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T K Harden - One of the best experts on this subject based on the ideXlab platform.

  • expression of a Cloned p2y purinergiC reCeptor that Couples to Phospholipase C
    Molecular Pharmacology, 1994
    Co-Authors: Theresa M Filtz, Jose L Boyer, Robert A Nicholas, T K Harden
    Abstract:

    P2Y purinergiC reCeptors previously have been shown to Couple either to aCtivation of Phospholipase C through a pertussis toxin-insensitive meChanism or to inhibition of adenylyl CyClase through pertussis toxin-sensitive members of the G1 family of G proteins. These and other pharmaCologiCal data strongly suggest that multiple P2Y purinergiC reCeptors exist. Webb et al. [FEBS Lett. 324:219-225 (1993)] Cloned a CDNA that, when expressed in frog ooCytes, displayed the general pharmaCologiCal CharaCteristiCs of a P2Y purinergiC reCeptor but whose seCond messenger linkage was not resolved. We have now Cloned the meleagrid (turkey) homologue of the previously Cloned ChiCk P2Y purinergiC reCeptor and have stably expressed it in a heterologous human Cell line (1321N1 astroCytoma Cells) to establish its signaling properties. The purinergiC reCeptor agonist 2-methylthio-ATP (2MeSATP) stimulated a marked aCtivation of Phospholipase C in 1321N1 Cells stably expressing the meleagrid reCeptor. The order of potenCy of a series of analogues of ATP and ADP for stimulation of Phospholipase C by the reCeptor expressed in 1321N1 Cells [2MeSATP = 2-methylthio-ADP > adenosine 5'-O-(2-thio)diphosphate > ADP > 2-Chloro-ATP = adenosine 5'-O-(3-thio)triphosphate > or = ATP > adenylyl-imidodiphosphate > UTP] was similar to that observed for P2Y purinergiC reCeptors in turkey erythroCytes and many other tissues and was markedly different from those of the P2U and P2X purinergiC reCeptor subtypes. Stimulation of inositol lipid hydrolysis by P2Y purinergiC agonists was not affeCted by preinCubation of Cells with pertussis toxin. In Contrast to its marked effeCts on Phospholipase C aCtivity, 2MeSATP Caused only a small and variable inhibition of CAMP aCCumulation. RibonuClease proteCtion analysis of turkey tissues showed that this P2Y purinergiC reCeptor is most highly expressed in blood and brain. Taken together, these results indiCate that a Phospholipase-C-aCtivating P2Y purinergiC reCeptor has been Cloned and stably expressed in 1321N1 astroCytoma Cells.

  • beta gamma subunit aCtivation of g protein regulated Phospholipase C
    Journal of Biological Chemistry, 1992
    Co-Authors: Jose L Boyer, G L Waldo, T K Harden
    Abstract:

    AbstraCt The availability of purified G alpha 11 and the G-protein-regulated Phospholipase C from turkey erythroCytes has allowed an examination of the direCt effeCts of G-protein beta gamma-subunit on the Components of the inositol lipid signaling system. ReConstitution of purified turkey erythroCyte or bovine brain beta gamma-subunit into phospholipid vesiCles Containing G alpha 11 inhibited AlF4- induCed aCtivation of Phospholipase C. However, beta gamma-subunit at higher ConCentrations inCreased Phospholipase C aCtivity. This stimulatory effeCt of beta gamma-subunit on Phospholipase C did not require the presenCe of the alpha-subunit. G alpha o had no effeCt on the CatalytiC aCtivity of Phospholipase C. However, CoreConstitution of G alpha o and beta gamma-subunit shifted to the right the ConCentration-effeCt Curve for beta gamma-subunit-promoted aCtivation of Phospholipase C. As was observed with G alpha 11, the inCrease in aCtivity observed in the presenCe of beta gamma-subunit oCCurred as an inCrease in the maximal aCtivity and with no Change in the apparent affinity for Ca2+ for Phospholipase C aCtivation. The ConCentration dependenCe of G alpha 11 for aCtivation of turkey erythroCyte Phospholipase C and bovine brain Phospholipase C-beta, as well as the ConCentration dependenCe of the two enzymes for aCtivation by G alpha 11, were very similar. In Contrast, beta gamma-subunit was a muCh less effeCtive aCtivator of bovine brain Phospholipase C-beta than the turkey erythroCyte enzyme. The observation of direCt effeCts of free beta gamma-subunit on Phospholipase C extend the possibilities for reCeptor-mediated regulation of this signaling pathway.

Gary J Fisher - One of the best experts on this subject based on the ideXlab platform.

  • reConstitution of thromboxane a2 reCeptor stimulated phosphoinositide hydrolysis in isolated platelet membranes involvement of phosphoinositide speCifiC Phospholipase C β and gtp binding protein gq
    Biochemical Journal, 1993
    Co-Authors: Joseph J Baldassare, Alan Tarver, Patricia A Henderson, W M Mackin, B Sahagan, Gary J Fisher
    Abstract:

    ACtivation of human platelets by the araChidoniC aCid metabolite thromboxane A2 and the thromboxane A2 mimiC U46619 is mediated through phosphoinositide-speCifiC Phospholipase C-Catalysed hydrolysis of phosphoinositides. We have established Conditions to reConstitute U46619-stimulated phosphoinositide breakdown by addition of guanine nuCleotides and soluble platelet Phospholipase C aCtivities to isolated 32P-labelled membranes. ReCeptor-aCtivated phosphoinositide hydrolysis was observed in the presenCe of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 miCroM and 500 nM respeCtively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet Cytosol and their ability to reConstitute GTP[S]-dependent and GTP/U46619-dependent phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not Phospholipase C-gamma 1 or -gamma 2, Catalysed phosphoinositide breakdown in the presenCe of GTP[S]. In Contrast, only Phospholipase C-beta was able to reConstitute GTP-dependent U46619-induCed hydrolysis. The partiCipation of GTP-regulatory proteins in the reConstitution of GTP[S]- and GTP/U46619-induCed phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeriC GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reConstitution of phosphoinositide hydrolysis with Phospholipase C-beta. In Contrast GTP[S]-stimulated hydrolysis by Phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the funCtional speCifiCity of GTP-binding proteins and Phospholipase C isoenzymes in mediating agonist-induCed phosphoinositide hydrolysis in human platelets.

  • reConstitution of thromboxane a2 reCeptor stimulated phosphoinositide hydrolysis in isolated platelet membranes involvement of phosphoinositide speCifiC Phospholipase C beta and gtp binding protein gq
    Biochemical Journal, 1993
    Co-Authors: Joseph J Baldassare, Alan Tarver, Patricia A Henderson, W M Mackin, B Sahagan, Gary J Fisher
    Abstract:

    ACtivation of human platelets by the araChidoniC aCid metabolite thromboxane A2 and the thromboxane A2 mimiC U46619 is mediated through phosphoinositide-speCifiC Phospholipase C-Catalysed hydrolysis of phosphoinositides. We have established Conditions to reConstitute U46619-stimulated phosphoinositide breakdown by addition of guanine nuCleotides and soluble platelet Phospholipase C aCtivities to isolated 32P-labelled membranes. ReCeptor-aCtivated phosphoinositide hydrolysis was observed in the presenCe of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 miCroM and 500 nM respeCtively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet Cytosol and their ability to reConstitute GTP[S]-dependent and GTP/U46619-dependent phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not Phospholipase C-gamma 1 or -gamma 2, Catalysed phosphoinositide breakdown in the presenCe of GTP[S]. In Contrast, only Phospholipase C-beta was able to reConstitute GTP-dependent U46619-induCed hydrolysis. The partiCipation of GTP-regulatory proteins in the reConstitution of GTP[S]- and GTP/U46619-induCed phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeriC GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reConstitution of phosphoinositide hydrolysis with Phospholipase C-beta. In Contrast GTP[S]-stimulated hydrolysis by Phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the funCtional speCifiCity of GTP-binding proteins and Phospholipase C isoenzymes in mediating agonist-induCed phosphoinositide hydrolysis in human platelets.

Jose L Boyer - One of the best experts on this subject based on the ideXlab platform.

  • expression of a Cloned p2y purinergiC reCeptor that Couples to Phospholipase C
    Molecular Pharmacology, 1994
    Co-Authors: Theresa M Filtz, Jose L Boyer, Robert A Nicholas, T K Harden
    Abstract:

    P2Y purinergiC reCeptors previously have been shown to Couple either to aCtivation of Phospholipase C through a pertussis toxin-insensitive meChanism or to inhibition of adenylyl CyClase through pertussis toxin-sensitive members of the G1 family of G proteins. These and other pharmaCologiCal data strongly suggest that multiple P2Y purinergiC reCeptors exist. Webb et al. [FEBS Lett. 324:219-225 (1993)] Cloned a CDNA that, when expressed in frog ooCytes, displayed the general pharmaCologiCal CharaCteristiCs of a P2Y purinergiC reCeptor but whose seCond messenger linkage was not resolved. We have now Cloned the meleagrid (turkey) homologue of the previously Cloned ChiCk P2Y purinergiC reCeptor and have stably expressed it in a heterologous human Cell line (1321N1 astroCytoma Cells) to establish its signaling properties. The purinergiC reCeptor agonist 2-methylthio-ATP (2MeSATP) stimulated a marked aCtivation of Phospholipase C in 1321N1 Cells stably expressing the meleagrid reCeptor. The order of potenCy of a series of analogues of ATP and ADP for stimulation of Phospholipase C by the reCeptor expressed in 1321N1 Cells [2MeSATP = 2-methylthio-ADP > adenosine 5'-O-(2-thio)diphosphate > ADP > 2-Chloro-ATP = adenosine 5'-O-(3-thio)triphosphate > or = ATP > adenylyl-imidodiphosphate > UTP] was similar to that observed for P2Y purinergiC reCeptors in turkey erythroCytes and many other tissues and was markedly different from those of the P2U and P2X purinergiC reCeptor subtypes. Stimulation of inositol lipid hydrolysis by P2Y purinergiC agonists was not affeCted by preinCubation of Cells with pertussis toxin. In Contrast to its marked effeCts on Phospholipase C aCtivity, 2MeSATP Caused only a small and variable inhibition of CAMP aCCumulation. RibonuClease proteCtion analysis of turkey tissues showed that this P2Y purinergiC reCeptor is most highly expressed in blood and brain. Taken together, these results indiCate that a Phospholipase-C-aCtivating P2Y purinergiC reCeptor has been Cloned and stably expressed in 1321N1 astroCytoma Cells.

  • beta gamma subunit aCtivation of g protein regulated Phospholipase C
    Journal of Biological Chemistry, 1992
    Co-Authors: Jose L Boyer, G L Waldo, T K Harden
    Abstract:

    AbstraCt The availability of purified G alpha 11 and the G-protein-regulated Phospholipase C from turkey erythroCytes has allowed an examination of the direCt effeCts of G-protein beta gamma-subunit on the Components of the inositol lipid signaling system. ReConstitution of purified turkey erythroCyte or bovine brain beta gamma-subunit into phospholipid vesiCles Containing G alpha 11 inhibited AlF4- induCed aCtivation of Phospholipase C. However, beta gamma-subunit at higher ConCentrations inCreased Phospholipase C aCtivity. This stimulatory effeCt of beta gamma-subunit on Phospholipase C did not require the presenCe of the alpha-subunit. G alpha o had no effeCt on the CatalytiC aCtivity of Phospholipase C. However, CoreConstitution of G alpha o and beta gamma-subunit shifted to the right the ConCentration-effeCt Curve for beta gamma-subunit-promoted aCtivation of Phospholipase C. As was observed with G alpha 11, the inCrease in aCtivity observed in the presenCe of beta gamma-subunit oCCurred as an inCrease in the maximal aCtivity and with no Change in the apparent affinity for Ca2+ for Phospholipase C aCtivation. The ConCentration dependenCe of G alpha 11 for aCtivation of turkey erythroCyte Phospholipase C and bovine brain Phospholipase C-beta, as well as the ConCentration dependenCe of the two enzymes for aCtivation by G alpha 11, were very similar. In Contrast, beta gamma-subunit was a muCh less effeCtive aCtivator of bovine brain Phospholipase C-beta than the turkey erythroCyte enzyme. The observation of direCt effeCts of free beta gamma-subunit on Phospholipase C extend the possibilities for reCeptor-mediated regulation of this signaling pathway.

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

  • gα15 and gα16 Couple a wide variety of reCeptors to Phospholipase C
    Journal of Biological Chemistry, 1995
    Co-Authors: Stefan Offermanns, Melvin I. Simon
    Abstract:

