Phosphorylation and regulation of a G protein–coupled receptor by protein kinase CK2

Abstract

We demonstrate a role for protein kinase casein kinase 2 (CK2) in the phosphorylation and regulation of the M3-muscarinic receptor in transfected cells and cerebellar granule neurons. On agonist occupation, specific subsets of receptor phosphoacceptor sites (which include the SASSDEED motif in the third intracellular loop) are phosphorylated by CK2. Receptor phosphorylation mediated by CK2 specifically regulates receptor coupling to the Jun-kinase pathway. Importantly, other phosphorylation-dependent receptor processes are regulated by kinases distinct from CK2. We conclude that G protein–coupled receptors (GPCRs) can be phosphorylated in an agonist-dependent fashion by protein kinases from a diverse range of kinase families, not just the GPCR kinases, and that receptor phosphorylation by a defined kinase determines a specific signalling outcome. Furthermore, we demonstrate that the M3-muscarinic receptor can be differentially phosphorylated in different cell types, indicating that phosphorylation is a flexible regulatory process where the sites that are phosphorylated, and hence the signalling outcome, are dependent on the cell type in which the receptor is expressed.

M 3 -muscarinic receptor promotes insulin release via receptor phosphorylation/arrestin-dependent activation of protein kinase D1

“…Our previous studies on the human M 3 -muscarinic receptor have established that the receptor is phosphorylated on multiple serines within the third intracellular loop (10,12). In the present study, mutation of 15 of these serine residues to alanine in the mouse M 3 -muscarinic receptor resulted in a receptor (termed M3 phos-neg ) (Fig.…”

The M 3 -muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner

“…The M 3 -muscarinic receptor can be phosphorylated by a number of protein kinases including members of the GRK family (26,27) as well as protein kinase CK2 (18) and CK1α (17). In this respect M 3 -muscarinic receptors are similar to many other GPCRs where phosphorylation at multiple sites by more than one protein kinase has been observed (30).…”

“…Nearly all GPCRs are rapidly phosphorylated in response to agonist stimulation at multiple sites on the intracellular loops and C-terminal tail (16). The M 3 -muscarinic receptor is no exception, being phosphorylated by a number of protein kinases at serine clusters contained within the third intracellular loop (17,18). We tested the possibility that M 3 -muscarinic receptor phosphorylation was important in the mechanism of action of this receptor in physiological responses.…”

“…We next tested whether removal of the phosphoacceptor sites on the M 3 -muscarinic receptor affected the ability of the receptor to recruit arrestin. These experiments were conducted on the human M 3 -muscarinic receptor where we have shown previously that mutation of the phospho-acceptor sites (comparable to those of the mouse receptor) resulted in a reduction in both agonist-mediated receptor phosphorylation and receptor internalization (18). In an assay for the recruitment of β-arrestin, the phosphorylation-deficient M 3 -muscarinic receptor showed a significant reduction in both the potency and efficacy of arrestinrecruitment in response to the full agonists methacholine and acetylcholine ( Fig.…”

“…Although it was clear from our previous studies that the M 3 -muscarinic receptor was phosphorylated at multiple sites (18), not just serine 384 , we nevertheless investigated the phosphorylation at this single site as an exemplar of receptor phosphorylation. Immunoprecipitation of the M 3 -muscarinic receptor from wild-type hippocampus followed by Western blotting using the anti-phosphoserine 384 antibody revealed that the receptor was phosphorylated at serine 384 in the basal state in the mouse hippocampus (Fig.…”