICAM-1 at levels comparable to wild-type CD11a/CD18, indicating that the residues in these chimeras which differ in human and murine I-domains may not play a critical role in LFA-1 binding to ICAM-1. A series of point mutations of residues that are conserved between murine and human CD11a I-domains, as well as between CD11b and CD11c, were also generated. Substitution of alanine for proline at position 192 in the human CD11a I-domain abrogated adhesion of LFA-1 to ICAM-1. Antibody binding data suggested that this was due to conformational changes within the I-domain. Mutation of the aspartic acids at positions 137 and 239 to either alanine or lysine completely destroyed ICAM-1 binding. The conformation of LFA-1 alanine mutants was not significantly altered. This suggests that these aspartic acids are required for binding of human LFA-1 to human ICAM-1.


Subject(s)
Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Animals , Cell Adhesion/genetics , Cell Adhesion/physiology , Chlorides/pharmacology , DNA Mutational Analysis , Epitopes , Humans , Ligands , Lymphocyte Function-Associated Antigen-1/genetics , Lymphocyte Function-Associated Antigen-1/immunology , Magnesium Chloride/pharmacology , Manganese Compounds/pharmacology , Mice , Point Mutation , Protein Binding/drug effects , Recombinant Fusion Proteins/metabolism , Species Specificity , Structure-Activity Relationship
13.
Bone ; 15(6): 639-46, 1994.
Article in English | MEDLINE | ID: mdl-7532981

ABSTRACT

Laminins may be encountered by osteoclasts and their precursors in basement membranes when they migrate from periosteal vasculature during skeletal development and in pathological situations. We have examined the recognition by osteoclasts of intact laminins and their proteolytic derivatives, and analysed the mechanism of adhesion. Rat osteoclasts fail to bind intact mouse Engelbreth-Holm-Swarm (EHS) laminin (3% adhesion relative to adhesion to foetal calf serum proteins) and bind only weakly to native human placental laminin (13%) or human merosin (9%). Pepsin treatment of native mouse EHS and human laminins increased osteoclast adhesion. Rat osteoclasts adhered to mouse EHS laminin-derived P1 fragment (70%), but failed to bind the E8 fragment, which contains adhesion sites recognised by some integrins. Binding to human and mouse P1 laminins was abolished by treatment with RGD-containing peptides and required divalent cations, but not by YIGSR peptide. Combinations of monoclonal antibodies to rat beta 3 and alpha v integrins reduced binding to P1 fragment by 91% and to human laminin by 72%, demonstrating that the major integrin involved in rat osteoclast adhesion to proteolysed laminin is alpha v beta 3. Antiserum to beta 1 integrin inhibited adhesion to human laminin by 40%, but to P1 fragment by only 8%; this suggests that beta 1 integrins(s) contribute to osteoclast adhesion to human laminin but probably not to P1 fragment. The involvement of alpha v beta 3 integrin was confirmed using a recombinant human alpha v beta 3 solid phase binding assay, alpha v beta 3 bound to mouse P1 fragment and proteolytically digested human laminin, but not intact laminins.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Integrins/metabolism , Laminin/metabolism , Osteoclasts/metabolism , Peptide Fragments/metabolism , Receptors, Laminin/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Binding, Competitive , Cations, Divalent , Cell Adhesion/drug effects , Humans , Integrins/isolation & purification , Laminin/chemistry , Mice , Molecular Sequence Data , Oligopeptides/metabolism , Peptide Fragments/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex , Rats , Receptors, Cytoadhesin/isolation & purification , Receptors, Cytoadhesin/metabolism , Receptors, Vitronectin , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Snake Venoms/metabolism
14.
Mol Biol Cell ; 3(9): 989-98, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1421580

ABSTRACT

We demonstrate an example of signal transduction by an integrin and have begun to define the pathway through which this signaling is achieved. We constructed a stably transfected derivative of 293 cells (ATCC 1573) that expresses the platelet integrin GPIIbIIIa (alpha IIb beta 3). This cell line, clone B, adheres to and spreads on fibrinogen, a ligand for alpha IIb beta 3, while the parent cell line does not. Stimulation of these cells either by adhesion to fibrinogen or with antiserum directed against alpha IIb beta 3 results in induction of calcium oscillations, followed by tyrosine phosphorylation of at least one protein of molecular weight approximately 125 kDa. We establish that this phosphorylation, as well as the morphological rearrangements, requires the mobilization of calcium.


