Expression of a mutant Gi2 alpha subunit inhibits ATP and thrombin stimulation of cytoplasmic phospholipase A2-mediated arachidonic acid release independent of Ca2+ and mitogen-activated protein kinase regulation.
The 85-kDa cytoplasmic phospholipase A2 (cPLA2) is the major hormone and growth factor-regulated enzyme that catalyzes release of arachidonic acid in mammalian cells. Activation of cPLA2 requires elevation of intracellular Ca2+ and the phosphorylation of the cPLA2 enzyme by mitogen-activated protein (MAP) kinase. Down-regulation of protein kinase C by phorbol esters or pertussis toxin catalyzed ADP-ribosylation of Gi proteins inhibits thrombin and ATP receptor-stimulated MAP kinase and arachidonic acid release, indicating that functional protein kinase C and Gi proteins are required for G protein regulation of arachidonic acid release. A mutant G alpha i2 subunit having Gly203 mutated to Thr (alpha i2G203T) inhibited thrombin and ATP receptor stimulation of arachidonic acid release independent of adenylyl cyclase inhibition, Ca2+ mobilization, and MAP kinase activation. Overexpression of the wild-type alpha i2 polypeptide or the inactive mutant alpha i2G204A (Gly204 mutated to Ala) polypeptide had no effect on thrombin or ATP receptor stimulation of arachidonic acid release. The phenotype observed with expression of the mutant alpha i2G203T polypeptide defines a role for Gi2 in the control of cPLA2 activity and subsequent arachidonic acid release in addition to the regulation of intracellular Ca2+ levels and MAP kinase activity.
Winitz
S
S
Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
Gupta
S K
SK
Qian
N X
NX
Heasley
L E
LE
Nemenoff
R A
RA
Johnson
G L
GL
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002772:Cholera Toxin / Q000494:pharmacology; D002999:Clone Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D014162:Transfection
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D010743:Phospholipids; D016718:Arachidonic Acid; D000255:Adenosine Triphosphate; D002772:Cholera Toxin; D000242:Cyclic AMP; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D013917:Thrombin; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium