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J. Biol. Chem., Vol. 280, Issue 26, 25048-25059, July 1, 2005

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Functional Selectivity of G Protein Signaling by Agonist Peptides and Thrombin for the Protease-activated Receptor-1^*

Joseph N. McLaughlin, Lixin Shen, Michael Holinstat, Joshua D. Brooks, Emmanuele DiBenedetto, and Heidi E. Hamm

From the Department of Pharmacology, Vanderbilt University Medical Center, Department of Mathematics and Center for Biomathematics, Vanderbilt University, Nashville, Tennessee 37232

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 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 G_12/13-mediated as chelation of G_q-mediated intracellular calcium with BAPTA-AM, pertussis toxin inhibition of G_i/o, or GM6001 inhibition of matrix metalloproteinase had no effect, whereas Y-27632 inhibition of the G_12/13-mediated Rho kinase abrogated the response. Similarly, calcium mobilization is G_q-mediated and independent of G_i/o and G_12/13 because pertussis toxin Y-27632 and had no effect, whereas U-73122 inhibition of phospholipase C- blocked the response. It is therefore likely that changes in permeability reflect G_12/13 activation, and changes in calcium reflect G_q activation, implying that the pharmacological differences between agonists are likely caused by the ability of the receptor to activate G_12/13 or G_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_q activation over G_12/13 by 800-fold.

Received for publication, December 15, 2004 , and in revised form, April 22, 2005.

^* This work was supported by National Institutes of Health Grant 5P01 HL60678 and 5R01 GM068953, Keck Futures Initiatives/NAKFI Sig05 (to H. E. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, 444 Robinson Research Bldg., 23rd Ave. South at Pierce, Nashville, TN 37232-6600. Tel.: 615-343-3533; Fax: 615-343-1084; E-mail: heidi.hamm{at}vanderbilt.edu.

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