Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 288, Issue 45, Pages 32553-32562Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.472373
Keywords
Bioluminescence Resonance Energy Transfer (BRET); G-protein-coupled Receptors (GPCR); Protein-Protein Interactions; Receptor Regulation; Thrombin; PAR1; PAR4; Protease-activated Receptor
Categories
Funding
- National Institutes of Health [HL098217]
- American Heart Association (AHA) [0865441D]
- AHA Scientist Development Grant [10SDG2600021]
- American Society for Hematology Scholar Award
- Cytometry and Imaging Microscopy Core Facility of the Case Comprehensive Cancer Center Grant [P30CA043703]
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Thrombin is a potent platelet agonist that activates platelets and other cells of the cardiovascular system by cleaving its G-protein-coupled receptors, protease-activated receptor 1 (PAR1), PAR4, or both. We now show that cleaving PAR1 and PAR4 with -thrombin induces heterodimer formation. PAR1-PAR4 heterodimers were not detected when unstimulated; however, when the cells were stimulated with 10 nm -thrombin, we were able to detect a strong interaction between PAR1 and PAR4 by bioluminescence resonance energy transfer. In contrast, activating the receptors without cleavage using PAR1 and PAR4 agonist peptides (TFLLRN and AYPGKF, respectively) did not enhance heterodimer formation. Preventing PAR1 or PAR4 cleavage with point mutations or hirugen also prevented the induction of heterodimers. To further characterize the PAR1-PAR4 interactions, we mapped the heterodimer interface by introducing point mutations in transmembrane helix 4 of PAR1 or PAR4 that prevented heterodimer formation. Finally, we show that mutations in PAR1 or PAR4 at the heterodimer interface prevented PAR1-assisted cleavage of PAR4. These data demonstrate that PAR1 and PAR4 require allosteric changes induced via receptor cleavage by -thrombin to mediate heterodimer formation, and we have determined the PAR1-PAR4 heterodimer interface. Our findings show that PAR1 and PAR4 have dynamic interactions on the cell surface that should be taken into account when developing and characterizing PAR antagonists.
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