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Originally published In Press as doi:10.1074/jbc.M412682200 on March 2, 2005

J. Biol. Chem., Vol. 280, Issue 19, 18950-18958, May 13, 2005
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Roles of Phosphorylation-dependent and -independent Mechanisms in the Regulation of M1 Muscarinic Acetylcholine Receptors by G Protein-coupled Receptor Kinase 2 in Hippocampal Neurons*

Jonathon M. Willets{ddagger}, Stefan R. Nahorski, and R. A. John Challiss

From the Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, LE1 9HN, United Kingdom

Received for publication, November 9, 2004 , and in revised form, February 16, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
When co-expressed with the inositol 1,4,5-trisphosphate biosensor eGFP-PHPLC{delta}, G protein-coupled receptor kinase 2 (GRK2) can suppress M1 muscarinic acetylcholine (mACh) receptor-mediated phospholipase C signaling in hippocampal neurons through a phosphorylation-independent mechanism, most likely involving the direct binding of the RGS homology domain of GRK2 to G{alpha}q/11. To define the importance of this mechanism in comparison with classical, phosphorylation-dependent receptor regulation by GRKs, we have examined M1 mACh receptor signaling in hippocampal neurons following depletion of GRK2 and also in the presence of non-G{alpha}q/11-binding GRK2 mutants. Depletion of neuronal GRK2 using an antisense strategy almost completely inhibited M1 mACh receptor desensitization without enhancing acute agonist-stimulated phospholipase C activity. By stimulating neurons with a submaximal agonist concentration before (R1) and after (R2) a period of exposure to a maximal agonist concentration, an index (R2/R1) of agonist-induced desensitization of signaling could be obtained. Co-transfection of neurons with either a non-G{alpha}q/11-binding (D110A) GRK2 mutant or the catalytically inactive D110A,K220RGRK2 did not suppress acute M1 mACh receptor-stimulated inositol 1,4,5-trisphosphate production. However, using the desensitization (R2/R1) protocol, it could be shown that expression of D110AGRK2 enhanced, whereas D110A,K220RGRK2 inhibited, agonist-induced M1 mACh receptor desensitization. In Chinese hamster ovary cells, the loss of G{alpha}q/11 binding did not affect the ability of the D110AGRK2 mutant to phosphorylate M1 mACh receptors, whereas expression of D110A,K220RGRK2 had no effect on receptor phosphorylation. These data indicate that in hippocampal neurons endogenous GRK2 is a key regulator of M1 mACh receptor signaling and that the regulatory process involves both phosphorylation-dependent and -independent mechanisms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite many years of investigation we still have an incomplete understanding of how cholinergic inputs modulate neuronal function in the hippocampus. Nevertheless, it has been clearly shown that cholinergic innervation of the hippocampus is widespread (1, 2) and that cholinergic deficits (caused by lesioning, pharmacological blockade, or gene knock-out) produce an array of disorders in learning and memory (3–5). Transgenic approaches have helped to define the key roles of M1 muscarinic acetylcholine (mACh)1 receptors in cholinergic regulation of hippocampal function (5–8), and gaining a better understanding of the physiological and pathophysiological regulation of this mACh receptor subtype in hippocampal neurons remains a key objective.

Desensitization of G protein-coupled receptor (GPCR) signaling following continuous or repeated agonist challenge is believed to be initiated by the phosphorylation of specific serine and/or threonine residues within the third intracellular loop and/or C-terminal tail of the receptor (9, 10) and is a crucial mechanism for reducing (or "switching") signaling (11). Receptor desensitization is usually mediated by either second messenger-activated kinases (e.g. protein kinase C (PKC)) or GPCR kinases (GRKs). Receptor phosphorylation leads to the recruitment of arrestin proteins, which bind and physically prevent interaction between the GPCR and G protein (10, 12). Receptor-arrestin complexes are also involved in the initiation of receptor internalization and may act as signaling scaffolds to assemble transduction pathways (12, 13).

Recently it has been shown that at least two members of the GRK family, GRK2 and GRK3, are able to suppress G{alpha}q/11-coupled receptor/phospholipase C (PLC) signaling even in the absence of kinase activity (14–16). GRK2-mediated, phosphorylation-independent receptor regulation has now been demonstrated not only in recombinant systems but also for endogenous receptors in cell lines (17) and in primary, cultured hippocampal neurons (18). Phosphorylation-independent receptor regulation by GRK2 is mediated through a specific interaction of GTP-loaded G{alpha}q/11 with the RGS homology (RH) domain situated at the N terminus of GRK2 (14, 15, 19). Mutational and crystallographic analyses have identified specific amino acid residues within the RH domain that mediate G{alpha}q binding that map almost exclusively to the {alpha}5 helix of the RH domain of GRK2 (19–21).

We have previously used confocal imaging to detect the translocation of eGFP-PHPLC{delta} as an IP3 biosensor (18, 22) to investigate M1 mACh receptor regulation of PLC activity in hippocampal neurons. Using this approach, we showed that GRK2 inhibits M1 mACh receptor signaling in hippocampal neurons via a phosphorylation-independent mechanism (18) most likely involving the direct binding of the RH domain of GRK2 to G{alpha}q/11-GTP. Although these data highlight a novel mechanism by which GRK2 can regulate M1 mACh receptor signaling in neurons, they do not establish whether GRK2-mediated receptor phosphorylation is also an important mechanism in M1 mACh receptor desensitization. Here we have used a number of approaches to delineate the mechanism by which M1 mACh receptor signaling is regulated by GRK2 in hippocampal neurons. Our data indicate that endogenous GRK2 is a key regulator of M1 mACh receptor signaling utilizing both phosphorylation-dependent and -independent mechanisms to regulate receptor activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfections—Hippocampal neurons from 1-day-old Lister-hooded rat pups were isolated as described previously (22, 23). Briefly, isolated hippocampi were dissociated with Pronase E (0.5 mg ml–1) and thermolysin (0.5 mg ml–1) in a HEPES-buffered salt solution (130 mM NaCl, 10 mM HEPES, 5.4 mM KCl, 1.0 mM MgSO4, 25 mM glucose, and 1.8 mM CaCl2, pH 7.2) for 30 min. Tissue fragments were further dissociated by trituration in HEPES-buffered salt solution containing DNase I (40 µg ml–1). Following centrifugation and further trituration, the cells were plated onto poly-D-lysine (50 µgml–1)-treated 25-mm glass coverslips. For the first 72 h, the cells were cultured in Neurobasal medium (Invitrogen) supplemented with B27 and 10% fetal calf serum. Cytosine arabinoside (5 µM) was added after 24 h, and after 72 h the cells were transferred to serum-free medium. The cultured neurons were transfected on day 5 with a 3:1 ratio of either vector-control or GRK constructs to eGFP-PHPLC{delta}, respectively, using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Typically, neuronal transfection rates of ≤5% were achieved using this technique. HEK293 and CHO-K1 cells were grown in {alpha}-minimal essential medium, supplemented with 10% fetal calf serum, penicillin (100 units ml–1), streptomycin (100 µg ml–1), and amphotericin B (2.5 µgml–1) (Invitrogen). All of the cells were maintained at 37 °C, under 5% CO2 in humidified conditions.

Measurement of PLC Activity in Neurons and Assessment of M1 mACh Receptor Desensitization—PLC activity was assessed using the agonist-stimulated translocation of eGFP-tagged pleckstrin homology domain of PLC{delta}1 (eGFP-PHPLC{delta}) and was visualized using an Olympus Optical FV500 scanning laser confocal IX70 inverted microscope as described previously (18, 22). All of the experiments were undertaken in the presence of tetrodotoxin (500 nM) to block action potential-dependent synaptic activity. M1 mACh receptor desensitization was assessed as described previously (18). Briefly, the neurons were challenged with an approximate EC50 concentration (10 µM, R1) of the mACh agonist methacholine (MCh) for 30 s, followed by a 5-min washout to allow recovery of PIP2, intracellular Ca2+ stores, and eGFP-PHPLC{delta} fluorescence to basal levels. Next a maximal concentration of MCh (100 µM) was applied for 1 min to induce receptor desensitization. Finally, following another 5-min washout, neurons were rechallenged with a second pulse of MCh (10 µM, R2) for a further 30 s. Receptor desensitization was determined as the reduction in peak IP3 formation in R2 when compared with R1.

M1 mACh Receptor Phosphorylation Studies—CHO-K1 cells were transfected with 1 µg of hemagglutinin-tagged (HA) rat M1 mACh receptor cDNA/well in the presence of 1 µg of vector control (pcDNA3), wild-type GRK2, D110AGRK2, K220RGRK2, or D110A, K220RGRK2 for 48 h. Confluent cells were loaded with [32P]orthophosphate (5 µCi ml–1; Amersham Biosciences) in phosphate-free Krebs buffer (118.6 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 4.2 mM NaHCO3, 1.3 mM CaCl2, 10 mM HEPES, and 11.7 mM glucose), pH 7.4. After 60 min at 37 °C, MCh (1 mM) was added to the cells for varying time periods. The cells were then solubilized as described previously (16), and the receptors were immunoprecipitated using a specific rat monoclonal anti-HA antibody (3F10; Roche Applied Science) and electrophoretically resolved as described previously (16). The autoradiograms were documented and analyzed using the GeneGenius system and software (Syngene, Cambridge, UK). To examine the effects of GRK2 manipulation on M1 mACh receptor phosphorylation, the cells were co-transfected with HA-tagged M1 mACh receptor and either wild-type GRK2, D110AGRK2, K220RGRK2, or D110A,K220RGRK2. Receptor expression was equalized following [3H]NMS binding prior to gel loading. For co-transfection experiments wild-type GRK2, D110AGRK2, K220RGRK2, or D110A,K220RGRK2 expression was determined by Western blotting using a rabbit polyclonal anti-GRK2 antibody (Santa Cruz, Santa Cruz, CA).

Assessment of Antisense Suppression of Endogenous GRK2 Expression—The ability of a specific antisense GRK2 construct (almost the full length of rat GRK2 cloned into pcDNA3 in an antisense direction) (23) to suppress endogenous GRK2 expression was initially determined after transfection of HEK293 cells with 1, 2, or 3 µg of either pcDNA3 (control) or antisense GRK2 using Lipofectamine 2000, according to the manufacturer's instructions. After 72 h the cells were lysed, and GRK2 expression was determined by Western blotting using a rabbit polyclonal anti-GRK2 antibody (Santa Cruz). To examine the specificity of the GRK2 antisense construct, the expression levels of GRK3 and GRK6 were also examined in the same samples by Western blotting using rabbit polyclonal anti-GRK3 and anti-GRK6 antibodies (Santa Cruz). To assess to what extent antisense treatment suppresses endogenous expression of GRK2 in neurons, hippocampal neurons were transfected with a 1:3 ratio of either eGFP and pcDNA3 (control) or eGFP and antisense GRK2. After 72 h, the neurons were fixed for 10 min using 4% paraformaldehyde prior to permeabilization with phosphate-buffered saline containing Triton X-100 (0.2%, for 5 min). Nonspecific antibody binding was blocked using phosphate-buffered saline containing Triton X-100-containing goat serum (10%, 30 min). The neurons were incubated overnight at 4 °C with a polyclonal anti-rabbit GRK2 antibody (Santa Cruz). After washing, the neurons were incubated with an Alexa Fluor 546 anti-rabbit IgG (Molecular Probes; 1:400, for 40 min) prior to washing and mounting. GRK2 staining was visualized using an Olympus Optical FV500 scanning laser confocal IX70 inverted microscope, using excitation at 488 nm for eGFP and 543 nm for Alexa Fluor 546. GRK2 expression was determined as the mean fluorescence intensity of all eGFP-expressing neurons.

Creation of Non-G{alpha}q-binding Mutants—The aspartate residue at position 110 of GRK2 was mutated to an alanine using QuikChange (Stratagene) following the manufacturer's instructions. Briefly, the following primers were used to create D110AGRK2 and D110A, K220RGRK2 mutants (forward primer, GTC TGC AGC CGA GAG ATC TTC GCG ACC TAC ATC ATG AAG GAG; reverse primer, CTC CTT CAT GAT GTA GGT CGC GAA GAT CTC TCG GCT GCA GAC), creating a new NruI endonuclease site, which was used to identify positive mutants. The presence of the correct mutation was determined following DNA sequencing. Wild-type GRK2, D110AGRK2, K220RGRK2, and D110A,K220RGRK2, were Myc-tagged following cloning into pcDNA3.1/Myc-His (Invitrogen) between the HindIII and EcoRI sites.

Assessment of GRK2/G{alpha}q Binding—The ability of wild-type GRK2, D110AGRK2, K220RGRK2, and D110A,K220RGRK2 to bind to G{alpha}q was determined as follows. HEK293 cells were transfected with either 2 µg of D110AGRK2, K220RGRK2, and D110A,K220RGRK2 or 1 µg of the constitutively active G{alpha}q mutant Q209LG{alpha}q using GeneJuice according to the manufacturer's instructions. After 48 h the cells were lysed on ice for 10 min with the following buffer: 20 mM Tris, pH 7.4, 100 mM NaCl, 3 mM MgCl2, 0.5 mM EDTA, 0.05% (v/v) Igepal, 0.2 mg/ml benzamidine, 0.1 mg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol. Insoluble material was pelleted by centrifugation (14,000 x g, 10 min, 4 °C). Supernatants from cells transfected with Q209LG{alpha}q were mixed with supernatants from cells expressing wild-type GRK2, D110AGRK2, K220RGRK2, or D110A, K220RGRK2. Following rolling for 1 h at 4 °C, the specific mouse monoclonal anti-Myc antibody 9E10 was added for 1 h at 4 °Cto immunoprecipitate Myc-tagged GRK2. Next 150 µl of protein A-Sepharose was added, and the samples were placed on a roller for 30 min at 4 °C. The samples were then washed three times with lysis buffer minus Igepal and EDTA, prior to resuspension in 2x SDS-PAGE loading buffer. The samples were heated for 3 min at 85 °C before separation by SDS-PAGE (10% acrylamide gel). Each sample (25 µl) was loaded onto a gel for G{alpha}q detection, and a further 10 µl was loaded onto another gel for detection of Myc expression. Separated protein was transferred to nitrocellulose, and G{alpha}q expression was detected using a rabbit polyclonal anti-G{alpha}q (1:2500 dilution); Myc expression was determined using a rabbit polyclonal anti-Myc antibody (1:1000 dilution; New England Biolabs). Protein expression was determined by the addition of ECL reagent (Amersham Biosciences) according to the manufacturer's instructions and exposure to Hyperfilm (Amersham Biosciences).

Measurement of Single Cell Ca2+ Concentration—Following loading with Fluo-3 AM (4 µM, 1 h), the neurons were excited at 488 nm, using an Olympus Optical FV500 scanning laser confocal IX70 inverted microscope. The cells were incubated at 37 °C using a temperature controller and microincubator (PDMI-2 and TC202A; Burleigh, UK) and perfused at 5 ml/min with Krebs buffer (119 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 4.2 mM NaHCO3, 10mM HEPES, 11.7 mM glucose, and 1.3 mM CaCl2, pH 7.4). The images were captured using an oil immersion x100 objective. Cytosolic Ca2+ levels were measured as the relative change in fluorescence detected in an area of interest as described previously (24). Drugs were applied via the perfusion line for the times stated under "Results."

Data Analysis—Concentration-response curves were fitted using nonlinear regression analysis using Prism, version 3.0 (GraphPad Software Inc., San Diego, CA). All of the data were analyzed using one or two-way analysis of variance, followed by Bonferroni's post-hoc test (Excel 5.0; Microsoft, Redmond, WA). Significance was accepted when p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Suppression of GRK2 Expression Using an Antisense GRK2 Construct—The GRK2 antisense construct used in this study has previously been shown to cause an approximate 75% suppression of endogenous GRK2 expression when stably expressed in NG108 –15 cells (25). To examine the effectiveness of the GRK2 antisense in a transient system, HEK293 cells were transfected with 1, 2, or 3 µg of GRK2 antisense (or the pcDNA3 vector) for 72 h. Western blot analysis showed that 1 µg of the GRK2 antisense was sufficient to cause an 80% suppression of endogenous GRK2 expression in HEK293 cells 72 h post-transfection (Fig. 1, A and B). The GRK2 antisense construct produced specific depletion of endogenous GRK2, because GRK3 and GRK6 expression levels were unaffected (Fig. 1A). Note that the GRK3 antibody used detects both GRK2 (upper band) and GRK3 (lower band) and that antisense GRK2 selectively depletes GRK2 without affecting GRK3 immunoreactivity (Fig. 1A). To determine whether antisense treatment was able to deplete endogenous GRK2 expression in neurons, the cultures were co-transfected with eGFP and pcDNA3 (control) or eGFP and antisense GRK2 for 72 h. Analysis of all neurons co-expressing eGFP indicated that the mean intensity of GRK2 immunoreactivity was reduced by ≥60% following antisense GRK2 treatment (Fig. 1, C and D), whereas GRK3 immunoreactivity was not affected (data not shown).



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  		FIG. 1.
Antisense GRK2 suppression of endogenous GRK2 expression in HEK293 cells and hippocampal neurons. HEK293 cells were transfected with 1, 2, or 3 µg of either pcDNA3 or antisense GRK2 (in pcDNA3). After 72 h the cells were lysed, and 40 µg of protein was loaded per lane for SDS-PAGE separation/Western transfer. A, Western blots showing lanes loaded with 1 µg pcDNA3 (lane 1) or 1 µg antisense GRK2 (lane 2) and GRK expression determined by immunoblotting with rabbit polyclonal antibodies recognizing GRK2, GRK3, or GRK6. Note that anti-GRK3 also detects GRK2, and it is the latter protein that is down-regulated in lane 2. The blots shown are representative of experiments performed on at least three separate occasions. B, densitometric analysis of GRK2 expression in HEK293 cells transfected with either pcDNA3 or antisense GRK2 constructs. The data are shown as the means ± S.E. for 5 separate experiments. The antisense GRK2 construct significantly suppressed endogenous GRK2 expression (**, p < 0.01) relative to pcDNA3-transfected HEK293 cells. Hippocampal neuronal cultures were transfected with a 1:3 ratio of eGFP and pcDNA3 or eGFP and antisense GRK2 for 72 h, prior to fixation and immunocytochemical analysis of GRK2 expression. C, representative images showing the effects of either pcDNA3 or antisense GRK2 on endogenous GRK2 expression. In both cases, the image on the left shows eGFP-transfected neurons, and that on the right shows GRK2 immunostaining. D, cumulative data showing mean intensity of GRK2 immunostaining for all neurons expressing eGFP and co-expressing either pcDNA3 or antisense GRK2. The data are represented as the mean intensities ± S.E. for 24 and 21 neurons for pcDNA3 and antisense GRK2 treatments, respectively. Antisense GRK2 treatment significantly suppressed endogenous GRK2 expression (**, p < 0.01) compared with pcDNA3-transfected neurons.

