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Alan V Smrcka - One of the best experts on this subject based on the ideXlab platform.

  • identifiCation of a region at the n terminus of Phospholipase C Beta 3 that interaCts with g protein Beta gamma subunits
    Biochemistry, 2000
    Co-Authors: Alastair J Barr, Hydar Ali, Bodduluri Haribabu, Ralph Snyderman, Alan V Smrcka
    Abstract:

    Members of the Phospholipase C-Beta (PLC-Beta) family of proteins are aCtivated either by G alpha or G Beta gamma subunits of heterotrimeriC G proteins. To define speCifiC regions of PLC-Beta 3 that are involved in binding and aCtivation by G Beta gamma, a series of fragments of PLC-Beta 3 as glutathione-S-transferase (GST) fusion proteins were produCed. A fragment enCompassing the N-terminal pleCkstrin homology (PH) domain and downstream sequenCe (GST-N) bound to G protein Beta 1 gamma 2 in an in vitro binding assay, and binding was inhibited by G protein alpha subunit, G alpha i1. This PLC-Beta 3 fragment also inhibited G Beta gamma-stimulated PLC-Beta aCtivity in a reConstitution system, while having no signifiCant effeCt on G alpha q-stimulated PLC-Beta 3 aCtivity. The N-terminal G Beta gamma binding region was delineated further to the first 180 amino aCids, and the sequenCe Asn150-Ser180, just distal to the PH domain, was found to be required for the interaCtion. Mutation of basiC residues 154Arg, 155Lys, 159Lys, and 161Lys to Glu within this region reduCed G Beta gamma binding affinity and speCifiCally reduCed the EC50 for G Beta gamma-dependent aCtivation of the mutant enzyme 3-fold. Basal aCtivity and G alpha q-dependent aCtivation of the enzyme were unaffeCted by the mutations. While these basiC residues may not direCtly mediate the interaCtion with G Beta gamma, the data provide evidenCe for an N-terminal G Beta gamma binding region of PLC-Beta 3 that is involved in aCtivation of the enzyme.

  • purifiCation and CharaCterization of reCombinant g16 alpha from sf9 Cells aCtivation of purified Phospholipase C isozymes by g protein alpha subunits
    Proceedings of the National Academy of Sciences of the United States of America, 1993
    Co-Authors: Tohru Kozasa, Melvin I. Simon, John R Hepler, Alan V Smrcka, Sue Goo Rhee, Paul C Sternweis, Alfred G Gilman
    Abstract:

    A CDNA enCoding G16 alpha, the alpha subunit of a heterotrimeriC guanine nuCleotide-binding protein, was expressed in Sf9 Cells using reCombinant baCulovirus. G16 alpha in membrane extraCts of Sf9 Cells aCtivated Phospholipase C-Beta 1 (PLC-Beta 1) in the presenCe of guanosine 5'-[gamma-thio]triphosphate; the system Could not be aCtivated by Al3+, Mg2+, and F-. The G16 alpha in the CytosoliC fraCtion of Sf9 Cells did not stimulate PLC-Beta 1. ConCurrent expression of the G-protein Beta gamma subunit Complex inCreased the amount of G16 alpha in Sf9 Cell membranes. The guanosine 5'-[gamma-thio]triphosphate-aCtivated form of G16 alpha was purified from Cholate extraCts of membranes from Cells expressing G16 alpha, and the G-protein Beta 2 and gamma 2 subunits. G16 alpha aCtivated PLC-Beta 1, PLC-Beta 2, and PLC-Beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated aCtivation of PLC-Beta 1 and PLC-Beta 3 greatly exCeeded that of PLC-Beta 2. G16 alpha did not aCtivate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to aCtivate the known isoforms of PLC-Beta.

  • reConstitution of agonist stimulated phosphatidylinositol 4 5 bisphosphate hydrolysis using purified m1 musCariniC reCeptor gq 11 and Phospholipase C Beta 1
    Journal of Biological Chemistry, 1992
    Co-Authors: Gabriel Berstein, Alan V Smrcka, Paul C Sternweis, Jonathan L Blank, John H Exton, Tsutomu Higashijima, Elliott M Ross
    Abstract:

    AbstraCt We desCribe the reConstitution using purified proteins of the m1 musCariniC CholinergiC pathway that aCtivates phosphatidylinositol 4,5-bisphosphate-speCifiC Phospholipase C via the G protein Gq/11. ReCombinant m1 musCariniC reCeptor was Co-reConstituted in lipid vesiCles with either hepatiC Gq/11 or with Cerebral alpha q/11 and Beta gamma subunits. The rate of [35S]GTP gamma S binding to the reConstituted vesiCles was stimulated 20-50-fold by agonist. Maximal reCeptor-Catalyzed binding was 7 mol of GTP gamma S bound per mol of reCeptor. The m2 musCariniC reCeptor was a poor aCtivator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesiCles indiCated that the reCeptor Could maintain up to 40% of the total Coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is Consistent with the rate prediCted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeriC G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indiCated that the reCeptor Catalyzed binding to both alpha q and alpha 11 with about equal effiCienCy. ReCeptor-Catalyzed aCtivation of Gq/11 by GTP gamma S, measured as the ability to aCtivate purified Phospholipase C-Beta 1, paralleled reCeptor-Catalyzed [35S]GTP gamma S binding. Co-reConstitution of reCeptor, Gq/11, and Phospholipase C-Beta 1 restored GTP gamma S-dependent CarbaChol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 reCeptor, Gq/11, and Phospholipase C-Beta 1 are thus suffiCient to initiate the hormonal inositol trisphosphate/diaCylglyCerol signaling pathway without additional proteins.

  • reConstitution of agonist stimulated phosphatidylinositol 4 5 bisphosphate hydrolysis using purified m1 musCariniC reCeptor gq 11 and Phospholipase C β
    Journal of Biological Chemistry, 1992
    Co-Authors: Gabriel Berstein, Alan V Smrcka, Paul C Sternweis, Jonathan L Blank, John H Exton, Tsutomu Higashijima, Elliott M Ross
    Abstract:

    AbstraCt We desCribe the reConstitution using purified proteins of the m1 musCariniC CholinergiC pathway that aCtivates phosphatidylinositol 4,5-bisphosphate-speCifiC Phospholipase C via the G protein Gq/11. ReCombinant m1 musCariniC reCeptor was Co-reConstituted in lipid vesiCles with either hepatiC Gq/11 or with Cerebral alpha q/11 and Beta gamma subunits. The rate of [35S]GTP gamma S binding to the reConstituted vesiCles was stimulated 20-50-fold by agonist. Maximal reCeptor-Catalyzed binding was 7 mol of GTP gamma S bound per mol of reCeptor. The m2 musCariniC reCeptor was a poor aCtivator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesiCles indiCated that the reCeptor Could maintain up to 40% of the total Coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is Consistent with the rate prediCted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeriC G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indiCated that the reCeptor Catalyzed binding to both alpha q and alpha 11 with about equal effiCienCy. ReCeptor-Catalyzed aCtivation of Gq/11 by GTP gamma S, measured as the ability to aCtivate purified Phospholipase C-Beta 1, paralleled reCeptor-Catalyzed [35S]GTP gamma S binding. Co-reConstitution of reCeptor, Gq/11, and Phospholipase C-Beta 1 restored GTP gamma S-dependent CarbaChol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 reCeptor, Gq/11, and Phospholipase C-Beta 1 are thus suffiCient to initiate the hormonal inositol trisphosphate/diaCylglyCerol signaling pathway without additional proteins.

Elliott M Ross - One of the best experts on this subject based on the ideXlab platform.

