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Protein kinase C deficiency blocks recovery from agonist-induced desensitization
1Department of Molecular Pharmacology, Diabetes & Metabolic Diseases Research Program, University Medical Center, SUNY/Stony Brook, Stony Brook, New York 11794-8651, USA.
Abstract:
Protein phosphorylation is central to agonist-induced attenuation of the function of G-protein-linked receptors. Stable expression of RNA antisense to specific protein kinase mRNAs permitted analysis of loss-of-function mutants of A431 human epidermoid carcinoma cells, lacking protein kinase A, protein kinase C, or beta-adrenergic receptor kinase. Deficiency of protein kinase C, but not the others, amplified rather than attenuated agonist-induced desensitization. In wild-type cells, the t1/2 for recovery from desensitization was approximately 25 min following removal of agonist. In the protein kinase C-deficient cells, no resensitization was observed even 60 min after agonist removal. Like protein kinase C-deficiency, inhibition of protein kinase C with bisindolylmaleimide or calphostin C blocked resensitization. Resensitization was suppressed by FK506, an inhibitor of protein phosphatase 2B, mimicking protein kinase C-deficiency, but in a non-additive manner. The data reveal protein kinase C and protein phosphatase 2B to be critical elements of resensitization.
Insights
Protein Kinase C (PKC) deficiency amplifies G-protein-linked receptor desensitization. PKC and Protein Phosphatase 2B are critical for receptor resensitization after agonist removal.
Area of Science:
- Cellular signaling pathways
- Receptor biology
- Biochemistry
Background:
- G-protein-linked receptors (GPCRs) mediate cellular responses to various stimuli.
- Agonist-induced desensitization is a key regulatory mechanism for GPCR function.
- Protein phosphorylation plays a crucial role in GPCR desensitization and resensitization.
Purpose of the Study:
- To investigate the role of specific protein kinases in agonist-induced desensitization and resensitization of GPCRs.
- To identify the key molecular players involved in the recovery of GPCR function after agonist exposure.
Main Methods:
- Utilized stable expression of antisense RNA to generate loss-of-function mutants in A431 cells lacking Protein Kinase A (PKA), Protein Kinase C (PKC), or beta-adrenergic receptor kinase (beta-ARK).
- Assessed agonist-induced desensitization and resensitization kinetics in wild-type and mutant cell lines.
- Employed specific inhibitors (bisindolylmaleimide, calphostin C, FK506) to probe the involvement of PKC and Protein Phosphatase 2B (PP2B) in resensitization.
Main Results:
- PKC deficiency amplified agonist-induced desensitization, unlike PKA or beta-ARK deficiency.
- Loss of PKC function abolished receptor resensitization, with no recovery observed even after 60 minutes of agonist removal.
- Inhibition of PKC or PP2B similarly blocked resensitization, indicating their critical roles.
- FK506, a PP2B inhibitor, suppressed resensitization in a non-additive manner with PKC deficiency.
Conclusions:
- Protein Kinase C is essential for the resensitization of G-protein-linked receptors following agonist stimulation.
- Protein Phosphatase 2B also plays a critical role in the resensitization process.
- These findings highlight the intricate interplay between protein kinases and phosphatases in regulating GPCR signaling dynamics.