Functional selectivity of G protein signaling by agonist peptides and thrombin for the protease-activated receptor-1.

McLaughlin JN, Shen L, Holinstat M, Brooks JD, Dibenedetto E, Hamm HE
J Biol Chem. 2005 280 (26): 25048-59

PMID: 15878870 · DOI:10.1074/jbc.M414090200

Thrombin activates protease-activated receptor-1 (PAR-1) by cleavage of the amino terminus to unmask a tethered ligand. Although peptide analogs can activate PAR-1, we show that the functional responses mediated via PAR-1 differ between the agonists. Thrombin caused endothelial monolayer permeability and mobilized intracellular calcium with EC(50) values of 0.1 and 1.7 nm, respectively. The opposite order of activation was observed for agonist peptide (SFLLRN-CONH(2) or TFLLRNKPDK) activation. The addition of inactivated thrombin did not affect agonist peptide signaling, suggesting that the differences in activation mechanisms are intramolecular in origin. Although activation of PAR-1 or PAR-2 by agonist peptides induced calcium mobilization, only PAR-1 activation affected barrier function. Induced barrier permeability is likely to be Galpha(12/13)-mediated as chelation of Galpha(q)-mediated intracellular calcium with BAPTA-AM, pertussis toxin inhibition of Galpha(i/o), or GM6001 inhibition of matrix metalloproteinase had no effect, whereas Y-27632 inhibition of the Galpha(12/13)-mediated Rho kinase abrogated the response. Similarly, calcium mobilization is Galpha(q)-mediated and independent of Galpha(i/o) and Galpha(12/13) because pertussis toxin Y-27632 and had no effect, whereas U-73122 inhibition of phospholipase C-beta blocked the response. It is therefore likely that changes in permeability reflect Galpha(12/13) activation, and changes in calcium reflect Galpha(q) activation, implying that the pharmacological differences between agonists are likely caused by the ability of the receptor to activate Galpha(12/13) or Galpha(q). This functional selectivity was characterized quantitatively by a mathematical model describing each step leading to Rho activation and/or calcium mobilization. This model provides an estimate that peptide activation alters receptor/G protein binding to favor Galpha(q) activation over Galpha(12/13) by approximately 800-fold.

MeSH Terms (34)

Actins Adenosine Diphosphate Amides Calcium Cells, Cultured Chelating Agents Dipeptides Dose-Response Relationship, Drug Egtazic Acid Electric Impedance Endothelium, Vascular Enzyme Inhibitors GTP-Binding Protein alpha Subunits, G12-G13 GTP-Binding Protein alpha Subunits, Gq-G11 GTP-Binding Proteins Humans Intracellular Signaling Peptides and Proteins Kinetics Ligands Matrix Metalloproteinase Inhibitors Microcirculation Models, Biological Models, Theoretical Peptides Pertussis Toxin Protease Inhibitors Protein-Serine-Threonine Kinases Protein Binding Pyridines Receptor, PAR-1 rho-Associated Kinases Signal Transduction Thrombin Time Factors

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