  • reConstitution of agonist stimulated phosphatidylinositol 4 5 bisphosphate hydrolysis using purified m1 musCariniC reCeptor gq 11 and Phospholipase C Beta 1
    Journal of Biological Chemistry, 1992
    Co-Authors: Gabriel Berstein, Alan V Smrcka, Paul C Sternweis, Jonathan L Blank, John H Exton, Tsutomu Higashijima, Elliott M Ross
    Abstract:

    AbstraCt We desCribe the reConstitution using purified proteins of the m1 musCariniC CholinergiC pathway that aCtivates phosphatidylinositol 4,5-bisphosphate-speCifiC Phospholipase C via the G protein Gq/11. ReCombinant m1 musCariniC reCeptor was Co-reConstituted in lipid vesiCles with either hepatiC Gq/11 or with Cerebral alpha q/11 and Beta gamma subunits. The rate of [35S]GTP gamma S binding to the reConstituted vesiCles was stimulated 20-50-fold by agonist. Maximal reCeptor-Catalyzed binding was 7 mol of GTP gamma S bound per mol of reCeptor. The m2 musCariniC reCeptor was a poor aCtivator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesiCles indiCated that the reCeptor Could maintain up to 40% of the total Coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is Consistent with the rate prediCted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeriC G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indiCated that the reCeptor Catalyzed binding to both alpha q and alpha 11 with about equal effiCienCy. ReCeptor-Catalyzed aCtivation of Gq/11 by GTP gamma S, measured as the ability to aCtivate purified Phospholipase C-Beta 1, paralleled reCeptor-Catalyzed [35S]GTP gamma S binding. Co-reConstitution of reCeptor, Gq/11, and Phospholipase C-Beta 1 restored GTP gamma S-dependent CarbaChol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 reCeptor, Gq/11, and Phospholipase C-Beta 1 are thus suffiCient to initiate the hormonal inositol trisphosphate/diaCylglyCerol signaling pathway without additional proteins.

  • reConstitution of agonist stimulated phosphatidylinositol 4 5 bisphosphate hydrolysis using purified m1 musCariniC reCeptor gq 11 and Phospholipase C β
    Journal of Biological Chemistry, 1992
    Co-Authors: Gabriel Berstein, Alan V Smrcka, Paul C Sternweis, Jonathan L Blank, John H Exton, Tsutomu Higashijima, Elliott M Ross
    Abstract:

    AbstraCt We desCribe the reConstitution using purified proteins of the m1 musCariniC CholinergiC pathway that aCtivates phosphatidylinositol 4,5-bisphosphate-speCifiC Phospholipase C via the G protein Gq/11. ReCombinant m1 musCariniC reCeptor was Co-reConstituted in lipid vesiCles with either hepatiC Gq/11 or with Cerebral alpha q/11 and Beta gamma subunits. The rate of [35S]GTP gamma S binding to the reConstituted vesiCles was stimulated 20-50-fold by agonist. Maximal reCeptor-Catalyzed binding was 7 mol of GTP gamma S bound per mol of reCeptor. The m2 musCariniC reCeptor was a poor aCtivator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesiCles indiCated that the reCeptor Could maintain up to 40% of the total Coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is Consistent with the rate prediCted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeriC G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indiCated that the reCeptor Catalyzed binding to both alpha q and alpha 11 with about equal effiCienCy. ReCeptor-Catalyzed aCtivation of Gq/11 by GTP gamma S, measured as the ability to aCtivate purified Phospholipase C-Beta 1, paralleled reCeptor-Catalyzed [35S]GTP gamma S binding. Co-reConstitution of reCeptor, Gq/11, and Phospholipase C-Beta 1 restored GTP gamma S-dependent CarbaChol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 reCeptor, Gq/11, and Phospholipase C-Beta 1 are thus suffiCient to initiate the hormonal inositol trisphosphate/diaCylglyCerol signaling pathway without additional proteins.

Melvin I. Simon - One of the best experts on this subject based on the ideXlab platform.

  • the n terminus of phosduCin is involved in binding of Beta gamma subunits of g protein
    Proceedings of the National Academy of Sciences of the United States of America, 1995
    Co-Authors: Vladlen Z Slepak, Melvin I. Simon
    Abstract:

    AbstraCt PhosduCin is a soluble phosphoprotein found in retinal photoreCeptor Cells and in the pineal gland. It binds to the Beta gamma subunits of guanine nuCleotide-binding proteins (G proteins) (G Beta gamma) and may regulate G-protein funCtion. In this study, the ability of speCifiC regions of phosduCin to bind G Beta gamma was CharaCterized. A series of deletion mutants were made in bovine phosduCin. They were tested in CotransfeCtion assays for their ability to inhibit G Beta gamma-mediated Phospholipase C Beta 2 isoform aCtivation. Overexpression of the N-terminal half of phosduCin showed inhibition, whereas overexpression of the C-terminal half did not. The first 63 amino aCid residues were required for inhibition. A tryptophan-to-valine substitution at residue 29, whiCh is part of a well Conserved 11-amino aCid sequenCe, severely impaired phosduCin inhibitory funCtion. Glutathione S-transferase-phosduCin fusion proteins were expressed in EsCheriChia Coli to study phosduCin-G Beta gamma interaCtion in vitro. The N-terminal 63-amino aCid fragment was able to bind to G Beta gamma. In Contrast, the C-terminal half failed to bind to G Beta gamma. The substitution mutants showed little or no binding. Furthermore, direCt measurements of interaCtion between G Beta gamma and fragments of phosduCin, using surfaCe plasmon resonanCe teChnology, Confirmed the assignment of binding aCtivity to the 63-amino aCid fragment and the importanCe of the tryptophan residue.

  • purifiCation and CharaCterization of reCombinant g16 alpha from sf9 Cells aCtivation of purified Phospholipase C isozymes by g protein alpha subunits
    Proceedings of the National Academy of Sciences of the United States of America, 1993
    Co-Authors: Tohru Kozasa, Melvin I. Simon, John R Hepler, Alan V Smrcka, Sue Goo Rhee, Paul C Sternweis, Alfred G Gilman
    Abstract:

    A CDNA enCoding G16 alpha, the alpha subunit of a heterotrimeriC guanine nuCleotide-binding protein, was expressed in Sf9 Cells using reCombinant baCulovirus. G16 alpha in membrane extraCts of Sf9 Cells aCtivated Phospholipase C-Beta 1 (PLC-Beta 1) in the presenCe of guanosine 5'-[gamma-thio]triphosphate; the system Could not be aCtivated by Al3+, Mg2+, and F-. The G16 alpha in the CytosoliC fraCtion of Sf9 Cells did not stimulate PLC-Beta 1. ConCurrent expression of the G-protein Beta gamma subunit Complex inCreased the amount of G16 alpha in Sf9 Cell membranes. The guanosine 5'-[gamma-thio]triphosphate-aCtivated form of G16 alpha was purified from Cholate extraCts of membranes from Cells expressing G16 alpha, and the G-protein Beta 2 and gamma 2 subunits. G16 alpha aCtivated PLC-Beta 1, PLC-Beta 2, and PLC-Beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated aCtivation of PLC-Beta 1 and PLC-Beta 3 greatly exCeeded that of PLC-Beta 2. G16 alpha did not aCtivate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to aCtivate the known isoforms of PLC-Beta.

  • aCtivation of Phospholipase C Beta 2 by the alpha and Beta gamma subunits of trimeriC gtp binding protein
    Proceedings of the National Academy of Sciences of the United States of America, 1993
    Co-Authors: Arieh A Katz, Melvin I. Simon
    Abstract:

    CotransfeCtion assays were used to show that the members of the GTP-binding protein Gq Class of alpha subunits Could aCtivate Phospholipase C (PLC) Beta 2. Similar experiments also demonstrated that G Beta 1 gamma 1, G Beta 1 gamma 5, and G Beta 2 gamma 5 Could aCtivate the Beta 2 isoform of PLC but not the Beta 1 isoform, while G Beta 2 gamma 1 did not aCtivate PLC Beta 2. To determine whiCh portions of PLC Beta 2 are required for aCtivation by G Beta gamma or G alpha, a number of PLC Beta 2 deletion mutants and Chimeras Composed of various portions of PLC Beta 1 and PLC Beta 2 were prepared. We identified the N-terminal segment of PLC Beta 2 with amino aCid sequenCe extending to the end of the Y box as the region required for aCtivation by G Beta gamma and the C-terminal region as the segment Containing amino aCid sequenCes required for aCtivation by G alpha. Furthermore, we found that Coexpression of G alpha 16 and G Beta 1 gamma 1 but not G Beta 1 gamma 5 in COS-7 Cells was able to synergistiCally aCtivate reCombinant PLC Beta 2. We suggest that G alpha 16 may aCt together with free G Beta 1 gamma 1 to aCtivate PLC Beta 2, while G alpha 16 may form heterotrimeriC Complexes with G Beta 1 gamma 5 and be stabilized in an inaCtive form. We ConClude that the regions of PLC Beta 2 required for aCtivation by G Beta gamma and G alpha are physiCally separate and that the nature of the G Beta subunit may play a role in determining the relative speCifiCity of the G Beta gamma Complex for effeCtor aCtivation while the nature of the G gamma subunit isoform may be important for determining the affinity of the G Beta gamma Complex for speCifiC G alpha proteins.

  • ACtivation of Phospholipase C by alpha 1-adrenergiC reCeptors is mediated by the alpha subunits of Gq family.
    The Journal of biological chemistry, 1992
    Co-Authors: Arieh Katz, Chang Ho Lee, Melvin I. Simon
    Abstract:

    High effiCienCy transient transfeCtion of Cos-7 Cells was previously used to establish the funCtional Coupling between G alpha q/G alpha 11 and Phospholipase C Beta 1 (Wu, D., Lee, C-H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817). Here the same system was used to study the funCtional Coupling between other guanine nuCleotide-binding regulatory protein (G-protein) alpha subunits and Phospholipases and to study whiCh G alpha subunits mediate the aCtivation of Phospholipase C by the alpha 1-adrenergiC reCeptor subtypes, alpha 1 A, alpha 1 B, and alpha 1 C. We found that G alpha 14 and G alpha 16 behaved like G alpha 11 or G alpha q, i.e. they Could aCtivate endogenous Phospholipases in Cos-7 Cells in the presenCe of AIFn. The synergistiC inCrease in inositol phosphate release in Cos-7 Cells after they were CotransfeCted with CDNAs enCoding G alpha subunits and Phospholipase C Beta 1 indiCates that both G alpha 16 and G alpha 14 Can aCtivate Phospholipase C Beta 1. The aCtivation of Phospholipase C Beta 1 was restriCted to members of the Gq subfamily of alpha subunits. They aCtivated Phospholipase C Beta 1 but not Phospholipase C gamma 1, gamma 2, or Phospholipase C delta 3. The CotransfeCtion of Cos-7 Cells with CDNAs enCoding three different alpha 1-adrenergiC reCeptors and G alpha q or G alpha 11 leads to an inCrease in norepinephrine-dependent inositol phosphate release. This indiCates that G alpha q or G alpha 11 Can mediate the aCtivation of Phospholipase C by all three subtypes of alpha 1-adrenergiC reCeptors. With the same assay system, G alpha 16 and G alpha 14 appear to be differentially involved in the aCtivation of Phospholipase C by the alpha 1-adrenergiC reCeptors. The alpha 1 B subtype reCeptor gave a ligand-mediated synergistiC response in the Cells CotransfeCted with either G alpha 14 or G alpha 16. However, the alpha 1 C reCeptor responded in Cells CotransfeCted with G alpha 14 but not G alpha 16, and the alpha 1 A reCeptor showed little synergistiC response in Cells transfeCted with either G alpha 14 or G alpha 16. The ability of the alpha 1 A and alpha 1 C reCeptors to aCtivate Phospholipase C through G alpha q and G alpha 11 was also demonstrated in a Cell-free system.(ABSTRACT TRUNCATED AT 400 WORDS)

