title: Serotonin-2A homodimers are needed for signalling via both phospholipase A2and phospholipase C in transfected CHO cells.
abstract: Different ligands differentially activate phospholipase A2(PLA2) and phospholipase C (PLC) signalling pathways that are coupled to the serotonin 2A(5-HT2A) receptor, a class-A G-protein coupled receptor (GPCR). The serotonin 5-HT2Areceptor has been shown to be expressed as a homodimer displaying some ligands negative cooperativity between protomers in the PLA2signalling pathway. We hypothesized that the homodimeric complex is the minimum functional unit required for activation of the PLA2and PLC pathways by the serotonin 5-HT2Areceptor. To investigate this hypothesis, we partially blocked the serotonin 5-HT2Areceptors with ritanserin and measured PLA2and PLC activity simultaneously. We subsequently added the competitive antagonist spiperone to release the inactivator through a crosstalk mechanism and thus allow the dimer to return to a reactive state. Partial inactivation of the homodimer by ritanserin binding decreased the activity of the receptor by 59±13% and 70±4% in the PLA2and PLC pathways respectively (P<0.001), with no difference in the potency of the serotonin (5-HT) was observed. The subsequent binding of spiperone released ritanserin due to the crosstalk between protomers and recovery of the receptor activity to 74±7% and 72±4%. Negative cooperativity between protomers in the dimer was maintained during arachidonic acid (AA) release after blocking ritanserin, as indicated by the biphasic inhibition curves for clozapine over 1μM serotonin (5-HT) in these conditions. These findings provide evidence that serotonin 5-HT2Areceptors must be expressed as homodimers in order to activate both the PLA2and PLC signalling pathways.
authors.affiliation: BioFarma Research Group, Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CiMUS), Universidade de Santiago de Compostela, Avenida de Barcelona 22, 15782 Santiago de Compostela, Spain.
authors.forename: Alba
authors.initials: A
authors.lastname: Iglesias
authors.affiliation: BioFarma Research Group, Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CiMUS), Universidade de Santiago de Compostela, Avenida de Barcelona 22, 15782 Santiago de Compostela, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Cimadevila
authors.affiliation: BioFarma Research Group, Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CiMUS), Universidade de Santiago de Compostela, Avenida de Barcelona 22, 15782 Santiago de Compostela, Spain.
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Cadavid
authors.affiliation: BioFarma Research Group, Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CiMUS), Universidade de Santiago de Compostela, Avenida de Barcelona 22, 15782 Santiago de Compostela, Spain. Electronic address: mabel.loza@usc.es.
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Loza
authors.affiliation: BioFarma Research Group, Centro Singular de Investigación en Medicina Molecular y Enfermedades Crónicas (CiMUS), Universidade de Santiago de Compostela, Avenida de Barcelona 22, 15782 Santiago de Compostela, Spain. Electronic address: pepo.brea@usc.es.
authors.forename: José
authors.initials: J
authors.lastname: Brea
mesh_terms: D000818:Animals; D016466:CHO Cells; D003024:Clozapine / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D054467:Phospholipases A2 / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D044402:Receptor, Serotonin, 5-HT2A / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology; D058830:Serotonin 5-HT2 Receptor Antagonists / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D044402:Receptor, Serotonin, 5-HT2A; D058830:Serotonin 5-HT2 Receptor Antagonists; D012701:Serotonin; D054467:Phospholipases A2; D010738:Type C Phospholipases; D003024:Clozapine
keywords: Crosstalk; Homodimer; Minimum functional unit; Phospholipase A(2); Phospholipase C; Ritanserin (PubChem CID: 5074); Serotonin 2(A) receptor; Serotonin hydrochloride (PubChem CID: 160436), Clozapine (PubChem CID: 2818), Spiperone (PubChem CID: 5265)