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

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 EC50 values of 0.1 and 1.7 nM, respectively. The opposite order of activation was observed for agonist peptide (SFLLRN-CONH2 or TFLL-RNKPDK) 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 G alpha(12/13)-mediated as chelation of G alpha(q)-mediated intracellular calcium with BAPTA-AM, pertussis toxin inhibition of G alpha(i/o), or GM6001 inhibition of matrix metalloproteinase had no effect, whereas Y-27632 inhibition of the G alpha(12/13)-mediated Rho kinase abrogated the response. Similarly, calcium mobilization is G alpha(q)-mediated and independent of G alpha(i/o) and G alpha(12/13) because pertussis toxin and Y-27632 had no effect, whereas U-73122 inhibition of phospholipase C-beta blocked the response. It is therefore likely that changes in permeability reflect G alpha(12/13) activation, and changes in calcium reflect G alpha(q) activation, implying that the pharmacological differences between agonists are likely caused by the ability of the receptor to activate G alpha(12/13) or G alpha(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 G alpha(q) activation over G alpha(12/13) by similar to 800-fold.