To identify novel regulators of G␣ o , the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active G␣ o as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an aminoterminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1-and hRGS-G␣-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of G␣ i1-3 , G␣ o , G␣ z , or G␣ q but not G␣ s were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free G␣ i2 and G␣ o under pre-steady-state conditions, and on M2-muscarinic receptor-activated G␣ i1 , G␣ i2 , G␣ i3 , G␣ z , and G␣ in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G z , RGS17 exhibited limited selectivity for G o among G i /G o proteins. All RZ family members reduced dopamine-D2/ G␣ i -mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/G␣ q -mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G i/o , G z , and G q over G s to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors. Regulators of G-protein signaling (RGS)1 proteins accelerate the intrinsic GTPase activity of heterotrimeric G-protein G␣ subunits. All RGS proteins contain a conserved RGS core domain, which is an interaction site for the G␣ subunits (1-3).There are more than 30 human RGS or RGS-like proteins that are classified into six subfamilies based upon their sequence homology and that have conserved functional and targeting domains outside of the RGS domain. For instance, a membrane-targeting domain immediately proximal to the RGS core domain directs small RGS proteins such as RGS1-5 and -16 to the cell membrane (4). Putative nuclear localization signals have been found within (5) and also outside of the RGS core domain (6), and this may direct certain RGS subtypes to the nucleus (7,8). RZ family members, such as RGSZ1 and RGS-G␣-interacting protein (GAIP), contain a cysteine-rich motif that may serve as a palmitoylation site for membrane association (9, 10). A more recent study showed that RZ family members also serve as adapter proteins for G␣ subunit degradation (11). The cysteine-rich motif interacts with the leucinerich region of GAIP-interacting protein N terminus, an E3 ubiquitin ligase responsible for G␣ i3 degradation. Recently RGSZ1 and RGS6 have been shown to associate with SCG-10, a protein involved in neuronal differentiation (12, 13), and the G z GTPase-activating protein (GAP) effect...
Galpha(i)-coupled receptors comprise a diverse family of receptors that induce transformation by largely unknown mechanisms. We previously found that the Galpha(i)-coupled dopamine-D2short (D2S) receptor transforms Balb-D2S cells via Gαi3. To identify new Gαi effectors, a yeast twohybrid screen was done using constitutively active Gαi3-Q204L as bait, and tumor necrosis factoralpha (TNFα)-induced protein 8 (TNFAIP8, SCC-S2/NDED/GG2-1) was identified. In contrast, TNFAIP8-related TIPE1 and TIPE2 showed a very weak interaction with Gαi3. In yeast mating, in vitro pull-down, co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays, TNFAIP8 preferentially interacted with activated Gαi proteins, consistent with direct Gαi-TNFAIP8 coupling. Over-expression or depletion of TNFAIP8 using antisense constructs in Balb-D2S cells did not affect D2S-induced signaling to Gαi-dependent inhibition of cAMP. In contrast, antisense depletion of TNFAIP8 completely inhibited spontaneous and D2S-induced foci formation, consistent with a role for TNFAIP8 in Gαi-dependent transformation. To address possible mechanisms, the effect of D2S signaling via TNFAIP8 on TNFα action was examined. D2S receptor activation inhibited TNFα-induced cell death in Balb-D2S cells, but not in cells depleted of TNFAIP8. However, depletion of TNFAIP8 did not prevent D2S-induced inhibition of TNFα-mediated caspase activation, suggesting that D2S/TNFAIP8-induced protection from TNFα-induced cell death is caspase-independent. The data suggest that Gαi-TNFAIP8-mediated rescue of pre-oncogenic cells enhances progression to oncogenic transformation, providing a selective target to inhibit cellular transformation.Heterotrimeric G proteins (composed of Gα and Gβγ subunits) mediate intracellular signaling of a wide variety of receptors (Bockaert et al., 2002;Fredriksson et al., 2003;Wise et al., 2004), but traditional effectors such as adenylyl cyclase (AC) do not explain all of their actions (Albert and Robillard, 2002;Siderovski and Willard, 2005;Dorsam and Gutkind, 2007;Dave et al., 2009). The "inhibitory" G proteins (Gi1, Gi2, Gi3, and Go) couple to inhibition of AC in nearly all cell types, yet can stimulate cell proliferation and transformation in mesenchymal cell types, but inhibit these processes in neuroendocrine cells (Albert and Robillard, 2002;Dorsam and Gutkind, 2007). In neuroendocrine cells, the Gi/Go-coupled dopamine-D2short (D2S) receptor inhibits AC, phospholipase C (PLC), and mitogen-activated protein kinase (MAPK; ERK1/2), leading to decreased hormone CIHR Author ManuscriptCIHR Author Manuscript CIHR Author Manuscript synthesis, secretion, and reduced cell proliferation (Albert, 1994(Albert, , 2002Albert et al., 1997;Banihashemi and Albert, 2002). Paradoxically, in mesenchymal cells such as Balb/c-3T3 fibroblasts, D2S receptors stimulate calcium mobilization and MAPK activation, leading to increased cell proliferation and transformation (Ghahremani et al., 2000; Albert and Robillard, 2002). These D2S-induced res...
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