Subject(s)
Blood Platelets/metabolism , Calcium/metabolism , Integrins/metabolism , Signal Transduction , Tyrosine/metabolism , Cell Adhesion , Cell Line , Cell Size , Fibrinogen , Periodicity , Phosphorylation , Transfection
15.
J Biol Chem ; 265(28): 17126-31, 1990 Oct 05.
Article in English | MEDLINE | ID: mdl-2211615

ABSTRACT

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


Subject(s)
DNA, Neoplasm/genetics , Integrins/genetics , Amino Acid Sequence , Base Sequence , Blotting, Northern , Cell Line , Cloning, Molecular , DNA, Neoplasm/isolation & purification , Humans , Integrins/isolation & purification , Macromolecular Substances , Molecular Sequence Data , Neoplasms , Oligonucleotide Probes , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Sequence Homology, Nucleic Acid
16.
J Biol Chem ; 265(11): 5938-41, 1990 Apr 15.
Article in English | MEDLINE | ID: mdl-1690718

ABSTRACT

We describe a novel integrin heterodimer on the surface of the human embryonic kidney cell line 293. This receptor is comprised of alpha v and beta 1 subunits, each of which has been previously found in association with other integrin subunits. This alpha v.beta 1 complex was identified as the predominant vitronectin receptor (VnR) on the surface of 293 cells by immunoprecipitation with antibodies raised against the alpha v subunit. Polymerase chain reaction analysis detected mRNAs for alpha v and beta 1 subunits while no evidence was obtained for beta 2, beta 3, or alpha IIb integrin subunit mRNA. Immunoprecipitation of surface-iodinated proteins with antibodies to alpha v gave bands of 150 and 120 kDa. The 120-kDa band reacted with antibodies to beta 1 in immunoblotting experiments. 293 cells adhere to vitronectin, fibronectin, laminin, and collagen IV, while von Willebrand factor and fibrinogen, known ligands of the VnR (alpha v.beta 3), did not support adhesion. A polyclonal antibody directed against both subunits of the VnR (alpha v, beta 3) inhibits attachment of 293 cells to vitronectin but not to other adhesive proteins. A beta 1-specific monoclonal inhibited attachment to fibronectin, laminin, and collagen IV, known ligands of beta 1 integrins, as well as vitronectin. This novel (alpha v. beta 1) VnR thus appears to mediate cell adhesion exclusively to vitronectin, in contrast to previously described VnRs which have multiple ligands.


Subject(s)
Integrins/metabolism , Receptors, Immunologic/metabolism , Antibodies , Antigen-Antibody Complex , Cell Adhesion , Cell Line , Embryo, Mammalian , Extracellular Matrix/immunology , Fibronectins/metabolism , Humans , Integrins/genetics , Integrins/isolation & purification , Kidney , Macromolecular Substances , Molecular Weight , Polymerase Chain Reaction , RNA, Messenger , Receptors, Fibronectin , Receptors, Vitronectin
17.
J Biol Chem ; 264(32): 18859-62, 1989 Nov 15.
Article in English | MEDLINE | ID: mdl-2808396

ABSTRACT

The ability of cDNAs encoding the human platelet glycoprotein IIbIIIa to be expressed and assembled into a functional integrin receptor was assessed by transient transfection into a human cell line. Transfection of full length cDNAs resulted in synthesis of high levels of integrin subunits which appear to be stable within the cell for several days. Coexpression of both subunits resulted in a proteolytically processed form of GPIIb that associated with GPIIIa as a heterodimeric complex as the cell surface. Transport to the cell surface required association of these subunits with each other or with endogenous integrin subunits. When expressed alone, the GPIIb subunit remained intracellular, while the GPIIIa subunit was found to complex with endogenous proteins and was mobilized to the cell surface. The GPIIbIIIa receptor complex facilitated attachment of cells to known ligands for GPIIbIIIa: fibrinogen, vitronectin, and von Willebrand factor. This adhesion was sensitive to inhibition by the peptide GRGDV and the monoclonal antibody AP2, known inhibitors of platelet aggregation


