Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters

C. D. Smith, R. J. Uhing, R. Snyderman

Research output: Contribution to journalArticle

110 Citations (Scopus)

Abstract

Polymorphonuclear leukocytes (PMNs) activate phospholipase C via a guanine nucleotide regulatory (G) protein. Pretreatment with the PMNs with pertussis toxin (PT) or 4-β-phorbol 12-myristate 13-acetate (PMA) inhibited chemoattractant-induced inositol trisphosphate generation. To determine the loci of inhibition by PT and PMA, G protein-mediated reactions in PMN plasma membranes were examined. Plasma membranes prepared from untreated and PMA-treated PMNs demonstrated equivalent ability of a GTP analogue to suppress high affinity binding of the chemoattractant-N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) to its receptor. The rate, but not the extent, of high affinity binding of GTPγ[35S] to untreated PMN membranes was stimulated up to 2-fold by preincubation with 1 μM fMet-Leu-Phe. The ability of fMet-Leu-Phe to stimulate the rate of GTPγS binding was absent in membranes prepared from PT-treated PMNs, but remained intact in membranes from PMA-treated cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) via phospholipase C could be activated in untreated PMN membranes by either fMet-Leu-Phe plus GTP or GTPγS alone at low concentrations of Ca2+ (0.1-1 μM). Membranes prepared from PT-treated PMNs degraded PIP2 upon exposure to GTPγS, but not fMet-Leu-Phe plus GTP. In contrast, membranes prepared from phorbol ester-treated PMNs did not hydrolyze PIP2 when incubated with GTPγS. Treatment with PT or PMA did not affect the ability of 1 mM Ca2+ to activate PIP2 hydrolysis in PMN membranes, indicating that neither treatment directly inactivated phospholipase C. Therefore, PT appears to block coupling of the chemoattractant receptors to G protein activation, while phorbol esters disrupt coupling of the activated G protein to phospholipase C. The phorbol-ester-mediated effect may mimic a negative feedback signal induced by protein kinase C activation by diacylglycerol generated upon activation of phospholipase C.

Original languageEnglish (US)
Pages (from-to)6121-6127
Number of pages7
JournalJournal of Biological Chemistry
Volume262
Issue number13
StatePublished - Jan 1 1987

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Type C Phospholipases
Phorbol Esters
Pertussis Toxin
methionyl-leucyl-phenylalanine
Neutrophils
Nucleotides
Chemical activation
Membranes
Guanosine Triphosphate
Acetates
GTP-Binding Proteins
Proteins
Chemotactic Factors
Cell membranes
G12-G13 GTP-Binding Protein alpha Subunits
Hydrolysis
Cell Membrane
Formyl Peptide Receptor
N-Formylmethionine Leucyl-Phenylalanine
Guanine Nucleotides

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{276ed8310b664e01a80a525c98b97aa9,
title = "Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters",
abstract = "Polymorphonuclear leukocytes (PMNs) activate phospholipase C via a guanine nucleotide regulatory (G) protein. Pretreatment with the PMNs with pertussis toxin (PT) or 4-β-phorbol 12-myristate 13-acetate (PMA) inhibited chemoattractant-induced inositol trisphosphate generation. To determine the loci of inhibition by PT and PMA, G protein-mediated reactions in PMN plasma membranes were examined. Plasma membranes prepared from untreated and PMA-treated PMNs demonstrated equivalent ability of a GTP analogue to suppress high affinity binding of the chemoattractant-N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) to its receptor. The rate, but not the extent, of high affinity binding of GTPγ[35S] to untreated PMN membranes was stimulated up to 2-fold by preincubation with 1 μM fMet-Leu-Phe. The ability of fMet-Leu-Phe to stimulate the rate of GTPγS binding was absent in membranes prepared from PT-treated PMNs, but remained intact in membranes from PMA-treated cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) via phospholipase C could be activated in untreated PMN membranes by either fMet-Leu-Phe plus GTP or GTPγS alone at low concentrations of Ca2+ (0.1-1 μM). Membranes prepared from PT-treated PMNs degraded PIP2 upon exposure to GTPγS, but not fMet-Leu-Phe plus GTP. In contrast, membranes prepared from phorbol ester-treated PMNs did not hydrolyze PIP2 when incubated with GTPγS. Treatment with PT or PMA did not affect the ability of 1 mM Ca2+ to activate PIP2 hydrolysis in PMN membranes, indicating that neither treatment directly inactivated phospholipase C. Therefore, PT appears to block coupling of the chemoattractant receptors to G protein activation, while phorbol esters disrupt coupling of the activated G protein to phospholipase C. The phorbol-ester-mediated effect may mimic a negative feedback signal induced by protein kinase C activation by diacylglycerol generated upon activation of phospholipase C.",
author = "Smith, {C. D.} and Uhing, {R. J.} and R. Snyderman",
year = "1987",
month = "1",
day = "1",
language = "English (US)",
volume = "262",
pages = "6121--6127",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
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}

Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters. / Smith, C. D.; Uhing, R. J.; Snyderman, R.

