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J C Loftus

Publications and source records attributed to J C Loftus.

At least 37 records · Page 2Linked to original sources
PubMed1994

Mutation of a ligand binding domain of beta 3 integrin. Integral role of oxygenated residues in alpha IIb beta 3 (GPIIb-IIIa) receptor function.

A single amino acid substitution in beta 3 (Asp119 --> Tyr) abrogates the ligand binding function of beta 3 integrins and alters the divalent cation conformation of the platelet integrin alpha IIb beta 3 (GPIIb-IIIa). This aspartic acid residue resides within a conserved cluster of oxygenated residues that may provide ligands for the coordination of divalent cations. To assign function to the other oxygenated residues in this group (Ser121, Ser123, Asp126, Asp127, and Ser130), each of these amino acids in beta 3 was individually substituted by alanine. None of these amino acid substitutions altered heterodimer formation or surface expression. However, the substitutions had differential effects on receptor function. Substitution at positions Asp119 or Ser121 produced a complete loss of receptor function. Cells expressing these mutants failed to adhere to fibrinogen, failed to bind activation-independent ligand-mimetic peptides, and did not bind the ligand-mimetic mAb PAC1 following activation of the receptor. Similarly, cells expressing beta 3 with a substitution at Ser123 also failed to adhere to fibrinogen and did not bind RGD peptide or mAb PAC1. These cells did retain the capacity to bind an alpha IIb beta 3-specific, high affinity peptidomimetic, but occupancy did not induce the conformational change from resting to activated state observed following occupancy of the wild type receptor. Substitution at positions Asp126, Asp127, or Ser130 had no effect on ligand binding function. These data indicate that Asp119, along with Ser121 and Ser123, plays an integral role in the ligand binding function of alpha IIb beta 3.

Amino Acid Sequence↗
PubMed1994

Integrin cytoplasmic domains mediate inside-out signal transduction.

We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.

Amino Acid Sequence↗
PubMed1993

Platelet integrins.

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Amino Acid Sequence↗
PubMed1993

Distinct functions of integrin alpha and beta subunit cytoplasmic domains in cell spreading and formation of focal adhesions.

Integrin-mediated cell adhesion often results in cell spreading and the formation of focal adhesions. We exploited the capacity of recombinant human alpha IIb beta 3 integrin to endow heterologous cells with the ability to adhere and spread on fibrinogen to study the role of integrin cytoplasmic domains in initiation of cell spreading and focal adhesions. The same constructs were also used to analyze the role of the cytoplasmic domains in maintenance of the fidelity of the integrin repertoire at focal adhesions. Truncation mutants of the cytoplasmic domain of alpha IIb did not interfere with the ability of alpha IIb beta 3 to initiate cell spreading and form focal adhesions. Nevertheless, deletion of the alpha IIb cytoplasmic domain allowed indiscriminate recruitment of alpha IIb beta 3 to focal adhesions formed by other integrins. Truncation of the beta 3 subunit cytoplasmic domain abolished cell spreading mediated by alpha IIb beta 3 and also abrogated recruitment of alpha IIb beta 3 to focal adhesions. This truncation also dramatically impaired the ability of alpha IIb beta 3 to mediate the contraction of fibrin gels. In contrast, the beta 3 subunit cytoplasmic truncation did not reduce the fibrinogen binding affinity of alpha IIb beta 3. Thus, the integrin beta 3 subunit cytoplasmic domain is necessary and sufficient for initiation of cell spreading and focal adhesion formation. Further, the beta 3 cytoplasmic domain is required for the transmission of intracellular contractile forces to fibrin gels. The alpha subunit cytoplasmic domain maintains the fidelity of recruitment of the integrins to focal adhesions and thus regulates their repertoire of integrins.

Amino Acid Sequence↗
PubMed1992

Characterization of a gain of function mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa).

Integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) is a prototype of integrins involved in cellular adhesive functions. As part of a structure-function analysis of this molecule, we constructed a mutant, designated alpha IIb beta 3 (beta 1-2), by replacing 6 amino acids within a putative ligand binding domain of the beta 3 subunit with sequences derived from beta 1. The alteration did not affect the capacity of beta 3(beta 1-2) to combine with transfected alpha IIb, nor did it cause it to combine with endogenous alpha 5. Integrin alpha IIb beta 3(beta 1-2) was in a "resting" state on Chinese hamster ovary cells as judged by minimal binding of an activation-specific anti-alpha IIb beta 3, PAC1. Nevertheless, cells expressing alpha IIb beta 3(beta 1-2) spontaneously bound fibrinogen with low affinity (Ka = (4.85 +/- 0.84) x 10(6) M-1). Activation with an anti-beta 3 antibody (monoclonal antibody 62) resulted in a 10-fold increase in fibrinogen binding affinity (Ka = (4.55 +/- 0.77) x 10(7) M-1), which was 3-fold greater than fibrinogen binding to activated wild type alpha IIb beta 3 (Ka = (1.66 +/- 0.33) x 10(7) M-1, F = 7.46, p = 0.008). The mutant receptor also bound fibrinogen mimetic peptide ligands with enhanced affinity as measured by the conformation-specific antibody, anti-LIBS1. This indicates that the increased affinity for fibrinogen was caused by enhanced interaction of alpha IIb beta 3(beta 1-2) with known recognition sequences in fibrinogen. Thus, this gain of function mutant augments ligand binding function, supporting a role for this region of the beta subunit in ligand binding to integrins.

Amino Acid Sequence↗
PubMed1992

The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb-IIIa on nonactivated platelets is accessible to high-affinity macromolecules.

We have characterized a murine IgG monoclonal antibody, OP-G2, specific for platelet glycoprotein (GP) IIb-IIIa (alpha IIb beta 3). OP-G2 Fab fragments inhibit fibrinogen-mediated platelet aggregation and competitively inhibit adenosine diphosphate-induced binding of 125I-fibrinogen to washed platelets. OP-G2 binding to GPIIb-IIIa is specifically inhibited by RGD-containing peptides but not the fibrinogen gamma-chain carboxy-terminal peptide, and OP-G2 Fab fragments, like RGD-containing peptides, alter the conformation of GPIIb-IIIa resulting in the expression of a ligand-induced binding site (LIBS) recognized by PMI-1. OP-G2 fails to bind to the recombinant Cam variant of GPIIb-IIIa (alpha III beta 3Cam) wherein an Asp119 to Tyr119 substitution in GPIIIa abrogates the ability to recognize RGD. These data indicate that OP-G2 recognizes an epitope at or in very close proximity to the RGD recognition site of GPIIb-IIIa and that, in every aspect tested, OP-G2 behaves like a macromolecular RGD ligand. Interestingly, two-color flow cytometry shows that OP-G2 IgG can bind to nonactivated platelets. Quantitative binding assays indicate that nonactivated platelets bind approximately 50,000 125I-OP-G2 molecules/platelet. Furthermore, the affinity of OP-G2 for platelets activated with thrombin is roughly fivefold higher (nonactivated, kd = 24.8 nmol/L; activated, kd = 4.9 nmol/L). These results suggest that the RGD recognition site of GPIIb-IIIa is available to macromolecules that contain RGD even on nonactivated platelets, provided that the affinity of the ligand is adequate.

Amino Acid Sequence↗
PubMed1992

A spontaneous mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site.

This work characterizes a mutant integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) from a thrombasthenic patient, ET, whose platelets fail to aggregate in response to stimuli. The nature of defect was defined by the reduced ability of synthetic peptide ligands, corresponding to the carboxyl terminus of the fibrinogen gamma chain (gamma 402-411) and Arg-Gly-Asp (RGD), to increase the binding of the occupancy-dependent anti-LIBS1 antibody to mutant alpha IIb beta 3 and the reduced binding of mutant alpha IIb beta 3 to an immobilized RGD peptide. In addition, ET's platelets failed to bind the ligand-mimetic monoclonal anti-alpha IIb beta 3, PAC1. DNA sequence analysis of amplified ET genomic DNA revealed a single G----A base change which encoded substitution of R214 by Q in mature beta 3. Introduction of this point mutation into recombinant wild type alpha IIb beta 3 expressed in Chinese hamster ovary cells reproduced the ET platelet alpha IIb beta 3 deficits in binding of fibrinogen, mAb PAC1, and synthetic peptide ligands. Furthermore, substitution of R214 by Q in the synthetic peptide containing the sequence of beta 3(211-222) resulted in decreased ability of this peptide to block fibrinogen binding to purified alpha IIb beta 3. These findings suggest that substitution of beta 3 R214 by Q is responsible for the functional defect in alpha IIb beta 3 and that R214 is proximal to or part of a ligand binding domain in alpha IIb beta 3.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and preliminary characterization of a novel cytoplasmic antigen recognized by myasthenia gravis sera.

A cDNA clone was isolated by screening of a lambda gt11 endothelial expression library with serum from a patient with myasthenia gravis (MG). Rabbit antisera raised against the recombinant protein and human MG sera reactive with the clone immunoblotted an M(r) integral of 250,000 polypeptide (gravin) present in endothelial cells and several adherent cells. Gravin was not detected in platelets, leukocytes, U937, or human erythroleukemic (HEL) cell lines, but was expressed in HEL cells after induction with phorbol myristate acetate. Northern blot analysis showed two transcripts of approximately 6.7 and 8.4 kb in endothelial cells but not U937 or HEL cells. Indirect immunofluorescence of permeabilized cells revealed a trabecular network of gravin staining with a distinct linear component. Antibodies to gravin, were present in sera from 22:72 (31%) of MG patients. In contrast 0:50 normal sera and 1:72 sera from patients with other autoimmune diseases contained antigravin antibodies. Gravin is not likely to be a nonerythroid spectrin, talin, myosin, or actin-binding protein based on the lack of reactivity of antigravin with these polypeptides in immunoblots. The nucleotide sequence of the immunoreactive clone indicated that it encodes a highly acidic polypeptide fragment that contains the carboxyl terminus of the protein. Neither amino acid nor nucleotide sequences were present in Genbank, EMBL, or Swissprot databases as of March, 1992. These data indicate that gravin is an inducible, cell type-specific cytoplasmic protein and that auto-antibodies to gravin may be highly specific for MG.

Amino Acid Sequence↗
PubMed1991

Platelet glycoprotein IIb-IIIa (alpha IIb beta 3 integrin) confers fibrinogen- and activation-dependent aggregation on heterologous cells.

To analyze molecular mechanisms of platelet aggregation, we have studied the aggregation of Chinese hamster ovary (CHO) cells expressing between 1 and 4 x 10(5) recombinant human glycoprotein (GP) IIb-IIIa molecules per cell (A5 cells). These cells aggregated as measured by the disappearance of single cells during rotary agitation. Aggregation was dependent on the presence of extracellular fibrinogen (approximately 500 nmol/L) and divalent cations, and required prior activation of the GPIIb-IIIa. A synthetic peptide (GRGDSP) and monoclonal anti-GPIIb-IIIa antibody (2G12) that block platelet aggregation also blocked aggregation of these cells. Parent CHO cells or those expressing recombinant GPIIb-IIIa containing a point mutation that causes variant thrombasthenia both failed to aggregate when stimulated in the presence of fibrinogen. These data show that GPIIb-IIIa is the only unique platelet surface component required for aggregation.

Animals↗
PubMed1991

Autoantibodies to the presumptive cytoplasmic domain of platelet glycoprotein IIIa in patients with chronic immune thrombocytopenic purpura.

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to autoantibodies against platelets that result in their destruction. In some patients, these autoantibodies bind to platelet glycoprotein (GP) IIIa. With the aim of better defining the antigenic epitopes, plasma from 13 selected patients with chronic ITP known to have anti-GPIIb/IIIa autoantibodies was tested for reactivity with nine synthetic peptides corresponding to different regions of the GP IIIa molecule. Of these plasmas, five bound significantly (P less than .001) with either peptide 8 (amino acids 721-744) or peptide 9 (amino acids 742-762), which together form most of the carboxyterminal region presumed to be the cytoplasmic domain. Three of these positive plasmas, were tested further. In two of these positive plasmas, the anti-peptide antibodies represented greater than 80% of the detectable circulating autoantibody. To further evaluate the importance of the carboxyterminal region as an antigenic site, the chronic ITP plasmas were tested against Chinese hamster ovary cells transfected with GPIIb and either whole GPIIIa or GPIIIa lacking amino acids 728 to 762. Ten of the 13 plasmas required the presence of this region for significant autoantibody binding. We conclude that the carboxyterminal region is an important area for stimulating antiplatelet autoantibody formation in some patients with chronic ITP. It is not known whether these autoantibodies to the presumed cytoplasmic domain play an important role in the pathogenesis of the disease or occur as a secondary phenomenon during the course of platelet destruction.

Amino Acid Sequence↗
PubMed1991

Ligands "activate" integrin alpha IIb beta 3 (platelet GPIIb-IIIa).

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) binds fibrinogen via recognition sequences such as Arg-Gly-Asp (RGD). Fibrinogen binding requires agonist activation of platelets, whereas the binding of short synthetic RGD peptides does not. We now find that RGD peptide binding leads to changes in alpha IIb beta 3 that are associated with acquisition of high affinity fibrinogen-binding function (activation) and subsequent platelet aggregation. The structural specificities for peptide activation and for inhibition of ligand binding are similar, indicating that both are consequences of occupancy of the same site(s) on alpha IIb beta 3. Thus, the RGD sequence is a trigger of high affinity ligand binding to alpha IIb beta 3, and certain RGD-mimetics are partial agonists as well as competitive antagonists of integrin function.

Amino Acid Sequence↗
PubMed1991

Ligand bridging mediates integrin alpha IIb beta 3 (platelet GPIIB-IIIA) dependent homotypic and heterotypic cell-cell interactions.

The aggregation of cells bearing recombinant integrin alpha IIb beta 3 (platelet GPIIb-IIIa) has been analyzed by two-color flow cytometry. As in normal platelets, aggregation requires functional alpha IIb beta 3, "activation" of alpha IIb beta 3, and fibrinogen (fg) binding to alpha IIb beta 3. Cellular aggregation required that both interacting cells express functional alpha IIb beta 3, because a binding defective mutant, alpha IIb beta 3 (D119----Y), failed to support interaction with wild type alpha IIb beta 3-bearing cells. In addition, cells bearing resting alpha IIb beta 3 were incorporated into aggregates formed by cells bearing a constitutively active mutant, alpha IIb beta 3 (beta 1-2), indicating that only one of the cells in an interacting pair must be activated. Finally, heterotypic interactions occurred between cells bearing activated alpha IIb beta 3 and cells bearing alpha V beta 3, a fg-binding integrin present on endothelial and tumor cells. Thus, ligand bridging between fg-binding integrins represents a mechanism of cell-cell interaction, cells bearing resting alpha IIb beta 3 (e.g., resting platelets) may be incorporated into aggregates formed by cells bearing activated alpha IIb beta 3, and alpha IIb beta 3 mediates heterotypic interactions with cells bearing other fg receptors.

Animals↗
PubMed1990

A beta 3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation.

The ligand-binding function of integrin adhesion receptors depends on divalent cations. A mutant alpha IIb beta 3 integrin (platelet gpIIb/IIIa) that lacks ligand recognition shows immunologic evidence of a perturbed interaction with divalent cations. This was found to be caused by a G----T mutation that resulted in an Asp119----Tyr119 substitution in the beta 3 subunit. This residue is proximal to bound ligand and is in a conserved region among integrins that are enriched in oxygenated residues. The spacing of these residues aligns with the calcium-binding residues in EF hand proteins, suggesting interaction with receptor-bound divalent cation as a mechanism of ligand binding common to all integrins.

Amino Acid Sequence↗
PubMed1990

Affinity modulation of the alpha IIb beta 3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor.

To analyze the basis of affinity modulation of integrin function, we studied cloned stable Chinese hamster ovary cell lines expressing recombinant integrins of the beta 3 family (alpha IIb beta 3 and alpha v beta 3). Antigenic and peptide recognition specificities of the recombinant receptors resembled those of the native receptors found in platelets or endothelial cells. The alpha IIb beta 3-expressing cell line (A5) bound RGD peptides and immobilized fibrinogen (Fg) but not soluble fibrinogen or the activation-specific monoclonal anti-alpha IIb beta 3 (PAC1), indicating that it was in the affinity state found on resting platelets. Several platelet agonists failed to alter the affinity state of ("activate") recombinant alpha IIb beta 3. The binding of soluble Fg and PAC1, however, was stimulated in both platelets and A5 cells by addition of IgG papain-digestion products (Fab) fragments of certain beta 3-specific monoclonal antibodies. These antibodies stimulated PAC1 binding to platelets fixed under conditions rendering them unresponsive to other agonists. Addition of these antibodies to detergent-solubilized alpha IIb beta 3 also stimulated specific Fg binding. These data demonstrate that certain anti-beta 3 antibodies activate alpha IIb beta 3 by acting directly on the receptor, possibly by altering its conformation. Furthermore, they indicate that the activation state of alpha IIb beta 3 is a property of the receptor itself rather than of the surrounding cell membrane microenvironment.

Animals↗