RGS protein modulation of neuronal Gaq-mediated signaling

Hdl Handle:
http://hdl.handle.net/10675.2/344556
Title:
RGS protein modulation of neuronal Gaq-mediated signaling
Authors:
Clark, Michael A.
Abstract:
The decay of excitatory postsynaptic potentials (EPSPs) in neurons is much shorter than the predicted lifetime of activated G aq and thus GTPase activating proteins (GAPs) are thought to accelerate inactivation. PLCp and regulators of G-protein signaling (RGS) proteins act as GAPs toward G aq in vitro. Thus, it is unknown which of these GAPs determine the fast decay rate observed in neuronal EPSP termination. We therefore test the hypothesis that endogenous RGS proteins regulate the kinetics of Gaqmediated signaling in cultured rat cerebellar granule neurons (CGNs). Electrophysiological recordings of G aq-regulated standing outward potassium currents (Ik(SO>) were performed using mutant RGS-insensitive (RGSi) G aq chimeras. RGS insensitivity was determined by these mutants’ inability to recruit RGS2-EGFP from the nucleus of HEK293 cells to the plasma membrane despite additional mutations that render constitutive activity. Recovery from Ik(so) inhibition mediated by RGSi mutants was 5-fold slower than their wild type counterparts, confirming the necessity of native RGS proteins for rapid termination of G aq mediated signals. In addition to regulating decay of EPSPs, evidence suggests RGS proteins control activation kinetics of G-protein-mediated signals by undetermined mechanisms. We therefore hypothesized that native RGS proteins regulate onset kinetics by acting as physical scaffolds to increase the availability of activated receptors to inactive G aq, bypassing cellular diffusional limitations. Fluorescence recovery after photobleaching (FRAP) was used to determine the diffusional mobility of G aq signaling components in the presence or absence of extracellular crosslinking reagents. Even though M3R recruited RGS2-EGFP to the plasma membrane in HEK293 cells, interaction between these two proteins was extremely transient, as RGS2-EGFP diffusion was unaltered upon immobilization of ECFP-M3R. In addition, G aq-EGFP constrained diffusion when interacting with ECFP-M3R was not further slowed by RGS2 expression. Finally, RGS proteins may act as kinetic scaffolds whereby RGS-accelerated GAP activity leads to multiple rounds of activation/inactivation per receptor-Gaq binding event. A mutation causing increased intrinsic GTPase activity of G aq significantly restored onset rates of G aq activation ( I k (s o > inhibition) in the background of RGS-insensitivity. These results indicate a minimal physical scaffolding function of RGS2 and provide evidence for native RGS protein-mediated kinetic scaffolding contributing to fast G aq activation kinetics observed in CGNs.
Affiliation:
Department of Pharmacology and Toxicology
Issue Date:
May-2006
URI:
http://hdl.handle.net/10675.2/344556
Additional Links:
http://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/304954361?accountid=12365
Type:
Dissertation
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorClark, Michael A.en
dc.date.accessioned2015-02-17T22:51:37Z-
dc.date.available2015-02-17T22:51:37Z-
dc.date.issued2006-05-
dc.identifier.urihttp://hdl.handle.net/10675.2/344556-
dc.description.abstractThe decay of excitatory postsynaptic potentials (EPSPs) in neurons is much shorter than the predicted lifetime of activated G aq and thus GTPase activating proteins (GAPs) are thought to accelerate inactivation. PLCp and regulators of G-protein signaling (RGS) proteins act as GAPs toward G aq in vitro. Thus, it is unknown which of these GAPs determine the fast decay rate observed in neuronal EPSP termination. We therefore test the hypothesis that endogenous RGS proteins regulate the kinetics of Gaqmediated signaling in cultured rat cerebellar granule neurons (CGNs). Electrophysiological recordings of G aq-regulated standing outward potassium currents (Ik(SO>) were performed using mutant RGS-insensitive (RGSi) G aq chimeras. RGS insensitivity was determined by these mutants’ inability to recruit RGS2-EGFP from the nucleus of HEK293 cells to the plasma membrane despite additional mutations that render constitutive activity. Recovery from Ik(so) inhibition mediated by RGSi mutants was 5-fold slower than their wild type counterparts, confirming the necessity of native RGS proteins for rapid termination of G aq mediated signals. In addition to regulating decay of EPSPs, evidence suggests RGS proteins control activation kinetics of G-protein-mediated signals by undetermined mechanisms. We therefore hypothesized that native RGS proteins regulate onset kinetics by acting as physical scaffolds to increase the availability of activated receptors to inactive G aq, bypassing cellular diffusional limitations. Fluorescence recovery after photobleaching (FRAP) was used to determine the diffusional mobility of G aq signaling components in the presence or absence of extracellular crosslinking reagents. Even though M3R recruited RGS2-EGFP to the plasma membrane in HEK293 cells, interaction between these two proteins was extremely transient, as RGS2-EGFP diffusion was unaltered upon immobilization of ECFP-M3R. In addition, G aq-EGFP constrained diffusion when interacting with ECFP-M3R was not further slowed by RGS2 expression. Finally, RGS proteins may act as kinetic scaffolds whereby RGS-accelerated GAP activity leads to multiple rounds of activation/inactivation per receptor-Gaq binding event. A mutation causing increased intrinsic GTPase activity of G aq significantly restored onset rates of G aq activation ( I k (s o > inhibition) in the background of RGS-insensitivity. These results indicate a minimal physical scaffolding function of RGS2 and provide evidence for native RGS protein-mediated kinetic scaffolding contributing to fast G aq activation kinetics observed in CGNs.en
dc.relation.urlhttp://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/304954361?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.subjectGaqen
dc.subjectRGSen
dc.subjectEPSPen
dc.subjectGTP hydrolysisen
dc.subjectPLCen
dc.subjectStanding Outward Potassium Currenten
dc.subjectTASKen
dc.subjectFRAPen
dc.titleRGS protein modulation of neuronal Gaq-mediated signalingen
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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