In: Journal of Biological Chemistry, Vol. 262, No. 13, 01.01.1987, p. 6121-6127.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters

AU - Smith, C. D.

AU - Uhing, R. J.

AU - Snyderman, R.

PY - 1987/1/1

Y1 - 1987/1/1

N2 - Polymorphonuclear leukocytes (PMNs) activate phospholipase C via a guanine nucleotide regulatory (G) protein. Pretreatment with the PMNs with pertussis toxin (PT) or 4-β-phorbol 12-myristate 13-acetate (PMA) inhibited chemoattractant-induced inositol trisphosphate generation. To determine the loci of inhibition by PT and PMA, G protein-mediated reactions in PMN plasma membranes were examined. Plasma membranes prepared from untreated and PMA-treated PMNs demonstrated equivalent ability of a GTP analogue to suppress high affinity binding of the chemoattractant-N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) to its receptor. The rate, but not the extent, of high affinity binding of GTPγ[35S] to untreated PMN membranes was stimulated up to 2-fold by preincubation with 1 μM fMet-Leu-Phe. The ability of fMet-Leu-Phe to stimulate the rate of GTPγS binding was absent in membranes prepared from PT-treated PMNs, but remained intact in membranes from PMA-treated cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) via phospholipase C could be activated in untreated PMN membranes by either fMet-Leu-Phe plus GTP or GTPγS alone at low concentrations of Ca2+ (0.1-1 μM). Membranes prepared from PT-treated PMNs degraded PIP2 upon exposure to GTPγS, but not fMet-Leu-Phe plus GTP. In contrast, membranes prepared from phorbol ester-treated PMNs did not hydrolyze PIP2 when incubated with GTPγS. Treatment with PT or PMA did not affect the ability of 1 mM Ca2+ to activate PIP2 hydrolysis in PMN membranes, indicating that neither treatment directly inactivated phospholipase C. Therefore, PT appears to block coupling of the chemoattractant receptors to G protein activation, while phorbol esters disrupt coupling of the activated G protein to phospholipase C. The phorbol-ester-mediated effect may mimic a negative feedback signal induced by protein kinase C activation by diacylglycerol generated upon activation of phospholipase C.

AB - Polymorphonuclear leukocytes (PMNs) activate phospholipase C via a guanine nucleotide regulatory (G) protein. Pretreatment with the PMNs with pertussis toxin (PT) or 4-β-phorbol 12-myristate 13-acetate (PMA) inhibited chemoattractant-induced inositol trisphosphate generation. To determine the loci of inhibition by PT and PMA, G protein-mediated reactions in PMN plasma membranes were examined. Plasma membranes prepared from untreated and PMA-treated PMNs demonstrated equivalent ability of a GTP analogue to suppress high affinity binding of the chemoattractant-N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) to its receptor. The rate, but not the extent, of high affinity binding of GTPγ[35S] to untreated PMN membranes was stimulated up to 2-fold by preincubation with 1 μM fMet-Leu-Phe. The ability of fMet-Leu-Phe to stimulate the rate of GTPγS binding was absent in membranes prepared from PT-treated PMNs, but remained intact in membranes from PMA-treated cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) via phospholipase C could be activated in untreated PMN membranes by either fMet-Leu-Phe plus GTP or GTPγS alone at low concentrations of Ca2+ (0.1-1 μM). Membranes prepared from PT-treated PMNs degraded PIP2 upon exposure to GTPγS, but not fMet-Leu-Phe plus GTP. In contrast, membranes prepared from phorbol ester-treated PMNs did not hydrolyze PIP2 when incubated with GTPγS. Treatment with PT or PMA did not affect the ability of 1 mM Ca2+ to activate PIP2 hydrolysis in PMN membranes, indicating that neither treatment directly inactivated phospholipase C. Therefore, PT appears to block coupling of the chemoattractant receptors to G protein activation, while phorbol esters disrupt coupling of the activated G protein to phospholipase C. The phorbol-ester-mediated effect may mimic a negative feedback signal induced by protein kinase C activation by diacylglycerol generated upon activation of phospholipase C.

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