Differential regulation of formyl peptide and platelet-activating factor receptors. Role of phospholipase Cbeta3 phosphorylation by protein kinase A.
Formylated peptides (e.g. n-formyl-Met-Leu-Phe (fMLP)) and platelet-activating factor (PAF) mediate chemotactic and cytotoxic responses in leukocytes through receptors coupled to G proteins that activate phospholipase C (PLC). In RBL-2H3 cells, fMLP utilizes a pertussis toxin (ptx)-sensitive G protein to activate PLC, whereas PAF utilizes a ptx-insensitive G protein. Here we demonstrate that fMLP, but not PAF, enhanced intracellular cAMP levels via a ptx-sensitive mechanism. Protein kinase A (PKA) inhibition by H-89 enhanced inositol phosphate formation stimulated by fMLP but not PAF. Furthermore, a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) inhibited phosphoinositide hydrolysis and secretion stimulated by fMLP but not PAF. Both cpt-cAMP and fMLP stimulated PLCbeta3 phosphorylation in intact RBL cells. The purified catalytic subunit of PKA phosphorylated PLCbeta3 immunoprecipitated from RBL cell lysate. Pretreatment of intact cells with cpt-cAMP and fMLP, but not PAF, resulted in an inhibition of subsequent PLCbeta3 phosphorylation by PKA in vitro. These data demonstrate that fMLP receptor, which couples to a ptx-sensitive G protein, activates both PLC and cAMP production. The resulting PKA activation phosphorylates PLCbeta3 and appears to block the ability of Gbetagamma to activate PLC. Thus, both fMLP and PAF generate stimulatory signals for PLCbeta3, but only fMLP produces a PKA-dependent inhibitory signal. This suggests a novel mechanism for the bidirectional regulation of receptors which activate PLC by ptx-sensitive G proteins.
Ali
H
H
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA. ali00001@mc.duke.edu
Sozzani
S
S
Fisher
I
I
Barr
A J
AJ
Richardson
R M
RM
Haribabu
B
B
Snyderman
R
R
D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D054799:Phospholipase C beta; D010766:Phosphorylation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D010738:Type C Phospholipases / Q000378:metabolism*
D007527:Isoenzymes; D010716:Phosphatidylinositols; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D020558:GTP Phosphohydrolases