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• W2017848367 abstract "When co-expressed with the inositol 1,4,5-trisphosphate biosensor eGFP-PHPLCδ, 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 Gαq/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-Gαq/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-Gαq/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 Gαq/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. When co-expressed with the inositol 1,4,5-trisphosphate biosensor eGFP-PHPLCδ, 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 Gαq/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-Gαq/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-Gαq/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 Gαq/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. 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 (1Frotscher M. Leranth C. J. Comp. Neurol. 1985; 239: 237-246Crossref PubMed Scopus (515) Google Scholar, 2Wainer B.H. Levey A.I. Rye D.B. Mesulam M.M. Mufson E.J. Neurosci. Lett. 1985; 54: 45-52Crossref PubMed Scopus (264) Google Scholar) and that cholinergic deficits (caused by lesioning, pharmacological blockade, or gene knock-out) produce an array of disorders in learning and memory (3Power A.E. Vazdarjanova A. McGaugh J.L. Neurobiol. Learning Memory. 2003; 80: 178-193Crossref PubMed Scopus (225) Google Scholar, 4Ovsepian S.V. Anwyl R. Rowan M.J. Eur. J. Neurosci. 2004; 20: 1267-1275Crossref PubMed Scopus (129) Google Scholar, 5Wess J. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 423-450Crossref PubMed Scopus (319) Google Scholar). Transgenic approaches have helped to define the key roles of M1 muscarinic acetylcholine (mACh) 1The 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.1The 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. receptors in cholinergic regulation of hippocampal function (5Wess J. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 423-450Crossref PubMed Scopus (319) Google Scholar, 6Hamilton S.E. Loose M.D. Qi M. Levey A.I. Hille B. McKnight G.S. Idzerda R.L. Nathanson N.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13311-13316Crossref PubMed Scopus (308) Google Scholar, 7Miyakawa T. Yamada M. Dattaroy A. Wess J. J. Neurosci. 2001; 21: 5239-5250Crossref PubMed Google Scholar, 8Anagnostaras S.G. Murphy G.G. Hamilton S.E. Mitchell S.L. Rahnama N.P. Nathanson N.M. Silva A.J. Nat. Neurosci. 2003; 6: 51-58Crossref PubMed Scopus (439) Google Scholar), 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 (9Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1080) Google Scholar, 10Ferguson S.S.G. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar) and is a crucial mechanism for reducing (or “switching”) signaling (11Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2064) Google Scholar). 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 (10Ferguson S.S.G. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar, 12Shenoy S.K. Lefkowitz R.J. Biochem. J. 2003; 375: 503-515Crossref PubMed Scopus (333) Google Scholar). Receptor-arrestin complexes are also involved in the initiation of receptor internalization and may act as signaling scaffolds to assemble transduction pathways (12Shenoy S.K. Lefkowitz R.J. Biochem. J. 2003; 375: 503-515Crossref PubMed Scopus (333) Google Scholar, 13Gurevich V.V. Gurevich E.V. Trends Pharmacol. Sci. 2004; 25: 105-111Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar).Recently it has been shown that at least two members of the GRK family, GRK2 and GRK3, are able to suppress Gαq/11-coupled receptor/phospholipase C (PLC) signaling even in the absence of kinase activity (14Carman C.V. Parent J.L. Day P.W. Pronin A.N. Sternweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 15Sallese M. Salvatore L. D'Urbano E. Sala G. Storto M. Launey T. Nicoletti F. Knöpfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Crossref PubMed Scopus (112) Google Scholar, 16Willets J.M. Challiss R.A.J. Kelly E. Nahorski S.R. Mol. Pharmacol. 2001; 60: 321-330Crossref PubMed Scopus (41) Google Scholar). GRK2-mediated, phosphorylation-independent receptor regulation has now been demonstrated not only in recombinant systems but also for endogenous receptors in cell lines (17Willets J.M. Mistry R. Nahorski S.R. Challiss R.A.J. Mol. Pharmacol. 2003; 64: 1059-1068Crossref PubMed Scopus (29) Google Scholar) and in primary, cultured hippocampal neurons (18Willets J.M. Nash M.S. Challiss R.A.J. Nahorski S.R. J. Neurosci. 2004; 24: 4157-4162Crossref PubMed Scopus (37) Google Scholar). Phosphorylation-independent receptor regulation by GRK2 is mediated through a specific interaction of GTP-loaded Gαq/11 with the RGS homology (RH) domain situated at the N terminus of GRK2 (14Carman C.V. Parent J.L. Day P.W. Pronin A.N. Sternweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 15Sallese M. Salvatore L. D'Urbano E. Sala G. Storto M. Launey T. Nicoletti F. Knöpfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Crossref PubMed Scopus (112) Google Scholar, 19Sterne-Marr R. Tesmer J.J. Day P.W. Stracquatanio R.P. Cilente J.A. O'Connor K.E. Pronin A.N. Benovic J.L. Wedegaertner P.B. J. Biol. Chem. 2003; 278: 6050-6058Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Mutational and crystallographic analyses have identified specific amino acid residues within the RH domain that mediate Gαq binding that map almost exclusively to the α5 helix of the RH domain of GRK2 (19Sterne-Marr R. Tesmer J.J. Day P.W. Stracquatanio R.P. Cilente J.A. O'Connor K.E. Pronin A.N. Benovic J.L. Wedegaertner P.B. J. Biol. Chem. 2003; 278: 6050-6058Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 20Lodowski D.T. Pitcher J.A. Capel W.D. Lefkowitz R.J. Tesmer J.J.G. Science. 2003; 300: 1256-1262Crossref PubMed Scopus (310) Google Scholar, 21Dhami G.K. Dale L.B. Anborgh P.H. O'Connor-Halligan K.E. Sterne-Marr R. Ferguson S.S.G. J. Biol. Chem. 2004; 279: 16614-16620Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar).We have previously used confocal imaging to detect the translocation of eGFP-PHPLCδ as an IP3 biosensor (18Willets J.M. Nash M.S. Challiss R.A.J. Nahorski S.R. J. Neurosci. 2004; 24: 4157-4162Crossref PubMed Scopus (37) Google Scholar, 22Nash M.S. Willets J.M. Billups B. Challiss R.A.J. Nahorski S.R. J. Biol. Chem. 2004; 279: 49036-49044Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) 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 (18Willets J.M. Nash M.S. Challiss R.A.J. Nahorski S.R. J. Neurosci. 2004; 24: 4157-4162Crossref PubMed Scopus (37) Google Scholar) most likely involving the direct binding of the RH domain of GRK2 to Gαq/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 PROCEDURESCell Culture and Transfections—Hippocampal neurons from 1-day-old Lister-hooded rat pups were isolated as described previously (22Nash M.S. Willets J.M. Billups B. Challiss R.A.J. Nahorski S.R. J. Biol. Chem. 2004; 279: 49036-49044Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 23Schell M.J. Erneux C. Irvine R.F. J. Biol. Chem. 2001; 276: 37537-37546Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). 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-PHPLCδ, 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 α-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 PLCδ1 (eGFP-PHPLCδ) and was visualized using an Olympus Optical FV500 scanning laser confocal IX70 inverted microscope as described previously (18Willets J.M. Nash M.S. Challiss R.A.J. Nahorski S.R. J. Neurosci. 2004; 24: 4157-4162Crossref PubMed Scopus (37) Google Scholar, 22Nash M.S. Willets J.M. Billups B. Challiss R.A.J. Nahorski S.R. J. Biol. Chem. 2004; 279: 49036-49044Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). 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 (18Willets J.M. Nash M.S. Challiss R.A.J. Nahorski S.R. J. Neurosci. 2004; 24: 4157-4162Crossref PubMed Scopus (37) Google Scholar). 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-PHPLCδ 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 (16Willets J.M. Challiss R.A.J. Kelly E. Nahorski S.R. Mol. Pharmacol. 2001; 60: 321-330Crossref PubMed Scopus (41) Google Scholar), and the receptors were immunoprecipitated using a specific rat monoclonal anti-HA antibody (3F10; Roche Applied Science) and electrophoretically resolved as described previously (16Willets J.M. Challiss R.A.J. Kelly E. Nahorski S.R. Mol. Pharmacol. 2001; 60: 321-330Crossref PubMed Scopus (41) Google Scholar). 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) (23Schell M.J. Erneux C. Irvine R.F. J. Biol. Chem. 2001; 276: 37537-37546Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) 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-Gαq-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/Gαq Binding—The ability of wild-type GRK2, D110AGRK2, K220RGRK2, and D110A,K220RGRK2 to bind to Gαq 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 Gαq mutant Q209LGαq 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 × g, 10 min, 4 °C). Supernatants from cells transfected with Q209LGαq 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 2× 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 Gαq detection, and a further 10 μl was loaded onto another gel for detection of Myc expression. Separated protein was transferred to nitrocellulose, and Gαq expression was detected using a rabbit polyclonal anti-Gαq (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 ×100 objective. Cytosolic Ca2+ levels were measured as the relative change in fluorescence detected in an area of interest as described previously (24Willets J.M. Parent J.L. Benovic J.L. Kelly E. J. Neurochem. 1999; 73: 1781-1789PubMed Google Scholar). 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.RESULTSSuppression 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 (25Willets J.M. Kelly E. Eur. J. Pharmacol. 2001; 431: 133-141Crossref PubMed Scopus (24) Google Scholar). 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).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.Fig. 2Effects of antisense GRK2 on M1 mACh receptor signaling in hippocampal neurons. Hippocampal neurons were transfected with a 1:3 ratio of eGFP-PHPLCδ and either pcDNA3 or antisense GRK2 for at least 72 h. A, concentration-response curves for eGFP-PHPLCδ translocations stimulated by a single 30-s application of MCh at the indicated concentrations in neurons expressing either pcDNA3 (▪) or antisense GRK2 (□). 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 Gβγ subunits to the C-terminal domain (Gly-495 to Leu-689) of bovine GRK2 (26Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar, 27Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar). Because Gβγ subunits are able to directly stimulate PLC signaling (27Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar, 28Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar), overexpression of GRK2 may lead to inhibition of PLC signaling through sequestration of free Gβγ subunits. To assess whether inhibition of PLC signaling seen with the overexpression of GRK2 or K220RGRK2 was due to sequestration of free Gβγ subunits, the C-terminal 194 amino acids of GRK2 (GRK2-ct) were co-expressed with eGFP-PHPLCδ. 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 Gαq/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 Gαq/11 (19Sterne-Marr R. Tesmer J.J. Day P.W. Stracquatanio R.P. Cilente J.A. O'Connor K.E. Pronin A.N. Benovic J.L. Wedegaertner P.B. J. Biol. Chem. 2003; 278: 6050-6058Abstract Full Text Full Text PDF PubMed Scopus (97) Goog" @default.
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