Abstract
The C-terminal portion of Gα proteins plays a key role in their selective activation by cognate receptors. α2A-Adrenoceptors (α2A-ARs) can differentially inhibit or stimulate adenylyl cyclases by the activation of distinct Gi/o and Gs protein families. The implication of the C-terminal portion of Gαo and Gαs proteins in their activation by α2A-ARs was analyzed by constructing mutant Gαo proteins in which each of the last five amino acid positions were exchanged for those corresponding to a Gαs protein. Agonist-dependent, pertussis toxin-resistant binding of guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) revealed that the degree of positive efficacy of clonidine was highly dependent on the presence of a Gαo protein-derived Gly amino acid as the −3 residue at the C-terminal portion of the protein. In contrast, antagonist properties for clonidine were observed for those mutants carrying a Gαs protein-derived Glu residue at this position. (−)-Epinephrine yielded almost similar maximal [35S]GTPγS binding responses, but its potency was decreased 22- to 150-fold at the −3 Glu containing mutant Gαo proteins compared with those mutants containing a Gly. A 9- to 39-fold increase in the α2A-AR agonist equilibrium dissociation constants further reflected changes in the Gα protein-induced α2A-AR state mediated by the specific Gly to Glu mutation in the C-terminal portion of the Gαo protein. The present data emphasize the unique role of the −3 position at the Gα protein C-terminal portion, independent of its surrounding peptidic environment, in constraining a structure favorable for activated receptor interaction and transmission of the mutation-induced conformational change from the Gαo protein to the α2A-AR.
Footnotes
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↵1 Present address: Department of Biotechnology, Tokyo University of Agriculture and Technology, 113-0032 Tokyo, Japan.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- α2A-AR
- α2A-adrenoceptor
- wt
- wild-type
- [35S]GTPγS
- guanosine 5′-O-(3-[35S]thio)triphosphate
- PTX
- Bordetella pertussis toxin
- PCR
- polymerase chain reaction
- RX 821002
- 2-(2-methoxy-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-imidazole
- UK 14304
- 5-bromo-6-(2-imidazoline-2-ylamino)quinoxaline tartrate
- Received December 6, 2000.
- Accepted June 11, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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