title: Functional selectivity of G protein signaling by agonist peptides and thrombin for the protease-activated receptor-1.
abstract: 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.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: Joseph N
authors.initials: JN
authors.lastname: McLaughlin
authors.forename: Lixin
authors.initials: L
authors.lastname: Shen
authors.forename: Michael
authors.initials: M
authors.lastname: Holinstat
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Brooks
authors.forename: Emmanuele
authors.initials: E
authors.lastname: Dibenedetto
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Hamm
mesh_terms: D000199:Actins / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000577:Amides / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008024:Ligands; D061965:Matrix Metalloproteinase Inhibitors; D008833:Microcirculation; D008954:Models, Biological; D008962:Models, Theoretical; D010455:Peptides / Q000737:chemistry*; D037342:Pertussis Toxin / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000737:chemistry* / Q000502:physiology; D015398:Signal Transduction; D013917:Thrombin / Q000737:chemistry*; D013997:Time Factors; D054460:rho-Associated Kinases
chemical_list: D000199:Actins; D000577:Amides; D002614:Chelating Agents; D004151:Dipeptides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D010455:Peptides; D011480:Protease Inhibitors; D011725:Pyridines; D044463:Receptor, PAR-1; C108830:Y 27632; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D037342:Pertussis Toxin; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D013917:Thrombin; D019204:GTP-Binding Proteins; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium