Reciprocal modulation of alpha(2A)-adrenoceptor and G(alpha o) protein states as determined by carboxy-terminal mutagenesis of a G(alpha o) protein.
The C-terminal portion of G(alpha) proteins plays a key role in their selective activation by cognate receptors. alpha(2A)-Adrenoceptors (alpha(2A)-ARs) can differentially inhibit or stimulate adenylyl cyclases by the activation of distinct G(i/o) and G(s) protein families. The implication of the C-terminal portion of G(alpha o) and G(alphas) proteins in their activation by alpha(2A)-ARs was analyzed by constructing mutant G(alpha o) proteins in which each of the last five amino acid positions were exchanged for those corresponding to a G(alphas) protein. Agonist-dependent, pertussis toxin-resistant binding of guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) revealed that the degree of positive efficacy of clonidine was highly dependent on the presence of a G(alpha o) 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 G(alphas) protein-derived Glu residue at this position. (-)-Epinephrine yielded almost similar maximal [(35)S]GTP gamma S binding responses, but its potency was decreased 22- to 150-fold at the -3 Glu containing mutant G(alpha o) proteins compared with those mutants containing a Gly. A 9- to 39-fold increase in the alpha(2A)-AR agonist equilibrium dissociation constants further reflected changes in the G(alpha) protein-induced alpha(2A)-AR state mediated by the specific Gly to Glu mutation in the C-terminal portion of the G(alpha o) protein. The present data emphasize the unique role of the -3 position at the G(alpha) 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 G(alpha o) protein to the alpha(2A)-AR.
Wurch
T
T
Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, Castres, France.
Okuda
J
J
Pauwels
P J
PJ
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D000316:Adrenergic alpha-Agonists; D018341:Receptors, Adrenergic, alpha-2; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins