Abstract
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.
Highlights
The use of the green fluorescent protein (GFP)1 from the jellyfish Aequoria victoria has had an enormous impact on cell biology [1,2,3,4,5]
Palmitoylation and myristoylation consensus sites were removed from Gi/o ␣ subunits (Gi1␣, Gi2␣, Gi3␣, and GoA␣) and a mutation introduced at Cys؊4 rendering the subunit resistant to pertussis toxin
Cloning techniques have enabled GFP to be fused with a protein of interest, and localization of such fusion proteins can be studied in a dynamic fashion in living cells using fluorescence microscopy
Summary
The use of the green fluorescent protein (GFP)1 from the jellyfish Aequoria victoria has had an enormous impact on cell biology [1,2,3,4,5]. Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein ␣ subunits. We further investigated the membrane localization of these proteins and by performing cell fractionation studies from monoclonal isolates of HEK293 cells expressing Gi3␣-CFP and GoA␣-
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