Joseph C. Loftus

β1 Integrins Participate in the Hypertrophic Response of Rat Ventricular Myocytes

Abstract—Multiple signaling pathways have been implicated in the hypertrophic response of ventricular myocytes, yet the importance of cell-matrix interactions has not been extensively examined. Integrins are cell-surface molecules that link the extracellular matrix to the cellular cytoskeleton. They can function as cell signaling molecules and transducers of mechanical information in noncardiac cells. Given these properties and their abundance in cardiac cells, we evaluated the hypothesis that β1 integrin function is involved in the α1-adrenergic mediated hypertrophic response of neonatal rat ventricular myocytes. The hypertrophic response of this model required interaction with extracellular matrix proteins. Specificity of these results was confirmed by demonstrating that ventricular myocytes plated onto an anti–β1 integrin antibody supported the hypertrophic gene response. Adenovirus-mediated overexpression of β1 integrin augmented the myocyte hypertrophic response when assessed by protein synthesis and...

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.

A Molecular Basis for Affinity Modulation of Fab Ligand Binding to Integrin αIIbβ3

The Arg-Gly-Asp (RGD) sequence within the third complementarity-determining region (CDR3) of the heavy chain (H3) is responsible for the binding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the platelet integrin αIIbβ3. AP7 binding is minimally influenced by the conformational state of this receptor, whereas PAC1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. To study the molecular basis for this functional difference, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of the analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (VH domain + Cγ1 domain) segments containing these H3 loop sequences were expressed as active Fab molecules by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing Fd and AP7 κ chain cDNA. Replacement of the entire AP7 H3 loop with that from PAC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified αIIbβ3 in a manner identical to that of PAC1.1. Identical results were obtained when solely the sequences flanking the amino side of RGD within the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibited saturable but submaximal binding to activated gel-filtered platelets. Relative to AP7, the number of AP7.3 or PAC1.1 Fab molecules bound per platelet was 17% in the presence of 1 mM Ca2+ + 1 mM Mg2+ or 40% in the presence of 10 μM Mn2+. The ratio of Fab molecules bound after versus before activation (mean ± S.D.; n = 3) was: for AP7.3, 9.8 ± 0.6; for PAC1.1, 8.8 ± 0.3; and for AP7, 1.4 ± 0.2. In addition, AP7 bound to the stably expressed integrin mutant αIIbβ3(S123A), whereas AP7.3 and PAC1 did not. Because AP7.3 behaves in every respect like PAC1.1, we conclude that the ability of RGD-based ligands to distinguish activated from resting conformations of the integrin αIIbβ3 can be regulated by limited amino acid sequences immediately adjacent to the RGD tripeptide. Furthermore, those Fab molecules that exhibit increased selectivity for the activated conformation of αIIbβ3 bind to a subpopulation of this integrin on platelets that is modulated by divalent cations.

Erratum: Integrin|[ndash]|ligand binding properties governcell migration speed through cell|[ndash]|substratum adhesiveness

This corrects the article DOI: 10.1038/385537a0

A mutation in the integrin αIIb subunit that selectively inhibits αIIbβ3 receptor function

The αIIb and αv integrins have been shown to play a significant role in a variety of disease processes. αIIbβ3 is a platelet-specific fibrinogen receptor that is critical for thrombosis and hemostasis. Determination of the basis of ligand recognition by αIIbβ3 is essential for modulation of platelet function. To identify αIIb residues involved in αIIbβ3 ligand binding function, cells expressing a constitutively active variant of αIIbβ3 were randomly mutagenized and selected for loss of αIIbβ3 ligand binding function. One mutant isolated in this manner contained a single amino acid substitution at position 96 in αIIb (Ser96→Leu). Cells expressing this αIIb mutant did not bind the ligand mimetic antibody PAC1 or adhere to fibrinogen. In addition, the mutant receptor did not bind to an RGD affinity matrix. Substitution of conserved serine residues at position 1 in β strand A of all seven repeats of αIIb similarly inhibited ligand binding to αIIbβ3. αIIb S96 maps to the central cavity of the β-propeller fold of the a IIb subunit immediately adjacent to a structurally important sequence at the center of the α and β subunit interface. In contrast, substitution of the analogous residues in a αv or a α4 did not disrupt the ligand binding function of αvβ3 or α4β1. These data support a potential unique structural or mechanistic role for this residue in αIIbβ3 receptor function.