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Phosphorylation of muscarinic receptors: regulation by G proteins
1Department of Biochemistry, University of Tokyo, Japan.
Abstract:
Effects of G proteins on the phosphorylation of muscarinic receptors (mAChRs) have been examined. Cerebral but not atrial mAChRs were phosphorylated by any one of three types of protein kinase C and 4-6 mol of phosphate were incorporated per mol of mAChR, mostly in the 12-14 kDa from the carboxyterminus. Atrial mAChRs were better substrates of cAMP-dependent protein kinase than cerebral mAChRs. Phosphorylation of mAChRs by protein kinase C or cAMP-dependent protein kinase was not dependent on the presence of agonists and G proteins except that a slight inhibition by G proteins was observed probably because G proteins were also substrates of the two kinases. Agonist-dependent phosphorylation of atrial mAChRs or recombinant human mAChRs (m2 subtype) by a kinase (mAChR kinase), which is the same or very similar to beta adrenergic receptor kinase (beta ARK), was found to be regulated by the G proteins in a dual manner; stimulation by G protein beta gamma subunits and inhibition by G protein alpha beta gamma trimer. The inhibition by the G protein trimer is restored by addition of guanine nucleotides and is considered to be due to the formation of a ternary complex of agonist, mAChR and guanine nucleotide free G proteins. The stimulation by G protein beta gamma subunits was also observed for the light- or agonist-dependent phosphorylation of rhodopsin and beta AR by the mAChR kinase but not for the light-dependent phosphorylation of rhodopsin by rhodopsin kinase. The phosphorylation by beta ARK 1 was also found to be stimulated by G protein beta gamma subunits. The beta gamma subunit is considered to interact with the extra 130 amino acid residue carboxyterminal tail of beta ARK, which does not exist in rhodopsin kinase, and the interaction results in the activation of the kinase. We may assume that the G protein coupled receptor kinase is an effector of G protein beta gamma subunits and that one of the functions of beta gamma subunits is to stimulate the phosphorylation of G protein coupled receptors thereby facilitating their desensitization.
Insights
G protein beta gamma subunits stimulate G protein-coupled receptor kinase activity, promoting receptor phosphorylation and desensitization. This highlights a key regulatory mechanism in cellular signaling pathways.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Signaling
Background:
- Muscarinic receptors (mAChRs) are G protein-coupled receptors involved in various physiological processes.
- Receptor phosphorylation is a critical mechanism for regulating receptor activity and desensitization.
- The role of G proteins in modulating receptor phosphorylation has been an area of active investigation.
Purpose of the Study:
- To investigate the effects of G proteins on the phosphorylation of muscarinic receptors (mAChRs).
- To elucidate the specific roles of different G protein subunits and kinases in mAChR phosphorylation.
- To understand the regulatory mechanisms underlying agonist-dependent phosphorylation of mAChRs.
Main Methods:
- Phosphorylation assays using cerebral and atrial mAChRs with various protein kinases (Protein Kinase C, cAMP-dependent protein kinase).
- Characterization of mAChR phosphorylation sites and stoichiometry.
- Investigation of G protein regulation using recombinant mAChRs (m2 subtype) and beta-adrenergic receptor kinase (beta ARK).
Main Results:
- Cerebral and atrial mAChRs exhibit differential phosphorylation by Protein Kinase C and cAMP-dependent protein kinase.
- Agonist-dependent phosphorylation of mAChRs by mAChR kinase (similar to beta ARK) is regulated by G proteins.
- G protein beta gamma subunits stimulate mAChR kinase activity, while G protein trimers inhibit it, suggesting a dual regulatory role.
Conclusions:
- G protein beta gamma subunits act as effectors for G protein-coupled receptor kinases (GPCRKs).
- GPCRKs, stimulated by beta gamma subunits, facilitate the phosphorylation and desensitization of G protein-coupled receptors.
- This study reveals a novel mechanism for G protein-mediated regulation of receptor function.