  • members of the gq alpha subunit gene family aCtivate Phospholipase C Beta isozymes
    Journal of Biological Chemistry, 1992
    Co-Authors: Chang Ho Lee, Sue Goo Rhee, D Park, Melvin I. Simon
    Abstract:

    The relative speCifiCities of members of the Gα_q family of GTP-binding proteins were tested for their ability to aCtivate different phosphoinositide-speCifiC Phospholipase C (PI-PLC) β isozymes. Cos-7 Cells were transfeCted with CDNA Corresponding to Gα_q, Gα_(11), Gα_(14), and Gα_(16). Most of the reCombinant protein was bound to the Cell membrane and these membranes were washed to elute endogenous PI-PLC aCtivity. The membrane preparation was reConstituted with purified preparations of the PI-PLC β isozymes and guanosine 5'-O-thiotriphosphate (GTPyS)-stimulated enzyme aCtivity was measured. All four proteins of the Gα_q family were found to stimulate PI-PLC β1, with Gα_q and Gα_(11) being most effiCient. On the other hand, Gα_(16) was found to most effeCtively aCtivate PI-PLC β2, while Gα_q, Gα_(11), and Gα_(14) showed less stimulation. SpeCifiC anti- Gα_(16) antibody bloCked the stimulation of both PI-PLC β1 and PI-PLC β2 in the enriChed membrane fraCtion. We ConClude that there is speCifiCity in the interaCtion of different members of the G_q family with different PI-PLC β effeCtors. This speCifiCity may be important in generating tissue- or reCeptor-speCifiC responses in vivo.

Gabriel Berstein - One of the best experts on this subject based on the ideXlab platform.

  • reConstitution of agonist stimulated phosphatidylinositol 4 5 bisphosphate hydrolysis using purified m1 musCariniC reCeptor gq 11 and Phospholipase C Beta 1
    Journal of Biological Chemistry, 1992
    Co-Authors: Gabriel Berstein, Alan V Smrcka, Paul C Sternweis, Jonathan L Blank, John H Exton, Tsutomu Higashijima, Elliott M Ross
    Abstract:

    AbstraCt We desCribe the reConstitution using purified proteins of the m1 musCariniC CholinergiC pathway that aCtivates phosphatidylinositol 4,5-bisphosphate-speCifiC Phospholipase C via the G protein Gq/11. ReCombinant m1 musCariniC reCeptor was Co-reConstituted in lipid vesiCles with either hepatiC Gq/11 or with Cerebral alpha q/11 and Beta gamma subunits. The rate of [35S]GTP gamma S binding to the reConstituted vesiCles was stimulated 20-50-fold by agonist. Maximal reCeptor-Catalyzed binding was 7 mol of GTP gamma S bound per mol of reCeptor. The m2 musCariniC reCeptor was a poor aCtivator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesiCles indiCated that the reCeptor Could maintain up to 40% of the total Coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is Consistent with the rate prediCted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeriC G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indiCated that the reCeptor Catalyzed binding to both alpha q and alpha 11 with about equal effiCienCy. ReCeptor-Catalyzed aCtivation of Gq/11 by GTP gamma S, measured as the ability to aCtivate purified Phospholipase C-Beta 1, paralleled reCeptor-Catalyzed [35S]GTP gamma S binding. Co-reConstitution of reCeptor, Gq/11, and Phospholipase C-Beta 1 restored GTP gamma S-dependent CarbaChol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 reCeptor, Gq/11, and Phospholipase C-Beta 1 are thus suffiCient to initiate the hormonal inositol trisphosphate/diaCylglyCerol signaling pathway without additional proteins.

