RGS proteins comprise a family of
proteins named for their ability to negatively regulate
heterotrimeric G protein signaling. Biochemical studies suggest that members of this
protein family act as
GTPase-activating proteins for certain Galpha subunits, thereby accelerating the turn-off mechanism of Galpha and terminating signaling by both Galpha and Gbetagamma subunits. In the present study, we used confocal microscopy to examine the intracellular distribution of several
RGS proteins in COS-7 cells expressing RGS-
green fluorescent protein (GFP) fusion
proteins and in cells expressing
RGS proteins endogenously. RGS2 and RGS10 accumulated in the nucleus of COS-7 cells transfected with GFP constructs of these
proteins. In contrast, RGS4 and RGS16 accumulated in the cytoplasm of COS-7 transfectants. As observed in COS-7 cells, RGS4 exhibited cytoplasmic localization in mouse
neuroblastoma cells, and RGS10 exhibited nuclear localization in human
glioma cells. Deletion or
alanine substitution of an N-terminal
leucine repeat motif present in both RGS4 and RGS16, a domain identified as a nuclear export sequence in HIV Rev and other
proteins, promoted nuclear localization of these
proteins in COS-7 cells. In agreement with this observation, treatment of mouse
neuroblastoma cells with
leptomycin B to inhibit
nuclear protein export by exportin1 resulted in accumulation of RGS4 in the nucleus of these cells. GFP fusions of RGS domains of
RGS proteins localized in the nucleus, suggesting that nuclear localization of
RGS proteins results from nuclear targeting via RGS domain sequences. RGSZ, which shares with RGS-GAIP a
cysteine-rich string in its N-terminal region, localized to the Golgi complex in COS-7 cells. Deletion of the N-terminal domain of RGSZ that includes the
cysteine motif promoted nuclear localization of RGSZ. None of the
RGS proteins examined were localized at the plasma membrane. These results demonstrate that
RGS proteins localize in the nucleus, the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle
proteins.
RGS proteins localize differentially within cells as a result of structural differences among these
proteins that do not appear to be important determinants for their
G protein-regulating activities. These findings suggest involvement of
RGS proteins in more complex cellular functions than currently envisioned.