Abstract
Fusion proteins allow for the analysis of receptor/G protein coupling under defined conditions. The β2-adrenoceptor (β2AR) fused to the long splice variant of Gsα (GsαL) exhibits a higher apparent constitutive activity than the β2-adrenoceptor fused to the short splice variant of Gsα (GsαS). Experimentally, this results in higher efficacy and potency of partial agonists and in higher efficacy of inverse agonists at the β2AR fused to GsαL relative to the β2AR fused to GsαS, indicating that the agonist-free β2AR and the β2AR occupied by partial agonists promote GDP dissociation from GsαL more efficiently than from GsαS. In fact, the GDP affinity of GsαS fused to the β2AR is higher than the GDP affinity of GsαL fused to the β2AR. We asked the question whether the histamine H2-receptor (H2R) exhibits similar coupling to Gsα splice variants as the β2AR. To address this question, we studied H2R-Gsα fusion proteins expressed in Sf9 cells. In contrast to β2AR-Gsα fusion proteins, the potencies and efficacies of partial agonists and the efficacies of inverse agonists were similar at the H2R fused to GsαL and GsαS as assessed by guanosine-5′-O-(3-thio)triphosphate binding and/or steady-state GTPase activity. However, the time course analysis of guanosine-5′-O-(3-thio)triphosphate binding indicated that GsαS fused to the H2R possesses a higher GDP-affinity than GsαL fused to the H2R. Our data show that the H2R fused to GsαL and GsαS possesses similar constitutive activity and is insensitive to differences in GDP affinity of Gsα splice variants. Thus, GDP affinity of G proteins does not generally determine constitutive activity of receptors.
Footnotes
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↵1 Current address: Quintiles Inc., Kansas City, MO 64134.
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This work was supported by a New Faculty Award of The University of Kansas (R.S.), the National Institutes of Health COBRE award 1 P20 RR15563-01, and matching support from The State of Kansas and The University of Kansas (R.S.), grants of the Fonds der Chemischen Industrie (A.B.), and the Deutscher Akademischer Austauschdienst within the international network “Medicinal Chemistry” (214/IQN-röd) (A.B.).
- Abbreviations:
- GPCR
- G protein-coupled receptor
- Gα
- nonspecified G-protein α-subunit
- Gs proteins
- family of G proteins that mediates adenylyl cyclase activation
- β2AR
- β2-adrenoceptor
- Gsα
- nonspecified Gsα protein
- GsαL
- long splice variant of Gsα
- GsαS
- short splice variant of Gsα
- β2AR-GsαL
- fusion protein containing the β2AR and the long splice variant of Gsα
- β2AR-GsαS
- fusion protein containing the β2AR and the short splice variant of Gsα
- H2R
- histamine H2-receptor
- HIS
- histamine
- BET
- betahistine
- CIM
- cimetidine
- RAN
- ranitidine
- ZOL
- zolantidine
- TIO
- tiotidine
- FAM
- famotidine
- H2R-GsαS
- fusion protein containing the H2R and the short splice variant of Gsα
- H2R-GsαL
- fusion protein containing the H2R and the long splice variant of Gsα
- IMP
- impromidine
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- PCR
- polymerase chain reaction
- PAGE
- polyacrylamide gel electrophoresis
- Received May 30, 2001.
- Accepted September 11, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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