Subject(s)
Fibrinogen/metabolism , Platelet Membrane Glycoproteins/genetics , Base Sequence , Blotting, Western , Cell Adhesion , Cell Line , DNA/genetics , Humans , Macromolecular Substances , Molecular Sequence Data , Oligonucleotide Probes , Platelet Membrane Glycoproteins/metabolism , Protein Binding , Recombinant Proteins/metabolism , Transfection
18.
Dev Biol ; 134(1): 30-7, 1989 Jul.
Article in English | MEDLINE | ID: mdl-2471662

ABSTRACT

The survival of sympathetic and sensory neurons is known to be controlled by nerve growth factor (NGF) supplied by the targets of innervation, yet little is known about how target NGF synthesis is regulated. We have investigated the pattern of NGF mRNA expression in developing rat heart ventricle using a sensitive RNA blotting procedure. We find that the concentration of NGF mRNA increases steadily from Embryonic Day 17 to peak levels at 10-14 days postnatal and then declines about twofold and stabilizes at the level found in adults. The rise in NGF mRNA concentration correlates with the arrival and differentiation of sympathetic nerve terminals in the heart and the cessation of sympathetic cell death. To assess the role of innervating sympathetic neurons in regulating NGF mRNA expression, neonatal rats were sympathectomized by treatment with 6-hydroxydopamine and heart ventricles were assayed for NGF message. Although this treatment reduced ventricle norepinephrine content by 82%, no significant change in NGF mRNA concentration was observed. These results suggest that the developmental program of NGF mRNA production in the heart is not influenced by innervating sympathetic neurons.


Subject(s)
Heart/growth & development , Nerve Growth Factors/genetics , RNA, Messenger/metabolism , Sympathectomy, Chemical , Aging/metabolism , Animals , Female , Heart/embryology , Heart/innervation , Heart Ventricles/metabolism , Hydroxydopamines , Male , Neurons/physiology , Norepinephrine/metabolism , Oxidopamine , Rats , Rats, Inbred Strains , Substance P/metabolism , Sympathetic Nervous System/physiology
19.
Science ; 234(4774): 352-5, 1986 Oct 17.
Article in English | MEDLINE | ID: mdl-3764415

ABSTRACT

The regulation of nerve growth factor (NGF) protein and NGF messenger RNA (mRNA) in the developing rat brain has been studied to assess the hypothesis that NGF supports the differentiation of cholinergic neurons in the basal forebrain. In the adult, the major targets of these neurons, the hippocampus and neocortex, contain the highest concentrations of NGF mRNA, but comparatively low ratios of NGF protein to its mRNA. In contrast, a high concentration of NGF protein and a low concentration of NGF mRNA were seen in the basal forebrain, consistent with retrograde transport of NGF protein into this region from the neocortex and hippocampus. In these two target regions NGF and NGF mRNA were barely detectable at birth, their concentrations increased to a peak at day 21, and then NGF mRNA, but not NGF protein, declined threefold by day 35. NGF accumulation in the basal forebrain paralleled that in the target regions and preceded an increase in choline acetyltransferase, suggesting that the differentiation of cholinergic projection neurons is indeed regulated by retrogradely transported NGF. In addition, high ratios of NGF protein to NGF mRNA, comparable to that in the basal forebrain, were seen in the olfactory bulb and cerebellum, suggesting that NGF may be transported into these regions by unidentified neurons.


Subject(s)
Brain/growth & development , Nerve Growth Factors/biosynthesis , Animals , Brain/metabolism , Brain Chemistry , Cerebellum/analysis , Cerebral Cortex/analysis , Hippocampus/analysis , Nerve Growth Factors/analysis , Nerve Growth Factors/genetics , RNA, Messenger/analysis , Rats , Rats, Inbred Strains
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