Activation-induced subcellular redistribution of G alpha(s) is dependent upon its unique N-terminus.
The heterotrimeric G protein subunit, alpha(s), 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 alpha(s) in this translocation in cultured cells. alpha(s) contains a single site for palmitoylation in its N-terminus, and this was replaced by different plasma membrane targeting motifs. These N-terminal alpha(s) 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 beta-AR, these N-terminal alpha(s) 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 alpha(s) and its single site of palmitoylation for facilitating activation-induced translocation and provide insight into the mechanism of this G protein trafficking event.
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Manimekalai M
MM
Thiyagarajan
Eve
E
Bigras
Hubert H M
HH
Van Tol
Terence E
TE
Hébert
Daniel S
DS
Evanko
Philip B
PB
Wedegaertner
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane; D000242:Cyclic AMP / Q000096:biosynthesis; D017931:DNA Primers; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016296:Mutagenesis; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism*; D014981:Xenopus
D017931:DNA Primers; D000242:Cyclic AMP; D020962:Heterotrimeric GTP-Binding Proteins