 
Effects of GRK2 Suppression on M1 mACh Receptor Signaling in Hippocampal Neurons—To examine the effects of GRK2 on M1 mACh receptor signaling endogenous GRK2 was specifically depleted following transfection with antisense GRK2 for 72 h. Expression of antisense GRK2 did not affect IP3 production stimulated by an acute agonist addition when compared with pcDNA3-transfected, control neurons (Fig. 2A). In agreement with our previous findings using the agonist pretreatment protocol, which compares responses to submaximal agonist concentrations before (R1) and after (R2) a maximal agonist application (100 µM, for 60 s), R2 was ~40% less than R1 following desensitization with 100 µM MCh in the presence of pcDNA3 (Fig. 2, B and D). However, in neurons expressing the antisense GRK2, R2 was virtually identical to R1, indicating that down-regulation of the endogenous GRK2 protein largely prevents M1 mACh receptor desensitization (Fig. 2, C and D). These data strongly suggest that GRK2 is a key endogenous mediator of M1 mACh receptor desensitization in hippocampal neurons.



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  		FIG. 2.
Effects of antisense GRK2 on M1 mACh receptor signaling in hippocampal neurons. Hippocampal neurons were transfected with a 1:3 ratio of eGFP-PHPLC{delta} and either pcDNA3 or antisense GRK2 for at least 72 h. A, concentration-response curves for eGFP-PHPLC{delta} translocations stimulated by a single 30-s application of MCh at the indicated concentrations in neurons expressing either pcDNA3 ({blacksquare}) or antisense GRK2 ({square}). Representative traces showing the desensitization protocol for M1 mACh receptor signaling (R1, R2 = 10 µM MCh for 30 s; MChmax = 100 µM MCh for 60 s) in hippocampal neurons transfected with pcDNA3 (B) or antisense GRK2 (C). D, cumulative data showing a significant reduction (**, p < 0.01) in the extent of desensitization of the M1 mACh receptor in hippocampal neurons expressing antisense GRK2 (n = 14) compared with pcDNA3 (n = 16). The data are shown as the means ± S.E. for the percentage of change in R2 relative to the R1 response.

 
A C-terminal Fragment of GRK2 (GRK2-ct) Does Not Alter M1 mACh Receptor Signaling—Agonist activation of receptors leads to a translocation of GRK2 to the plasma membrane, a process that requires binding of free G{beta}{gamma} subunits to the C-terminal domain (Gly-495 to Leu-689) of bovine GRK2 (26, 27). Because G{beta}{gamma} subunits are able to directly stimulate PLC signaling (27, 28), overexpression of GRK2 may lead to inhibition of PLC signaling through sequestration of free G{beta}{gamma} subunits. To assess whether inhibition of PLC signaling seen with the overexpression of GRK2 or K220RGRK2 was due to sequestration of free G{beta}{gamma} subunits, the C-terminal 194 amino acids of GRK2 (GRK2-ct) were co-expressed with eGFP-PHPLC{delta}. Overexpression of GRK2-ct, confirmed by Western blotting (data not shown), did not affect the acute IP3 response to single concentrations of MCh (3, 10 or 100 µM, for 30 s; data not shown), and had no effect on the R2/R1 ratio assessed using the M1 mACh receptor desensitization protocol (data not shown).

Does M1 mACh Receptor Phosphorylation by GRK2 Mediate Receptor Desensitization?—Suppression of endogenous GRK2 expression reverses M1 mACh receptor desensitization in hippocampal neurons; however, it is unclear whether this is due to inhibition of PLC signaling via binding of G{alpha}q/11 and/or GRK2-mediated receptor phosphorylation. To determine whether GRK2-mediated receptor phosphorylation is required for M1 mACh receptor desensitization, we introduced a single point mutation D110A to create both GRK2 and K220RGRK2 mutants, which are incapable of binding G{alpha}q/11 (19). When transiently transfected, expression levels of the GRK2, D110AGRK2, K220RGRK2, and D110A,K220RGRK2 constructs were similar in HEK293 cells (data not shown). To assess GRK2/G{alpha}q/11 binding, Myc-tagged GRK2, D110AGRK2, K220RGRK2, and D110A,K220RGRK2 proteins were mixed with constitutively active Q209LG{alpha}q. Immunoprecipitation of Myc-tagged the GRK2 constructs followed by immunoblotting for G{alpha}q indicated a strong binding of G{alpha}q to wild-type GRK2 and K220RGRK2 (Fig. 3A). However, introduction of the D110A mutation prevented Q209LG{alpha}q binding to D110AGRK2 or D110A,K220RGRK2 (Fig. 3A). Initial experiments indicated that the acute agonist-stimulated IP3 response was unaffected when D110AGRK2 or D110A,K220RGRK2 proteins were expressed, but in common with our previous findings (18), introduction of K220RGRK2 caused a marked suppression of this response in hippocampal neurons (Fig. 3B). These data suggest that without an intact RH domain, GRK2 cannot inhibit M1 mACh receptor signaling through binding to GTP-bound activated G{alpha}q/11. As the acute agonist-stimulated IP3 response was normal in D110AGRK2- or D110A,K220RGRK2-expressing neurons, we could now assess whether GRK2-mediated phosphorylation is necessary for M1 mACh receptor desensitization. Assessing the R2/R1 ratio using the M1 mACh receptor desensitization protocol, it could be shown that expression of D110AGRK2 significantly enhances M1 mACh receptor desensitization, whereas expression of the catalytically inactive D110A,K220RGRK2 protein significantly diminishes M1 mACh receptor desensitization (Fig. 3C). These data indicate that GRK2-mediated phosphorylation is required for the agonist-dependent attenuation of the IP3 signal mediated by the M1 mACh receptor in hippocampal neurons.



