Abundance and stability of complexes containing inactive G protein-coupled receptors and G proteins.
G protein-coupled receptors (GPCRs) interact directly with heterotrimeric G proteins to transduce physiological signals. Early studies of this interaction concluded that GPCRs (R) and G proteins (G) collide with each other randomly after receptor activation and that R-G complexes are transient. More recent studies have suggested that inactive R and G are preassembled (precoupled) as stable R-G complexes. Here we examine the stability of complexes formed between cyan fluorescent protein-labeled alpha(2A)-adrenoreceptors (C-alpha2ARs) and G proteins in cells using fluorescence recovery after photobleaching (FRAP). Labeled G proteins diffused in the plasma membrane with equal mobility in the absence and presence of immobile C-alpha2ARs. Immobile C-alpha2ARs activated labeled G proteins, demonstrating functional coupling without stable physical association. In contrast, a stable R-G interaction was detected when G proteins were deprived of nucleotides and C-alpha2ARs were active, as predicted by the ternary complex model. Overexpression of regulator of G protein signaling 4 (RGS4) accelerated the onset of effector activation but did not detectably alter the interaction between C-alpha2ARs and G proteins. We conclude that at most a small fraction of C-alpha2ARs and G proteins exist as R-G complexes at any moment.
Qin
Kou
K
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30809, USA.
Sethi
Pooja R
PR
Lambert
Nevin A
NA
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004355:Drug Stability; D036681:Fluorescence Recovery After Photobleaching; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D046912:Multiprotein Complexes; D020710:RGS Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
C547768:ADRA2A protein, human; C485182:Cyan Fluorescent Protein; D046912:Multiprotein Complexes; D020710:RGS Proteins; D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C098799:RGS4 protein; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins