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Determinants of physical and functional coupling between Thromboxane A2 receptor and Gαq
Author(s) -
Chakraborty Raja,
Xu Bing,
Bhullar Rajinder Pal,
Chelikani Prashen
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1031.20
Subject(s) - g protein coupled receptor , heterotrimeric g protein , g protein , receptor , chemistry , cysteine , thromboxane receptor , thromboxane a2 , biochemistry , microbiology and biotechnology , biology , enzyme
G protein coupled receptors (GPCRs) interact with heterotrimeric G proteins and initiate a wide variety of signaling pathways. The molecular nature of GPCR‐G protein interaction in the clinically important thromboxane A2 receptor (TP) is poorly understood. TP activates its cognate G‐protein (Gαq) in response to binding of thromboxane and mediates vasoconstriction and thrombosis. Although quite a large number of biophysical and biochemical studies have shown the possible regions and/or residues important in GPCR‐G protein interactions here we attempt to elucidate the determinants of physical and functional coupling between TP and Gαq. We have constructed TP with minimal number of cysteines by replacing 4 of the endogenous and non‐essential cysteines (4‐Cys). From molecular modeling analysis, the TP amino acids predicted to interact with Gαq were replaced with cysteines one at a time, using the 4‐Cys TP as the base receptor. Disulphide cross linking between TP cysteine mutants and C‐terminal Gαq cysteine mutants are being pursued using Copper phenanthroline in presence of TP specific agonist U46619 under reducing and non ‐ reducing conditions. The crosslinked regions will be analyzed by western blots using TP and Gαq specific antibodies. The results will enable us to understand the dynamics of GPCR‐G protein interactions. Supported by MHRC, MICH/faculty of dentistry Fellowship to RC, HSF and MHRC to PC