Mutant thrombin receptor activating peptide induces unique coupling of PAR‐1 receptor to G proteins.

Mechanisms of functional selectivity at the protease activated receptor‐1 (PAR‐1) were studied by addressing how binding interactions with synthetic peptide mutants affect ligand function. Recent work showed that activation of PAR‐1 with agonist peptides rather than thrombin favors Ca 2+ mobilization over barrier permeability in Human Microvascular Endothelial cells (HMEC; McLaughlin et al., JBC, 2005). These results suggest that some PAR‐1 ligands are functionally selective and can differentially activate G protein subfamilies. Elucidation of the specific partners involved in endothelial barrier function could provide novel targets for therapeutic intervention in cardiovascular disease. Our current work with peptide mutants suggests that specific positions along the thrombin receptor activating peptide can selectively affect receptor activated second messenger signaling. Specifically, SFLLRN and SFALRN were tested in HMEC on several effector pathways (e.g. Ca 2+ mobilization and barrier disruption) to determine G protein selectivity. SFALRN exhibited similar potency and intrinsic activity for Gα q mediated Ca 2+ mobilization relative to SFLLRN. Conversely, SFALRN elicited strikingly different results than thrombin or SFLLRN at Rho mediated barrier disruption. A stair‐case functional effect was observed, suggesting differential recruitment and activation of G proteins induced by SFALRN with respect to thrombin or SFLLRN. Subsequent studies provide evidence to support SFALRN induced coupling with both Gα 12/13 and Gα i/o proteins at barrier function. These studies suggest that a structure‐based understanding of interaction not only at the ligand‐receptor interface but also at the receptor‐G protein interface will yield important mechanistic insights.