  • reConstitution of agonist stimulated phosphatidylinositol 4 5 bisphosphate hydrolysis using purified m1 musCariniC reCeptor gq 11 and Phospholipase C β
    Journal of Biological Chemistry, 1992
    Co-Authors: Gabriel Berstein, Alan V Smrcka, Paul C Sternweis, Jonathan L Blank, John H Exton, Tsutomu Higashijima, Elliott M Ross
    Abstract:

    AbstraCt We desCribe the reConstitution using purified proteins of the m1 musCariniC CholinergiC pathway that aCtivates phosphatidylinositol 4,5-bisphosphate-speCifiC Phospholipase C via the G protein Gq/11. ReCombinant m1 musCariniC reCeptor was Co-reConstituted in lipid vesiCles with either hepatiC Gq/11 or with Cerebral alpha q/11 and Beta gamma subunits. The rate of [35S]GTP gamma S binding to the reConstituted vesiCles was stimulated 20-50-fold by agonist. Maximal reCeptor-Catalyzed binding was 7 mol of GTP gamma S bound per mol of reCeptor. The m2 musCariniC reCeptor was a poor aCtivator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesiCles indiCated that the reCeptor Could maintain up to 40% of the total Coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is Consistent with the rate prediCted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeriC G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indiCated that the reCeptor Catalyzed binding to both alpha q and alpha 11 with about equal effiCienCy. ReCeptor-Catalyzed aCtivation of Gq/11 by GTP gamma S, measured as the ability to aCtivate purified Phospholipase C-Beta 1, paralleled reCeptor-Catalyzed [35S]GTP gamma S binding. Co-reConstitution of reCeptor, Gq/11, and Phospholipase C-Beta 1 restored GTP gamma S-dependent CarbaChol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 reCeptor, Gq/11, and Phospholipase C-Beta 1 are thus suffiCient to initiate the hormonal inositol trisphosphate/diaCylglyCerol signaling pathway without additional proteins.

Paul C Sternweis - One of the best experts on this subject based on the ideXlab platform.

  • purifiCation and CharaCterization of reCombinant g16 alpha from sf9 Cells aCtivation of purified Phospholipase C isozymes by g protein alpha subunits
    Proceedings of the National Academy of Sciences of the United States of America, 1993
    Co-Authors: Tohru Kozasa, Melvin I. Simon, John R Hepler, Alan V Smrcka, Sue Goo Rhee, Paul C Sternweis, Alfred G Gilman
    Abstract:

    A CDNA enCoding G16 alpha, the alpha subunit of a heterotrimeriC guanine nuCleotide-binding protein, was expressed in Sf9 Cells using reCombinant baCulovirus. G16 alpha in membrane extraCts of Sf9 Cells aCtivated Phospholipase C-Beta 1 (PLC-Beta 1) in the presenCe of guanosine 5'-[gamma-thio]triphosphate; the system Could not be aCtivated by Al3+, Mg2+, and F-. The G16 alpha in the CytosoliC fraCtion of Sf9 Cells did not stimulate PLC-Beta 1. ConCurrent expression of the G-protein Beta gamma subunit Complex inCreased the amount of G16 alpha in Sf9 Cell membranes. The guanosine 5'-[gamma-thio]triphosphate-aCtivated form of G16 alpha was purified from Cholate extraCts of membranes from Cells expressing G16 alpha, and the G-protein Beta 2 and gamma 2 subunits. G16 alpha aCtivated PLC-Beta 1, PLC-Beta 2, and PLC-Beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated aCtivation of PLC-Beta 1 and PLC-Beta 3 greatly exCeeded that of PLC-Beta 2. G16 alpha did not aCtivate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to aCtivate the known isoforms of PLC-Beta.

