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Author Notes:

Correspondence: John R. Hepler, Ph.D., 1510 Clifton Road, G205 Rollins Research Center, Atlanta, GA 30322; Email: jhepler@emory.edu; Tel: +1 404 727 8192; Fax: +1 404 727 0365

Acknowledgments: We would like to thank Dr. Randy Hall (Emory University) for allowing us to use the Amaxa Nucleofector II.

The authors also are indebted to Jon Waters for skillful technical assistance.

Subject:

Research Funding:

The research was supported by American Heart Association Pre-doctoral Fellowship Grant 0715465B (KLM), R01NS049195 (JRH), R01NS37112 (JRH), R01NS039419 (SFT), R01GM053536 (AVS), a PhRMA Foundation Pre-doctoral Pharmacology/Toxicology Fellowship (CPV), and NIH Pharmacological Sciences Training Grant (T32 GM008602, SG).

Keywords:

  • Protease-activated Receptor
  • PAR1
  • astrocytes
  • thrombin
  • G protein
  • Gq
  • G11
  • Go
  • G12/13
  • phospholipase Cε
  • PLCε

Protease-Activated Receptor 1 (PAR1) coupling to Gq/11 but not to Gi/o or G12/13 is mediated by discrete amino acids within the receptor second intracellular loop

Tools:

Journal Title:

Cellular Signalling

Volume:

Volume 24, Number 6

Publisher:

, Pages 1351-1360

Type of Work:

Article | Post-print: After Peer Review

Abstract:

Protease-activated receptor 1 (PAR1) is an unusual GPCR that interacts with multiple G protein subfamilies (Gq/11, Gi/o, and G12/13) and their linked signaling pathways to regulate a broad range of pathophysiological processes. However, the molecular mechanisms whereby PAR1 interacts with multiple G proteins are not well understood. Whether PAR1 interacts with various G proteins at the same, different, or overlapping binding sites is not known. Here we investigated the functional and specific binding interactions between PAR1 and representative members of the Gq/11, Gi/o, and G12/13 subfamilies. We report that Gq/11 physically and functionally interacts with specific amino acids within the second intracellular (i2) loop of PAR1. We identified five amino acids within the PAR1 i2 loop that, when mutated individually, each markedly reduced PAR1 activation of linked inositol phosphate formation in transfected COS-7 cells (functional PAR1-null cells). Among these mutations, only R205A completely abolished direct Gq/11 binding to PAR1 and also PAR1-directed inositol phosphate and calcium mobilization in COS-7 cells and PAR1−/− primary astrocytes. In stark contrast, none of the PAR1 i2 loop mutations disrupted direct PAR1 binding to either Go or G12, or their functional coupling to linked pertussis toxin-sensitive ERK phosphorylation and C3 toxin-sensitive Rho activation, respectively. In astrocytes, our findings suggest that PAR1-directed calcium signaling involves a newly appreciated Gq/11-PLCε pathway. In summary, we have identified key molecular determinants for PAR1 interactions with Gq/11, and our findings support a model where Gq/11, Gi/o or G12/13 each bind to distinct sites within the cytoplasmic regions of PAR1.

Copyright information:

© 2012 Elsevier Inc. All rights reserved.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported License (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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