Distinct roles for Gα(i)2, Gα(i)3, and Gβγ in modulation of forskolin- or G(s)-mediated cAMP accumulation and calcium mobilization by dopamine D2S receptors

Abstract

Previous studies have shown that a single G protein-coupled receptor can regulate different effector systems by signaling through multiple subtypes of heterotrimeric G proteins. In LD2S fibroblast cells, the dopamine D2S receptor couples to pertussis toxin (PTX)-sensitive G(i)/G(o) proteins to inhibit forskolin- or prostaglandin E1-stimulated cAMP production and to stimulate calcium mobilization. To analyze the role of distinct Gα(i/o) protein subtypes, LD2S cells were stably transfected with a series of PTX- insensitive Gα(i/o) protein Cys → Ser point mutants and assayed for D2S receptor signaling after PTX treatment. The level of expression of the transfected Gα mutant subunits was similar to the endogenous level of the most abundant Gα(i/o) proteins (Gα(o), Gα(i)3). D2S receptor-mediated inhibition of forskolin-stimulated cAMP production was retained only in clones expressing mutant Gα(i)2. In contrast, the D2S receptor utilized Gα(i)3 to inhibit PGE1-induced (G(s)-coupled) enhancement of cAMP production. Following stable or transient transfection, no single or pair set of mutant Gα(i/o) subtypes rescued the D2S-mediated calcium response following PTX pretreatment. On the other hand, in LD2S cells stably transfected with GRK-CT, a receptor kinase fragment that specifically antagonizes Gβγ, subunit activity, D2S receptor-mediated calcium mobilization was blocked. The observed specificity of Gα(i)2 and Gα(i)3 for different states of adenylyl cyclase activation suggests a higher level of specificity for interaction of Gα(i) subunits with forskolin- versus G(s)- activated states of adenylyl cyclase than has been previously appreciated.