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Mutational analysis of the potential phosphorylation sites for protein kinase C on the CCK(A) receptor
R L Smeets1, M A Fouraux, W Pouwels
1Department of Biochemistry, University of Nijmegen, The Netherlands.
Abstract:
1. Many G protein-coupled receptors contain potential phosphorylation sites for protein kinase C (PKC), the exact role of which is poorly understood. In the present study, a mutant cholecystokininA (CCK(A)) receptor was generated in which the four consensus sites for PKC action were changed in an alanine. Both the wild-type (CCK(A)WT) and mutant (CCK(A)MT) receptor were stably expressed in Chinese hamster ovary (CHO) cells. 2. Binding of [3H]-cholecystokinin-(26-33)-peptide amide (CCK-8) to membranes prepared from CHO-CCK(A)WT cells and CHO-CCK(A)MT cells revealed no difference in binding affinity (Kd values of 0.72 nM and 0.86 nM CCK-8, respectively). 3. The dose-response curves for CCK-8-induced cyclic AMP accumulation and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation were shifted to the left in CHO-CCK(A)MT cells. This leftward shift was mimicked by the potent inhibitor of protein kinase activity, staurosporine. However, the effect of staurosporine was restricted to CHO-CCK(A)WT cells. This demonstrates that attenuation of CCK-8-induced activation of adenylyl cyclase and phospholipase C-beta involves a staurosporine-sensitive kinase, which acts directly at the potential sites of PKC action on the CCK(A) receptor in CCK-8-stimulated CHO-CCK(A)WT cells. 4. The potent PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), evoked a rightward shift of the dose-response curve for CCK-8-induced cyclic AMP accumulation in CHO-CCK(A)WT cells but not CHO-CCK(A)MT cells. This is in agreement with the idea that PKC acts directly at the CCK(A) receptor to attenuate adenylyl cyclase activation. 5. In contrast, TPA evoked a rightward shift of the dose-response curve for CCK-8-induced Ins(1,4,5)P3 formation in both cell lines. This demonstrates that high-level PKC activation inhibits CCK-8-induced Ins(1,4,5)P3 formation also at a post-receptor site. 6. TPA inhibition of agonist-induced Ca2+ mobilization was only partly reversed in CHO-CCK(A)MT cells. TPA also inhibited Ca2+ mobilization in response to the G protein activator, Mas-7. These findings are in agreement with the idea that partial reversal of agonist-induced Ca2+ mobilization is due to the presence of an additional site of PKC inhibition downstream of the receptor and that the mutant receptor itself is not inhibited by the action of PKC. 7. The data presented demonstrate that the predicted sites for PKC action on the CCK(A) receptor are the only sites involved in TPA-induced uncoupling of the receptor from its G proteins. In addition, the present study unveils a post-receptor site of PKC action, the physiological relevance of which may be that it provides a means for the cell to inhibit phospholipase C-beta activation by receptors that are not phosphorylated by PKC.
Insights
Protein kinase C (PKC) phosphorylation sites on the cholecystokinin A (CCK(A)) receptor directly regulate adenylyl cyclase and phospholipase C-beta activation. This study identifies both direct and post-receptor mechanisms of PKC-mediated signaling attenuation in CCK(A) receptor function.
Area of Science:
- Molecular Cell Biology
- G Protein-Coupled Receptor Signaling
- Enzymology
Background:
- G protein-coupled receptors (GPCRs) possess numerous phosphorylation sites, but the precise role of protein kinase C (PKC) in their function remains largely unelucidated.
- The cholecystokinin A (CCK(A)) receptor is a key mediator of gastrointestinal and neuronal signaling, with potential regulatory sites for PKC.
Purpose of the Study:
- To investigate the role of specific PKC phosphorylation sites on the CCK(A) receptor in mediating cellular responses to cholecystokinin-8 (CCK-8).
- To differentiate between direct, receptor-level effects and indirect, post-receptor effects of PKC activation on CCK(A) receptor signaling pathways.
Main Methods:
- Generation of a mutant CCK(A) receptor (CCK(A)MT) with alanine substitutions at four putative PKC phosphorylation sites.
- Stable expression of wild-type (CCK(A)WT) and CCK(A)MT receptors in Chinese hamster ovary (CHO) cells.
- Assessment of CCK-8 binding affinity, cyclic AMP accumulation, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation, and Ca2+ mobilization in response to CCK-8, PKC activators (TPA), and kinase inhibitors (staurosporine).
Main Results:
- Mutation of PKC sites did not alter CCK-8 binding affinity but significantly affected CCK-8-induced signaling.
- PKC activation by TPA attenuated CCK-8-induced adenylyl cyclase activation specifically in CCK(A)WT cells, indicating direct receptor phosphorylation.
- PKC activation inhibited Ins(1,4,5)P3 formation and Ca2+ mobilization via both direct (receptor-dependent) and post-receptor mechanisms, with partial reversal in the mutant receptor.
Conclusions:
- The identified PKC phosphorylation sites on the CCK(A) receptor are crucial for its desensitization and uncoupling from G proteins.
- PKC exerts dual inhibitory control over CCK(A) receptor signaling, acting both directly on the receptor and at a post-receptor level to modulate phospholipase C-beta activity.