Ric-8A Catalyzes Guanine Nucleotide Exchange on Gαi1 Bound to the GPR/GoLoco Exchange Inhibitor AGS3

Abstract

Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including Gα subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various Gα·GDP subunits and forms a stable nucleotide-free Ric-8A:Gα complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes Gα subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for Gα·GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:Gαil and that of Ric-8A with the AGS3-C:Gαil·GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:Gαi1·GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:Gαi1·GDP. Subsequent dissociation of AGS3-C and GDP from Gαi1 yields a stable nucleotide free Ric-8A·Gαi1 complex that, in the presence of GTP, dissociates to yield Ric-8A and Gαi1·GTP. AGS3-C does not induce dissociation of the Ric-8A·Gαi1 complex, even when present at very high concentrations. The action of Ric-8A on AGS3:Gαi1·GDP ensures unidirectional activation of Gα subunits that cannot be reversed by AGS3. Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including Gα subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various Gα·GDP subunits and forms a stable nucleotide-free Ric-8A:Gα complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes Gα subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for Gα·GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:Gαil and that of Ric-8A with the AGS3-C:Gαil·GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:Gαi1·GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:Gαi1·GDP. Subsequent dissociation of AGS3-C and GDP from Gαi1 yields a stable nucleotide free Ric-8A·Gαi1 complex that, in the presence of GTP, dissociates to yield Ric-8A and Gαi1·GTP. AGS3-C does not induce dissociation of the Ric-8A·Gαi1 complex, even when present at very high concentrations. The action of Ric-8A on AGS3:Gαi1·GDP ensures unidirectional activation of Gα subunits that cannot be reversed by AGS3. Canonical G-protein signaling pathways are activated when agonist-bound heptahelical receptors, acting as guanine nucleotide exchange factors (GEFs), 2The abbreviations used are:GEFguanine nucleotide exchange factorAGSactivator of G-protein signalingRic-8Aresistance to inhibitors of cholinesterase 8AGPRG-protein regulatory motifRGSregulators of G-protein signalingNuMAnuclear mitotic apparatus proteinGDIguanine nucleotide dissociation inhibitorGTPγSguanosine 5′-[γ-thio]triphosphatePMSFphenylmethanesulfonyl fluorideGSTglutathione S-transferaseTEVtobacco etch virusIMACimmobilized metal ion affinity chromatographyCFPcyan fluorescent proteinYFPyellow fluorescent proteinFRETfluorescence resonance energy transferDTTdithiothreitolFPLCfast protein liquid chromatographyITCisothermal titration calorimetry. 2The abbreviations used are:GEFguanine nucleotide exchange factorAGSactivator of G-protein signalingRic-8Aresistance to inhibitors of cholinesterase 8AGPRG-protein regulatory motifRGSregulators of G-protein signalingNuMAnuclear mitotic apparatus proteinGDIguanine nucleotide dissociation inhibitorGTPγSguanosine 5′-[γ-thio]triphosphatePMSFphenylmethanesulfonyl fluorideGSTglutathione S-transferaseTEVtobacco etch virusIMACimmobilized metal ion affinity chromatographyCFPcyan fluorescent proteinYFPyellow fluorescent proteinFRETfluorescence resonance energy transferDTTdithiothreitolFPLCfast protein liquid chromatographyITCisothermal titration calorimetry. promote the exchange of GDP for GTP on Gα subunits present in Gα·GDP:Gβγ heterotrimers (1Gilman A.G. 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Biol. 1987; Full Text PDF PubMed Google Scholar). in the presence of to a to a The and at by of a in of the of of Gαi1 guanine nucleotide exchange at of in the presence of to and containing various of The at for and fluorescence at at as as a function of The of in the presence of of AGS3-C and to and for Gαi1 binding in the presence of stopped-flow to the and of of of with of of and of in and a cell of a stopped-flow apparatus at at a for of the fluorescence a at The of the to be when used with a A control by mixing of of with of the The by used as the to the A stopped-flow mixing to of with of The of the from the of with as of of and of in and and the to for GTP of in the with the cell of the stopped-flow at for at a The of GDP release from the complex by mixing of of with of both in release of and and fluorescence at at A control by mixing of AGS3-C of with of both in release in the of the of GDP by that not in complex with binding to of of protein in release a stopped-flow and the fluorescence at at are of mixing of at to regions of Ric-8A that that residues of Ric-8A to and C. J. that, the Ric-8A that be in A.M. Gilman A.G. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google not at high yield in E. the Ric-8A to as be in E. to yields of of of protein for Gαi1 used for the that the of Gαi1 exchange of GDP for with that by Ric-8A from cell to a the nucleotide exchange of Gαi1 A.M. Gilman A.G. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Ric-8A, binds to release of forms a nucleotide-free complex. Upon of the complex dissociates to free and Gαi1 not The of AGS3 of (AGS3-C; as a GDI and forms a stable complex N. A. Lanier S.M. J. Biol. Full Text Full Text PDF PubMed Scopus (55) Google Scholar, A. Sprang S.R. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Ric-8A can induce dissociation of from we with a complex of AGS3-C with for at The by of the that AGS3-C from the complex to a complex the that AGS3-C dissociation of Gαi1 from we AGS3-C with complex and the as complex on the that nucleotide-free complex does not dissociate in the presence of even at to of containing and and that can Gαi1 from the complex with on the does not a stable complex with Gαi1 to titration to the of interactions AGS3-C and that binding of to AGS3-C and that molecules of to of in with A. Sprang S.R. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google isothermal titration of AGS3-C a containing complex significant of of to Gαi1 by in fluorescence at K.M. P.C. Gilman A.G. J. Biol. 1987; Full Text PDF PubMed Google Scholar). The in fluorescence of binding to Gαi1 in the presence of of that in the of with binding to by Gαi1 A.M. Gilman A.G. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). in fluorescence of changes at the of fluorescence for binding for in by However, binding that catalyzes binding to at a to that for Gαi1 The of binding to by this and the the of binding to Gαi1 and the in the presence of of that the can be used to the of as a Gαi1 subunits the of exchange of GDP for at various of in the presence of a of of a of and a of for the binding of to Gαi1 in the presence of a inhibitor of exchange of GDP by on of the of guanine nucleotide exchange as a function of Gαi1 and binding by fluorescence as in in the of AGS3-C The that to AGS3-C from complex that as a may function as inhibitor of Gαi1 guanine nucleotide exchange that high of AGS3-C and the protein as inhibitors of Ric-8A Gilman A.G. Sci. A. 2005; PubMed Scopus Google Scholar). the of AGS3-C as inhibitor of and to the of we the of exchange of GDP for at of the presence of the for nucleotide exchange the not AGS3-C as a inhibitor of that AGS3-C and may to on The by AGS3-C the that to the complex and a transient Gαi1 complex. this we that the and a complex of with AGS3-C in of a transient at the fluorescence of and proteins in E. to and used in binding the of gel and binding and fluorescence assays, we that and and GDI A rapid mixing to the of with of proteins in in the cell of a stopped-flow and the of at of at The of mixing The of the and fluorescence of a in fluorescence to a by a by a in fluorescence to the The and of the of the to be on the of the of the of a of and a for the of The and at a of this to dissociation of the complex the release of AGS3 and GDP from A control of and fluorescence at the only to the fluorescence the rapid mixing of complex with significant fluorescence changes at the not to the nucleotide-free complex the of with of a of GTP with the release of and in allow of the AGS3:Gαi1·GDP complex and transient with as A mixing such that a of with in a for to of free and GDP as in the in the as that in The of the with GTP in the cell of the stopped-flow in a in to in in the and of in this to rapid of and free of GTP to of GTP to yield of with with to of free with the complex, by of and dissociation of the ternary complex by release of GDP from Gαi1 and of complex. with dissociation of from Gαi1 and from with of Because GTP present in of this of can for the of the of cycle of in the dissociation of the AGS3-C from and dissociation of GDP from Gαi1 by we rapid mixing to we the release of GDP from Gαi1 as a function of fluorescence that binding of AGS3-C to does not induce a in fluorescence at not AGS3-C stabilizes the fluorescence of and AGS3-C that, of a nucleotide-free complex with a in the fluorescence of Gαi1 of not with this we the of GDP release from a complex of of of and we that at a of of a of to the complex the of fluorescence to The processes in include of with and dissociation of AGS3 from the transient complex with and Gαi1 as as GDP dissociation from the that Ric-8A, a for and G-protein subunits, to exchange of GTP for GDP from to the complex Gilman A.G. Sci. A. 2005; PubMed Scopus Google Scholar). AGS3, of