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  		FIG. 3.
Effects of non-G{alpha}q/11binding and/or kinase-dead GRK2 mutants on M1 mACh receptor signaling in HEK293 cells and hippocampal neurons. A, the ability of Myc-tagged wild-type GRK2 (lane 3), D110AGRK2 (lane 4), K220RGRK2 (lane 5), and D110A, K220RGRK2 (lane 6) to bind to a constitutively active Q209LG{alpha}q protein was assessed in HEK293 cells. Myc-tagged GRK2 constructs were mixed in vitro with Q209LG{alpha}q and incubated for 1 h at 4 °C (see "Experimental Procedures"). Myc-GRK2 constructs were immunoprecipitated using a monoclonal anti-Myc antibody. Following SDS-PAGE separation/Western transfer, G{alpha}q protein was visualized a specific rabbit polyclonal anti-G{alpha}q antibody. Immunoprecipitation efficiency of the different of GRK2 constructs was assessed using a specific rabbit anti-Myc antibody. B, MCh-stimulated IP3 generation was assessed in hippocampal neurons transfected with a 1:3 ratio of eGFP-PHPLC{delta} to pcDNA3, D110AGRK2, D110A,K220RGRK2, or K220RGRK2 for 48 h. Expression of pcDNA3, D110AGRK2, or D110A,K220RGRK2 did not affect the IP3 response to 3, 10, or 100 µM MCh, whereas the K220RGRK2 construct essentially abolished the response to MCh. The data are shown as the peak increases in cytosolic fluorescence and are presented as the means ± S.E. for 12–24 neurons in each case. C, cumulative data showing the effects of expressing the D110AGRK2 or D110A,K220RGRK2 constructs on M1 mACh receptor desensitization in hippocampal neurons. The desensitization (R1/MChmax/R2) protocol was performed as described under "Experimental Procedures." The data are presented as the means ± S.E., for 8–11 neurons taken from at least three separate hippocampal preparations. Expression of the D110AGRK2 construct significantly increased desensitization (*, p < 0.05), whereas the D110A, K220RGRK2 construct markedly decreased (**, p < 0.01) the degree of receptor desensitization. IP, immunoprecipitation; WB, Western blot.

 
Agonist-stimulated Phosphorylation of the M1 mACh Receptor—Definitive assessment of whether inhibition of the G{alpha}q/11-GRK2 binding interaction alters agonist-stimulated M1 mACh receptor phosphorylation in hippocampal neurons should ideally be made in this neuronal preparation. However, because of the lack of a suitable antibody capable of immunoprecipitating the endogenous rat M1 mACh receptor, we have expressed HA-tagged M1 mACh receptors in CHO cells to provide an insight into this key question. Preliminary studies indicated that agonist-stimulated M1 mACh receptor phosphorylation peaks at approximately 10 min, and therefore this time was used for subsequent experiments (Fig. 4A). The rate of phosphorylation of the rat M1 mACh receptor was comparable with that observed previously for the human M1 mACh receptor stably expressed in CHO cells (29). MCh-stimulated M1 mACh receptor phosphorylation was increased approximately 2-fold in the presence of wild-type GRK2 or D110AGRK2 (Fig. 4, B and E) compared with receptor phosphorylation observed in vector (pcDNA3)-transfected cells. Expression of kinase-dead K220RGRK2, or D110A,K220RGRK2 mutants had no effect upon MCh-stimulated M1 mACh receptor phosphorylation (Fig. 4, C and E). These data confirm the lack of catalytic activity in the K220R mutants but also suggest that, at least in CHO cells, endogenous GRK2 may not be responsible for agonist-stimulated M1 mACh receptor phosphorylation.



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  		FIG. 4.
Effect of non-G{alpha}q binding GRK2 mutants on M1 mACh receptor phosphorylation. CHO cells were transfected with HA-tagged rat M1 mACh receptor (1 µg) and 1 µg of pcDNA3, wild-type GRK2, D110AGRK2, K220RGRK2, or D110A,K220RGRK2 for 48 h, prior to loading cells with [32P]orthophosphate for 60 min. The cells were stimulated with MCh (1 mM) and processed to visualize M1 mACh receptor phosphorylation as described under "Experimental Procedures". A, representative autoradiogram showing the time course of agonist-stimulated of M1 mACh receptor phosphorylation. Lane 1, basal MCh stimulation; lane 2, 1-min MCh stimulation; lane 3, 5-min MCh stimulation; lane 4, 10-min MCh stimulation; lane 5, 20-min MCh stimulation. B, representative autoradiogram showing M1 mACh receptor phosphorylation in cells overexpressing wild-type GRK2 or D110AGRK2 following a 10-min application of MCh (1 mM). The lanes shown are pcDNA3-transfected cells (lanes 1 and 2), wild-type GRK2 (lanes 3 and 4), or D110AGRK2 (lanes 5 and 6). C, representative autoradiogram showing MCh-stimulated (1 mM, for 10 min) M1 mACh receptor phosphorylation in the presence of pcDNA3 (lanes1 and 2), K220RGRK2 (lanes 3 and 4), or K220R,D110AGRK2 (lanes 5 and 6). D, for all of the phosphorylation experiments, the expression levels of GRK2 in CHO cells were determined. The data shown are representative Western blots of GRK2 expression levels in CHO cells transfected with either pcDNA3 (lanes 1 and 4), GRK2 (lane 2), D110AGRK2 (lane 3), K220RGRK2 (lane 5), and D110A,K220RGRK2 (lane 6). E, densitometric analysis of agonist-stimulated M1 mACh receptor phosphorylation is shown for basal (filled bars) and MCh-stimulated (1 mM, 10 min, open bars) cells. The data are shown as mean absorbances ± S.E. for three to five separate experiments. Statistically significant differences from the response to MCh observed in pcDNA3 transfected are indicated (*, p < 0.05).

 
Effects of Expression of D110A-GRK2 and D110A,K220RGRK2 on M3 mACh Receptor Signaling in SH-SY5Y Cells—We have previously shown that GRK2 can inhibit M3 mACh receptor signaling in SH-SY5Y cells in a phosphorylation-independent manner (17). K220RGRK2 does not inhibit agonist-stimulated M3 mACh receptor phosphorylation, indicating that endogenous GRK2 does not phosphorylate the endogenous M3 mACh receptor in SH-SY5Y cells (17). Transient transfection of SH-SY5Y cells with K220RGRK2 inhibited the acute IP3 response to a single concentration of MCh (100 µM, for 60 s); however, no inhibition of agonist-stimulated IP3 production was observed in the presence of the D110AGRK2 or D110A, K220RGRK2 proteins (Fig. 5A). To examine whether expression of the non-G{alpha}q/11-binding GRK2 mutants affects M3 mACh receptor desensitization, we applied the pretreatment protocol where 10 µM MCh was added for 1 min during R1 and R2, and the desensitizing concentration of MCh (100 µM) was applied for 3 min, with 5-min wash periods between MCh additions (17). In agreement with our previous data, the R2 response was attenuated by approximately 40% compared with R1 in vector (pcDNA3)-transfected cells. Expression of either D110AGRK2 or D110A, K220RGRK2 proteins in SH-SY5Y cells had no effect on R2/R1 ratios (Fig. 5B), indicating that GRK2 is unlikely to play a significant role in M3 mACh receptor desensitization in this human neuroblastoma cell line.



