It is now established that most of the ∼800 G-protein-coupled receptors (GPCRs) are regulated by phosphorylation in a process that results in the recruitment of arrestins, leading to receptor desensitization and the activation of arrestin-dependent processes. This generalized view of GPCR regulation, however, does not provide an adequate mechanism for the control of tissue-specific GPCR signalling. Here, we review the evidence that GPCR phosphorylation is, in fact, a flexible and dynamic regulatory process in which GPCRs are phosphorylated in a unique manner that is associated with the cell type in which the receptor is expressed. In this scenario, phosphorylation offers a mechanism of regulating the signalling outcome of GPCRs that can be tailored to meet a specific physiological role.

G-protein-coupled receptors are hyper-phosphorylated in a process that controls receptor coupling to downstream signaling pathways. The pattern of receptor phosphorylation has been proposed to generate a “bar code” that can be varied in a tissue-specific manner to direct physiologically relevant receptor signaling. If such a mechanism existed, receptors would be expected to be phosphorylated in a cell/tissue-specific manner. Using tryptic phosphopeptide maps, mass spectrometry, and phospho-specific antibodies, it was determined here that the prototypical Gq/11-coupled M3-muscarinic receptor was indeed differentially phosphorylated in various cell and tissue types supporting a role for differential receptor phosphorylation in directing tissue-specific signaling. Furthermore, the phosphorylation profile of the M3-muscarinic receptor was also dependent on the stimulus. Full and partial agonists to the M3-muscarinic receptor were observed to direct phosphorylation preferentially to specific sites. This hitherto unappreciated property of ligands raises the possibility that one mechanism underlying ligand bias/functional selectivity, a process where ligands direct receptors to preferred signaling pathways, may be centered on the capacity of ligands to promote receptor phosphorylation at specific sites.

Platelet-derived growth factor (PDGF) and sphingosine 1-phosphate (S1P) act via PDGF␤ receptor-S1P 1 receptor complexes in airway smooth muscle cells to promote mitogenic signaling. Several lines of evidence support this conclusion. First, both receptors were co-immunoprecipitated from cell lysates with specific anti-S1P 1 antibodies, indicating that they form a complex. Second, treatment of airway smooth muscle cells with PDGF stimulated the phosphorylation of p42/p44 MAPK, and this phosphorylated p42/p44 MAPK associates with the PDGF␤ receptor-S1P 1 receptor complex. Third, treatment of cells with antisense S1P 1 receptor plasmid construct reduced the PDGF-and S1P-dependent activation of p42/p44 MAPK. Fourth, S1P and/or PDGF induced the formation of endocytic vesicles containing both PDGF␤ receptors and S1P 1 receptors, which was required for activation of the p42/ p44 MAPK pathway. PDGF does not induce the release of S1P, suggesting the absence of a sequential mechanism. However, sphingosine kinase 1 is constitutively exported from cells and supports activation of p42/p44 MAPK by exogenous sphingosine. Thus, the presentation of sphingosine from other cell types and its conversion to S1P by the kinase exported from airway smooth muscle cells might enable S1P to act with PDGF on the PDGF␤ receptor-S1P 1 receptor complex to induce biological responses in vivo. These data provide further evidence for a novel mechanism for G-protein-coupled receptor and receptor tyrosine kinase signal integration that is distinct from the transactivation of receptor tyrosine kinases by G-proteincoupled receptor agonists and/or sequential release and action of S1P in response to PDGF. Sphingosine 1-phosphate (S1P)1 is a bioactive lysolipid that has been proposed to have both intracellular and extracellular actions (1). To date, five closely related G-protein-coupled receptors (GPCR), termed S1P 1 -S1P 5 (2) (and formerly named EDG1, EDG5/AGR16/H218, EDG3, EDG6 and EDG8/nrg-1, respectively) have been identified as high affinity S1P receptors (3-9). Further characterization studies confirmed the S1P 1 receptor to be a GPCR with high affinity for S1P that stimulates p42/p44 MAPK and inhibits adenylyl cyclase in cells (10 -13). The S1P 2 and S1P 3 receptors also have high affinity for S1P (14) and are linked via G q to phospholipase C and calcium mobilization and p42/p44 MAPK activation (14, 15) and via G 12 and G 13 to Rhoguanine nucleotide factor and Rho activation. The S1P 4 receptor is lymphoid specific and, in common with the S1P 5 receptor, uses G i/o and G 12 to signal (6, 8).The S1P 1 receptor is implicated in regulating smooth muscle cell migration, proliferation, and vascular maturation. Insight into the function of the S1P 1 receptor was obtained by studies showing that disruption of the s1p 1 gene by homologous recombination in mice results in extensive intra-embryonic hemorrhaging and intrauterine (16). This is caused by incomplete vascular maturation due to the failure of mural cells, vascular smooth muscle cells, and per...

Degeneration of the cholinergic system is considered to be the underlying pathology that results in the cognitive deficit in Alzheimer's disease. This pathology is thought to be linked to a loss of signaling through the cholinergic M 1 -muscarinic receptor subtype. However, recent studies have cast doubt on whether this is the primary receptor mediating cholinergic-hippocampal learning and memory. The current study offers an alternative mechanism involving the M 3 -muscarinic receptor that is expressed in numerous brain regions including the hippocampus. We demonstrate here that M 3 -muscarinic receptor knockout mice show a deficit in fear conditioning learning and memory. The mechanism used by the M 3 -muscarinic receptor in this process involves receptor phosphorylation because a knockin mouse strain expressing a phosphorylation-deficient receptor mutant also shows a deficit in fear conditioning. Consistent with a role for receptor phosphorylation, we demonstrate that the M 3 -muscarinic receptor is phosphorylated in the hippocampus following agonist treatment and following fear conditioning training. Importantly, the phosphorylation-deficient M 3 -muscarinic receptor was coupled normally to G q/11 -signaling but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. It can, therefore, be concluded that M 3 -muscarinic receptor-dependent learning and memory depends, at least in part, on receptor phosphorylation/arrestin signaling. This study opens the potential for biased M 3 -muscarinic receptor ligands that direct phosphorylation/arrestin-dependent (non-G protein) signaling as being beneficial in cognitive disorders.A mong the multitude of physiological responses regulated by G protein-coupled receptors (GPCRs), one of the most intriguing is the ability of this superfamily of cell-surface receptors to regulate neurological and behavioral processes such as learning and memory (1-4). The members of the muscarinic acetylcholine receptor family are prominent among the GPCR subtypes associated with cognitive function because lesions in cholinergic innervations to the hippocampus and other brain areas are widely thought to underlie the cognitive deficit observed in Alzheimer's disease (5). Whereas the M 1 -muscarinic receptor subtype has been proposed to be the subtype associated with acetylcholine-mediated cognition (6, 7), recent gene-knockout experiments have cast doubt on the direct role of this receptor subtype in learning and memory (1,8). This has been reinforced by the discovery of a novel selective M 1 -muscarinic receptor antagonist that was effective in blocking M 1 -muscarinic receptor-mediated seizures in vivo but had no effect on hippocampal-based contextual fear conditioning (9). In addition, recent studies using an M 1 -muscarinic receptorpositive allosteric modulator, BQCA, have suggested that M 1 -muscarinic receptors can mediate learning and memory through an indirect mechanism by stimulating the prefrontal cortex (10). There is some con...

The activity of G protein-coupled receptors is regulated via hyperphosphorylation following agonist stimulation. Despite the universal nature of this regulatory process, the physiological impact of receptor phosphorylation remains poorly studied. To address this question, we have generated a knock-in mouse strain that expresses a phosphorylation-deficient mutant of the M 3 -muscarinic receptor, a prototypical G q/11 -coupled receptor. This mutant mouse strain was used here to investigate the role of M 3 -muscarinic receptor phosphorylation in the regulation of insulin secretion from pancreatic islets. Importantly, the phosphorylation deficient receptor coupled to G q/11 -signaling pathways but was uncoupled from phosphorylation-dependent processes, such as receptor internalization and β-arrestin recruitment. The knock-in mice showed impaired glucose tolerance and insulin secretion, indicating that M 3 -muscarinic receptors expressed on pancreatic islets regulate glucose homeostasis via receptor phosphorylation-/arrestin-dependent signaling. The mechanism centers on the activation of protein kinase D1, which operates downstream of the recruitment of β-arrestin to the phosphorylated M 3 -muscarinic receptor. In conclusion, our findings support the unique concept that M 3 -muscarinic receptor-mediated augmentation of sustained insulin release is largely independent of G protein-coupling but involves phosphorylation-/ arrestin-dependent coupling of the receptor to protein kinase D1. G-protein coupled receptor | ligand biasT he vast majority of G protein-coupled receptors (GPCRs) respond to agonist occupation by becoming rapidly hyperphosphorylated within intracellular domains (1-3). This process not only leads to the uncoupling of the receptor from its cognate G proteins, but also allows for the activation of G proteinindependent signaling, a process that is driven largely by the recruitment of β-arrestin adaptor proteins (4-7). As a consequence, GPCRs regulate an extensive array of signaling pathways and biological responses (3). G protein-independent signaling pathways have mostly been studied in recombinant systems. However, the current challenge is to understand to what extent these processes are involved in the regulation of key physiological responses.In the present study, we examined the in vivo role of GPCR phosphorylation by generating a knock-in mouse strain expressing a phosphorylation-deficient GPCR. Specifically, we used the M 3 -muscarinic acetylcholine receptor, a prototypic G q/11 -coupled GPCR, as a model system (8, 9). We and others have previously demonstrated that the M 3 -muscarinic receptor is rapidly phosphorylated on agonist occupation by a range of protein kinases that include members of the GPCR kinase (GRK) family, as well as casein kinase 1α and protein kinase CK2 (10-13). To define the potential physiological role of M 3 -muscarinic receptor phosphorylation, we generated a mouse strain where the wild-type M 3 -muscarinic receptor gene had been replaced by a phosphorylationdeficient mutan...