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Role of phospholipase Cbeta3 phosphorylation in the desensitization of cellular responses to platelet-activating
1Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA. ali00001@mc.duke.edu
Abstract:
Platelet-activating factor (PAF) stimulates a diverse array of cellular responses through receptors coupled to G proteins that activate phospholipase C (PLC). Truncation of the cytoplasmic tail of the receptor to remove phosphorylation sites (mutant PAF receptor, mPAFR) results in enhancement of PAF-stimulated responses. Here we demonstrate that PAF or phorbol 12-myristate 13-acetate (PMA) pretreatment inhibited wild type PAFR-induced PLC-mediated responses by approximately 90%, whereas these responses to the phosphorylation-deficient mPAFR were inhibited by approximately 50%, despite normal G protein coupling, suggesting a distal inhibitory locus. PAF and PMA, as well as a membrane permeable cyclic AMP analog, stimulated phosphorylation of PLCbeta3. A protein kinase C (PKC) inhibitor blocked phosphorylation of PLCbeta3 stimulated by PAF and PMA but not by cAMP. Activation of protein kinase A (PKA) by cAMP did not result in inhibition of Ca2+ mobilization stimulated by PAF. In contrast, cAMP did inhibit the response to formylpeptide chemoattractant receptor. These data suggest that homologous desensitization of PAF-mediated responses is regulated via phosphorylation at two levels in the signaling pathway, one at the receptor and the other at PLCbeta3 mediated by PKC but not by PKA. Phosphorylation of PLCbeta3 by PKA could explain the inhibition of formylpeptide chemoattractant receptor signaling by cAMP. As PAF and formylpeptide chemoattractant receptors activate PLC via different G proteins, phosphorylation of PLCbeta3 by PKC and PKA could provide distinct regulatory control for classes of G protein-coupled receptors.
Insights
Platelet-activating factor (PAF) desensitization involves receptor and PLCbeta3 phosphorylation. Protein kinase C mediates this, not protein kinase A, offering distinct regulatory control for G protein-coupled receptors.
Area of Science:
- Cellular signaling
- Molecular biology
- Immunology
Background:
- Platelet-activating factor (PAF) triggers cellular responses via G protein-coupled receptors activating phospholipase C (PLC).
- Mutant PAF receptors (mPAFR) lacking cytoplasmic tail phosphorylation sites show enhanced PAF responses.
- Understanding PAF receptor desensitization mechanisms is crucial for cellular signaling research.
Purpose of the Study:
- To investigate the molecular mechanisms underlying Platelet-activating factor (PAF) receptor desensitization.
- To determine the role of receptor and phospholipase C (PLC) phosphorylation in PAF-mediated signaling.
- To elucidate the involvement of protein kinase C (PKC) and protein kinase A (PKA) in PAF desensitization.
Main Methods:
- Utilized wild type and mutant PAF receptors (mPAFR) to study PAF-stimulated responses.
- Assessed the impact of Platelet-activating factor (PAF) and phorbol 12-myristate 13-acetate (PMA) pretreatment on PLC-mediated signaling.
- Investigated the phosphorylation of PLCbeta3 stimulated by PAF, PMA, and cyclic AMP (cAMP) analogs.
- Employed protein kinase C (PKC) inhibitors and protein kinase A (PKA) activators to probe signaling pathways.
Main Results:
- PAF and PMA pretreatment significantly inhibited wild type PAF receptor-induced PLC responses, with less inhibition observed for the phosphorylation-deficient mPAFR.
- Both PAF and PMA, along with a cAMP analog, stimulated PLCbeta3 phosphorylation.
- PKC inhibition blocked PAF- and PMA-stimulated PLCbeta3 phosphorylation, while PKA activation by cAMP did not inhibit PAF-stimulated calcium mobilization but did inhibit formylpeptide chemoattractant receptor signaling.
- These findings suggest homologous desensitization occurs at two levels: the receptor and PLCbeta3, with PKC mediating the latter.
Conclusions:
- Homologous desensitization of Platelet-activating factor (PAF) responses is regulated by phosphorylation at both the receptor and PLCbeta3 levels.
- Protein kinase C (PKC) plays a key role in PLCbeta3 phosphorylation during PAF desensitization, whereas Protein kinase A (PKA) does not.
- Differential phosphorylation of PLCbeta3 by PKC and PKA provides distinct regulatory control for different classes of G protein-coupled receptors, including PAF and formylpeptide chemoattractant receptors.