Molecular mapping of thrombin-receptor interactions

Ayala, YM Cantwell, AM Rose, T Bush, LA Arosio, D Di Cera, E

Ym. Ayala et al., Molecular mapping of thrombin-receptor interactions, PROTEINS, 45(2), 2001, pp. 107-116

33

INGLESE

Article

Biochemistry & Biophysics

PROTEINS-STRUCTURE FUNCTION AND GENETICS

0887-3585 → ACNP

45

2

2001

107 - 116

ISI

0887-3585(20011101)45:2<107:MMOTI>2.0.ZU;2-A

In addition to its procoagulant and anticoagulant roles in the blood coagul ation cascade, thrombin works as a signaling molecule when it interacts wit h the G-protein coupled receptors PAR1, PAR3, and PAR4. We have mapped the thrombin epitopes responsible for these interactions using enzymatic assays and Ala scanning mutagenesis. The epitopes overlap considerably, and are a lmost identical to those of fibrinogen and fibrin, but a few unanticipated differences are uncovered that help explain the higher (90-fold) specificit y of PAR1 relative to PAR3 and PAR4. The most critical residues for the int eraction with the PARs are located around the active site where mutations a ffect recognition in the order PAR4 > PAR3 > PAR1 Other important residues for PAR binding cluster in a small area of exosite I where mutations affect recognition in the order PAR1 > PAR3 > PAR4. Owing to this hierarchy of ef fects, the mutation W215A selectively compromises PAR4 cleavage, whereas th e mutation R67A abrogates the higher specificity of PAR1 relative to PAR3 a nd PAR4. 3D models of thrombin complexed with PAR1, PAR3, and PAR4 are cons tructed and account for the perturbations documented by the mutagenesis stu dies. (C) 2001 Wiley-Liss, Inc.