FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034785658> ?p ?o ?g. }

• W2034785658 endingPage "36710" @default.
• W2034785658 startingPage "36698" @default.
• W2034785658 abstract "Heterotrimeric G-protein Gα subunits and GoLoco motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco motif proteins bind selectively to the inhibitory subclass (Gαi) of Gα subunits, and thus it is assumed that a Gαi·GoLoco motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Gαi subunits that renders a selective loss-of-function for GoLoco motif binding, namely an asparagine-to-isoleucine substitution in the αD-αE loop of the Gα helical domain. This GoLoco-insensitivity (“GLi”) mutation prevented Gαi1 association with all human GoLoco motif proteins and abrogated interaction between the Caenorhabditis elegans Gα subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Gαi subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gβγ dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Gαi subunits unable to recruit GoLoco motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Gαi subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Gαi·GoLoco motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco motifs in receptor-mediated signal transduction. Heterotrimeric G-protein Gα subunits and GoLoco motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco motif proteins bind selectively to the inhibitory subclass (Gαi) of Gα subunits, and thus it is assumed that a Gαi·GoLoco motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Gαi subunits that renders a selective loss-of-function for GoLoco motif binding, namely an asparagine-to-isoleucine substitution in the αD-αE loop of the Gα helical domain. This GoLoco-insensitivity (“GLi”) mutation prevented Gαi1 association with all human GoLoco motif proteins and abrogated interaction between the Caenorhabditis elegans Gα subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Gαi subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gβγ dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Gαi subunits unable to recruit GoLoco motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Gαi subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Gαi·GoLoco motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco motifs in receptor-mediated signal transduction. Seven transmembrane-domain receptors (7TMRs) 2The abbreviations used are: 7TMRseven transmembrane domain receptoraaamino acidCFPcyan fluorescent proteinGLGoLocoGLiGoLoco-insensitiveGPSMG-protein signaling modulatorGSTglutathione S-transferaseGTPγSguanosine 5′-3-O-(thio)triphosphateHAhemagglutinin epitope tagKT3SV40 large T antigen-derived epitope tagmRFPmonomeric red fluorescent proteinMECA5-N-methylcarboxamidoadenosinemPmillipolarization unit of measurementMTmicrotubulePCP-2Purkinje cell protein 2PTXpertussis toxinRGSregulator of G-protein signalingSPRsurface plasmon resonanceYFPvenus yellow fluorescent proteinNEnorepinephrinePDBProtein Data BankFITCfluorescein isothiocyanateGDIguanine nucleotide dissociation inhibitorSCGsuperior cervical ganglionTEA-OHtetraethylammonium hydroxideMDCKMadin-Darby canine kidney 2The abbreviations used are: 7TMRseven transmembrane domain receptoraaamino acidCFPcyan fluorescent proteinGLGoLocoGLiGoLoco-insensitiveGPSMG-protein signaling modulatorGSTglutathione S-transferaseGTPγSguanosine 5′-3-O-(thio)triphosphateHAhemagglutinin epitope tagKT3SV40 large T antigen-derived epitope tagmRFPmonomeric red fluorescent proteinMECA5-N-methylcarboxamidoadenosinemPmillipolarization unit of measurementMTmicrotubulePCP-2Purkinje cell protein 2PTXpertussis toxinRGSregulator of G-protein signalingSPRsurface plasmon resonanceYFPvenus yellow fluorescent proteinNEnorepinephrinePDBProtein Data BankFITCfluorescein isothiocyanateGDIguanine nucleotide dissociation inhibitorSCGsuperior cervical ganglionTEA-OHtetraethylammonium hydroxideMDCKMadin-Darby canine kidney mediate the actions of various extracellular sensory, hormonal, and metabolic stimuli (1Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell Biol. 2002; 3: 639-650Crossref PubMed Scopus (2063) Google Scholar). Among the signaling components coupled to the intracytosolic side of 7TMRs are the heterotrimeric G-proteins: molecular switches composed of a guanine nucleotide-binding Gα subunit and a Gβγ dimer that transduce 7TMR activation into intracellular modulation of multiple different effectors, including adenylyl cyclases, ion channels, cyclic nucleotide phosphodiesterases, and phospholipase C isoforms (2Johnston C.A. Siderovski D.P. Mol. Pharmacol. 2007; 72: 219-230Crossref PubMed Scopus (109) Google Scholar, 3Siderovski D.P. Willard F.S. Int. J. Biol. Sci. 2005; 1: 51-66Crossref PubMed Scopus (324) Google Scholar). 7TMR-promoted activation of Gαβγ causes Gα to exchange the more abundant GTP for bound GDP, which in turn causes Gα·GTP and Gβγ to dissociate. Gα·GTP and Gβγ are then free to regulate effector systems that alter cell physiology (4Cabrera-Vera T.M. Vanhauwe J. Thomas T.O. Medkova M. Preininger A. Mazzoni M.R. Hamm H.E. Endocr. Rev. 2003; 24: 765-781Crossref PubMed Scopus (508) Google Scholar, 5Wettschureck N. Offermanns S. Physiol. Rev. 2005; 85: 1159-1204Crossref PubMed Scopus (798) Google Scholar). This classical 7TMR-initiated G-protein nucleotide cycle is reset by intrinsic GTP hydrolysis activity possessed by the Gα subunit.An evolutionarily conserved role for Gα subunits of the adenylyl cyclase inhibitory (Gαi) subfamily has recently been identified in the control of mitotic spindle orientation in cell divisions that generate cellular diversity during organismal development (6Hampoelz B. Knoblich J.A. Cell. 2004; 119: 453-456Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 7Willard F.S. Kimple R.J. Siderovski D.P. Annu. Rev. Biochem. 2004; 73: 925-951Crossref PubMed Scopus (161) Google Scholar). Studies of asymmetric cell division in Caenorhabditis elegans embryos and Drosophila melanogaster embryonic neuroblasts have identified initial steps of this process as generation of cell polarity and segregation of various cell fate determinants to different sides of the polarized cell (8Betschinger J. Knoblich J.A. Curr. Biol. 2004; 14: R674-R685Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar); the mitotic spindle is then positioned to facilitate appropriate distribution of determinants to daughter cells during chromosomal segregation and cytokinesis. An integral part of the cellular machinery underlying accurate spindle positioning is the involvement of heterotrimeric G-protein Gα and Gβγ subunits in a manner considered independent of 7TMR activation and instead involving RIC-8 (a cytosolic guanine nucleotide exchange factor), GoLoco motif 3The GoLoco motif is also referred to as the G-protein regulatory motif (79Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). 3The GoLoco motif is also referred to as the G-protein regulatory motif (79Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). proteins (such as GPSM2/LGN, Pins, and GPR-1/2 that act as GDP dissociation inhibitors), and GTPase-accelerating proteins (“GAPs”; i.e. RGS proteins) (6Hampoelz B. Knoblich J.A. Cell. 2004; 119: 453-456Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 7Willard F.S. Kimple R.J. Siderovski D.P. Annu. Rev. Biochem. 2004; 73: 925-951Crossref PubMed Scopus (161) Google Scholar, 8Betschinger J. Knoblich J.A. Curr. Biol. 2004; 14: R674-R685Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 9Afshar K. Willard F.S. Colombo K. Johnston C.A. McCudden C.R. Siderovski D.P. Gonczy P. Cell. 2004; 119: 219-230Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 10Colombo K. Grill S.W. Kimple R.J. Willard F.S. Siderovski D.P. Gonczy P. Science. 2003; 300: 1957-1961Crossref PubMed Scopus (224) Google Scholar, 11Gotta M. Ahringer J. Nat. Cell Biol. 2001; 3: 297-300Crossref PubMed Scopus (216) Google Scholar, 12Hess H.A. Roper J.C. Grill S.W. Koelle M.R. Cell. 2004; 119: 209-218Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 13Srinivasan D.G. Fisk R.M. Xu H. van den Heuvel S. Genes Dev. 2003; 17: 1225-1239Crossref PubMed Scopus (179) Google Scholar). Vertebrate neuroepithelial progenitors use the same cellular machinery to modulate mitotic spindle orientation controlling the balance between asymmetric cell divisions that drive differentiation and planar divisions that favor maintenance and expansion of the neuroepithelial architecture (14Sanada K. Tsai L.H. Cell. 2005; 122: 119-131Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 15Konno D. Shioi G. Shitamukai A. Mori A. Kiyonari H. Miyata T. Matsuzaki F. Nat. Cell Biol. 2008; 10: 93-101Crossref PubMed Scopus (384) Google Scholar, 16Morin X. Jaouen F. Durbec P. Nat. Neurosci. 2007; 10: 1440-1448Crossref PubMed Scopus (193) Google Scholar). Similarly, an analogous mechanism appears to operate in the stratification and differentiation of mammalian skin (17Lechler T. Fuchs E. Nature. 2005; 437: 275-280Crossref PubMed Scopus (751) Google Scholar).An essential feature of the various emerging models of G-protein nucleotide cycling in mitotic spindle positioning is the requirement for a Gαi·GoLoco motif complex. For example, in our working model of C. elegans asymmetric cell division controlled by the Gα subunits GOA-1 and GPA-16 (18Afshar K. Willard F.S. Colombo K. Siderovski D.P. Gonczy P. Development (Camb.). 2005; 132: 4449-4459Crossref PubMed Scopus (68) Google Scholar, 19Johnston C.A. Afshar K. Snyder J.T. Tall G.G. Gonczy P. Siderovski D.P. Willard F.S. J. Biol. Chem. 2008; 283: 21550-21558Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), it is the Gα·GDP/GPR-1/2 complex that activates the generation of astral microtubule (MT) force on mitotic spindle poles, whereas in a competing model (3Siderovski D.P. Willard F.S. Int. J. Biol. Sci. 2005; 1: 51-66Crossref PubMed Scopus (324) Google Scholar, 12Hess H.A. Roper J.C. Grill S.W. Koelle M.R. Cell. 2004; 119: 209-218Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 20Wilkie T.M. Kinch L. Curr. Biol. 2005; 15: R843-R854Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), the Gα·GDP/GoLoco motif complex is required for the nucleotide exchange (“GEF”) activity for RIC-8, thereby generating Gα·GTP as the presumed active form of the G-protein (12Hess H.A. Roper J.C. Grill S.W. Koelle M.R. Cell. 2004; 119: 209-218Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Tall G.G. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16584-16589Crossref PubMed Scopus (74) Google Scholar, 22Thomas C.J. Tall G.G. Adhikari A. Sprang S.R. J. Biol. Chem. 2008; 283: 23150-23160Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). However, it has not been formally established that the Gα/GoLoco motif interaction is required per se for the function of Gα subunits and GoLoco motif proteins in mitotic spindle positioning. For example, both models of C. elegans asymmetric cell division have been generated primarily by correlating various genetic phenotype data, including loss of pulling forces upon RNA interference-mediated knockdown of goa-1/gpa-16 or gpr-1/2 expression (9Afshar K. Willard F.S. Colombo K. Johnston C.A. McCudden C.R. Siderovski D.P. Gonczy P. Cell. 2004; 119: 219-230Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 10Colombo K. Grill S.W. Kimple R.J. Willard F.S. Siderovski D.P. Gonczy P. Science. 2003; 300: 1957-1961Crossref PubMed Scopus (224) Google Scholar, 11Gotta M. Ahringer J. Nat. Cell Biol. 2001; 3: 297-300Crossref PubMed Scopus (216) Google Scholar, 18Afshar K. Willard F.S. Colombo K. Siderovski D.P. Gonczy P. Development (Camb.). 2005; 132: 4449-4459Crossref PubMed Scopus (68) Google Scholar, 19Johnston C.A. Afshar K. Snyder J.T. Tall G.G. Gonczy P. Siderovski D.P. Willard F.S. J. Biol. Chem. 2008; 283: 21550-21558Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). These phenotypic results, although suggestive of a critical function for a Gα·GoLoco protein complex, might alternatively reflect separate and distinct functions of Gα subunits and the multidomain GPR-1/2 proteins in parallel pathways culminating in MT force generation, given that both classes of proteins have other binding partners and established functions. Furthermore, it remains unresolved as to whether Gβγ is an independent signaling entity in this system or merely a buffer of free Gα·GDP levels (14Sanada K. Tsai L.H. Cell. 2005; 122: 119-131Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 23Yu F. Cai Y. Kaushik R. Yang X. Chia W. J. Cell Biol. 2003; 162: 623-633Crossref PubMed Scopus (98) Google Scholar, 24Fuse N. Hisata K. Katzen A.L. Matsuzaki F. Curr. Biol. 2003; 13: 947-954Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar).To provide a tool to address these questions, we sought to design a variant Gα subunit that will not interact with GoLoco motifs and yet retain wild type interactions with guanine nucleotides, 7TMRs, Gβγ subunits, Gα effectors, and RGS proteins. Here we describe and validate a single point mutation that renders Gαi subunits unable to bind GoLoco motif proteins, yet preserves all other aspects of Gα function. Furthermore, we use this GoLoco-insensitivity (“GLi”) mutation to demonstrate that direct Gα/GoLoco motif interaction is required for the Gα-dependent modulation of MT dynamics during mitotic spindle positioning.EXPERIMENTAL PROCEDURESMaterials—All peptides were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) group protection, high pressure liquid chromatography-purified, and validated by mass spectrometry at the Tufts University Core Facility (Medford, MA). Fluorescent guanine nucleotides were from Invitrogen. Anti-KT3 antibody MMS-125P was from Covance (Berkeley, CA). Unless elsewhere specified, all additional reagents were of the highest quality obtainable from Sigma or Fisher.Molecular Biology—The expression vectors pcDNA3.1 human Gαi1 (Missouri Science and Technology cDNA Resource Center), pPROEXHTb human Gαi1 (25Kimple R.J. De Vries L. Tronchere H. Behe C.I. Morris R.A. Gist Farquhar M. Siderovski D.P. J. Biol. Chem. 2001; 276: 29275-29281Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), pcDNA3.1 human Gαi1-KT3 (26Kimple R.J. Willard F.S. Hains M.D. Jones M.B. Nweke G.K. Siderovski D.P. Biochem. J. 2004; 378: 801-808Crossref PubMed Scopus (49) Google Scholar), pCI rat Gαi1(C352G) (27Ikeda S.R. Jeong S.W. Methods Enzymol. 2004; 389: 170-189Crossref PubMed Scopus (23) Google Scholar), pCI rat Gαi2(C353G) (27Ikeda S.R. Jeong S.W. Methods Enzymol. 2004; 389: 170-189Crossref PubMed Scopus (23) Google Scholar), pCI rat Gαi3(C352G) (27Ikeda S.R. Jeong S.W. Methods Enzymol. 2004; 389: 170-189Crossref PubMed Scopus (23) Google Scholar), and pPROEXHTb GOA-1 (encoding aa 28-351) (9Afshar K. Willard F.S. Colombo K. Johnston C.A. McCudden C.R. Siderovski D.P. Gonczy P. Cell. 2004; 119: 219-230Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) were each subjected to site-directed mutagenesis to create N149I or N150I variants. All mutagenesis was performed using the QuikChange system (Stratagene, La Jolla, CA). The mammalian expression vectors pCI bovine Gβ1 and pCI bovine Gγ2 are described in Ref. 28Ikeda S.R. Nature. 1996; 380: 255-258Crossref PubMed Scopus (706) Google Scholar, and pCI rat mGluR2 is described in Ref. 29Kammermeier P.J. Davis M.I. Ikeda S.R. Mol. Pharmacol. 2003; 63: 183-191Crossref PubMed Scopus (21) Google Scholar. pK mammalian expression vectors and derivatives thereof (including venus yellow fluorescent protein (YFP) fusion (30Nagai T. Ibata K. Park E.S. Kubota M. Mikoshiba K. Miyawaki A. Nat. Biotechnol. 2002; 20: 87-90Crossref PubMed Scopus (2156) Google Scholar) (pK-VENUS), monomeric red fluorescent protein (mRFP) fusion (31Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7877-7882Crossref PubMed Scopus (1984) Google Scholar) (pK-mRFP), and 3× HA tag fusion (pK-HA3)), originated from the Macara laboratory (University of Virginia, VA) and are derived from pRK5 (BD Biosciences). pK-YFP-GPSM2 and pK-Gαi1-YFP are described in Ref. 32Du Q. Macara I.G. Cell. 2004; 119: 503-516Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar. Wild type and N149I pK-Gαi1-YFP, pK-Gαi1-mRFP, and pK-Gαi1-HA3 were made by PCR amplification of pcDNA3.1(Gαi1, wild type and N149I) and subcloning into the XbaI sites of pK-VENUS, pK-mRFP, and pK-HA3 respectively. To construct pK-GPSM1-YFP, mouse GPSM1 cDNA was PCR-amplified and subcloned into the BamHI/EcoRI sites of pK-VENUS. A pFLAG expression construct encoding the adenosine A2A receptor fused to venus-enhanced YFP is described in Ref. 33Vidi P.A. Chemel B.R. Hu C.D. Watts V.J. Mol. Pharmacol. 2008; 74: 544-551Crossref PubMed Scopus (80) Google Scholar. C. elegans RGS-7 in pBluescript was provided by Pierre Gonczy (ISREC, Lausanne, Switzerland). DNA encoding the predicted minimal RGS domain of RGS-7 (aa 667-808 of RGS-7A (12Hess H.A. Roper J.C. Grill S.W. Koelle M.R. Cell. 2004; 119: 209-218Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar)) was cloned into pPROEXHTb using heterostagger PCR (34Willard F.S. Kimple A.J. Johnston C.A. Siderovski D.P. Anal. Biochem. 2005; 340: 341-351Crossref PubMed Scopus (38) Google Scholar). All DNA constructs were verified by DNA sequencing.Protein Purification—GST fusion proteins were purified to homogeneity using standard methods (34Willard F.S. Kimple A.J. Johnston C.A. Siderovski D.P. Anal. Biochem. 2005; 340: 341-351Crossref PubMed Scopus (38) Google Scholar, 35Willard F.S. Siderovski D.P. Methods Enzymol. 2004; 389: 320-338Crossref PubMed Scopus (34) Google Scholar). The GST-GoLoco motif fusion proteins purified were rat GPSM1(GL1234, aa 361-650 (36De Vries L. Fischer T. Tronchere H. Brothers G.M. Strockbine B. Siderovski D.P. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14364-14369Crossref PubMed Scopus (131) Google Scholar)), human GPSM2 (GL1234, aa 481-657 (37McCudden C.R. Willard F.S. Kimple R.J. Johnston C.A. Hains M.D. Jones M.B. Siderovski D.P. Biochim. Biophys. Acta. 2005; 1745: 254-264Crossref PubMed Scopus (38) Google Scholar)), human GPSM3/G18 (GL123, aa 61-160 (26Kimple R.J. Willard F.S. Hains M.D. Jones M.B. Nweke G.K. Siderovski D.P. Biochem. J. 2004; 378: 801-808Crossref PubMed Scopus (49) Google Scholar)), human PCP-2/GPSM4 (GL12, full-length (38Willard F.S. McCudden C.R. Siderovski D.P. Cell. Signal. 2006; 18: 1226-1234Crossref PubMed Scopus (20) Google Scholar)), rat RGS12 (aa 1184-1228 (25Kimple R.J. De Vries L. Tronchere H. Behe C.I. Morris R.A. Gist Farquhar M. Siderovski D.P. J. Biol. Chem. 2001; 276: 29275-29281Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar)), rat RGS14 (aa 496-531 (39Kimple R.J. Kimple M.E. Betts L. Sondek J. Siderovski D.P. Nature. 2002; 416: 878-881Crossref PubMed Scopus (206) Google Scholar)), Rap1GAP1a (aa 1-34 (40Willard F.S. Low A.B. McCudden C.R. Siderovski D.P. Cell. Signal. 2007; 19: 428-438Crossref PubMed Scopus (14) Google Scholar)), and Rap1GAP1b (aa 25-65 (40Willard F.S. Low A.B. McCudden C.R. Siderovski D.P. Cell. Signal. 2007; 19: 428-438Crossref PubMed Scopus (14) Google Scholar)) (see also supplemental Fig. S1 for a graphical representation). Gα subunits were purified to homogeneity using previously described methods, including the removal of His6 tags by tobacco etch virus protease cleavage (25Kimple R.J. De Vries L. Tronchere H. Behe C.I. Morris R.A. Gist Farquhar M. Siderovski D.P. J. Biol. Chem. 2001; 276: 29275-29281Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 34Willard F.S. Kimple A.J. Johnston C.A. Siderovski D.P. Anal. Biochem. 2005; 340: 341-351Crossref PubMed Scopus (38) Google Scholar, 35Willard F.S. Siderovski D.P. Methods Enzymol. 2004; 389: 320-338Crossref PubMed Scopus (34) Google Scholar). The specific activities of wild type and N149I Gαi1 were determined using [35S]GTPγS binding (mean ± S.E. of mol of GTPγS bound per mol of Gαi1) as follows: wild type, 0.93 ± 0.02; N149I 0.93 ± 0.02. C. elegans RGS-7, also with its His6 tag removed, was purified to homogeneity using methods standard for other RGS domains (41Soundararajan M. Willard F.S. Kimple A.J. Turnbull A.P. Ball L.J. Schoch G.A. Gileadi C. Fedorov O.Y. Dowler E.F. Higman V.A. Hutsell S.Q. Sundstrom M. Doyle D.A. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 6457-6462Crossref PubMed Scopus (132) Google Scholar).Surface Plasmon Resonance—Surface plasmon resonance analysis of GoLoco motif/Gα interactions was conducted as described in Refs. 25Kimple R.J. De Vries L. Tronchere H. Behe C.I. Morris R.A. Gist Farquhar M. Siderovski D.P. J. Biol. Chem. 2001; 276: 29275-29281Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 38Willard F.S. McCudden C.R. Siderovski D.P. Cell. Signal. 2006; 18: 1226-1234Crossref PubMed Scopus (20) Google Scholar.Fluorescence Anisotropy—Fluorescence anisotropic assays of Gα binding to FITC-labeled GoLoco motif peptides was conducted as described in Ref. 42Kimple A.J. Yasgar A. Hughes M. Jadhav A. Willard F.S. Muller R.E. Austin C.P. Inglese J. Ibeanu G.C. Siderovski D.P. Simeonov A. Comb. Chem. High Throughput Screen. 2008; 11: 396-409Crossref PubMed Scopus (25) Google Scholar for Fig. 2 and Fig. 7 and as described in Ref. 40Willard F.S. Low A.B. McCudden C.R. Siderovski D.P. Cell. Signal. 2007; 19: 428-438Crossref PubMed Scopus (14) Google Scholar for Fig. 3. A minor modification was the use of a 5 nm final concentration of the FITC-RGS14, FITC-RGS12, FITC-GPSM2(GL2), and FITC-KB-1753 peptides. FITC-RGS12 is described in Ref. 42Kimple A.J. Yasgar A. Hughes M. Jadhav A. Willard F.S. Muller R.E. Austin C.P. Inglese J. Ibeanu G.C. Siderovski D.P. Simeonov A. Comb. Chem. High Throughput Screen. 2008; 11: 396-409Crossref PubMed Scopus (25) Google Scholar. FITC-GPSM2(GL2) is described in Ref. 40Willard F.S. Low A.B. McCudden C.R. Siderovski D.P. Cell. Signal. 2007; 19: 428-438Crossref PubMed Scopus (14) Google Scholar. FITC-KB-1753 is described in Ref. 43Johnston C.A. Lobanova E.S. Shavkunov A.S. Low J. Ramer J.K. Blaesius R. Fredericks Z. Willard F.S. Kuhlman B. Arshavsky V.Y. Siderovski D.P. Biochemistry. 2006; 45: 11390-11400Crossref PubMed Scopus (25) Google Scholar. The FITC-RGS14 peptide included amino acids 496-531 of rat RGS14 (FITC-β-alanine-S-DIEGLVELLNRVQSSGAHDQRGLLRKEDLVLPEFLQ-NH2). Anisotropy data are presented as millipolarization units (mP) following data analysis as described in Ref. 42Kimple A.J. Yasgar A. Hughes M. Jadhav A. Willard F.S. Muller R.E. Austin C.P. Inglese J. Ibeanu G.C. Siderovski D.P. Simeonov A. Comb. Chem. High Throughput Screen. 2008; 11: 396-409Crossref PubMed Scopus (25) Google Scholar.FIGURE 7GoLoco-insensitive Gαil has normal interactions with the effector adenylyl cyclase and the effector-mimetic peptide KB-1753. A, affinity of wild type and N149I Gαi1 proteins for the Gα-effector mimetic peptide KB-1753 (43Johnston C.A. Lobanova E.S. Shavkunov A.S. Low J. Ramer J.K. Blaesius R. Fredericks Z. Willard F.S. Kuhlman B. Arshavsky V.Y. Siderovski D.P. Biochemistry. 2006; 45: 11390-11400Crossref PubMed Scopus (25) Google Scholar) was measured using fluorescence anisotropy. 5 nm FITC-KB-1753 peptide was mixed with increasing amounts of Gαi1 proteins, and equilibrium fluorescence anisotropy was measured. Data are presented as the mean ± S.E.M. of triplicate determinations. Dissociation constants were determined by nonlinear regression: wild type Gαi1·GDP (24.1 ± 4 μm), wild type Gαi1·GDP·AlF4− (294 ± 40 nm), N149I Gαi1·GDP (13.0 ± 2 μm), N149I Gαi1·GDP·AlF4− (311 ± 40 nm). B, cells were transiently transfected with cDNA encoding Gαi1(Q204L,C352G), Gαi1(N149I,Q204L,C352G), or pcDNA3.1(+) as a vector control, with the adenosine A2A receptor. Cyclic AMP accumulation was stimulated with 1 μm MECA for 15 min at 37 °C. Data represent the mean ± S.E.M. of four independent experiments in duplicate. *, p < 0.05; **, p < 0.01 compared with A2A-R + empty vector transfection under matched stimulation (basal or MECA), one-way analysis of variance followed by Dunnett's post hoc test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3N149I substitution is a loss-of-function Gα mutation for all mammalian GoLoco motifs. The universality of the Gαi1 N149I mutation was analyzed by surface plasmon resonance. GST fusion proteins of the GoLoco motifs of GPSM1(GL1,2,3,4) (A); GPSM2(GL1,2,3,4) (B); GPSM3(GL1,2,3) (C); PCP-2(GL1,2) (D); RGS12 (E); Rap1GAP1a (F); and Rap1GAP1b (G) were immobilized on SPR biosensor surfaces. 1 μm wild type Gαi1·GDP (blue), 1 μm wild type Gαi1·GDP·AlF4− (red), 10 μm N149I Gαi1·GDP (green), and 10 μm N149I Gαi1·GDP·AlF4− (black) were separately injected over biosensor surfaces. Binding curves were obtained by subtracting nonspecific binding to GST alone. H, affinity of wild type and N149I Gαi1 proteins for GPSM2(GL2) was measured using fluorescence anisotropy. 5 nm FITC-GPSM(GL2) peptide was mixed with increasing amounts of Gαi1 proteins, and equilibrium fluorescence anisotropy was measured. Data are expressed as millipolarization units as described in Ref. 42Kimple A.J. Yasgar A. Hughes M. Jadhav A. Willard F.S. Muller R.E. Austin C.P. Inglese J. Ibeanu G.C. Siderovski D.P. Simeonov A. Comb. Chem. High Throughput Screen. 2008; 11: 396-409Crossref PubMed Scopus (25) Google Scholar. Dissociation constants were determined by nonlinear regression as follows: wild type Gαi1·GDP (150 ± 20 nm) and N149I Gαi1·GDP (>99 μm). I, affinity of wild type and N149I Gαi1 proteins for the RGS12 GoLoco motif was measured using fluorescence anisotropy. 5 nm FITC-RGS12 peptide was mixed with increasing amounts of Gαi1 proteins and equilibrium fluorescence anisotropy was measured. Data are expressed as millipolarization units as described in Ref. 42Kimple A.J. Yasgar A. Hughes M. Jadhav A. Willard F.S. Muller R.E. Austin C.P. Inglese J. Ibeanu G.C. Siderovski D.P. Simeonov A. Comb. Chem. High Throughput Screen. 2008; 11: 396-409Crossref PubMed Scopus (25) Google Scholar. Dissociation constants were determined by nonlinear regression: wild type Gαi1·GDP (44 ± 6 nm), N149I Gαi1·GDP (9.3 ± 0.5 μm). RU, resonance units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nucleotide Binding and Hydrolysis Assays—[35S]GTPγS binding and [γ-32P]GTP hydrolysis assays were conducted as described in Ref. 9Afshar K. Willard F.S. Colombo K. Johnston C.A. McCudden C.R. Siderovski D.P. Gonczy P. Cell. 2004; 119: 219-230Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 44Willard F.S. Siderovski D.P. Biochem. Biophys. Res. Commun. 2006; 339: 1107-1112Crossref PubMed Scopus (12) Google Scholar. [35S]GTPγS binding was used to measure GPR-1/2-mediated GDI activity on GOA-1 as described in Refs. 40Willard F.S. Low A.B. McCudden C.R. Siderovski D.P. Cell. Signal. 2007; 19: 428-438Crossref PubMed Scopus (14) Google Scholar, 44Willard F.S. Siderovski D.P. Biochem. Biophys. Res. Commun. 2006; 339: 1107-1112Crossref PubMed Scopus (12) Google Scholar. The GPR-1/2 peptide (aa 423-461) is described in Refs. 9Afshar K. Willard F.S. Colombo K. Johnston C.A. McCudden C.R. Siderovski D.P. Gonczy P. Cell. 2004; 119: 219-230Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 42Kimple A.J. Yasgar A. Hughes M. Jadhav A. Willard F.S. Muller R.E. Austin C.P. Inglese J. Ibeanu G.C. Siderovski D.P. Simeonov A. Comb. Chem. High Throughput Screen. 2008; 11: 396-409Crossref PubMed Scopus (25) Google Scholar. BODIPYFL-GTPγS binding assays were used to quantify GoLoco motif-promoted Gαi1 GDI activity, as" @default.
• W2034785658 created "2016-06-24" @default.
• W2034785658 creator A5001549606 @default.
• W2034785658 creator A5004277635 @default.
• W2034785658 creator A5009204513 @default.
• W2034785658 creator A5034889874 @default.
