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.
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
D Scott
DS
Witherow
Vladlen Z
VZ
Slepak
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