binds to a GDP-bound Gα and nucleotide release H. S.G. Lanier S.M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S.M. Biol. Cell. 2004; PubMed Google Scholar). the of Ric-8A Gilman A.G. Sci. A. 2005; PubMed Scopus Google Scholar). in that AGS3-C can Ric-8A These to Ric-8A with AGS3-C in binding to as by Gilman A.G. Sci. A. 2005; PubMed Scopus Google Scholar), to to the AGS3:Gαi1·GDP complex. The a the function of Ric-8A action The of Ric-8A cannot be reversed by AGS3 the of the G-protein and gel that the complex dissociates in the presence of the complex does not dissociate in the presence of the and indicate that of with yields GDP and nucleotide-free complex. The high affinity of for nucleotide-free Gαi1 and the affinity of AGS3-C the are the of this The in Gαi1 in the nucleotide-free of this However, the of isothermal titration of with AGS3-C that AGS3-C does not to nucleotide-free Ric-8A·Gαi1 Ric-8A to promote the exchange of GDP for GTP that of GTP A.M. Gilman A.G. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). used Gαi1 for The of nucleotide exchange to be for and the to by proteins Gilman A.G. Sci. A. 2005; PubMed Scopus Google Scholar). Thus, not from the be for with The complex a stable and of this that dissociates in the presence of GTP to yield and Gαi1·GTP. The that catalyzes binding to Gαi1 with a for of and a AGS3-C a inhibitor of and does not the for The for AGS3-C to be not in AGS3-C a inhibitor of Ric-8A with to Gαi1 the that Ric-8A and AGS3 to on that that with to a ternary complex with a of The the of to the fluorescence The transient in that at present a of the of Because does not to can only from binding of to present in a complex. the that from interactions and that not to the of the transient ternary complex of and the protein used in to molecules of Gαi1 may be to to the in However, the of that from binding of to of the Gαi1 molecules on may and the Gαi1 molecules may not be for with of even a to of in of to the be to dissociation of a from the complex. of of the complex a in of the complex to release of GDP from Gαi1 molecules to induce such a of nucleotide-free with of Gαi1 fluorescence and of to the complex with a that only the when binds to and nucleotide from free of the by to the at Ric-8A dissociation of GDP from as by (in for of be with the of and GDP release the complex a that in the release of AGS3-C from the complex and dissociation of GDP from dissociation of Gαi1 molecules from the AGS3-C by GDP release from Gαi1 molecules in the complex. for the from the that of may be A. Sprang S.R. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). mixing indicate that the nucleotide-free Gαi1 in complex to nucleotide when by from as in to the G-protein be that, the that only the G-protein Ric-8A can to both free and The for the of Ric-8A Ric-8A and AGS3-C are of a transient complex with Gαi1 that the at binding The of the of to interactions the and the and of Gαi1 the and the M.E. L. J. Nature. PubMed Scopus Google Scholar). Because Ric-8A and AGS3-C a transient complex with Ric-8A to with at that outside of this Because the of Ric-8A for Gαi1 not by that the binding for the proteins on Gαi1 not The for receptors, release of GDP from include residues of Gα that are from the switch regions 2007; PubMed Scopus Google Scholar, Rev. Cell Biol. 2008; PubMed Scopus Google Scholar, N. A.M. Sci. A. 2007; PubMed Scopus Google Scholar). with G-protein AGS3 and Gβγ both to the switch of the GDP-bound of Gα to nucleotide release (6Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1009) Google Scholar, M.E. L. J. Nature. PubMed Scopus Google Scholar). to that the for Ric-8A and are the Ric-8A a of acting on free and subunits A.M. Gilman A.G. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Because Ric-8A a cell protein that cannot nucleotide exchange of A.M. Gilman A.G. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), does not compete with Ric-8A may as the of Gαi1 subunits in a that during cell this pathway, Ric-8A may receptor-independent by GDP GTP be by the action of proteins, such as C. that of GTP J.C. Cell. 2004; 119: Full Text Full Text PDF PubMed Scopus Google Scholar). this activation and may be to the release and of the protein from the AGS3 to to the cell with Cell. 2004; 119: Full Text Full Text PDF PubMed Scopus Google Scholar). Ric-8A has in to from control of in interactions with that this cycle be responsible for generation of the microtubule pulling forces that the mitotic Ric-8A to on subunits that are on AGS3 that G-protein for and rapid activation The unidirectional of the by the that AGS3 cannot with complex GDP with Gα·GTP that are to and for and to Gilman for of this with