Complexes of the G protein subunit gbeta 5 with the regulators of G protein signaling RGS7 and RGS9. Characterization in native tissues and in transfected cells.
A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G protein pathways through a direct interaction with Galpha subunits and stimulation of GTP hydrolysis. An RGS subfamily including RGS6, -7, -9, and -11, which contain a characteristic Ggamma -like domain, also has the unique ability to interact with the G protein beta subunit Gbeta(5). Here, we examined the behavior of Gbeta(5), RGS7, RGS9, and Galpha in tissue extracts using immunoprecipitation and conventional chromatography. Native Gbeta(5) and RGS7 from brain, as well as photoreceptor-specific Gbeta(5)L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gbeta(5) nor Gbeta(5)-free RGS could be detected. Co-expression in COS-7 cells of Gbeta(5) dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gbeta(5):RGS stoichiometry in a manner similar to Gbetagamma complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysis of native Gbeta(5)-RGS and their coupled expression argue that in vivo, Gbeta(5) and Ggamma-like domain-containing RGSs only exist as heterodimers. Native Gbeta(5)-RGS7 did not co-precipitate or co-purify with Galpha(o) or Galpha(q); nor did Gbeta(5)L-RGS9 with Galpha(t). However, in transfected cells, RGS7 and Gbeta(5)-RGS7 inhibited Galpha(q)-mediated Ca(2+) response to muscarinic M3 receptor activation. Thus, Gbeta(5)-RGS dimers differ from other RGS proteins in that they do not bind to Galpha with high affinity, but they can still inhibit G protein signaling.
Witherow
D S
DS
Department of Molecular and Cellular Pharmacology and the Neuroscience Program, University of Miami School of Medicine, Miami, Florida 33136, USA.
Wang
Q
Q
Levay
K
K
Cabrera
J L
JL
Chen
J
J
Willars
G B
GB
Slepak
V Z
VZ
D000818:Animals; D001921:Brain / Q000378:metabolism*; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D044387:GTP-Binding Protein beta Subunits; D019204:GTP-Binding Proteins / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016210:Methacholine Chloride / Q000494:pharmacology; D011485:Protein Binding; D020710:RGS Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014158:Transcription, Genetic; D014162:Transfection
D044387:GTP-Binding Protein beta Subunits; C490610:Gnb5 protein, rat; D020710:RGS Proteins; D012333:RNA, Messenger; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; C492038:Rgs7 protein, mouse; C115771:Rgs7 protein, rat; C116925:regulator of g-protein signaling 9; D016210:Methacholine Chloride; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins