Differential association modes of the thrombin receptor PAR1 with Galphai1, Galpha12, and beta-arrestin 1.
Although many G protein-coupled receptors (GPCRs) are known to activate multiple signaling pathways by coupling to different types of G proteins or by promoting G protein-independent events, how this occurs remains unclear. Using bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer, we provide evidence for protease-activated receptor 1 (PAR(1)) forming preassembled complexes with Galphai1 but not Galpha12. PAR(1) activation appears to rapidly induce transient Galphai1 activation (t(1/2) = 4.13 s) but late and stable recruitment of Galpha12 (t(1/2) = 8.8 min) in parallel with beta-arrestin 1 (t(1/2) = 7.5 min). However, there is no significant difference in the potency of the agonist-dependent response between Galphai1, Galpha12, and beta-arrestin 1 (EC(50) values 0.48, 0.30, and 0.15 nM, respectively). Although it seems beta-arrestin 1 is recruited to preassembled PAR(1)-Galphai1 complexes, this appears unlikely with Galpha12, suggesting 2 distinct receptor populations. Of note, we observed a different Galpha12 association mode with other GPCRs, indicating that preassembly and association dynamics may be specific properties of a receptor-G protein pair. Furthermore, the Galpha C terminus appears to play different roles in the distinct association modes. Consequently, G protein preassembly or recruitment may constitute novel mechanisms for controlling the kinetics and multitude of GPCR signaling pathways.
Ayoub
Mohammed Akli
MA
CNRS UMR5203, INSERM U661, Universités Montpellier I and II, Institut de Génomique Fonctionnelle, Département de Pharmacologie Moléculaire, 141, rue de la Cardonille, Montpellier F-34094 Cedex 5, France.
Trinquet
Eric
E
Pfleger
Kevin D G
KD
Pin
Jean-Philippe
JP
D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay; D031541:Fluorescence Resonance Energy Transfer; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism*; D000071557:beta-Arrestins
D019390:Arrestins; D044463:Receptor, PAR-1; D000071557:beta-Arrestins; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D019206:GTP-Binding Protein alpha Subunits, Gi-Go