Distinct Roles for Gαi2, Gαi3, and Gβγ in Modulation of Forskolin- or Gs-mediated cAMP Accumulation and Calcium Mobilization by Dopamine D2S Receptors

Mohammad H Ghahremani,Peihua Cheng,Paola M.C Lembo,Paul R Albert

doi:10.1074/jbc.274.14.9238

Mohammad H Ghahremani, Peihua Cheng + Show 2 more

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https://doi.org/10.1074/jbc.274.14.9238

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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 Gi/Go proteins to inhibit forskolin- or prostaglandin E1-stimulated cAMP production and to stimulate calcium mobilization. To analyze the role of distinct Galphai/o protein subtypes, LD2S cells were stably transfected with a series of PTX-insensitive Galphai/o protein Cys --> Ser point mutants and assayed for D2S receptor signaling after PTX treatment. The level of expression of the transfected Galpha mutant subunits was similar to the endogenous level of the most abundant Galphai/o proteins (Galphao, Galphai3). D2S receptor-mediated inhibition of forskolin-stimulated cAMP production was retained only in clones expressing mutant Galphai2. In contrast, the D2S receptor utilized Galphai3 to inhibit PGE1-induced (Gs-coupled) enhancement of cAMP production. Following stable or transient transfection, no single or pair set of mutant Galphai/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 Gbeta gamma subunit activity, D2S receptor-mediated calcium mobilization was blocked. The observed specificity of Galphai2 and Galphai3 for different states of adenylyl cyclase activation suggests a higher level of specificity for interaction of Galphai subunits with forskolin- versus Gs-activated states of adenylyl cyclase than has been previously appreciated.

Highlights

A wide variety of physiological functions and pathological conditions are regulated by hormones and neurotransmitters which transduce intracellular signals by coupling to heterotrimeric guanine nucleotide-binding proteins (G proteins).1 Upon receptor activation, G proteins dissociate into G␣ and G␤␥
Expression of Mutant G␣i/o Subtypes in LD2S Cells—In order to investigate the importance of individual G␣ subtypes in dopamine-mediated responses, pertussis toxin (PTX)-insensitive mutants of G␣i/o were generated and stably transfected into LD2S cells (LtkϪ cells stably transfected with the rat D2S receptor cDNA)
Dopamine (10 ␮M) inhibited forskolin-stimulated cAMP accumulation in these cells by 84.5 Ϯ 12.2% (n ϭ 5), an action that was mimicked by apomorphine (1 ␮M, not shown) and was largely reversed by pretreatment with PTX (Fig. 2A), indicating the involvement of Gi/o proteins

Summary

Introduction

A wide variety of physiological functions and pathological conditions are regulated by hormones and neurotransmitters which transduce intracellular signals by coupling to heterotrimeric guanine nucleotide-binding proteins (G proteins). Upon receptor activation, G proteins dissociate into G␣ and G␤␥. By contrast, when expressed in cells of mesenchymal lineage (e.g. LtkϪ fibroblast or Chinese hamster ovary cells), the same receptor mediates stimulation of phospholipase C activity to induce calcium mobilization, and activation of the mitogen-activated protein kinase cascade leading to enhanced gene transcription and cell proliferation (8, 14, 17, 19 –23). These findings suggest that the same receptor mediates different cellular responses depending on the repertoire of cell-specific effectors that are expressed. These results indicate a strong dependence on G␣i subtype for D2S-mediated inhibition of AC that is not observed for stimulation of calcium mobilization

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