Protease-activated receptor-4 uses dual prolines and an anionic retention motif for thrombin recognition and cleavage.
Thrombin activation of human platelets is mediated by the high-affinity PAR1 (protease-activated receptor-1) and the low-affinity PAR4 receptor. PAR1 and PAR4 exhibit markedly disparate kinetics of activation that likely reflect differences in the macromolecular association of thrombin with their respective N-terminal extracellular domains (exodomains). Here we examine the mechanism of initial thrombin binding and cleavage of the high- and low-affinity PAR exodomains using steady-state kinetic analyses. We showed that the PAR4 exodomain lacks the functional hirudin-like sequence found in PAR1 and does not bind exosite I to cause allosteric activation or inhibition of thrombin. Instead, PAR4 contains an anionic cluster, Asp(57)...Asp(59) ...Glu(62)...Asp(65) (DDED), in its exodomain, which slows the dissociation of PAR4 from the cationic thrombin. The analogous anionic residues in the PAR1 exodomain do not influence affinity for thrombin. Although PAR4 is cleaved more slowly than PAR1 on the cell surface, peptides containing the PAR4 P(4)-P(1) active-site-interacting sequence, Pro(45)-Ala-Pro-Arg (PAPR), are efficiently cleaved due to the optimal placement of dual prolines at positions P(4) and P(2). In comparison, thrombin has low affinity and slow cleavage rates for peptides that have a P(3) proline as occurs in human PAR3. Thus, to compensate for the lack of exosite I binding, PAR4 utilizes proline residues in its P(4)-P(1) sequence to provide high-affinity interactions with the active site and an anionic cluster to slow dissociation from the cationic thrombin.
D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions / Q000032:analysis; D001665:Binding Sites; D019556:COS Cells; D006629:Hirudins / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011392:Proline / Q000032:analysis*; D017434:Protein Structure, Tertiary; D044463:Receptor, PAR-1 / Q000737:chemistry / Q000378:metabolism; D018179:Receptors, Thrombin / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000502:physiology
D000838:Anions; D006629:Hirudins; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D014364:Tryptophan; D011392:Proline; D013917:Thrombin; C112908:protease-activated receptor 4