  • reConstitution of agonist stimulated phosphatidylinositol 4 5 bisphosphate hydrolysis using purified m1 musCariniC reCeptor gq 11 and Phospholipase C Beta 1
    Journal of Biological Chemistry, 1992
    Co-Authors: Gabriel Berstein, Alan V Smrcka, Paul C Sternweis, Jonathan L Blank, John H Exton, Tsutomu Higashijima, Elliott M Ross
    Abstract:

    AbstraCt We desCribe the reConstitution using purified proteins of the m1 musCariniC CholinergiC pathway that aCtivates phosphatidylinositol 4,5-bisphosphate-speCifiC Phospholipase C via the G protein Gq/11. ReCombinant m1 musCariniC reCeptor was Co-reConstituted in lipid vesiCles with either hepatiC Gq/11 or with Cerebral alpha q/11 and Beta gamma subunits. The rate of [35S]GTP gamma S binding to the reConstituted vesiCles was stimulated 20-50-fold by agonist. Maximal reCeptor-Catalyzed binding was 7 mol of GTP gamma S bound per mol of reCeptor. The m2 musCariniC reCeptor was a poor aCtivator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesiCles indiCated that the reCeptor Could maintain up to 40% of the total Coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is Consistent with the rate prediCted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeriC G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indiCated that the reCeptor Catalyzed binding to both alpha q and alpha 11 with about equal effiCienCy. ReCeptor-Catalyzed aCtivation of Gq/11 by GTP gamma S, measured as the ability to aCtivate purified Phospholipase C-Beta 1, paralleled reCeptor-Catalyzed [35S]GTP gamma S binding. Co-reConstitution of reCeptor, Gq/11, and Phospholipase C-Beta 1 restored GTP gamma S-dependent CarbaChol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 reCeptor, Gq/11, and Phospholipase C-Beta 1 are thus suffiCient to initiate the hormonal inositol trisphosphate/diaCylglyCerol signaling pathway without additional proteins.

  • reConstitution of agonist stimulated phosphatidylinositol 4 5 bisphosphate hydrolysis using purified m1 musCariniC reCeptor gq 11 and Phospholipase C β
    Journal of Biological Chemistry, 1992
    Co-Authors: Gabriel Berstein, Alan V Smrcka, Paul C Sternweis, Jonathan L Blank, John H Exton, Tsutomu Higashijima, Elliott M Ross
    Abstract:

    AbstraCt We desCribe the reConstitution using purified proteins of the m1 musCariniC CholinergiC pathway that aCtivates phosphatidylinositol 4,5-bisphosphate-speCifiC Phospholipase C via the G protein Gq/11. ReCombinant m1 musCariniC reCeptor was Co-reConstituted in lipid vesiCles with either hepatiC Gq/11 or with Cerebral alpha q/11 and Beta gamma subunits. The rate of [35S]GTP gamma S binding to the reConstituted vesiCles was stimulated 20-50-fold by agonist. Maximal reCeptor-Catalyzed binding was 7 mol of GTP gamma S bound per mol of reCeptor. The m2 musCariniC reCeptor was a poor aCtivator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesiCles indiCated that the reCeptor Could maintain up to 40% of the total Coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is Consistent with the rate prediCted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeriC G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indiCated that the reCeptor Catalyzed binding to both alpha q and alpha 11 with about equal effiCienCy. ReCeptor-Catalyzed aCtivation of Gq/11 by GTP gamma S, measured as the ability to aCtivate purified Phospholipase C-Beta 1, paralleled reCeptor-Catalyzed [35S]GTP gamma S binding. Co-reConstitution of reCeptor, Gq/11, and Phospholipase C-Beta 1 restored GTP gamma S-dependent CarbaChol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 reCeptor, Gq/11, and Phospholipase C-Beta 1 are thus suffiCient to initiate the hormonal inositol trisphosphate/diaCylglyCerol signaling pathway without additional proteins.