G-protein-coupled receptor phosphorylation: where, when and by whom

Abstract

Almost all G-protein coupled receptors (GPCRs) are regulated by phosphorylation and this process is a key event in determining the signalling properties of this receptor super-family. Receptors are multiply phosphorylated at sites that can occur throughout the intracellular regions of the receptor. This diversity of phospho-acceptor sites together with a lack of consensus phosphorylation sequences has led to the suggestion that the precise site of phosphorylation is not important in the phosphorylation-dependent regulation of GPCR function but rather it is the increase in bulk negative charge of the intracellular face of the receptor which is the significant factor. This review investigates the possibility that the multi-site nature of GPCR phosphorylation reflects the importance of specific phosphorylation events which mediate distinct signalling outcomes. In this way receptor phosphorylation may provide for a flexible regulatory mechanism that can be tailored in a tissue specific manner to regulate physiological processes. By understanding the flexible nature of GPCR phosphorylation if may be possible to develop agonists or allosteric modulators that promote a subset of phosphorylation events on the target GPCR and thereby restrict the action of the drug to a particular receptor mediated signalling response.

Figure 1

Interaction of arrestin with an activated and phosphorylated G-protein-coupled receptor. (1) Agonist binding to the receptor results in a conformational change that particularly affects the orientation of transmembrane helices III and VI. (2) This conformational change is thought to result in as yet undefined changes in the intracellular loop regions and C-terminal tail that unmasks phosphorylation sites and results in multi-site phosphorylation (P). (3) The active conformation of the receptor is detected by the activation sensor on arrestin. The activation sensor consists of multiple interaction sites situated on the concave surface of the arrestin molecule that are able to engage with the active conformation of the receptor. Also, phosphorylated residues are shown initially interacting with residues (lysines) situated on the outer surface of the arrestin molecule. (4) Arrestin binds with high affinity to the receptor and adopts an active conformation through further interactions with the activated receptor via the activation sensor and by phosphates interacting with the phosphate sensor situated in the polar core. The active conformation of arrestin is proposed to involve re-orientation of the two domains of arrestin.

Figure 2

Summary of protein kinases known to phosphorylate G-protein-coupled receptors (GPCRs). Shown are protein kinases that have been demonstrated to phosphorylate GPCRs and the main functional consequences of the phosphorylation event.

Figure 3

M₃-muscarinic receptors expressed in Chinese hamster ovary (CHO) cells and cerebellar granule neurons are differentially phosphorylated. Cerebellar granule neurons were cultured from 6-day-old neonatal mice and maintained for 6 days before being labelled for phosphorylation studies. CHO cells expressing the recombinant mouse M₃-muscarinic receptor or cerebellar granule neurons were labelled with [³²P]orthophosphate for 1–2 h (100–200 μCi ml⁻¹). The cells were then stimulated for 5 min with the muscarinic agonist methacholine (100 μM). Cells were then solubilized and the M₃-muscarinic receptor immunoprecipitated using a receptor-specific antibody. The immunoprecipitate was resolved by 8% SDS-PAGE and the radioactive band associated with the receptor was excised. The receptor band was then digested with trypsin and the resulting peptides resolved in the first dimension by electrophoresis and in the second dimension by thin layer chromatography. The position of the radioactive phospho-peptides was then determined in a phospho-imager. The numbered peptides are those that run in the same position and represent phosphorylation sites that are the same in the two cell types. The arrows represent phosphorylation events that are specific to the cell in which the receptor is expressed. The experiment shown was adapted from Torrecilla et al. (2007) where details of the methods can be obtained.