Phospholipase C, Ca2+, and calmodulin signaling are required for 5-HT2A receptor-mediated transamidation of Rac1 by transglutaminase

Ying Dai, Nichole L. Dudek, Qian Li, Nancy A. Muma

Research output: Contribution to journalArticle

Abstract

Rationale: Serotonin and especially serotonin 2A (5-HT2A) receptor signaling are important in the etiology and treatment of schizophrenia and affective disorders. We previously reported a novel 5-HT2A receptor effector, increased transglutaminase (TGase)-catalyzed transamidation, and activation of the small G protein Rac1 in A1A1v cells, a rat embryonic cortical cell line. Objectives: In this study, we explore the signaling pathway involved in 5-HT2A receptor-mediated Rac1 transamidation. Methods: A1A1v cells were pretreated with pharmacological inhibitors of phospholipase C (PLC) or calmodulin (CaM), and then stimulated by the 5-HT2A receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI). Intracellular Ca2+ concentration and TGase-modified Rac1 transamidation were monitored. The effect of manipulation of intracellular Ca2+ by a Ca2+ ionophore or a chelating agent on Rac1 transamidation was also evaluated. Results: In cells pretreated with a PLC inhibitor U73122, DOI-stimulated increases in the intracellular Ca2+ concentration and TGase-modified Rac1 were significantly attenuated as compared to those pretreated with U73343, an inactive analog. The membrane-permeant Ca2+ chelator, BAPTA-AM strongly reduced TGase-catalyzed Rac1 transamidation upon DOI stimulation. Conversely, the Ca2+ ionophore ionomycin, at a concentration that induced an elevation of cytosolic Ca2+ to a level comparable to cells treated with DOI, produced an increase in TGase-modified Rac1 without 5-HT 2A receptor activation. Moreover, the CaM inhibitor W-7, significantly decreased Rac1 transamidation in a dose-dependent manner in DOI-treated cells. Conclusions: These results indicate that 5-HT2A receptor-coupled PLC activation and subsequent Ca2+ and CaM signaling are necessary for TGase-catalyzed Rac1 transamidation, and an increase in intracellular Ca2+ is sufficient to induce Rac1 transamidation.

Original languageEnglish (US)
Pages (from-to)403-412
Number of pages10
JournalPsychopharmacology
Volume213
Issue number2-3
DOIs
StatePublished - Feb 2011
Externally publishedYes

Fingerprint

Receptor, Serotonin, 5-HT2A
Transglutaminases
Type C Phospholipases
Calmodulin
Ionophores
Chelating Agents
Serotonin 5-HT2 Receptor Agonists
Ionomycin
Monomeric GTP-Binding Proteins
Mood Disorders
Serotonin
Schizophrenia
4-iodoamphetamine
Pharmacology
Cell Line
Membranes

Keywords

  • 5-HT receptor
  • A1A1v cells
  • Calcium
  • Calmodulin
  • Phospholipase C
  • Rac1
  • Serotonin
  • Serotonylation
  • Small G proteins
  • Transamidation
  • Transglutaminase

ASJC Scopus subject areas

  • Pharmacology

Cite this

Phospholipase C, Ca2+, and calmodulin signaling are required for 5-HT2A receptor-mediated transamidation of Rac1 by transglutaminase. / Dai, Ying; Dudek, Nichole L.; Li, Qian; Muma, Nancy A.

In: Psychopharmacology, Vol. 213, No. 2-3, 02.2011, p. 403-412.

Research output: Contribution to journalArticle

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abstract = "Rationale: Serotonin and especially serotonin 2A (5-HT2A) receptor signaling are important in the etiology and treatment of schizophrenia and affective disorders. We previously reported a novel 5-HT2A receptor effector, increased transglutaminase (TGase)-catalyzed transamidation, and activation of the small G protein Rac1 in A1A1v cells, a rat embryonic cortical cell line. Objectives: In this study, we explore the signaling pathway involved in 5-HT2A receptor-mediated Rac1 transamidation. Methods: A1A1v cells were pretreated with pharmacological inhibitors of phospholipase C (PLC) or calmodulin (CaM), and then stimulated by the 5-HT2A receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI). Intracellular Ca2+ concentration and TGase-modified Rac1 transamidation were monitored. The effect of manipulation of intracellular Ca2+ by a Ca2+ ionophore or a chelating agent on Rac1 transamidation was also evaluated. Results: In cells pretreated with a PLC inhibitor U73122, DOI-stimulated increases in the intracellular Ca2+ concentration and TGase-modified Rac1 were significantly attenuated as compared to those pretreated with U73343, an inactive analog. The membrane-permeant Ca2+ chelator, BAPTA-AM strongly reduced TGase-catalyzed Rac1 transamidation upon DOI stimulation. Conversely, the Ca2+ ionophore ionomycin, at a concentration that induced an elevation of cytosolic Ca2+ to a level comparable to cells treated with DOI, produced an increase in TGase-modified Rac1 without 5-HT 2A receptor activation. Moreover, the CaM inhibitor W-7, significantly decreased Rac1 transamidation in a dose-dependent manner in DOI-treated cells. Conclusions: These results indicate that 5-HT2A receptor-coupled PLC activation and subsequent Ca2+ and CaM signaling are necessary for TGase-catalyzed Rac1 transamidation, and an increase in intracellular Ca2+ is sufficient to induce Rac1 transamidation.",
keywords = "5-HT receptor, A1A1v cells, Calcium, Calmodulin, Phospholipase C, Rac1, Serotonin, Serotonylation, Small G proteins, Transamidation, Transglutaminase",
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T1 - Phospholipase C, Ca2+, and calmodulin signaling are required for 5-HT2A receptor-mediated transamidation of Rac1 by transglutaminase

