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dc.contributor.authorDigby, Gregory J.
dc.date.accessioned2014-05-29T20:46:39Z
dc.date.available2014-05-29T20:46:39Z
dc.date.issued2008-06en
dc.identifier.urihttp://hdl.handle.net/10675.2/317631
dc.description.abstractOn the basis of numerous studies using cell membranes, it is commonly assumed that active G protein heterotrimers physically dissociate into GTPbound Gα and Gβγ subunits. However, due to inadequate evidence in vivo, several groups question this hypothesis. To explore this problem, we have developed an assay that measures G protein dissociation in living cells. We examined protein mobility using fluorescence recovery after photobleaching (FRAP) and found that Gβ1γ2 subunits formed heterotrimers with inactive immobile Gα subunits. When we activated heterotrimers with receptors, Gβγ subunits released from Gα, suggesting that G protein heterotrimers physically dissociate in living cells. To our knowledge this is the first definitive measure of this event in vivo. When different Gα isoforms were compared, we found that Gαi/o subunits released Gβγ dimers more readily than Gαs subunits, suggesting that heterotrimers differentially dissociate. To determine if differential release of Gβγ is a mechanism for Gα specific activation of Gated inwardly rectifying potassium (GIRK) channels, we activated GIRKs in cells expressing GαoA or Gαs subunits. We found that GαoA heterotrimers were more effective activators of GIRK channels than Gαs heterotrimers when comparable amounts of each were available. Thus, since GαoA subunits also released Gβγ dimers more efficiently than Gαs subunits, we propose that differential dissociation provides a mechanism for Gα specific activation of GIRK. In addition to providing a clear demonstration of G protein dissociation, we also propose a more complete model of the G protein cycle where active G proteins are in continuous association-dissociation equilibrium. Accordingly, at any given time during activation, G protein heterotrimers can cycle through several dissociation-association events until GTP is hydrolyzed. A model of the G protein cycle where GαGTP + Gβγ and GαGTPGβγ are both present during activation is included.
dc.language.isoen_USen
dc.relation.urlhttp://search.proquest.com/docview/304402675?accountid=12365en
dc.subjectG proteinsen
dc.subjectDissociationen
dc.subjectG protein coupled receptorsen
dc.subjectGated Inwardly-Rectifying Potassium Channelen
dc.subjectFluorescence Recovery After Photobleachingen
dc.titlePhysical Dissociation of G Protein Heterotrimers in Living Cellsen
dc.typeDissertationen
dc.contributor.departmentDepartment of Neuroscience and Regenerative Medicineen
dc.description.advisorLambert, Nevin A.en
dc.description.degreeDoctor of Philosophy (Ph.D.)en
html.description.abstractOn the basis of numerous studies using cell membranes, it is commonly assumed that active G protein heterotrimers physically dissociate into GTPbound Gα and Gβγ subunits. However, due to inadequate evidence in vivo, several groups question this hypothesis. To explore this problem, we have developed an assay that measures G protein dissociation in living cells. We examined protein mobility using fluorescence recovery after photobleaching (FRAP) and found that Gβ1γ2 subunits formed heterotrimers with inactive immobile Gα subunits. When we activated heterotrimers with receptors, Gβγ subunits released from Gα, suggesting that G protein heterotrimers physically dissociate in living cells. To our knowledge this is the first definitive measure of this event in vivo. When different Gα isoforms were compared, we found that Gαi/o subunits released Gβγ dimers more readily than Gαs subunits, suggesting that heterotrimers differentially dissociate. To determine if differential release of Gβγ is a mechanism for Gα specific activation of Gated inwardly rectifying potassium (GIRK) channels, we activated GIRKs in cells expressing GαoA or Gαs subunits. We found that GαoA heterotrimers were more effective activators of GIRK channels than Gαs heterotrimers when comparable amounts of each were available. Thus, since GαoA subunits also released Gβγ dimers more efficiently than Gαs subunits, we propose that differential dissociation provides a mechanism for Gα specific activation of GIRK. In addition to providing a clear demonstration of G protein dissociation, we also propose a more complete model of the G protein cycle where active G proteins are in continuous association-dissociation equilibrium. Accordingly, at any given time during activation, G protein heterotrimers can cycle through several dissociation-association events until GTP is hydrolyzed. A model of the G protein cycle where GαGTP + Gβγ and GαGTPGβγ are both present during activation is included.


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