Protein-Protein Interaction between G protein-coupled receptors and heterotrimeric G proteins

Hdl Handle:
http://hdl.handle.net/10675.2/558707
Title:
Protein-Protein Interaction between G protein-coupled receptors and heterotrimeric G proteins
Authors:
Qin, Kou
Abstract:
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 (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.
Affiliation:
Department of Pharmacology and Toxicology
Issue Date:
Jan-2011
URI:
http://hdl.handle.net/10675.2/558707
Additional Links:
http://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/865312190?accountid=12365
Type:
Dissertation
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorQin, Kouen
dc.date.accessioned2015-06-30T21:56:17Zen
dc.date.available2015-06-30T21:56:17Zen
dc.date.issued2011-01en
dc.identifier.urihttp://hdl.handle.net/10675.2/558707en
dc.description.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.en
dc.relation.urlhttp://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/865312190?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectG protein coupled receptorsen
dc.subjectG proteinsen
dc.subjectCollision modelen
dc.subjectPreassembly modelen
dc.subjectPrecoupling modelen
dc.subjectProtein-protein interactionen
dc.subjectAdrenergic receptorsen
dc.subjectMuscarinic acetylcholine receptorsen
dc.subjectFluorescence Recovery After Photobleachingen
dc.subjectFRAPen
dc.titleProtein-Protein Interaction between G protein-coupled receptors and heterotrimeric G proteinsen
dc.typeDissertationen
dc.contributor.departmentDepartment of Pharmacology and Toxicologyen
dc.description.advisorLambert, Nevin A.en
dc.description.committeeNot Listeden
dc.description.degreeDoctor of Philosophy (Ph.D.)en
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