The principles by which components in the G-protein cycle (Gα, Gβλ and regulators of G-protein signalling) reach the plasma membrane (PM) is only partially understood.
DNA constructs fused in frame to fluorescent proteins (cyan (CFP), green (GFP) and yellow (YFP) fluorescent protein) were generated (Goα-CFP/GFP/YFP, Giα3-CFP, Gβ1-YFP, Gλ2-CFP, RGS8-YFP and GAIP-YFP) using vectors pECFP-N1, pEGFP-N1 and pEYFP-N1 (Clontech). In addition, the first 35 amino acids of RGS8-YFP were deleted (ΔN-RGS8) or fused in frame with YFP (Nonly-RGS8-YFP). These constructs were transfected into HEK293 cells and their subcellular localisation was studied using confocal microscopy.
G-protein α subunits with a dual palmitoylation or a palmitoylation/myristoylation signal trafficked independently to the PM. In contrast, localisation of Gβ1-YFP and Gλ2-CFP to the PM required coexpression of Goα and Gλ2 or Gβ1, respectively. RGS8-YFP distribution was mainly to the nucleus and cytoplasm while that of GAIP-YFP was largely to the cytosol. Nonly-RGS8-YFP expressing cells showed largely nuclear staining whereas the ΔNRGS8-YFP mutant was diffusely distributed through the cell. Cotransfection of Goα, Goα-CFP and Giα3-CFP with RGS8-YFP and GAIP-YFP resulted in significant translocation of RGS to the plasma membrane. Colocalisation analysis was performed and produced the following values (colocalisation coefficients expressed as means ± S.E.M.) for RGS8-YFP: RGS8-YFP in Goα-CFP, 0.599 ± 0.04 (n = 17); RGS8-YFP in Mem-CFP (a membrane targeted CFP used as a control), 0.102 ± 0.024 (n = 16, Student’s unpaired t test, P < 0.0001). ΔNRGS8-YFP and Nonly-RGS8-YFP did not localise to the PM with coexpression of either Goα-CFP or Goα. Colocalisation analysis was performed for GAIP-YFP: GAIP-YFP in Giα3-CFP, 0.605 ± 0.065 (n = 11, P < 0.001 vs. Mem-CFP, one-way ANOVA); GAIP-YFP in Goα-CFP, 0.553 ± 0.038 (n = 19, P < 0.001 vs. Mem-CFP); and GAIP-YFP in Mem-CFP, 0.228 ± 0.031 (n = 16). Finally, cotransfection of Go α-CFP with a point mutation (G184Œ{special}S) that interferes with RGS binding and action also prevents translocation of RGS8-YFP to the PM.
We conclude that the Gα subunit has a central role in translocation of the other components involved in the G-protein cycle to the PM. The N-terminus of RGS8 is necessary but not sufficient for PM localisation after coexpression with Gα.
This work was sponsored by the BHF and Wellcome Trust.