title: Functional domains of the mouse beta3-adrenoceptor associated with differential G protein coupling.
abstract: Alternative splicing of mouse beta3-adrenoceptor transcripts produces an additional receptor isoform (beta3b-adrenoceptor) with a C terminus comprising 17 amino acids distinct from the 13 in the known receptor (beta3a-adrenoceptor). We have shown that the beta3b-adrenoceptor couples to both Gs and Gi, whereas the beta3a-adrenoceptor couples only to Gs. To define the regions involved in this differential G protein coupling, we have compared wild-type, truncated, and mutant beta3-adrenoceptors. In Chinese hamster ovary cells expressing beta3-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 beta3a-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 beta3a-adrenoceptor, beta3b-adrenoceptor, and the truncated beta3-adrenoceptor. Treatment with beta3a-Tp10 (1 microM) caused cAMP responses to CL316243 in the beta3a-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 beta3a-adrenoceptor) did not alter responses or pertussis toxin sensitivity relative to the parent receptor. Surprisingly, a Ser388Ala/Ser389Ala mutant beta3b-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 beta3a-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 beta3a-adrenoceptor.
authors.affiliation: Department of Pharmacology, P.O. Box 13E, Monash University, Victoria 3800, Australia.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Sato
authors.forename: Dana S
authors.initials: DS
authors.lastname: Hutchinson
authors.forename: Tore
authors.initials: T
authors.lastname: Bengtsson
authors.forename: Anders
authors.initials: A
authors.lastname: Floren
authors.forename: Ulo
authors.initials: U
authors.lastname: Langel
authors.forename: Takahiro
authors.initials: T
authors.lastname: Horinouchi
authors.forename: Bronwyn A
authors.initials: BA
authors.lastname: Evans
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Summers
mesh_terms: D058667:Adrenergic beta-3 Receptor Agonists; D058673:Adrenergic beta-3 Receptor Antagonists; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D004149:Dioxoles / Q000494:pharmacology; D004337:Drug Carriers / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D051379:Mice; D008956:Models, Chemical; D009154:Mutation; D037342:Pertussis Toxin / Q000494:pharmacology; D011412:Propanolamines / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000494:pharmacology; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D022702:Receptors, Adrenergic, beta-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C097869:3-(2-ethylphenoxy)-1-(1,2,3,4-tetrahydronaphth-1-ylamino)-2-propanol oxalate; D058667:Adrenergic beta-3 Receptor Agonists; D058673:Adrenergic beta-3 Receptor Antagonists; D000319:Adrenergic beta-Antagonists; D004149:Dioxoles; D004337:Drug Carriers; D011412:Propanolamines; D020033:Protein Isoforms; D022702:Receptors, Adrenergic, beta-3; C076126:disodium (R,R)-5-(2-((2-(3-chlorophenyl)-2-hydroxyethyl)-amino)propyl)-1,3-benzodioxole-2,3-dicarboxylate; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs