Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 38
Filter
Add more filters










Publication year range
1.
Clin Exp Immunol ; 200(1): 53-60, 2020 04.
Article in English | MEDLINE | ID: mdl-31867717

ABSTRACT

Phospholipase C epsilon 1 (PLCε1) is a unique member of the phospholipase family, in that it also functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rap1. It is this function as a Rap1 GEF that gives PLCε1 an essential role in chemokine-mediated T cell adhesion. We have utilized a syngeneic tumor model, MC38 cells in C57BL/6 mice, and observed that tumors grow larger and more quickly in the absence of PLCε1. Single-cell analysis revealed an increased CD4+ /CD8+ ratio in the spleens, lymph nodes and tumors of PLCε1 knock-out tumor-bearing mice. T cells isolated from PLCε1 knock-out mice were less activated by multiple phenotypical parameters than those from wild-type mice. We additionally noted a decrease in expression of the chemokine receptors C-X-C chemokine receptor type 4 (CXCR4) and C-C motif chemokine receptor 4 (CCR4) on CD4+ T cells from the spleens, lymph nodes and tumors of PLCε1 knock-out mice compared to wild-type mice, and diminished migration of PLCε1-depleted CD3+ T cells towards stromal cell-derived factor (SDF)-1α. Based on these results, we conclude that PLCε1 is a potential regulator of tumor-infiltrating lymphocytes, functioning, at least in part, at the level of T cell trafficking and recruitment.


Subject(s)
Cell Movement/genetics , Gene Expression Regulation, Neoplastic , Neoplasms, Experimental/genetics , Phosphoinositide Phospholipase C/genetics , T-Lymphocytes/metabolism , Tumor Burden/genetics , Animals , Cell Line, Tumor , Chemokine CXCL12/genetics , Chemokine CXCL12/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Neoplasms, Experimental/immunology , Neoplasms, Experimental/metabolism , Phosphoinositide Phospholipase C/deficiency , Receptors, CCR4/genetics , Receptors, CXCR4/genetics
2.
Mol Biol Cell ; 26(6): 1188-98, 2015 Mar 15.
Article in English | MEDLINE | ID: mdl-25609085

ABSTRACT

We recently identified a novel GPCR-dependent pathway for regulation of cardiac hypertrophy that depends on Golgi phosphatidylinositol 4-phosphate (PI4P) hydrolysis by a specific isoform of phospholipase C (PLC), PLCε, at the nuclear envelope. How stimuli are transmitted from cell surface GPCRs to activation of perinuclear PLCε is not clear. Here we tested the role of G protein ßγ subunits. Gßγ inhibition blocked ET-1-stimulated Golgi PI4P depletion in neonatal and adult ventricular myocytes. Blocking Gßγ at the Golgi inhibited ET-1-dependent PI4P depletion and nuclear PKD activation. Translocation of Gßγ to the Golgi stimulated perinuclear Golgi PI4P depletion and nuclear PKD activation. Finally, blocking Gßγ at the Golgi or PM blocked ET-1-dependent cardiomyocyte hypertrophy. These data indicate that Gßγ regulation of the perinuclear Golgi PI4P pathway and a separate pathway at the PM is required for ET-1-stimulated hypertrophy, and the efficacy of Gßγ inhibition in preventing heart failure maybe due in part to its blocking both these pathways.


Subject(s)
GTP-Binding Protein beta Subunits/physiology , GTP-Binding Protein gamma Subunits/physiology , Golgi Apparatus/metabolism , Myocytes, Cardiac/physiology , Animals , Cardiomegaly/metabolism , Cells, Cultured , Hydrolysis , Phosphatidylinositol Phosphates , Protein Transport , Rats, Sprague-Dawley , Second Messenger Systems
3.
Cell Mol Life Sci ; 65(14): 2191-214, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18488142

ABSTRACT

G protein betagamma subunits are central participants in G protein-coupled receptor signaling pathways. They interact with receptors, G protein alpha subunits and downstream targets to coordinate multiple, different GPCR functions. Much is known about the biology of Gbetagamma subunits but mysteries remain. Here, we will review what is known about general aspects of structure and function of Gbetagamma as well as discuss emerging mechanisms for regulation of Gbetagamma signaling. Recent data suggest that Gbetagamma is a potential therapeutic drug target. Thus, a thorough understanding of the molecular and physiological functions of Gbetagamma has significant implications.


Subject(s)
GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Crystallography, X-Ray , Drug Evaluation, Preclinical , GTP-Binding Protein alpha Subunits/chemistry , GTP-Binding Protein gamma Subunits/chemistry , Humans , Models, Biological , Models, Molecular , Multiprotein Complexes , Peptide Mapping , Pertussis Toxin/toxicity , Protein Interaction Mapping , Receptors, G-Protein-Coupled/chemistry , Signal Transduction/drug effects , Static Electricity
4.
Mol Pharmacol ; 73(2): 410-8, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18006643

ABSTRACT

G protein betagamma subunit-dependent signaling is important for chemoattractant-dependent leukocyte chemotaxis. Selective small molecule targeting of phosphoinositide 3-kinase (PI3-kinase) gamma catalytic activity is a target of interest for anti-inflammatory pharmaceutical development. In this study, we examined whether small-molecule inhibition of Gbetagamma-dependent signaling, including Gbetagamma-dependent activation of PI3-kinase gamma and Rac1, could inhibit chemoattractant-dependent neutrophil migration in vitro and inflammation in vivo. Small-molecule Gbetagamma inhibitors suppressed fMLP-stimulated Rac activation, superoxide production, and PI3-kinase activation in differentiated HL60 cells. These compounds also blocked fMLP-dependent chemotaxis in HL60 cells and primary human neutrophils. Systemic administration inhibited paw edema and neutrophil infiltration in a mouse carrageenan-induced paw edema model. Overall, the data demonstrate that targeting Gbetagamma-regulation may be an effective anti-inflammation strategy.


Subject(s)
Cell Migration Inhibition , Chemotaxis, Leukocyte/physiology , GTP-Binding Protein beta Subunits/antagonists & inhibitors , GTP-Binding Protein gamma Subunits/antagonists & inhibitors , Neutrophils/pathology , Signal Transduction/physiology , Animals , Chemotaxis, Leukocyte/drug effects , Edema/metabolism , Edema/prevention & control , GTP-Binding Protein beta Subunits/genetics , GTP-Binding Protein beta Subunits/physiology , GTP-Binding Protein gamma Subunits/genetics , GTP-Binding Protein gamma Subunits/physiology , HL-60 Cells , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation/prevention & control , Mice , Neutrophils/drug effects , Neutrophils/metabolism , Protein Binding/drug effects , Protein Binding/physiology , Signal Transduction/drug effects , Xanthenes/metabolism , Xanthenes/pharmacology , Xanthenes/therapeutic use
5.
J Biol Chem ; 276(45): 41797-802, 2001 Nov 09.
Article in English | MEDLINE | ID: mdl-11546822

ABSTRACT

The G protein betagamma complex regulates a wide range of effectors, including the phospholipase Cbeta isozymes (PLCbetas). Prenyl modification of the gamma subunit is necessary for this activity. Evidence presented here supports a direct interaction between the G protein gamma subunit prenyl group and PLCbeta isozymes. A geranylgeranylated peptide corresponding to the C-terminal region of the gamma subunit type, gamma2, strongly inhibits stimulation of PLCbeta2 and PLCbeta3 activity by the betagamma complex. This effect is specific because the same peptide has no effect on stimulation of PLCbeta by an alpha subunit type, alphaq. Prenylation of the gamma peptide is required for its inhibitory effect. When interaction of prenylated gamma subunit peptide to fluorophore-tagged PLCbeta2 was examined by fluorescence spectroscopy, prenylated but not unprenylated peptide increased PLCbeta2 fluorescence emission energy, indicating direct binding of the prenyl moiety to PLCbeta. In addition, fluorescence resonance energy transfer was detected between fluorophore tagged PLCbeta and wild type betagamma complex but not an unprenylated mutant betagamma complex. We conclude that a major function of the gamma subunit prenyl group is to facilitate direct protein-protein interaction between the betagamma complex and an effector, phospholipase Cbeta.


Subject(s)
Heterotrimeric GTP-Binding Proteins/chemistry , Isoenzymes/chemistry , Protein Prenylation , Type C Phospholipases/chemistry , Heterotrimeric GTP-Binding Proteins/physiology , Phospholipase C beta
6.
EMBO J ; 20(4): 743-54, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11179219

ABSTRACT

Three classes of mammalian phosphoinositide-specific phospholipase C (PLC) have been characterized, PLCbeta, PLCgamma and PLCdelta, that are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLCepsilon, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C-terminal Ras-binding (RA) domains, RA1 and RA2. The RA2 domain binds H-Ras in a GTP-dependent manner, comparable with the Ras-binding domain of Raf-1; however, the RA1 domain binds H-Ras with a low affinity in a GTP-independent manner. While G(alpha)q, Gbetagamma or, surprisingly, H-Ras do not activate recombinant purified protein in vitro, constitutively active Q61L H-Ras stimulates PLC(epsilon) co-expressed in COS-7 cells in parallel with Ras binding. Deletion of either the RA1 or RA2 domain inhibits this activation. Site-directed mutagenesis of the RA2 domain or Ras demonstrates a conserved Ras-effector interaction and a unique profile of activation by Ras effector domain mutants. These studies identify a novel fourth class of mammalian PLC that is directly regulated by Ras and links two critical signaling pathways.


Subject(s)
Type C Phospholipases/metabolism , ras Proteins/metabolism , Amino Acid Sequence , Animals , COS Cells , Cloning, Molecular , Molecular Sequence Data , Mutagenesis , Phosphoinositide Phospholipase C , Protein Binding , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Sequence Homology, Amino Acid , Spodoptera , Type C Phospholipases/chemistry , Type C Phospholipases/genetics
7.
EMBO J ; 20(4): 767-76, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11179221

ABSTRACT

To understand the requirements for binding to G protein betagamma subunits, phage-displayed random peptide libraries were screened using immobilized biotinylated betagamma as the target. Selected peptides were grouped into four different families based on their sequence characteristics. One group (group I) had a clear conserved motif that has significant homology to peptides derived from phospholipase C beta (PLC beta) and to a short motif in phosducin that binds to G protein beta subunits. The other groups had weaker sequence homologies or no homology to the group I sequences. A synthetic peptide from the strongest consensus group blocked activation of PLC by G protein betagamma subunits. The peptide did not block betagamma-mediated inhibition of voltage-gated calcium channels and had little effect on betagamma-mediated inhibition of Gs-stimulated type I adenylate cyclase. Competition experiments indicated that peptides from all four families bound to a single site on betagamma. These peptides may bind to a protein-protein interaction 'hot spot' on the surface of betagamma subunits that is used by a subclass of effectors.


Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Amino Acid Sequence , Coliphages/chemistry , Heterotrimeric GTP-Binding Proteins/chemistry , Molecular Sequence Data , Peptides/metabolism , Potassium Channels/metabolism , Protein Binding , Sequence Homology, Amino Acid
8.
J Biol Chem ; 276(14): 11246-51, 2001 Apr 06.
Article in English | MEDLINE | ID: mdl-11145956

ABSTRACT

In previous work (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148-7154), we showed that overlapping peptides, N20K (Asn(564)-Lys(583)) and E20K (Glu(574)-Lys(593)), from the catalytic domain of phospholipase C (PLC) beta2 block Gbetagamma-dependent activation of PLC beta2. The peptides could also be directly cross-linked to betagamma subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to Gbeta(1) was inhibited by PLC beta2 but not by alpha(i1)(GDP), indicating that the peptide-binding site on beta(1) represents a binding site for PLC beta2 that does not overlap with the alpha(i1)-binding site. Here we identify the site of peptide cross-linking and thereby define a site for PLC beta2 interaction with beta subunits. Each of the 14 cysteine residues in beta(1) were altered to alanine. The ability of the PLC beta2-derived peptide to cross-link to each betagamma mutant was then analyzed to identify the reactive sulfhydryl moiety on the beta subunit required for the cross-linking reaction. We find that C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding to a region near cysteine 25 of beta(1) which is located in the amino-terminal coiled-coil region of beta(1) and identifies a PLC-binding site distinct from the alpha subunit interaction site.


Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Isoenzymes/metabolism , Type C Phospholipases/metabolism , Animals , Binding Sites , Cross-Linking Reagents , Heterotrimeric GTP-Binding Proteins/chemistry , Heterotrimeric GTP-Binding Proteins/genetics , Isoenzymes/chemistry , Isoenzymes/genetics , Mutation , Phospholipase C beta , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Type C Phospholipases/chemistry , Type C Phospholipases/genetics
9.
J Biol Chem ; 275(50): 38961-4, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11042163

ABSTRACT

Receptor stimulation of nucleotide exchange in a heterotrimeric G protein (alphabetagamma) is the primary event-modulating signaling by G proteins. The molecular mechanisms at the basis of this event and the role of the G protein subunits, especially the betagamma complex, in receptor activation are unclear. In a reconstituted system, a purified muscarinic receptor, M2, activates G protein heterotrimers alphai2beta1gamma5 and alphai2beta1gamma7 with equal efficacy. However, when the alpha subunit type is substituted with alphao, alphaobeta1gamma7 shows a 100% increase in M2-stimulated GTP hydrolysis compared with alphaobeta1gamma5. Using a sensitive assay based on betagamma complex stimulation of phospholipase C activity, we show that both beta1gamma5 and beta1gamma7 form heterotrimers equally well with alphao and alphai. These results indicate that the gamma subunit interaction with a receptor is critical for modulating nucleotide exchange and is influenced by the subunit-type composition of the heterotrimer.


Subject(s)
Heterotrimeric GTP-Binding Proteins/physiology , Receptors, Cell Surface/metabolism , Baculoviridae/metabolism , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine Triphosphate/metabolism , Hydrolysis , Kinetics , Protein Binding , Receptor, Muscarinic M2 , Receptors, Muscarinic/metabolism , Recombinant Proteins/metabolism , Signal Transduction , Time Factors , Type C Phospholipases/metabolism
10.
Biochemistry ; 39(37): 11340-7, 2000 Sep 19.
Article in English | MEDLINE | ID: mdl-10985779

ABSTRACT

When the beta(5) (short form) and gamma(2) subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged alpha(i) in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged alpha(i) and resulted in purification of beta(5)gamma(2) to near homogeneity. Subsequent anion-exchange chromatography of beta(5)gamma(2) resulted in resolution of beta(5) from gamma(2) and further purification of beta(5). The purified beta(5) eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of gamma. beta(5) monomer could be assembled with partially purified hexahistidine-tagged gamma(2) in vitro to form a functional dimer that could selectively activate PLC beta2 but not PLC beta3. alpha(o)-GDP inhibited activation of PLC beta2 by beta(5)gamma(2) supporting the idea that beta(5)gamma(2) can bind to alpha(o). beta(5) monomer and beta(5)gamma(2) only supported a small degree of ADP ribosylation of alpha(i) by pertussis toxin (PTX), but beta(5) monomer was able to compete for beta(1)gamma(2) binding to alpha(i) and alpha(o) to inhibit PTX-catalyzed ADP ribosylation. These data indicate that beta(5) functionally interacts with PTX-sensitive GDP alpha subunits and that beta(5) subunits can be assembled with gamma subunits in vitro to reconstitute activity and also support the idea that there are determinants on beta subunits that are selective for even very closely related effectors.


Subject(s)
GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , GTP-Binding Proteins/metabolism , Heterotrimeric GTP-Binding Proteins/metabolism , Adenosine Diphosphate Ribose/antagonists & inhibitors , Adenosine Diphosphate Ribose/metabolism , Animals , Baculoviridae/genetics , Chromatography, Gel , Chromatography, Ion Exchange , Detergents , Enzyme Activation , Enzyme Inhibitors/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go , GTP-Binding Protein alpha Subunits, Gq-G11 , Heterotrimeric GTP-Binding Proteins/biosynthesis , Heterotrimeric GTP-Binding Proteins/genetics , Heterotrimeric GTP-Binding Proteins/isolation & purification , Histidine/genetics , Hydrolysis , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Phospholipase C beta , Phosphoproteins/metabolism , Protein Binding/genetics , RGS Proteins , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spodoptera/genetics , Trypsin/metabolism , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolism
11.
Biochemistry ; 39(7): 1800-6, 2000 Feb 22.
Article in English | MEDLINE | ID: mdl-10677230

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.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go , GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , GTP-Binding Proteins/metabolism , Isoenzymes/metabolism , Peptide Fragments/metabolism , Type C Phospholipases/metabolism , Amino Acid Sequence , Amino Acid Substitution/genetics , Binding, Competitive/genetics , Enzyme Activation/genetics , GTP-Binding Protein alpha Subunit, Gi2 , GTP-Binding Proteins/physiology , Glutathione Transferase/genetics , Heterotrimeric GTP-Binding Proteins/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Molecular Sequence Data , Peptide Fragments/genetics , Phospholipase C beta , Point Mutation , Protein Binding/genetics , Proto-Oncogene Proteins/metabolism , Recombinant Fusion Proteins/chemical synthesis , Recombinant Fusion Proteins/metabolism , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/genetics
12.
Science ; 287(5455): 1046-9, 2000 Feb 11.
Article in English | MEDLINE | ID: mdl-10669417

ABSTRACT

The roles of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) in chemoattractant-elicited responses were studied in mice lacking these key enzymes. PI3Kgamma was required for chemoattractant-induced production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5)P3] and has an important role in chemoattractant-induced superoxide production and chemotaxis in mouse neutrophils and in production of T cell-independent antigen-specific antibodies composed of the immunoglobulin lambda light chain (TI-IglambdaL). The study of the mice lacking PLC-beta2 and -beta3 revealed that the PLC pathways have an important role in chemoattractant-mediated production of superoxide and regulation of protein kinases, but not chemotaxis. The PLC pathways also appear to inhibit the chemotactic activity induced by certain chemoattractants and to suppress TI-IglambdaL production.


Subject(s)
Chemotactic Factors/pharmacology , Chemotaxis, Leukocyte/physiology , Isoenzymes/metabolism , Neutrophils/physiology , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Type C Phospholipases/metabolism , Animals , B-Lymphocytes/immunology , Chemokine CCL4 , Immunoglobulin lambda-Chains/biosynthesis , Macrophage Inflammatory Proteins/pharmacology , Mice , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophil Infiltration , Neutrophils/metabolism , Peritonitis/immunology , Phosphatidylinositol Phosphates/metabolism , Phospholipase C beta , Phosphorylation , Skin Ulcer/pathology , Superoxides/metabolism
13.
Proc Natl Acad Sci U S A ; 96(18): 10385-90, 1999 Aug 31.
Article in English | MEDLINE | ID: mdl-10468617

ABSTRACT

Morphine and other micro opioids regulate a number of intracellular signaling pathways, including the one mediated by phospholipase C (PLC). By studying PLC beta3-deficient mice, we have established a strong link between PLC and mu opioid-mediated responses at both the behavioral and cellular levels. Mice lacking PLC beta3, when compared with the wild type, exhibited up to a 10-fold decrease in the ED(50) value for morphine in producing antinociception. The reduced ED(50) value was unlikely a result of changes in opioid receptor number or affinity because no differences were found in whole-brain B(max) and K(d) values for mu, kappa, and delta opioid receptors between wild-type and PLC beta3-null mice. We also found that opioid regulation of voltage-sensitive Ca(2+) channels in primary sensory neurons (dorsal root ganglion) was different between the two genotypes. Consistent with the behavioral findings, the specific mu agonist [D-Ala(2),(Me)Phe(4),Gly(ol)(5)]enkephalin (DAMGO) induced a greater whole-cell current reduction in a greater proportion of neurons isolated from the PLC beta3-null mice than from the wild type. In addition, reconstitution of recombinant PLC protein back into PLC beta3-deficient dorsal root ganglion neurons reduced DAMGO responses to those of wild-type neurons. In neurons of both genotypes, activation of protein kinase C with phorbol esters markedly reduced DAMGO-mediated Ca(2+) current reduction. These data demonstrate that PLC beta3 constitutes a significant pathway involved in negative modulation of mu opioid responses, perhaps via protein kinase C, and suggests the possibility that differences in opioid sensitivity among individuals could be, in part, because of genetic factors.


Subject(s)
Brain/metabolism , Enkephalins/pharmacology , Gene Expression Regulation, Enzymologic , Isoenzymes/genetics , Isoenzymes/metabolism , Morphine/pharmacology , Neurons, Afferent/physiology , Pain/genetics , Receptors, Opioid, mu/metabolism , Type C Phospholipases/genetics , Type C Phospholipases/metabolism , Animals , Calcium Channels/genetics , Cell Membrane/metabolism , Enkephalin, Ala(2)-MePhe(4)-Gly(5)- , Ganglia, Spinal/physiology , Gene Expression Regulation , Isoenzymes/deficiency , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Mice, Knockout , Neurons, Afferent/drug effects , Pain/physiopathology , Phospholipase C beta , Receptors, Opioid, delta/metabolism , Receptors, Opioid, kappa/metabolism , Type C Phospholipases/deficiency
14.
J Biol Chem ; 273(12): 7148-54, 1998 Mar 20.
Article in English | MEDLINE | ID: mdl-9507029

ABSTRACT

To delineate the specific regions of phospholipase C beta2 (PLC beta2) involved in binding and activation by G protein betagamma subunits, we synthesized peptides corresponding to segments of PLC beta2. Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the activation of PLC beta2 by betagamma subunits (IC50 50 and 150 microM, respectively), whereas two control peptides did not. N20K and E20K, but not the control peptides, inhibited betagamma-dependent ADP-ribosylation of Galphai1 by pertussis toxin and betagamma-dependent activation of phosphoinositide 3-kinase. To demonstrate direct binding of the peptides to betagamma subunits, the peptides were chemically cross-linked to purified beta1gamma2. N20K and E20K cross-linked to both beta1 and gamma2 subunits, whereas the control peptides did not. Cross-linking to beta and gamma was inhibited by incubation with excess PLC beta2 or PLC beta3, whereas cross-linking to gamma but not beta was inhibited by r-myr-alphai1. These data together demonstrate specificity of N20K and E20K for G betagamma binding and inhibition of effector activation by betagamma subunits. The results suggest that an overlapping region of the two active peptides, Glu-574-Lys-583, mimics a region of PLC beta2 that is involved in binding to betagamma subunits. Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to betagamma subunits. Alignment of these peptides with the three-dimensional structure from PLC delta1 identifies a putative alpha helical region on the surface of the catalytic domain of PLC beta2 that could interact with betagamma subunits.


Subject(s)
GTP-Binding Proteins/metabolism , Isoenzymes/metabolism , Type C Phospholipases/metabolism , Adenosine Diphosphate Ribose/metabolism , Amino Acid Sequence , Animals , Baculoviridae/genetics , Cross-Linking Reagents , Enzyme Activation , GTP-Binding Proteins/chemistry , Isoenzymes/antagonists & inhibitors , Molecular Sequence Data , Peptide Fragments/metabolism , Phospholipase C beta , Recombinant Proteins/metabolism , Sequence Alignment , Spodoptera , Type C Phospholipases/antagonists & inhibitors
15.
Cell ; 89(1): 105-14, 1997 Apr 04.
Article in English | MEDLINE | ID: mdl-9094719

ABSTRACT

Two highly similar, PtdIns(4,5)P2-selective, G beta gamma-activated PI3Ks were purified from pig neutrophil cytosol. Both were heterodimers, were composed of a 101 kDa protein and either a 120 kDa or a 117 kDa catalytic subunit, and were activated greater than 100-fold by G beta gammas. Peptide sequence-based oligonucleotide probes were used to clone cDNAs for the p120 and p101 species. The cDNA of p120 is highly related to p110 gamma, while the cDNA of p101 is not substantially related to anything in current databases. The proteins were expressed in and purified from insect and mammalian cells. They bound tightly to one another, both in vivo and in vitro, and in so doing, p101 amplified the effect of G beta gammas on the PI3K activity of p120 from less than 2-fold to greater than 100-fold.


Subject(s)
GTP-Binding Proteins/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Cytosol/chemistry , DNA, Complementary , Gene Expression Regulation, Enzymologic/physiology , Humans , Insecta , Leukemia, Promyelocytic, Acute , Mammals , Molecular Sequence Data , Phosphatidylinositol 3-Kinases , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Recombinant Proteins/genetics , Sensitivity and Specificity , Swine , Transfection , Tumor Cells, Cultured/chemistry , Tumor Cells, Cultured/enzymology
16.
J Biol Chem ; 271(41): 25071-8, 1996 Oct 11.
Article in English | MEDLINE | ID: mdl-8810260

ABSTRACT

Phospholipase C beta2 (PLC beta2) is activated by G protein betagamma subunits and calcium. The enzyme is soluble and its substrate, phosphatidylinositol 4,5-bisphosphate (PIP2), is present in phospholipid membranes. A potential mechanism for regulation of this enzyme is through influencing the equilibrium association of the enzyme with membrane surfaces. In this paper we describe a fluorescence resonance energy transfer (FRET) method for measuring the association of PLC beta2 with phospholipid bilayers. The method allows equilibrium measurements to be made under a variety of conditions, including those that support enzymatic activity and ability to be regulated by G proteins. Using this method it was found that PLC beta2 bound to vesicles containing anionic lipids and demonstrated a selective and unique interaction with PIP2-containing vesicles. The FRET data were corroborated with a centrifugation based method for estimating the affinity of PLC beta2 for vesicles. Apparently different modes of association of PLC beta2 with vesicles of different composition can be distinguished based on alterations in resonance energy transfer efficiency. Association of PLC beta2 with PIP2 vesicles requires an intact lipid bilayer, is blocked by neomycin, and is not affected by D-myo-inositol 1,4,5-trisphosphate (D-IP3). G protein betagamma subunits do not alter the affinity of PLC beta2 for lipid bilayers and at the PIP2 concentrations used to measure betagamma-dependent stimulation of PLC activity, the majority of the PLC beta2 is already associated with the vesicle surface. Furthermore, under conditions where betagamma subunits strongly activate PLC activity, the extent of association with vesicles is unaffected by betagamma subunits or calcium. These results indicate that activation of PLC beta2 by G protein betagamma subunits or Ca2+ in vitro does not involve translocation to the vesicle surface.


Subject(s)
GTP-Binding Proteins/metabolism , Isoenzymes/chemistry , Isoenzymes/metabolism , Lipid Bilayers , Phospholipids/metabolism , Type C Phospholipases/chemistry , Type C Phospholipases/metabolism , Animals , Cell Line , Cloning, Molecular , Detergents/pharmacology , Energy Transfer , Enzyme Activation , Isoenzymes/isolation & purification , Kinetics , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phospholipase C beta , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Spodoptera , Transfection , Type C Phospholipases/isolation & purification
17.
J Biol Chem ; 271(41): 25204-7, 1996 Oct 11.
Article in English | MEDLINE | ID: mdl-8810279

ABSTRACT

The signaling pathway leading from G protein-coupled chemoattractant receptors to the generation of oxidants by NADPH oxidase in human neutrophils requires the formation of the lipid mediator phosphatidylinositol 3,4,5-trisphosphate (PIP3). Two mechanisms through which PIP3 can be generated have been described in human leukocytes. One pathway involves the coupling of the src-related tyrosine kinase Lyn to the "classical" p85/p110 form of phosphatidylinositol 3-kinase. The second paradigm utilizes a novel form of phosphatidylinositol 3-kinase whose activity is directly regulated by G protein betagamma subunits. In this paper, we show that formation of PIP3 in chemoattractant-stimulated neutrophils is substantially attenuated by inhibitors that specifically block tyrosine kinase activity. These data suggest that the Lyn activation pathway plays a major role in the formation of this important lipid messenger during chemoattractant stimulation of human neutrophils.


Subject(s)
Chemotaxis, Leukocyte , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/physiology , Phosphatidylinositol Phosphates/blood , Protein-Tyrosine Kinases/blood , Signal Transduction , Enzyme Inhibitors/pharmacology , GTP-Binding Proteins/metabolism , Genistein , Humans , Isoflavones/pharmacology , Neutrophils/drug effects , Neutrophils/enzymology , Phosphatidylinositol 3-Kinases , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/blood , Phosphotransferases (Alcohol Group Acceptor)/isolation & purification , Protein-Tyrosine Kinases/antagonists & inhibitors
18.
J Biol Chem ; 271(23): 13430-4, 1996 Jun 07.
Article in English | MEDLINE | ID: mdl-8662841

ABSTRACT

Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PTx)-sensitive activation of phospholipase C (PLC) have been investigated using a cotransfection assay system in COS-7 cells. The abilities of the receptors for C5a and fMLP to activate PLC beta2 and PLC beta3 through the Gbetagamma subunits of endogenous Gi proteins in COS-7 cells were tested because both PLC beta2 and PLC beta3 were shown to be activated by the betagamma subunits of G proteins in in vitro reconstitution assays. Neither of the receptors can activate endogenous PLC beta3 or recombinant PLC beta3 in transfected COS-7 cells. However, both receptors can clearly activate PLC beta2 in a PTx-sensitive manner, suggesting that the receptors may interact with endogenous PTx-sensitive G proteins and activate PLC beta2 probably through the Gbetagamma subunits. These findings were further corroborated by the results that PLC beta3 could only be slightly activated by Gbeta1gamma1 or Gbeta1gamma5 in the cotransfection assay, whereas the Gbetagamma subunits strongly activated PLC beta2 under the same conditions. PLC beta3 can be activated by Galphaq, Galpha11, and Galpha16 in the cotransfection assay. In addition, the Ggamma2 and Ggamma3 mutants with substitution of the C-terminal Cys residue by a Ser residue, which can inhibit wild type Gbetagamma-mediated activation of PLC beta2, were able to inhibit C5a or fMLP-mediated activation of PLC beta2. These Ggamma mutants, however, showed little effect on m1-muscarinic receptor-mediated PLC activation, which is mediated by the Gq class of G proteins. These results all confirm that the Gbetagamma subunits are involved in PLC beta2 activation by the two chemoattractant receptors and suggest that in COS-7 cells activation of PLC beta3 by Gbetagamma may not be the primary pathway for the receptors.


Subject(s)
Antigens, CD/metabolism , Pertussis Toxin , Receptors, Complement/metabolism , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Type C Phospholipases/metabolism , Virulence Factors, Bordetella/pharmacology , Amino Acid Sequence , Animals , Antigens, CD/genetics , Cell Line , Enzyme Activation/drug effects , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Molecular Sequence Data , Receptor, Anaphylatoxin C5a , Receptors, Complement/genetics , Receptors, Formyl Peptide , Receptors, Immunologic/genetics , Receptors, Peptide/genetics , Signal Transduction , Transfection , Type C Phospholipases/genetics
19.
Biochemistry ; 35(23): 7499-505, 1996 Jun 11.
Article in English | MEDLINE | ID: mdl-8652528

ABSTRACT

The gamma subunit of heterotrimeric G proteins is isoprenylated and methylated on its carboxyl terminal cysteine residue. While retinal transducin is farnesylated, all other gamma subunits are modified by geranylgeranylation. An immobilized form of pig liver esterase (iPLE) is able to hydrolyze the methyl ester of a geranylgeranylated beta gamma isoform (beta 1 gamma 2). Since methylation is the only reversible reaction in the isoprenylation pathway, it could be a site of regulation of G protein activity. With both the methylated and demethylated beta 1 gamma 2 now available, the role of methylation for a geranylgeranylated heterotrimeric G protein may be addressed. Here, it is reported that methylation has no effect on the ability of beta gamma to interact with an alpha subunit, as probed by ADP-ribosylation studies with pertussis toxin, and has a small effect (less than 2-fold) on the ability of geranylgeranylated beta gamma to activate phosphatidylinositol-specific phospholipase C (PIPLC) and phosphoinositide 3 kinase (PI3K). In binding studies, demethylation only slightly decreased the ability of beta 1 gamma 2 to adhere to azolectin vesicles. Therefore, methylation of heterotrimeric G proteins appears to have only a minor effect in signal transduction processes which can be correlated to a decrease in hydrophobicity of the beta gamma subunit.


Subject(s)
GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Transducin/chemistry , Transducin/metabolism , Adenosine Diphosphate Ribose/metabolism , Animals , Cell Line , Cysteine , Enzymes, Immobilized/metabolism , Esterases/metabolism , Liver/enzymology , Macromolecular Substances , Methylation , Pertussis Toxin , Phosphatidylcholines , Phosphatidylinositol 3-Kinases , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Phospholipids , Phosphoric Diester Hydrolases/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Prenylation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spodoptera , Swine , Transfection , Virulence Factors, Bordetella/pharmacology
20.
Proc Natl Acad Sci U S A ; 93(7): 2964-8, 1996 Apr 02.
Article in English | MEDLINE | ID: mdl-8610151

ABSTRACT

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.


Subject(s)
GTP-Binding Proteins/metabolism , Type C Phospholipases/chemistry , Type C Phospholipases/metabolism , Amino Acid Sequence , Animals , Antigens, CD/biosynthesis , Antigens, CD/metabolism , Blotting, Western , Cell Line , Chlorocebus aethiops , DNA, Complementary , GTP-Binding Proteins/chemistry , Glutamic Acid , Humans , Isoenzymes/biosynthesis , Isoenzymes/chemistry , Isoenzymes/metabolism , Kidney , Macromolecular Substances , Mammals , Mutagenesis, Site-Directed , Point Mutation , Polymerase Chain Reaction , Receptor, Anaphylatoxin C5a , Receptors, Complement/biosynthesis , Receptors, Complement/metabolism , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Transfection , Type C Phospholipases/biosynthesis
SELECTION OF CITATIONS
SEARCH DETAIL
...