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  		FIG. 5.
Non-G{alpha}q/11 binding GRK2 mutants do not affect M3 mACh receptor desensitization in SH-SY5Y neuroblastoma cells. SH-SY5Y cells were transfected with a 1:3 ratio of eGFP-PHPLC{delta} and pcDNA3, D110AGRK2, K220RGRK2, or D110A,K220RGRK2 constructs. A, IP3 generation following acute MCh (100 µM, for 1 min) addition. The data are presented as peak IP3 responses and are shown as the means ± S.E. for 9–20 cells. Only expression of the K220RGRK2 construct significantly (**, p < 0.01) attenuated the IP3 response in SH-SY5Y cells. B, cumulative data showing the lack of effect of D110AGRK2 or D110A,K220RGRK2 expression on agonist-induced M3 mACh receptor desensitization in SH-SY5Y cells. The data are presented as R2/R1 ratios and shown as the means ± S.E., for 9–11 cells.

 
Role of {beta}-Arrestins in M1 mACh Receptor Signaling in Hippocampal Neurons—Initially, {beta}-arrestin expression was determined after transfection with eGFP-tagged bovine {beta}-arrestins 1 and 2. There was no detectable difference in the levels of expression of {beta}-arrestins 1 and 2 (Fig. 6A), and expression was evident in hippocampal neurons for at least 15 days in vitro (data not shown). Non-eGFP-tagged bovine {beta}-arrestin 1 and 2 constructs were co-transfected with eGFP-PHPLC{delta} and effects on M1 mACh receptor desensitization assessed using the R1/R2 protocol. Expression of {beta}-arrestin 2 significantly increased the extent of M1 mACh receptor desensitization, whereas expression of the {beta}-arrestin 1 protein had no effect (Fig. 6B). These data suggest that recruitment of {beta}-arrestin 2 may constitute an important step in M1 mACh receptor desensitization.



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  		FIG. 6.
Effects of {beta}-arrestin 1 and {beta}-arrestin 2 overexpression on agonist-stimulated M1 mACh receptor desensitization in hippocampal neurons. A, representative images showing expression of eGFP-tagged {beta} -arrestin 1 and {beta} -arrestin 2 in hippocampal neurons. B, neurons were transfected with a 1:3 ratio of eGFP-PHPLC{delta} with pcDNA3, {beta}-arrestin 1, or {beta}-arrestin 2. Agonist-mediated M1 mACh receptor desensitization was determined using the R1/MChmax/R2 protocol as described under "Experimental Procedures." The data are represented as the means ± S.E. for the percentages of change in R2/R1 response for 8–10 neurons from at least three separate preparations. Expression of {beta}-arrestin 2, but not {beta}-arrestin 1, significantly increased (**, p < 0.01) the degree of M1 mACh receptor desensitization compared with pcDNA3-transfected neurons.

 
Effects of Altered PKC Activity on M1 mACh Receptor Signaling in Hippocampal Neurons—To determine whether PKC is involved in the regulation of M1 mACh receptor signaling, we manipulated PKC activity using the protein kinase inhibitor staurosporine and PKC activator phorbol 12,13-dibutyrate (PDBu). To assess the acute effect of PDBu, the neurons were stimulated with two pulses of MCh (100 µM, 30 s) separated by a 10-min washout period during which PDBu (1 µM) was added for 3 min prior to the second pulse. This treatment resulted in a ≥50% suppression of the IP3 response compared with vehicle control (Fig. 7, A and B). The effect of PDBu was prevented by pretreatment with staurosporine (1 µM, added 15 min before the first MCh challenge; Fig. 7B). Staurosporine alone did not affect either the first or second response to MCh in hippocampal neurons (data not shown). Further, down-regulation of conventional and novel PKC isoenzymes through chronic PDBu (1 µM, 24 h) treatment did not affect M1 mACh receptor desensitization assessed using the R1/MChmax/R2 protocol. Thus, PKC down-regulation had no effect upon the acute MCh-stimulated IP3 response (data not shown) and also had no effect upon the R2/R1 ratio (Fig. 7C). In addition, preincubation of neurons with staurosporine (1 µM) also had no effect on the R2/R1 ratio (Fig. 7D). These data indicate that although acute PKC activation can inhibit PLC activity, PKC appears to play no role in the agonist-stimulated desensitization of M1 mACh receptor signaling in hippocampal neurons.



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  		FIG. 7.
The effects of altering PKC activity on M1 mACh receptor signaling in hippocampal neurons. A, representative traces showing hippocampal neurons stimulated with MCh (100 µM, 30 s), followed by a 5-min washout followed by rechallenge with MCh (100 µM, 30 s). The broken line shows the acute inhibitory effect of adding PDBu (1 µM) for 3 min before the second MCh challenge. B, cumulative data showing the inhibitory effect of acute PDBu addition (**, p < 0.01) and the complete reversal of this action by preincubation of neurons with staurosporine (Stauro; 1 µM) for 15 min before the first MCh challenge. Note that staurosporine per se did not affect the IP3 responses to MCh additions (data not shown). The data are presented as ratios of the initial peak response to MCh compared with a second challenge. C, effects PKC down-regulation in hippocampal neurons by PDBu (1 µM) treatment for 24 h, M1 mACh receptor desensitization was determined using the R1/MChmax/R2 protocol as described under "Experimental Procedures." The data are presented as the means ± S.E. for the percentage change in the R2/R1 response for nine neurons, from three separate preparations. D, lack of effect of PKC inhibition by staurosporine (1 µM, added 15 min before R1 MCh stimulation) on agonist-stimulated M1 mACh receptor desensitization. The data are shown as the means ± S.E. for the percentages of change in the R2/R1 response for five neurons from three separate preparations.

 
Effects of Manipulating [Ca2+]i on M1 mACh Receptor Desensitization in Hippocampal Neurons—Assessment of receptor desensitization using measurements of PLC activity must take into account the fact that this enzyme is modulated by other factors, including intracellular Ca2+ (28). To examine the possible role that Ca2+ plays in the desensitization of the M1 mACh receptor, we assessed the effect of the loading neurons with the Ca2+ chelator BAPTA-AM. Pretreatment with BAPTA-AM (30 µM, 20 min) caused a reduction in the peak IP3 response following MCh stimulation, with a greater effect being observed at 10 µM than at 30 µM MCh (Fig. 8A). However, M1 mACh receptor desensitization, assessed using the R1/MChmax/R2 protocol, was unaffected in BAPTA-loaded cells (Fig. 8B). To assess the effectiveness of BAPTA-AM pretreatment, intracellular Ca2+ levels were determined in neurons using the Ca2+ indicator Fluo-3. Agonist stimulation (MCh, 100 µM, 30 s) produced only a small rise in intracellular Ca2+ when compared with that elicited by depolarization with KCl (40 mM) for 1 min (Fig. 8C). Following depolarization, the Ca2+ response to a second addition of MCh (100 µM, 30 s) was often, although not invariably, increased (Fig. 8C). Pretreatment of neurons with BAPTA-AM resulted in complete attenuation of the MCh-stimulated [Ca2+]i response but only a modest attenuation of the depolarization-induced response (Fig. 8C).



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  		FIG. 8.
Effects of altering intracellular Ca2+ responses on M1 mACh receptor desensitization in hippocampal neurons. A, neurons were stimulated with either 10 or 30 µM MCh for 30 s (filled bars) prior to incubation with BAPTA-AM (30 µM, for 30 min). The neurons were then restimulated with the same concentration of MCh for 30 s (open bars). B, the effect of chelation of [Ca2+]i on M1 mACh receptor desensitization was assessed using the R1/MChmax/R2 protocol (see "Experimental Procedures") in the absence (filled bars) or presence (empty bars) of BAPTA (30 µM). The data are presented as the mean R2/R1 ratio ± S.E., for 6–21 neurons from at least three separate transfections. C, representative traces showing the effects of MCh (100 µM, for 30 s), K+ (40 mM, for 1 min), and a second addition of MCh (100 µM, for 30 s) on intracellular Ca2+ levels in Fluo-3-loaded neurons in the absence (solid line) or presence of BAPTA (30 µM, broken line). The observed changes in fluorescence are representative of 42–45 neurons for each condition taken from at least three separate cultures.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously shown using the IP3 biosensor eGFP-PHPLC{delta} that expression of GRK2 in hippocampal neurons leads to an almost complete suppression of M1 mACh receptor-mediated IP3 signaling and that this suppression is independent of kinase activity, because the catalytically inactive K220RGRK2 mutant also completely inhibited signaling (18). In common with a number of other groups (14, 15, 19, 21), we proposed that the suppression of signaling is a consequence of the direct interaction of the N-terminal RH domain of GRK2 with GTP-bound G{alpha}q/11. In the present study we have examined whether the catalytic activity of GRK2 acts as an alternative or additional mechanism for GRK2-mediated suppression of M1 mACh receptor signaling.

Because both GRK2 and K220RGRK2 are equally effective in suppressing M1 mACh receptor signaling (18), another approach was required to determine whether endogenous GRK2 is involved in M1 mACh receptor regulation. We have previously used a construct consisting of virtually the whole coding sequence for rat GRK2 cloned into pcDNA3 in an antisense direction. This construct was able to bring about a 75% depletion of endogenous GRK2 when stably expressed in NG108 –15 cells (24). Here, we have confirmed this finding by transfecting the GRK2 antisense construct into HEK293 cells and demonstrating that 72 h post-transfection, an 80% suppression of GRK2 could be observed without affecting the expression of other GRKs. In hippocampal neurons expression of the GRK2 antisense construct produced ≥60% depletion of endogenous GRK2 expression, which almost completely prevented the prestimulated desensitization of M1 mACh receptor-mediated IP3 signaling, implicating endogenous GRK2 as a key protein kinase responsible for attenuating the M1 mACh receptor response to agonist.

If GRK2 acts to suppress M1 mACh receptor signaling purely through a direct RH-G{alpha}q interaction, it would follow that depletion of endogenously expressed GRK2 in hippocampal neurons might lead to enhanced agonist sensitivity. However, no change in acute agonist-stimulated IP3 responses was observed in neurons transfected with the GRK2 antisense construct. It is possible that GRK3, the expression of which is unaffected by the presence of GRK2 antisense, may be able to compensate for the loss of GRK2 and thus prevent a change in the sensitivity of M1 mACh receptors to agonist stimulation from occurring. However, this would contrast with the inability of GRK3 to compensate in GRK2-depleted neurons in experiments evaluating agonist-mediated M1 mACh receptor desensitization. Although the use of the GRK2 antisense construct highlights the requirement for endogenous GRK2 in the regulation of M1 mACh receptor signaling in hippocampal neurons, this approach sheds little new light on the mechanism(s) involved.

Initially, GRK2 was thought to block PLC signaling because of its ability to bind free G{beta}{gamma} subunits, thus inhibiting G{beta}{gamma} stimulation of PLC{beta} isoenzymes (27, 30). However, as has previously been demonstrated in COS7 cells (27), M1 mACh receptor-stimulated IP3 formation is unaffected following overexpression of the C-terminal domain (Gly-495 to Leu-689) of bovine GRK2 in hippocampal neurons. These data suggest that G{beta}{gamma} subunits play little or no role in M1 mACh receptor-mediated PLC activation in this cell type. More recent studies have shown that the N-terminal RH domain of GRK2 is responsible for inhibiting G{alpha}q/11-coupled signaling to PLC (14, 15). Indeed, the key amino acids involved in this interaction have been mapped to the {alpha}5 helix of the N-terminal domain of GRK2 (19). Mutation of several individual amino acid residues within this region prevents G{alpha}q binding to GRK2 (19) and restores the ability of the G{alpha}q/11-coupled mGlu1a receptor to signal through PLC (21). In agreement with these findings, we show here not only that mutation of the RH domain prevented G{alpha}q binding, but also that the expression of the non-G{alpha}q-binding D110AGRK2 mutant in hippocampal neurons did not inhibit acute MCh-stimulated M1 mACh receptor signaling. Crucially, abolition of the GRK2 RH domain-G{alpha}q interaction restored the agonist-stimulated IP3 response and meant that we were able to show that expression of D110AGRK2 enhanced, whereas D110A,K220RGRK2 markedly decreased M1 mACh receptor desensitization in hippocampal neurons. Very recently Iwata et al. (31) have used similar single and double GRK2 point mutations to delineate the requirement for both the RH domain and receptor kinase activity in the rapid termination of signaling by recombinantly expressed H1 histamine receptors in HEK cells. Although it is difficult to make definitive conclusions regarding the effects of D110A mutants without directly determining their effects on M1 mACh receptor phosphorylation in hippocampal neurons, we believe our data are the first to point to GRK2-mediated phosphorylation being required to mediate the desensitization of an endogenously expressed receptor in a native neuronal background.