• W2034785658 creator A5042997375 @default.
• W2034785658 creator A5063371805 @default.
• W2034785658 creator A5065145502 @default.
• W2034785658 creator A5074003204 @default.
• W2034785658 creator A5075879139 @default.
• W2034785658 creator A5076023837 @default.
• W2034785658 creator A5079993897 @default.
• W2034785658 creator A5083011492 @default.
• W2034785658 creator A5085155960 @default.
• W2034785658 creator A5089279041 @default.
• W2034785658 date "2008-12-01" @default.
• W2034785658 modified "2023-10-05" @default.
• W2034785658 title "A Point Mutation to Gαi Selectively Blocks GoLoco Motif Binding" @default.
• W2034785658 cites W1524291730 @default.
• W2034785658 cites W1543897018 @default.
• W2034785658 cites W1544256708 @default.
• W2034785658 cites W1550019326 @default.
• W2034785658 cites W1564384722 @default.
• W2034785658 cites W1601676238 @default.
• W2034785658 cites W1603274157 @default.
• W2034785658 cites W1759476206 @default.
• W2034785658 cites W1971421184 @default.
• W2034785658 cites W1972148870 @default.
• W2034785658 cites W1972211504 @default.
• W2034785658 cites W1973620594 @default.
• W2034785658 cites W1976964790 @default.
• W2034785658 cites W1978847871 @default.
• W2034785658 cites W1978968660 @default.
• W2034785658 cites W1989711466 @default.
• W2034785658 cites W1990888281 @default.
• W2034785658 cites W1990969735 @default.
• W2034785658 cites W1993752846 @default.
• W2034785658 cites W1998207140 @default.
• W2034785658 cites W1999243747 @default.
• W2034785658 cites W2003067279 @default.
• W2034785658 cites W2003218087 @default.
• W2034785658 cites W2005614924 @default.
• W2034785658 cites W2005925880 @default.
• W2034785658 cites W2006259642 @default.
• W2034785658 cites W2018372554 @default.
• W2034785658 cites W2019593540 @default.
• W2034785658 cites W2026060763 @default.
• W2034785658 cites W2027084558 @default.
• W2034785658 cites W2027857326 @default.
• W2034785658 cites W2028945975 @default.
• W2034785658 cites W2029907860 @default.
• W2034785658 cites W2030185192 @default.
• W2034785658 cites W2034762571 @default.
• W2034785658 cites W2039210066 @default.
• W2034785658 cites W2043775294 @default.
• W2034785658 cites W2043799749 @default.
• W2034785658 cites W2048351462 @default.
• W2034785658 cites W2050395095 @default.
• W2034785658 cites W2050688574 @default.
• W2034785658 cites W2051144604 @default.
• W2034785658 cites W2058848453 @default.
• W2034785658 cites W2059841182 @default.
• W2034785658 cites W2059998696 @default.
• W2034785658 cites W2070667507 @default.
• W2034785658 cites W2073405845 @default.
• W2034785658 cites W2073557891 @default.
• W2034785658 cites W2074701657 @default.
• W2034785658 cites W2074785708 @default.
• W2034785658 cites W2074869718 @default.
• W2034785658 cites W2077835601 @default.
• W2034785658 cites W2078316320 @default.
• W2034785658 cites W2078775310 @default.
• W2034785658 cites W2083514564 @default.
• W2034785658 cites W2091404816 @default.
• W2034785658 cites W2092486392 @default.
• W2034785658 cites W2094473032 @default.
• W2034785658 cites W2097899984 @default.
• W2034785658 cites W2106702385 @default.
• W2034785658 cites W2107913200 @default.
• W2034785658 cites W2115734013 @default.
• W2034785658 cites W2127718743 @default.
• W2034785658 cites W2128810077 @default.
• W2034785658 cites W2130530387 @default.
• W2034785658 cites W2133185992 @default.
• W2034785658 cites W2135587043 @default.
• W2034785658 cites W2136535147 @default.
• W2034785658 cites W2136872433 @default.
• W2034785658 cites W2136906322 @default.
• W2034785658 cites W2147278325 @default.
• W2034785658 cites W2149353937 @default.
• W2034785658 cites W2152422855 @default.
• W2034785658 cites W2155994842 @default.
• W2034785658 cites W2161582173 @default.
• W2034785658 cites W2166080731 @default.
• W2034785658 cites W2169124753 @default.
• W2034785658 cites W2417987987 @default.
• W2034785658 cites W84256186 @default.

• next