Activation-Induced Subcellular Redistribution of Gαs Is Dependent upon Its Unique N-Terminus

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Abstract
The heterotrimeric G protein subunit, αs, can move reversibly from plasma membranes to cytoplasm in response to activation by GPCRs or activating mutations. We examined the importance of the unique N-terminus of αs in this translocation in cultured cells. αs contains a single site for palmitoylation in its N-terminus, and this was replaced by different plasma membrane targeting motifs. These N-terminal αs mutants were targeted properly to plasma membranes, capable of coupling activated GPCRs to effectors, and able to constitutively stimulate cAMP production when they also contained an activating mutation. However, when activated by a constitutively activating mutation or by agonist-activated β-AR, these N-terminal αs mutants failed, for the most part, to undergo redistribution from plasma membranes to cytoplasm, as assayed by immunofluorescence microscopy, or from a particulate to soluble fraction, as assayed by subcellular fractionation. These results highlight the importance of the extreme N-terminus of αs and its single site of palmitoylation for facilitating activation-induced translocation and provide insight into the mechanism of this G protein trafficking event.