FIGURE 2.

R7BP or GPR158 cause redistribution of RGS7 from cytoplasmic granules to plasma membrane. HEK293T cells were co-transfected with taggedRGS7 and Gβ₅, with or without FLAG-R7BP (A and B) or GPR158-Myc (A and C) plasmids. A, YFP-RGS7 was immunoprecipitated from the cell lysate, and the eluates were probed for RGS7, Gβ₅, and R7BP. B, immunoprecipitated YFP-RGS7 complex was probed for RGS7, Gβ₅, and GPR158. C, cells expressing YFP-RGS7 and Gβ₅ (top row), or these proteins together with FLAG-R7BP (middle row), or GPR158-Myc (bottom row) were analyzed by confocal microscopy. YFP-RGS7 was detected by epifluorescence, and R7BP or GPR158 were detected using antibodies against FLAG or Myc tag, respectively. D, quantification of the result in C. Regions of interest corresponding to plasma membrane (cell periphery) and cytosol were selected within a cell. Fluorescence intensities within these regions were measured using Leica LAS AF Lite image analysis software. The data show the means ± S.D. of the plasma membrane to cytosol ratio of the YFP-RGS7 fluorescence. ***, p <0.001. For every condition, we analyzed 20–25 cells in each of three independent transfection experiments. The ratio of 1:1 in the absence of R7BP and GPR158 represents the fluorescence distribution for cytosol-localized proteins.