AU - Dai, Ying

AU - Dudek, Nichole L.

AU - Li, Qian

AU - Muma, Nancy A.

PY - 2011/2

Y1 - 2011/2

N2 - Rationale: Serotonin and especially serotonin 2A (5-HT2A) receptor signaling are important in the etiology and treatment of schizophrenia and affective disorders. We previously reported a novel 5-HT2A receptor effector, increased transglutaminase (TGase)-catalyzed transamidation, and activation of the small G protein Rac1 in A1A1v cells, a rat embryonic cortical cell line. Objectives: In this study, we explore the signaling pathway involved in 5-HT2A receptor-mediated Rac1 transamidation. Methods: A1A1v cells were pretreated with pharmacological inhibitors of phospholipase C (PLC) or calmodulin (CaM), and then stimulated by the 5-HT2A receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI). Intracellular Ca2+ concentration and TGase-modified Rac1 transamidation were monitored. The effect of manipulation of intracellular Ca2+ by a Ca2+ ionophore or a chelating agent on Rac1 transamidation was also evaluated. Results: In cells pretreated with a PLC inhibitor U73122, DOI-stimulated increases in the intracellular Ca2+ concentration and TGase-modified Rac1 were significantly attenuated as compared to those pretreated with U73343, an inactive analog. The membrane-permeant Ca2+ chelator, BAPTA-AM strongly reduced TGase-catalyzed Rac1 transamidation upon DOI stimulation. Conversely, the Ca2+ ionophore ionomycin, at a concentration that induced an elevation of cytosolic Ca2+ to a level comparable to cells treated with DOI, produced an increase in TGase-modified Rac1 without 5-HT 2A receptor activation. Moreover, the CaM inhibitor W-7, significantly decreased Rac1 transamidation in a dose-dependent manner in DOI-treated cells. Conclusions: These results indicate that 5-HT2A receptor-coupled PLC activation and subsequent Ca2+ and CaM signaling are necessary for TGase-catalyzed Rac1 transamidation, and an increase in intracellular Ca2+ is sufficient to induce Rac1 transamidation.

AB - Rationale: Serotonin and especially serotonin 2A (5-HT2A) receptor signaling are important in the etiology and treatment of schizophrenia and affective disorders. We previously reported a novel 5-HT2A receptor effector, increased transglutaminase (TGase)-catalyzed transamidation, and activation of the small G protein Rac1 in A1A1v cells, a rat embryonic cortical cell line. Objectives: In this study, we explore the signaling pathway involved in 5-HT2A receptor-mediated Rac1 transamidation. Methods: A1A1v cells were pretreated with pharmacological inhibitors of phospholipase C (PLC) or calmodulin (CaM), and then stimulated by the 5-HT2A receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI). Intracellular Ca2+ concentration and TGase-modified Rac1 transamidation were monitored. The effect of manipulation of intracellular Ca2+ by a Ca2+ ionophore or a chelating agent on Rac1 transamidation was also evaluated. Results: In cells pretreated with a PLC inhibitor U73122, DOI-stimulated increases in the intracellular Ca2+ concentration and TGase-modified Rac1 were significantly attenuated as compared to those pretreated with U73343, an inactive analog. The membrane-permeant Ca2+ chelator, BAPTA-AM strongly reduced TGase-catalyzed Rac1 transamidation upon DOI stimulation. Conversely, the Ca2+ ionophore ionomycin, at a concentration that induced an elevation of cytosolic Ca2+ to a level comparable to cells treated with DOI, produced an increase in TGase-modified Rac1 without 5-HT 2A receptor activation. Moreover, the CaM inhibitor W-7, significantly decreased Rac1 transamidation in a dose-dependent manner in DOI-treated cells. Conclusions: These results indicate that 5-HT2A receptor-coupled PLC activation and subsequent Ca2+ and CaM signaling are necessary for TGase-catalyzed Rac1 transamidation, and an increase in intracellular Ca2+ is sufficient to induce Rac1 transamidation.

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KW - Phospholipase C

KW - Rac1

KW - Serotonin

KW - Serotonylation

KW - Small G proteins

KW - Transamidation

KW - Transglutaminase

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