FIGURE 5.
The L148S mutation does not abolish G-protein binding, and overexpression of G-proteins partially rescues the functional deficits of IL2-10 mutants. A, in E. coli, both WT and L148S mutant GST-hGPR54 IL2 fusion proteins co-purify with Gαq. GST alone does not co-purify with Gαq. B, co-immunoprecipitation experiments reveal that both TAP-WT hGPR54 and TAP-L148S hGPR54 proteins pull down Gαq and Gα15/16. C, overexpression of Gα15/16 DNA in L148S hGPR54-GFP cells leads to a concentration-dependent rescue of functional coupling, as measured by PI hydrolysis. L148S hGPR54 results (C) are normalized to the WT response to 1 μm KP-10, which was set at 100%. Inset, Western blot shows that increasing the amount of Gα15/16 DNA transfected increases the relative levels of Gα15/16 protein in L148S hGPR54-GFP cells. D, co-overexpression of L132S α1A-AR and Gαq modestly rescues the deficit in PI hydrolysis. The results presented in A and B are representative of three experiments, and C and D show the means ± S.E. of three experiments performed in triplicate.