| Regulators of G-protein Signaling (RGSs) are proteins which attenuate G-Protein coupled receptor (GPCR) signaling by acting as GAPs (GTPase activating proteins) for the Galpha subunit of the heterotrimeric G protein. Although RGSs have been clearly shown to bind the Galpha subunit of heterotrimeric G-proteins, recent studies have shown that RGS specificity occurs via receptor association. To examine possible RGS/GPCR interactions, we constructed a somatostatin 5 (SST5) receptor deletion mutant lacking most of its intracellular C-tail. Here we show that the activation of a GPCR-responsive FUS1 -LacZ reporter gene in yeast strains expressing full length WT SST5 receptor as well as the C-terminally truncated mutant were both inhibited by RGSs 1, 2, 5 and 16, suggesting that the C-tail does not play an integral role in RGS function. As an alternative approach to examine possible RGS/GPCR interactions, RGS function was analyzed via halo assay in yeast cells expressing different RGSs as well the C-tail of different GPCRs including mouse LPA 1, LPA4, Cbeta1, 5HT2A, beta 2AR and human SST5 receptors as well as the third intracellular loop of human SST5. The C-tails and the i3-loop were constructed as GFP fusions. Western blot analysis confirmed that the fusions were expressed in yeast. Of all the combinations of GPCR-C-tail-GFP fusions and RGSs expressed in yeast, only LPA4-GFP was able to interfere with RGS2 function. RGS function was also not inhibited by the expression of SST5-i3-GFP. This suggests that there is a high degree of specificity involved in dictating the interaction between RGSs and GPCRs. In a second study, we wanted to further characterize an immunoreactive RGS5 protein band which was detected from western blot analysis of extract from a yeast strain expressing RGS5 and that was double the size of RGS5. To examine the possibility that this band represents an RGS5 dimer, we examined the molecular weight of RGS5 protein in yeast cells expressing an RGS5-GFP fusion. Western blot analysis of yeast extract expressing GFP-tagged RGS5 detected a band at approximately 50 kDa (representing RGS5-GFP) and a second band at 100 kDa. This suggests that RGS5, like GPCRs, are capable of forming dimers. |
