Investigation of the role of the carboxyl terminal tails of the alpha and beta isoforms of the human thromboxane A2 receptor (TP) in mediating receptor : effector coupling
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|Title:||Investigation of the role of the carboxyl terminal tails of the alpha and beta isoforms of the human thromboxane A2 receptor (TP) in mediating receptor : effector coupling||Authors:||Walsh, Marie-Therese
Foley, John F.
Kinsella, B. Therese
|Permanent link:||http://hdl.handle.net/10197/3168||Date:||17-Apr-2000||Abstract:||We have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A2 receptor (TP) to Galpha16 and Galpha12 members of the Gq and G12 families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha10 or HEK.beta3, stably over-expressing TPalpha and TPbeta, respectively. Moreover, using HEK.TP-328 cells which over-expresses a variant of TP truncated at the point of divergence of TPalpha and TPbeta we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPalpha and TPbeta couple similarly to Galpha16 to affect increases in IP3 and mobilization of intracellular calcium ([Ca2+]i) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca2+]i mobilization in cells co-transfected with Galpha12, neither receptor generated corresponding increases in IP3 indicating that the Galpha12 mediated increases in [Ca2+]i do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca2+ channels reduced [Ca2+]i mobilization in TPalpha and TPbeta cells co-transfected with Galpha12 to approximately 40% of that mobilized in its absence whereas 3,4,5-trimethyloxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), an antagonist of intracellular Ca2+ release, had no effect on [Ca2+]i mobilization by either receptor isoform co-transfected with Galpha12. Despite the lack of differential coupling specificity by TPalpha and TPbeta, TP-328 signaled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Galpha11, Galpha12 or Galpha16 subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPalpha but not TPbeta, TP-328 coupled to GalphaS, leading to increased cAMP, rather than to Galphai. Whereas TP-328 signaled more efficiently in the absence of co-transfected G protein compared to the wild type TPalpha co-transfection of Galphas did not augment cAMP generation by TP-328. Hence, from these studies involving the wild type TPalpha, TPbeta and TP-328, we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Galpha11 and Galpha16 members of the Gq family or to Galpha12; it may play a role in determining GS versus Gi coupling and may act as a determinant of coupling efficiency.||Funding Details:||Health Research Board||Type of material:||Journal Article||Publisher:||Elsevier||Copyright (published version):||2000 Elsevier Science B.V.||Keywords:||Thromboxane A2 receptor;G12;G16;Gs;Signalling||Subject LCSH:||Thromboxanes
|DOI:||10.1016/S0167-4889(00)00031-8||Language:||en||Status of Item:||Peer reviewed|
|Appears in Collections:||Conway Institute Research Collection|
Biomolecular and Biomedical Science Research Collection
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