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CELLULAR AND MOLECULAR

Functional Domains of the Mouse ₃-Adrenoceptor Associated with Differential G Protein Coupling

Department of Pharmacology, Monash University, Victoria, Australia (M.S., D.S.H., T.H., B.A.E., R.J.S.); Department of Physiology, Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, Stockholm, Sweden (D.S.H., T.B.); and Department of Neurochemistry and Neurotoxicology, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden (A.F., U.L.)

Alternative splicing of mouse ₃-adrenoceptor transcripts produces an additional receptor isoform (_3b-adrenoceptor) with a C terminus comprising 17 amino acids distinct from the 13 in the known receptor (_3a-adrenoceptor). We have shown that the _3b-adrenoceptor couples to both G_s and G_i, whereas the _3a-adrenoceptor couples only to G_s. To define the regions involved in this differential G protein coupling, we have compared wild-type, truncated, and mutant ₃-adrenoceptors. In Chinese hamster ovary cells expressing ₃-adrenoceptors truncated at the splicing point, cAMP accumulation with CL316243 [GenBank] [(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate] increased by 59% following pretreatment with pertussis toxin, suggesting that the C-terminal region of the _3a-adrenoceptor inhibits coupling to G_i. We next utilized the cell-penetrating peptide Transportan 10 (Tp10) to introduce peptides comprising the different C-terminal tail fragments into cells expressing _3a-adrenoceptor, _3b-adrenoceptor, and the truncated ₃-adrenoceptor. Treatment with _3a-Tp10 (1 µM) caused cAMP responses to CL316243 [GenBank] in the _3a-adrenoceptor to become pertussis toxin-sensitive and display a 30% increase over control, whereas the other peptides did not affect any receptor. Mutation at a potential tyrosine phosphorylation site (Tyr392Ala _3a-adrenoceptor) did not alter responses or pertussis toxin sensitivity relative to the parent receptor. Surprisingly, a Ser388Ala/Ser389Ala mutant _3b-adrenoceptor became unresponsive to CL316243 [GenBank] while retaining an extracellular acidification rate response to SR59230A [3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate]. Our findings suggest that the _3a-adrenoceptor cannot couple to G_i because of conformational changes induced by a protein(s) that interacts with residues in the C-terminal tail or because this protein(s) affects the intracellular localization of the _3a-adrenoceptor.

Address correspondence to: Roger J. Summers, Department of Pharmacology, P.O. Box 13E, Monash University, Victoria 3800, Australia. E-mail: roger.summers{at}med.monash.edu.au

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