title: Co-expression of the beta2-adrenoceptor and dopamine D1-receptor with Gsalpha proteins in Sf9 insect cells: limitations in comparison with fusion proteins.
abstract: The G-protein G(salpha) exists in three isoforms, the G(salpha) splice variants G(salphashort) (G(salphaS)) and G(salphalong) (G(salphaL)), and the G-protein G(alphaolf) that is not only involved in olfactory signaling but also in extrapyramidal motor regulation. Studies with beta(2)-adrenoceptor (beta(2)AR)-G(salpha) fusion proteins showed that G(salpha) proteins activate adenylyl cyclase (AC) in the order of efficacy G(salphaS)>G(salphaL) approximately G(alphaolf) and that G(salpha) proteins confer the hallmarks of constitutive activity to the beta(2)AR in the order of efficacy G(salphaL)>G(alphaolf)>G(salphaS). However, it is unclear whether such differences between G(salpha) proteins also exist in the nonfused state. In the present study, we co-expressed the beta(2)AR and dopamine D(1)-receptor (D(1)R) with G(salpha) proteins at different ratios in Sf9 insect cells. In agreement with the fusion protein studies, nonfused G(alphaolf) was less efficient than nonfused G(salphaS) and G(salphaL) at activating AC, but otherwise, we did not observe differences between the three G(salpha) isoforms. Thus, it is much easier to dissect differences between G(salpha) isoforms using beta(2)AR-G(salpha) fusion proteins than nonfused G(salpha) isoforms.
authors.affiliation: Department of Pharmacology and Toxicology, The University of Kansas, 1251 Wescoe Hall Drive, Malott Hall, Room 5064, Lawrence, KS 66045-7582, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gille
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001552:Benzazepines / Q000493:pharmacokinetics; D001665:Binding Sites; D002460:Cell Line; D004082:Dihydroalprenolol / Q000493:pharmacokinetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D017447:Receptors, Dopamine D1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
chemical_list: D001552:Benzazepines; D018343:Receptors, Adrenergic, beta-2; D017447:Receptors, Dopamine D1; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004082:Dihydroalprenolol; D020558:GTP Phosphohydrolases; D019205:GTP-Binding Protein alpha Subunits, Gs