Crystallographic analysis of the structure of GRK2 in a complex with G{beta}{gamma} (20) has revealed that this GRK is able to bind simultaneously to receptor and G{alpha}q, in addition to G{beta}{gamma}, suggesting that both G{beta}{gamma}, and G{alpha}q binding might be required for the correct targeting of GRK2 to the receptor. We have shown here that in the absence of G{alpha}q binding, GRK2 is still able to phosphorylate the M1 mACh receptor, apparently as efficiently as wild-type GRK2, in CHO cells. These data suggest that the availability of G{beta}{gamma} subunits and receptor are sufficient to allow the efficient interaction with GRK2 binding and receptor phosphorylation. Indeed, RH domain mutation of GRK2, to eliminate the interaction with G{alpha}q/11, does not reduce the ability of GRK2 to phosphorylate rhodopsin (19), and inhibition of GRK2/G{alpha}q/11 interactions has also been shown not to alter the ability of GRK2 to bind avidly to the mGlu1a receptor (21).

Thus, we propose that in hippocampal neurons, M1 mACh receptors are subject to two distinct modes of regulation by GRK2, as well as being likely substrates for GRK6-mediated phosphorylation (18). We have also shown here that PKC- and Ca2+-dependent mechanisms do not appear to have a role in agonist-evoked M1 mACh receptor desensitization. GRK-mediated GPCR phosphorylation often acts as a cue for arrestin recruitment to the receptor (10, 30). At present, evidence implicating arrestin involvement in M1 mACh receptor signaling is contradictory, even with respect to data obtained in similar cell expression systems. Thus, Lee et al. (32) have reported that co-expression of M1 mACh receptors with {beta}-arrestin 1 or {beta}-arrestin 2 did not enhance receptor sequestration in HEK293-tsA201 cells. However, expression of the dominant-negative {beta}-arrestin 1 V53D mutant in HEK293 cells has been reported to suppress sequestration of M1 mACh receptors (33). In another report, antisense suppression of arrestin expression in HEK293 cells attenuated desensitization of the endogenously expressed mACh receptor but not the P2Y1 or P2Y2 receptors (34). In the present study we have provided evidence that M1 mACh receptor phosphorylation, mediated by GRK(s) (GRK2 and/or GRK6), promotes the selective binding of {beta}-arrestin 2, because receptor desensitization was enhanced in the presence of {beta}-arrestin 2 but not {beta}-arrestin 1. In agreement with our findings Santini et al. (35) found that in RBL-2H3 cells expressing physiological levels of M1 mACh receptors, agonist stimulation leads to the selective recruitment of {beta}-arrestin 2, but not {beta}-arrestin 1.

An increasing number of studies have now provided a substantial body of evidence for {beta}-arrestins not only facilitating receptor internalization but also scaffolding critical components of the ERK/JNK cascades, including Raf-1, MEK1, and ERK1/2 (36–38), and JNK3 (12, 39). Thus, M1 mACh receptor phosphorylation by GRK2 (and possibly other GRKs/protein kinases) and the recruitment of {beta}-arrestin 2 may not only attenuate phosphoinositide signaling but also act as a template for switching the signaling outputs of the receptor to alternative pathways. In the central nervous system this may provide a mechanism through which M1 mACh receptors can cause the sustained signaling necessary to bring about long term changes in neuronal activity (40, 41).

Despite the presence of a RH domain in the N terminus of all GRKs, at present only GRK2 and GRK3 (and recently GRK4) (42) have been shown to suppress GPCR signaling in a phosphorylation-independent manner (14, 15, 19, 43). In the case of GRK4, whether this action results from the RH domain binding to G{alpha}i/o or the {gamma}-aminobutyric acid, type B receptor remains to be established. With the exception of mGlu1a receptor, where evidence has been provided to support a model whereby GRK2 recruitment displaces G{alpha}q/11 binding to the receptor on agonist addition (21), little is known about the physiological significance of phosphorylation-independent regulation of G{alpha}q/11-coupled receptors. Indeed, as discussed above, structural analysis has revealed that GRK2 is able to bind G{alpha}q/11, G{beta}{gamma}, and receptor simultaneously, inferring that GRK2 might act to inhibit GPCR signaling by blocking the interaction of G{alpha}q/11 (and/or G{beta}{gamma}) with PLC{beta} prior to receptor phosphorylation and arrestin binding (20). Our data show that the RH domain of GRK2 can mediate a rapid attenuation of M1 mACh receptor-stimulated signaling events; however, in the absence of GRK2 RH domain-mediated signaling inhibition, the importance of receptor phosphorylation for the prolonged suppression (or signal switching) of M1 mACh receptor signaling could be unmasked. In conclusion, GRK2 has been shown to inhibit M1 mACh receptor signaling through phosphorylation-dependent and -independent mechanisms. It appears likely that these mechanisms are interactive and perhaps sequential with the phosphorylation-independent mechanism initially suppressing G{alpha}q/11/PLC interactions prior to receptor phosphorylation and recruitment of {beta}-arrestin 2, which can cause receptor internalization, perhaps to initiate alternative signaling cascades as a prelude to longer term plasticity of neuronal functions.


   FOOTNOTES
 
* This work is supported by Program Grant 062495 from the Well-come Trust of Great Britain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 44-116-252-3087; Fax: 44-116-252-5045; E-mail: jmw23{at}le.ac.uk.

1 The abbreviations used are: mACh, muscarinic acetylcholine; PLC, phosphoinositide-specific phospholipase C; GPCR, G protein-coupled receptor; PKC, protein kinase C; CHO, Chinese hamster ovary; MCh, methacholine; IP3, inositol 1,4,5-trisphosphate; GRK, G protein-coupled receptor kinase; RH, RGS homology; eGFP, enhanced green fluorescent protein; HA, hemagglutinin; PDBu, phorbol 12,13-dibutyrate; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; JNK, c-Jun N-terminal kinase; AM, acetoxymethyl ester; BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester. Back


   ACKNOWLEDGMENTS
 
We thank the following for the donation of constructs: {beta}-arrestin 1 and 2, Stuart Mundell (University of Bristol, UK); HA-tagged rat M1 mACh receptor, Ed Hulme (National Institute for Medical Research, London, UK); GRK2-ct, Robert Lefkowitz (Duke University); and Q209LG{alpha}q, John Hepler (Emory University). We also thank Roger James (University of Leicester, Leicester, UK) for the gift of the monoclonal 9E10 anti-Myc antibody, and Graeme Milligan (University of Glasgow, Glasgow, UK) for providing the polyclonal anti-G{alpha}q antibody.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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