    The murine G-protein α-subunit Gα15 and its human Counterpart Gα16 are expressed in a subset of hematopoietiC Cells, and they have been shown to regulate β-isoforms of inositide-speCifiC Phospholipase C. We studied the ability of a variety of reCeptors to interaCt with Gα15 and Gα16 by CotransfeCting reCeptors and G-protein α-subunits in COS-7 Cells. ACtivation of β2 adrenergiC and musCariniC M2 reCeptors in Cells expressing the reCeptors alone or together with Gαq, Gα11, or Gα14 led to a very small stimulation of endogenous Phospholipase C. However, when the reCeptors were Coexpressed with Gα15 and Gα16, addition of appropriate ligands Caused a severalfold inCrease in inositol phosphate produCtion whiCh was time- and dose-dependent. A similar aCtivation of Phospholipase C was observed when several other reCeptors whiCh were previously shown to Couple to members of the Gi and Gs family were Coexpressed with Gα15/16. In addition, stimulation of inositol phosphate formation via reCeptors naturally Coupled to Phospholipase C was enhanCed by CotransfeCtion of Gα15 and Gα16. These data demonstrate that Gα15 and Gα16 are unique in that they Can be aCtivated by a wide variety of G-protein-Coupled reCeptors. The ability of Gα15 and Gα16 to bypass the seleCtivity of reCeptor G-protein interaCtion Can be a useful tool to understand the meChanism of reCeptor-induCed G-protein aCtivation. In addition, the promisCuous behavior of Gα15 and Gα16 toward reCeptors may be helpful in finding ligands Corresponding to orphan reCeptors whose signaling properties are unknown.

  • 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)

Joseph J Baldassare - One of the best experts on this subject based on the ideXlab platform.

  • reConstitution of thromboxane a2 reCeptor stimulated phosphoinositide hydrolysis in isolated platelet membranes involvement of phosphoinositide speCifiC Phospholipase C β and gtp binding protein gq
    Biochemical Journal, 1993
    Co-Authors: Joseph J Baldassare, Alan Tarver, Patricia A Henderson, W M Mackin, B Sahagan, Gary J Fisher
    Abstract:

    ACtivation of human platelets by the araChidoniC aCid metabolite thromboxane A2 and the thromboxane A2 mimiC U46619 is mediated through phosphoinositide-speCifiC Phospholipase C-Catalysed hydrolysis of phosphoinositides. We have established Conditions to reConstitute U46619-stimulated phosphoinositide breakdown by addition of guanine nuCleotides and soluble platelet Phospholipase C aCtivities to isolated 32P-labelled membranes. ReCeptor-aCtivated phosphoinositide hydrolysis was observed in the presenCe of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 miCroM and 500 nM respeCtively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet Cytosol and their ability to reConstitute GTP[S]-dependent and GTP/U46619-dependent phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not Phospholipase C-gamma 1 or -gamma 2, Catalysed phosphoinositide breakdown in the presenCe of GTP[S]. In Contrast, only Phospholipase C-beta was able to reConstitute GTP-dependent U46619-induCed hydrolysis. The partiCipation of GTP-regulatory proteins in the reConstitution of GTP[S]- and GTP/U46619-induCed phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeriC GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reConstitution of phosphoinositide hydrolysis with Phospholipase C-beta. In Contrast GTP[S]-stimulated hydrolysis by Phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the funCtional speCifiCity of GTP-binding proteins and Phospholipase C isoenzymes in mediating agonist-induCed phosphoinositide hydrolysis in human platelets.

  • reConstitution of thromboxane a2 reCeptor stimulated phosphoinositide hydrolysis in isolated platelet membranes involvement of phosphoinositide speCifiC Phospholipase C beta and gtp binding protein gq
    Biochemical Journal, 1993
    Co-Authors: Joseph J Baldassare, Alan Tarver, Patricia A Henderson, W M Mackin, B Sahagan, Gary J Fisher
    Abstract:

    ACtivation of human platelets by the araChidoniC aCid metabolite thromboxane A2 and the thromboxane A2 mimiC U46619 is mediated through phosphoinositide-speCifiC Phospholipase C-Catalysed hydrolysis of phosphoinositides. We have established Conditions to reConstitute U46619-stimulated phosphoinositide breakdown by addition of guanine nuCleotides and soluble platelet Phospholipase C aCtivities to isolated 32P-labelled membranes. ReCeptor-aCtivated phosphoinositide hydrolysis was observed in the presenCe of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 miCroM and 500 nM respeCtively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet Cytosol and their ability to reConstitute GTP[S]-dependent and GTP/U46619-dependent phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not Phospholipase C-gamma 1 or -gamma 2, Catalysed phosphoinositide breakdown in the presenCe of GTP[S]. In Contrast, only Phospholipase C-beta was able to reConstitute GTP-dependent U46619-induCed hydrolysis. The partiCipation of GTP-regulatory proteins in the reConstitution of GTP[S]- and GTP/U46619-induCed phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeriC GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reConstitution of phosphoinositide hydrolysis with Phospholipase C-beta. In Contrast GTP[S]-stimulated hydrolysis by Phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the funCtional speCifiCity of GTP-binding proteins and Phospholipase C isoenzymes in mediating agonist-induCed phosphoinositide hydrolysis in human platelets.