Visualization of a functional Galpha q-green fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not be activation mediated by receptors or AlF4-.
To investigate how G protein alpha subunit localization is regulated under basal and activated conditions, we inserted green fluorescent protein (GFP) into an internal loop of Galpha(q). alpha(q)-GFP stimulates phospholipase C in response to activated receptors and inhibits betagamma-dependent activation of basal G protein-gated inwardly rectifying K(+) currents as effectively as alpha(q) does. Association of alpha(q)-GFP with the plasma membrane is reduced by mutational activation and eliminated by mutation of the alpha(q) palmitoylation sites, suggesting that alpha(q) must be in the inactive, palmitoylated state to be targeted to this location. We tested the effects of activation by receptors and by AlF(4)(-) on the localization of alpha(q)-GFP in cells expressing both alpha(q)-GFP and a protein kinase Cgamma-red fluorescent protein fusion that translocates to the plasma membrane in response to activation of G(q). In cells that clearly exhibit protein kinase Cgamma-red fluorescent protein translocation responses, relocalization of alpha(q)-GFP is not observed. Thus, under conditions associated with palmitate turnover and betagamma dissociation, alpha(q)-GFP remains associated with the plasma membrane. These results suggest that upon reaching the plasma membrane alpha(q) receives an anchoring signal in addition to palmitoylation and association with betagamma, or that during activation, one or both of these factors continues to retain alpha(q) in this location.
Hughes
T E
TE
Department of Ophthalmology Yale University School of Medicine, New Haven, Connecticut 06520-8026, USA.
Zhang
H
H
Logothetis
D E
DE
Berlot
C H
CH
D017607:Aluminum Compounds / Q000378:metabolism*; D002462:Cell Membrane; D005459:Fluorides / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D008958:Models, Molecular; D016296:Mutagenesis; D019308:Palmitic Acid / Q000378:metabolism*; D011487:Protein Conformation
D017607:Aluminum Compounds; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D005459:Fluorides; C032311:aluminum fluoride