The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gqalpha -dependent ERK-independent pathway.
The Ca(2+)-sensing receptor (CaR) stimulates a number of phospholipase activities, but the specific phospholipases and the mechanisms by which the CaR activates them are not defined. We investigated regulation of phospholipase A(2) (PLA(2)) by the Ca(2+)-sensing receptor (CaR) in human embryonic kidney 293 cells that express either the wild-type receptor or a nonfunctional mutant (R796W) CaR. The PLA(2) activity was attributable to cytosolic PLA(2) (cPLA(2)) based on its inhibition by arachidonyl trifluoromethyl ketone, lack of inhibition by bromoenol lactone, and enhancement of the CaR-stimulated phospholipase activity by coexpression of a cDNA encoding the 85-kDa human cPLA(2). No CaR-stimulated cPLA(2) activity was found in the cells that expressed the mutant CaR. Pertussis toxin treatment had a minimal effect on CaR-stimulated arachidonic acid release and the CaR-stimulated rise in intracellular Ca(2+) (Ca(2+)(i)), whereas inhibition of phospholipase C (PLC) with completely inhibited CaR-stimulated PLC and cPLA(2) activities. CaR-stimulated PLC activity was inhibited by expression of RGS4, an RGS (Regulator of G protein Signaling) protein that inhibits Galpha(q) activity. CaR-stimulated cPLA(2) activity was inhibited 80% by chelation of extracellular Ca(2+) and depletion of intracellular Ca(2+) with EGTA and inhibited 90% by treatment with W7, a calmodulin inhibitor, or with KN-93, an inhibitor of Ca(2+), calmodulin-dependent protein kinases. Chemical inhibitors of the ERK activator, MEK, and a dominant negative MEK, MEK(K97R), had no effect on CaR-stimulated cPLA(2) activity but inhibited CaR-stimulated ERK activity. These results demonstrate that the CaR activates cPLA(2) via a Galpha(q), PLC, Ca(2+)-CaM, and calmodulin-dependent protein kinase-dependent pathway that is independent the ERK pathway.
Division of Nephrology, Department of Medicine, University of Florida, Gainesville, Florida 32610, USA.
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ME
Handlogten
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C
Huang
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Shiraishi
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H
Awata
R T
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Miller
D016718:Arachidonic Acid / Q000378:metabolism; D001095:Arachidonic Acids / Q000494:pharmacology; D001596:Benzylamines / Q000494:pharmacology; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D005799:Genes, Dominant; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation; D009281:Naphthalenes / Q000494:pharmacology; D037342:Pertussis Toxin; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D011753:Pyrones / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D020710:RGS Proteins / Q000378:metabolism; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000096926:Benzenesulfonamides
D016718:Arachidonic Acid; D001095:Arachidonic Acids; D001596:Benzylamines; D002118:Calcium; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin; D018076:DNA, Complementary; D004533:Egtazic Acid; D004791:Enzyme Inhibitors; D004963:Estrenes; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D020928:Mitogen-Activated Protein Kinases; D009281:Naphthalenes; D037342:Pertussis Toxin; D010726:Phosphodiesterase Inhibitors; D010741:Phospholipases A; D054467:Phospholipases A2; D011493:Protein Kinase C; D011753:Pyrones; D011760:Pyrrolidinones; D020710:RGS Proteins; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D013449:Sulfonamides; D010738:Type C Phospholipases; D010566:Virulence Factors, Bordetella; C081565:arachidonyltrifluoromethane; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C072105:KN 93; D000096926:Benzenesulfonamides; C098799:RGS4 protein; C017967:W 7; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one