Protein-Protein Interaction between G protein-coupled receptors and heterotrimeric G proteins
AbstractG 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 (collision model). More recent studies have suggested that inactive R and G are preassembled as stable R-G complexes in cells (preassembly models). Using fluorescence recovery after photobleaching (FRAP) we examined the stability of complexes formed between cyan fluorescent protein-labeled a2Aadrenoreceptors (C-a2ARs) and G proteins in HEK293 cells. Labeled G proteins diffused in the plasma membrane with equal mobility in the absence and presence of immobile C- a2ARs. In contrast, a stable R-G interaction was detected when G proteins were deprived of nucleotides and C- a2ARs were active. Over-expression of regulator of G protein signaling 4 (RGS4) accelerated the onset of effector activation but did not alter the interaction between C- a2ARs and G proteins. At most a small fraction of C- a2ARs and G proteins exist as R-G complexes at any moment. However, applying similar technique and protocols, we demonstrated that immobilized M3R specifically decreases the mobility of Gaq heterotrimers on the plasma membranes of intact HEK293 cells, suggesting the existence of R-G preassembly. The C-terminus of M3R was determined to be both required and sufficient for preassembly. The M3R C-terminus contains a polybasic region (565KKKRRK570) located distal to the 8th a-helix domain. Substitution of this polybasic region with 6 electroneutral alanines (M3R6A) prevented preassembly. Permeabilization of cells with low ionic strength buffer resulted in enhanced R-G interaction, implicating electrostatic forces as a factor in the preassembly. We examined the functional properties of the mutant M3R6A, which showed decreases in acetylcholine potency compared with M3R. M3R6A produced active Gq at half the rate of M3R. Other Gq-coupled receptors, such as M1 and M5 muscarinic and a1a,a1b, aid adrenergic receptors, contain similar C-terminal polybasic regions. We found that both M5R and alb adrenoceptor (albAR) preassembled with Gq proteins. Our findings suggest that a polybasic regionmediated electrostatic mechanism could be a common mechanism of preassembly between Gq-coupled receptors and Gq proteins.
AffiliationDepartment of Pharmacology and Toxicology
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Regulators of G Protein Signaling (RGS Proteins) Regulate Presynaptic Inhibition at Rat Hippocampal SynapsesChen, Huanmian; Department of Pharmacology and Toxicology (2000-11)Presynaptic inhibition o f elicited neurotransmitter release mediated by G protein-coupled receptors (GPCRs) can develop and decay in a few seconds. This time course is too rapid to be accounted for by the intrinsic GTPase activity o f Ga subunits alone. Here we test the hypothesis that endogenous regulators o f G protein signaling (RGS proteins), which are GTPase activating proteins (GAPs) for Ga, are required for rapid, brief presynaptic inhibition. Endogenous G protein a subunits were uncoupled from GPCRs by treating hippocampal microisland cultures with pertussis toxin (PTX). Adenovirusmediated expression o f mutant PTX-insensitive (PTX-i) Gau.3 or Ga0 subunits rescued adenosine-induced presynaptic inhibition in neurons. Expression o f double mutant Gan or Ga0 subunits that were both PTX-insensitive and unable to bind RGS proteins (PTX/RGS-i) also rescued presynaptic inhibition. Presynaptic inhibition mediated by PTX/RGS-i subunits decayed much more slowly after agonist removal than that mediated by PTX-i subunits or native G proteins. The onset o f presynaptic inhibition mediated by PTX/RGS-i Ga0 was also slower than that mediated by PTX-i Ga0. In contrast, the onset o f presynaptic inhibition mediated by PTX/RGS-i Gan was similar to that mediated by PTX-i Gan. These results suggest that endogenous RGS proteins are present in presynaptic terminals and essential for fast recovery o f presynaptic inhibition. The effect o f endogenous RGS proteins on the onset o f presynaptic inhibition appears dependent on the particular Ga subunits involved. We also performed experiments to test whether the functions o f RGS proteins are sensitive to upregulation. Over-expression o f RGS8 in neurons without pretreatment o f PTX not only accelerated the time course o f the onset but also increased the steady state level o f presynaptic inhibition. Overexpression o f RGS4 also enhanced the steady state. These results suggest that RGS8 and probably RGS4 as well can be transported to presynaptic terminals and upregulate the activation o f Gy0 protein signaling. Interestingly, overexpression o f these RGS proteins failed to accelerate the recovery o f presynaptic inhibition, although it is well established that both RGS8 and RGS4 are strong GAPs for GcCj/0. This result suggests GAP activity for Gai/0 in presynaptic terminals is physiologically “ saturated” by endogenous RGS proteins.