Kok Choi Kong

Location, location, location...site-specific GPCR phosphorylation offers a mechanism for cell-type-specific signalling.

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.

Differential G-protein-coupled receptor phosphorylation provides evidence for a signaling bar code.

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.

The M3-muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner

Degeneration of the cholinergic system is considered to be the underlying pathology that results in the cognitive deficit in Alzheimers disease. This pathology is thought to be linked to a loss of signaling through the cholinergic M1-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 M3-muscarinic receptor that is expressed in numerous brain regions including the hippocampus. We demonstrate here that M3-muscarinic receptor knockout mice show a deficit in fear conditioning learning and memory. The mechanism used by the M3-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 M3-muscarinic receptor is phosphorylated in the hippocampus following agonist treatment and following fear conditioning training. Importantly, the phosphorylation-deficient M3-muscarinic receptor was coupled normally to Gq/11-signaling but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. It can, therefore, be concluded that M3-muscarinic receptor–dependent learning and memory depends, at least in part, on receptor phosphorylation/arrestin signaling. This study opens the potential for biased M3-muscarinic receptor ligands that direct phosphorylation/arrestin-dependent (non-G protein) signaling as being beneficial in cognitive disorders.

M3-muscarinic receptor promotes insulin release via receptor phosphorylation/arrestin-dependent activation of protein kinase D1.

The activity of G protein-coupled receptors is regulated via hyper-phosphorylation 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 M3-muscarinic receptor, a prototypical Gq/11-coupled receptor. This mutant mouse strain was used here to investigate the role of M3-muscarinic receptor phosphorylation in the regulation of insulin secretion from pancreatic islets. Importantly, the phosphorylation deficient receptor coupled to Gq/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 M3-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 M3-muscarinic receptor. In conclusion, our findings support the unique concept that M3-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.

Regulation of cell survival by lipid phosphate phosphatases involves the modulation of intracellular phosphatidic acid and sphingosine 1-phosphate pools

We have shown previously that LPPs (lipid phosphate phosphatases) reduce the stimulation of the p42/p44 MAPK (p42/p44 mitogen-activated protein kinase) pathway by the GPCR (G-protein-coupled receptor) agonists S1P (sphingosine 1-phosphate) and LPA (lysophosphatidic acid) in serum-deprived HEK-293 cells [Alderton, Darroch, Sambi, McKie, Ahmed, N. J. Pyne and S. Pyne (2001) J. Biol. Chem. 276, 13452-13460]. In the present study, we now show that this can be blocked by pretreating HEK-293 cells with the caspase 3/7 inhibitor, Ac-DEVD-CHO [N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde)]. Therefore LPP2 and LPP3 appear to regulate the apoptotic status of serum-deprived HEK-293 cells. This was supported further by: (i) caspase 3/7-catalysed cleavage of PARP [poly(ADP-ribose) polymerase] was increased in serum-deprived LPP2-overexpressing compared with vector-transfected HEK-293 cells; and (ii) serum-deprived LPP2- and LPP3-overexpressing cells exhibited limited intranucleosomal DNA laddering, which was absent in vector-transfected cells. Moreover, LPP2 reduced basal intracellular phosphatidic acid levels, whereas LPP3 decreased intracellular S1P in serum-deprived HEK-293 cells. LPP2 and LPP3 are constitutively co-localized with SK1 (sphingosine kinase 1) in cytoplasmic vesicles in HEK-293 cells. Moreover, LPP2 but not LPP3 prevents SK1 from being recruited to a perinuclear compartment upon induction of PLD1 (phospholipase D1) in CHO (Chinese-hamster ovary) cells. Taken together, these data are consistent with an important role for LPP2 and LPP3 in regulating an intracellular pool of PA and S1P respectively, that may govern the apoptotic status of the cell upon serum deprivation.

Cooperative mitogenic signaling by G protein-coupled receptors and growth factors is dependent on Gq/11

Previously we reported that the G protein‐coupled receptor (GPCR) agonist thrombin potentiated the mitogenic effect of epidermal growth factor (EGF) on human airway smooth muscle (ASM) by promoting sustained late‐phase activation of PI3K and p70S6K via a pathway dependent on Gβγ subunits of heterotrimeric G proteins. Here, we provide additional mechanistic insight and reveal the robustness of this phenomenon by demonstrating that H1 histamine and thromboxane receptors utilize the same mechanism to augment ASM growth via specific activation of the heterotrimeric G protein Gq/11. Thrombin, histamine, and U46619 all enhanced EGF‐stimulated [3H]‐thymidine incorporation as well as late‐phase Akt and p70S6K phosphorylation in ASM cultures. Heterologous expression of Gβγ sequestrants (GRK2CT‐GFP or GαiG203A), as well as GRK2NT‐GFP (an RGS protein for Gq/11) but neither p115RhoGEFRGS‐GFP (an RGS for G12/13) nor pertussis toxin pretreatment (inactivating Gi/o), attenuated the effects on both signaling and growth. Inhibition of Rho, Rho kinase, or Src, or modulation of arrestin expression did not significantly affect the cooperative signaling by EGF and any of the GPCR agonists. Thus, Gq/11‐coupled receptors are the principal GPCR subfamily mediating cooperative mitogenic signaling in ASM, acting through Gβγ‐dependent, and Src/arrestin‐independent activation of PI3K and p70S6K.—Kong, K. C., Billington, C. K., Gandhi, U., Panettieri, R. A., Penn, R. B. Cooperative mitogenic signaling by G protein‐coupled receptors and growth factors is dependent on Gq/11. FASEB J. 20, E880–E887 (2006)

Physiological role of G-protein coupled receptor phosphorylation.

