We have compared the ability of a number of -opioid receptor (MOPr) ligands to activate G proteins with their abilities to induce MOPr phosphorylation, to promote association of arrestin-3 and to cause MOPr internalization. For a model of G protein-coupled receptor (GPCR) activation where all agonists stabilize a single active conformation of the receptor, a close correlation between signaling outputs might be expected. Our results show that overall there is a very good correlation between efficacy for G protein activation and arrestin-3 recruitment, whereas a few agonists, in particular endomorphins 1 and 2, display apparent bias toward arrestin recruitment. The agonist-induced phosphorylation of MOPr at Ser 375 , considered a key step in MOPr regulation, and agonist-induced internalization of MOPr were each found to correlate well with arrestin-3 recruitment. These data indicate that for the majority of MOPr agonists the ability to induce receptor phosphorylation, arrestin-3 recruitment, and internalization can be predicted from their ability as agonists to activate G proteins. For the prototypic MOPr agonist morphine, its relatively weak ability to induce MOPr internalization can be explained by its low agonist efficacy.
The G i/o -coupled histamine H 4 receptor is highly expressed in hemopoietic cells and is a promising new target for the treatment of chronic inflammatory diseases. 1-[(5-Chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine (JNJ7777120) has been described as a selective antagonist at the H 4 receptor and is widely used to characterize the physiological role of the H 4 receptor. We have investigated the pharmacological properties of JNJ7777120 using two distinct downstream signaling measurements, G protein activation and -arrestin recruitment. The H 4 receptor agonists histamine and clobenpropit, but not JNJ7777120, were able to induce [35 S]GTP␥S binding in membranes prepared from U2OS-H 4 cells. Thioperamide, a dual H 3 /H 4 receptor antagonist, and JNJ7777120 were both able to inhibit the [ 35 S]GTP␥S binding induced by clobenpropit. Agonists and antagonists specific for other members of the histamine receptor family had no effect in this assay format. Histamine and clobenpropit increased -arrestin recruitment to the H 4 receptor in a concentration-dependent manner. This -arrestin recruitment could be inhibited by preincubation with thioperamide. We were surprised to find that preincubation with the H 4 -selective antagonist JNJ7777120 potentiated rather than antagonized the response to a submaximal concentration of clobenpropit. JNJ7777120 treatment alone resulted in an increase in -arrestin recruitment, which again could be inhibited by preincubation with thioperamide. Schild analysis demonstrated competitive antagonism between thioperamide and both clobenpropit and JNJ7777120. Histamine and clobenpropit had comparable potencies for both [ 35 S]GTP␥S binding and -arrestin recruitment, suggesting little difference in the levels of receptor reserve between the two assays. In conclusion, we have demonstrated that JNJ7777120 recruits -arrestin to the H 4 receptor, independent of G protein activation.
Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the -opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [DAla 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K ϩ current in a concentrationdependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K ϩ current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.
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...
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