A novel strategy to engineer functional fluorescent inhibitory G-protein alpha subunits.
Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein alpha subunits. We have designed G(i/o)alpha subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-protein-gated inwardly rectifying K(+) channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from G(i/o) alpha subunits (G(i1)alpha, G(i2)alpha, G(i3)alpha, and G(oA)alpha) and a mutation introduced at Cys(-4) rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed G(i/o)alpha protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant G(i/o) alpha subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the Gbetagamma dimer by the mutant Galpha subunit. Moreover, each subunit was able to support A1-mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged G(i3)alpha and G(oA)alpha alpha subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.
Leaney
Joanne L
JL
BHF Laboratories, Room 420, 4th Floor, and Centre for Clinical Pharmacology, Department of Medicine, University College London, 5 University Street, London WC1E 6JJ, United Kingdom.
Benians
Amy
A
Graves
Fiona M
FM
Tinker
Andrew
A
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism; D016366:Open Reading Frames; D019308:Palmitic Acid / Q000378:metabolism; D037342:Pertussis Toxin; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism; D011485:Protein Binding; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D018076:DNA, Complementary; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D010566:Virulence Factors, Bordetella; D019814:Myristic Acid; D005576:Colforsin; D019308:Palmitic Acid; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D011188:Potassium