It is now well established that G-protein coupled receptors (GPCRs) are hyper-phosphorylated following agonist occupation usually at serine and threonine residues contained on the third intracellular loop and C-terminal tail. After some 2 decades of intensive research, the nature of protein kinases involved in this process together with the signalling consequences of receptor phosphorylation has been firmly established. The major challenge that the field currently faces is placing all this information within a physiological context and determining to what extent does phosphoregulation of GPCRs impact on whole animal responses. In this chapter, we address this issue by describing how GPCR phosphorylation might vary depending on the cell type in which the receptor is expressed and how this might be employed to drive selective regulation of physiological responses.

The role of M(3)-muscarinic receptor signaling in insulin secretion.

Recently, M3-muscarinic receptor (M3R) has been identified as the bona fide receptor responsible for the cholinergic regulation of glucose-induced insulin release. The molecular mechanisms of such regulation have also begun to be unravelled. These include the conventional G protein-dependent pathways involving calcium mobilization and activation of protein kinase C. In addition, recent studies also provided evidence for G protein-independent pathways in the regulation of insulin secretion by M3R. These include phosphorylation/arrestin-dependent activation of protein kinase D1, Src family kinase-dependent activation of the sodium channel NALCN and the involvement of regulator of G protein signalling (RGS)-4. Time has now come to extend these studies which were done mainly in rodents to human and explore the potential for targeting such pathways at different levels for the treatment of diseases with impaired insulin secretion such as type II diabetes.

N-Methyl-d-aspartate Receptors Mediate the Phosphorylation and Desensitization of Muscarinic Receptors in Cerebellar Granule Neurons

Changes in synaptic strength mediated by ionotropic glutamate N-methyl-d-asparate (NMDA) receptors is generally considered to be the molecular mechanism underlying memory and learning. NMDA receptors themselves are subject to regulation through signaling pathways that are activated by G-protein-coupled receptors (GPCRs). In this study we investigate the ability of NMDA receptors to regulate the signaling of GPCRs by focusing on the Gq/11-coupled M3-muscarinic receptor expressed endogenously in mouse cerebellar granule neurons. We show that NMDA receptor activation results in the phosphorylation and desensitization of M3-muscarinic receptors through a mechanism dependent on NMDA-mediated calcium influx and the activity of calcium-calmodulin-dependent protein kinase II. Our study reveals a complex pattern of regulation where GPCRs (M3-muscarinic) and NMDA receptors can feedback on each other in a process that is likely to influence the threshold value of signaling networks involved in synaptic plasticity.

Exploiting functional domains of GRK2/3 to alter the competitive balance of pro- and anticontractile signaling in airway smooth muscle

To clarify the potential utility of targeting GRK2/3‐mediated desensitization as a means of manipulating airway smooth muscle (ASM) contractile state, we assessed the specificity of GRK2/3 regulation of procontractile and relaxant G‐protein‐coupled receptors in ASM. Functional domains of GRK2/3 were stably expressed, or siRNA‐mediated GRK2/3 knockdown was performed, in human ASM cultures, and agonist‐induced signaling was assessed. Regulation of contraction of murine tracheal rings expressing GRK2 C terminus was also assessed. GRK2/3 knockdown or expression of the GRK2 C terminus caused a significant (~30–90%) increase in maximal β‐agonist and histamine [phosphoinositide (PI) hydrolysis] signaling, without affecting the calculated EC50. GRK2 C‐terminal expression did not affect signaling by methacholine, thrombin, or LTD4. Expression of the GRK2 N terminus or kinase‐dead holo‐GRK2 diminished (~30–70%) both PI hydrolysis and Ca2+ mobilization by every Gq‐coupled receptor examined. Under conditions of GRK2 C‐terminal expression, β‐agonist inhibition of methacholine‐stimulated PI hydrolysis was greater. Finally, transgenic expression of the GRK2 C terminus in murine ASM enabled ~30–50% greater β‐agonist‐mediated relaxation of methacholine‐induced contraction. Collectively these data demonstrate the relative selectivity of GRKs for the β2AR in ASM and the ability to exploit GRK2/3 functional domains to render ASM hyporesponsive to contractile agents while increasing responsiveness to bronchodilating β‐agonist.—Deshpande, D. A., Yan, H., Kong, K.‐C., Tiegs, B. C., Morgan, S. J., Pera, T., Panettieri, R. A., Eckhart, A. D., Penn, R. B. Exploiting functional domains of GRK2/3 to alter the competitive balance of pro‐ and anticontractile signaling in airway smooth muscle. FASEB J. 28, 956–965 (2014). www.fasebj.org