Biochemical purification and functional analysis of complexes between the G-protein subunit Gbeta5 and RGS proteins.
Regulator of G-protein signaling (RGS) proteins of the R7 subfamily (RGS6, 7, 9, and 11) contain a unique Ggamma-like (GGL) domain that enables their association with the G-protein beta subunit Gbeta5. The existence of these complexes was demonstrated by their purification from native tissues as well as by reconstitution in vitro. According to pulse-chase analysis, Gbeta5 and RGS7 monomers undergo rapid proteolytic degradation in cells, whereas the dimer is stable. Studies of the functional role of Gbeta5-RGS dimers using GTPase activity, ion channel, and calcium mobilization assays showed that, similarly to other RGS proteins, they can negatively regulate G-protein-mediated signal transduction. Protein-protein interactions involving the Gbeta5-RGS7 complex can be studied in cells using fluorescence resonance energy transfer utilizing Gbeta5, RGS, and Galpha subunits fused to the cyan and yellow versions of green fluorescent protein.
Witherow
D Scott
DS
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
Slepak
Vladlen Z
VZ
D000818:Animals; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020710:RGS Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000737:chemistry; D015290:Second Messenger Systems / Q000502:physiology
D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; C490610:Gnb5 protein, rat; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins