The olfactory G protein G(alphaolf) possesses a lower GDP-affinity and deactivates more rapidly than G(salphashort): consequences for receptor-coupling and adenylyl cyclase activation.
The olfactory G protein G(alphaolf) differs from the short splice variant of G(salpha) (G(salphaS)) in 80 amino acids, but little is known about biochemical differences between G(alphaolf) and G(salphaS). We addressed this question by analyzing fusion proteins of the beta2-adrenoceptor (beta2AR) and G(alphaolf) and G(salphaS), respectively, using Sf9 insect cells as expression system. The fusion ensured defined receptor/G protein stoichiometry and efficient coupling. High-affinity agonist binding studies showed that G(alphaolf) possesses a lower GDP-affinity than G(salphaS) As a result, the agonist-free beta2AR and the beta2AR occupied by partial agonists were more efficient at promoting GDP-dissociation from G(alphaolf) than from G(salphaS) a assessed by guanosine 5'-O-(3-thiotriphosphate) binding, adenylyl cyclase (AC) activity and GTP hydrolysis. Basal AC activity in the absence of GTP was almost sixfold lower in membranes expressing beta2AR-G(alphaolf) than in membranes expressing beta2AR-G(salphaS) at similar levels, reflecting the lower abundance of G(alphaolf-GDP) relative to G(salphaS-GDP). The maximum agonist-stimulated AC activity with beta2AR-G(salphaS) was more than twofold higher than with beta2AR-G(alphaolf), but the relative agonist-stimulation of AC with beta2AR-G(alphaolf) was much greater than with beta2AR-G(salphaS). The difference in maximum AC activity can be explained by more rapid deactivation of G(alphaolf-GTP) by GTP hydrolysis and GTP dissociation relative to G(salphaS-GTP). Taken together, there are biochemical differences between G(alphaolf) and G(salphaS), supporting different roles of these G proteins in vivo.
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Department of Pharmacology and Toxicology, The University of Kansas, Lawrence, Kansas, USA.
Wenzel-Seifert
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Seifert
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D000262:Adenylyl Cyclases / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D004082:Dihydroalprenolol / Q000493:pharmacokinetics; D004280:Dobutamine / Q000494:pharmacology; D004809:Ephedrine / Q000494:pharmacology; D044385:GTP-Binding Protein alpha Subunits; D014644:Genetic Variation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007545:Isoproterenol / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D000318:Adrenergic beta-Agonists; D044385:GTP-Binding Protein alpha Subunits; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C084618:olfactory G protein subunit alpha olf; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004280:Dobutamine; D004082:Dihydroalprenolol; D006160:Guanosine Triphosphate; D020962:Heterotrimeric GTP-Binding Proteins; D000262:Adenylyl Cyclases; D004809:Ephedrine; D007545:Isoproterenol; C084581:dichloroisoproterenol