RT Journal Article SR Electronic T1 Functional Domains of the Mouse β3-Adrenoceptor Associated with Differential G Protein Coupling JF Journal of Pharmacology and Experimental Therapeutics JO J Pharmacol Exp Ther FD American Society for Pharmacology and Experimental Therapeutics SP 1354 OP 1361 DO 10.1124/jpet.105.091736 VO 315 IS 3 A1 Masaaki Sato A1 Dana S. Hutchinson A1 Tore Bengtsson A1 Anders Floren A1 Ülo Langel A1 Takahiro Horinouchi A1 Bronwyn A. Evans A1 Roger J. Summers YR 2005 UL http://jpet.aspetjournals.org/content/315/3/1354.abstract AB Alternative splicing of mouse β3-adrenoceptor transcripts produces an additional receptor isoform (β3b-adrenoceptor) with a C terminus comprising 17 amino acids distinct from the 13 in the known receptor (β3a-adrenoceptor). We have shown that the β3b-adrenoceptor couples to both Gs and Gi, whereas the β3a-adrenoceptor couples only to Gs. To define the regions involved in this differential G protein coupling, we have compared wild-type, truncated, and mutant β3-adrenoceptors. In Chinese hamster ovary cells expressing β3-adrenoceptors truncated at the splicing point, cAMP accumulation with CL316243 [(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 β3a-adrenoceptor inhibits coupling to Gi. We next utilized the cell-penetrating peptide Transportan 10 (Tp10) to introduce peptides comprising the different C-terminal tail fragments into cells expressing β3a-adrenoceptor, β3b-adrenoceptor, and the truncated β3-adrenoceptor. Treatment with β3a-Tp10 (1 μM) caused cAMP responses to CL316243 in the β3a-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 β3a-adrenoceptor) did not alter responses or pertussis toxin sensitivity relative to the parent receptor. Surprisingly, a Ser388Ala/Ser389Ala mutant β3b-adrenoceptor became unresponsive to CL316243 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 β3a-adrenoceptor cannot couple to Gi 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 β3a-adrenoceptor. The American Society for Pharmacology and Experimental Therapeutics