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Bimodal Regulation of the Human H1 Histamine Receptor by G Protein-coupled Receptor Kinase 2

2004; Elsevier BV; Volume: 280; Issue: 3 Linguagem: Inglês

10.1074/jbc.m408834200

ISSN

1083-351X

Autores

Ken Iwata, Jiansong Luo, Raymond B. Penn, Jeffrey Benovic,

Tópico(s)

Neuropeptides and Animal Physiology

Resumo

The H1 histamine receptor (H1HR) is a member of the G protein-coupled receptor superfamily and regulates numerous cellular functions through its activation of the Gq/11 subfamily of heterotrimeric G proteins. Although the H1HR has been shown to undergo desensitization in multiple cell types, the mechanisms underlying the regulation of H1HR signaling are poorly defined. To address this issue, we examined the effects of wild type and mutant G protein-coupled receptor kinases (GRKs) on the phosphorylation and signaling of human H1HR in HEK293 cells. Overexpression of GRK2 promoted H1HR phosphorylation in intact HEK293 cells and completely inhibited inositol phosphate production stimulated by H1HR, whereas GRK5 and GRK6 had lesser effects on H1HR phosphorylation and signaling. Interestingly, catalytically inactive GRK2 (GRK2-K220R) also significantly attenuated H1HR-mediated inositol phosphate production, as did an N-terminal fragment of GRK2 previously characterized as a regulator of G protein signaling (RGS) protein for Gαq/11. Disruption of this RGS function in holo-GRK2 by mutation (GRK2-D110A) partially reversed the quenching effect of GRK2, whereas deletion of both the kinase activity and RGS function (GRK2-D110A/K220R) effectively relieved the inhibition of inositol phosphate generation. To evaluate the role of endogenous GRKs on H1HR regulation, we used small interfering RNAs to selectively target GRK2 and GRK5, two of the primary GRKs expressed in HEK293 cells. A GRK2-specific small interfering RNA effectively reduced GRK2 expression and resulted in a significant increase in histamine-promoted calcium flux. In contrast, knockdown of GRK5 expression was without effect on H1HR signaling. These findings demonstrate that GRK2 is the principal kinase mediating H1 histamine receptor desensitization in HEK293 cells and suggest that rapid termination of H1HR signaling is mediated by both the kinase activity and RGS function of GRK2. The H1 histamine receptor (H1HR) is a member of the G protein-coupled receptor superfamily and regulates numerous cellular functions through its activation of the Gq/11 subfamily of heterotrimeric G proteins. Although the H1HR has been shown to undergo desensitization in multiple cell types, the mechanisms underlying the regulation of H1HR signaling are poorly defined. To address this issue, we examined the effects of wild type and mutant G protein-coupled receptor kinases (GRKs) on the phosphorylation and signaling of human H1HR in HEK293 cells. Overexpression of GRK2 promoted H1HR phosphorylation in intact HEK293 cells and completely inhibited inositol phosphate production stimulated by H1HR, whereas GRK5 and GRK6 had lesser effects on H1HR phosphorylation and signaling. Interestingly, catalytically inactive GRK2 (GRK2-K220R) also significantly attenuated H1HR-mediated inositol phosphate production, as did an N-terminal fragment of GRK2 previously characterized as a regulator of G protein signaling (RGS) protein for Gαq/11. Disruption of this RGS function in holo-GRK2 by mutation (GRK2-D110A) partially reversed the quenching effect of GRK2, whereas deletion of both the kinase activity and RGS function (GRK2-D110A/K220R) effectively relieved the inhibition of inositol phosphate generation. To evaluate the role of endogenous GRKs on H1HR regulation, we used small interfering RNAs to selectively target GRK2 and GRK5, two of the primary GRKs expressed in HEK293 cells. A GRK2-specific small interfering RNA effectively reduced GRK2 expression and resulted in a significant increase in histamine-promoted calcium flux. In contrast, knockdown of GRK5 expression was without effect on H1HR signaling. These findings demonstrate that GRK2 is the principal kinase mediating H1 histamine receptor desensitization in HEK293 cells and suggest that rapid termination of H1HR signaling is mediated by both the kinase activity and RGS function of GRK2. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HA, hemagglutinin; H1HR, H1 histamine receptor; IP, inositol phosphate; PKC, protein kinase C; PLC, phospholipase C; RGS, regulator of G protein signaling; siRNA, small interfering RNA; DMEM, Dulbecco's modified Eagle's medium; SC, scrambled. comprise a superfamily of seven transmembrane-spanning receptors that transduce extracellular signals into discrete intracellular signals to regulate cell functions. GPCR signaling is regulated not only by ligand availability but also by complex mechanisms that regulate receptor responsiveness to their cognate stimuli. The regulatory process of desensitization utilizes a wide variety of regulatory proteins that interact with a given GPCR to render it hyporesponsive to agonists. For the majority of GPCRs, desensitization caused by agonist exposure is mediated by one or more members of a family of GPCR kinases (GRKs) that phosphorylate the agonist-occupied receptor and promote the subsequent binding of arrestin molecules (1Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar, 2Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar, 3Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (452) Google Scholar). Arrestin binding to GPCRs disrupts receptor activation of heterotrimeric G proteins and can also initiate the process of receptor internalization, which can lead to either GPCR recycling or degradation (1Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar, 2Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar, 3Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (452) Google Scholar). However, studies to date clearly demonstrate that the propensity for a particular GPCR to be regulated by second messenger-dependent kinases, GRKs, or arrestins is receptor-specific. The H1 histamine receptor (H1HR) mediates the functional effects of histamine in multiple cell types through activation of the Gq/11 heterotrimeric G protein and its downstream effector phospholipase C (PLC). Stimulation of the H1HR-Gq/11-PLC pathway results in the synthesis of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (4Leurs R. Smit M.J. Timmerman H. Pharmacol. Ther. 1995; 66: 413-463Crossref PubMed Scopus (344) Google Scholar), which in turn stimulate an increase in intracellular Ca2+ and the activation of protein kinase C (PKC). These histamine-induced intracellular messengers promote diverse functions in multiple cell types, including smooth muscle and nonsmooth muscle contraction (4Leurs R. 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Rev. 1998; 50: 515-596PubMed Google Scholar), both of which can contribute to inflammation and inflammatory disease processes (reviewed in Refs. 4Leurs R. Smit M.J. Timmerman H. Pharmacol. Ther. 1995; 66: 413-463Crossref PubMed Scopus (344) Google Scholar, 5Hill S.J. Ganellin C.R. Timmerman H. Schwartz J.C. Shankley N.P. Young J.M. Schunack W. Levi R. Haas H.L. Pharmacol. Rev. 1997; 49: 253-278PubMed Google Scholar, and 10Barnes P.J. Chung K.F. Page C.P. Pharmacol. Rev. 1998; 50: 515-596PubMed Google Scholar, 11Jutel M. Watanabe T. Klunker S. Akdis M. Thomet O.A. Malolepszy J. Zak-Nejmark T. Koga R. Kobayashi T. Blaser K. Akdis C.A. Nature. 2001; 413: 420-425Crossref PubMed Scopus (507) Google Scholar, 12Billington C.K. Penn R.B. Respir. Res. 2003; 4: 2Crossref PubMed Google Scholar). Numerous studies have demonstrated that both endogenously expressed as well as heterologously expressed H1HRs exhibit hyporesponsiveness/desensitization when exposed to either PKC-activating agents or histamine (7Murray R.K. Bennett C.F. Fluharty S.J. Kotlikoff M.I. Am. J. Physiol. 1989; 257: L209-L216PubMed Google Scholar, 13Fujimoto K. Ohta K. Kangawa K. Kikkawa U. Ogino S. Fukui H. Mol. Pharmacol. 1999; 55: 735-742PubMed Google Scholar, 14Smit M.J. Timmerman H. Hijzelendoorn J.C. Fukui H. Leurs R. Br. J. Pharmacol. 1996; 117: 1071-1080Crossref PubMed Scopus (60) Google Scholar, 15McCreath G. Hall I.P. Hill S.J. Br. J. Pharmacol. 1994; 113: 823-830Crossref PubMed Scopus (22) Google Scholar, 16Hishinuma S. Ogura K. J. Neurochem. 2000; 75: 772-781Crossref PubMed Scopus (22) Google Scholar, 17Hishinuma S. Naiki A. Tsuga H. Young J.M. J. Neurochem. 1998; 71: 2626-2633Crossref PubMed Scopus (15) Google Scholar, 18Pype J.L. Mak J.C. Dupont L.J. Verleden G.M. Barnes P.J. Br. J. Pharmacol. 1998; 125: 1477-1484Crossref PubMed Scopus (18) Google Scholar, 19Smit M.J. Bloemers S.M. Leurs R. Tertoolen L.G. Bast A. de Laat S.W. Timmerman H. Br. J. Pharmacol. 1992; 107: 448-455Crossref PubMed Scopus (57) Google Scholar) and that agonist-specific desensitization can be associated with H1HR internalization or down-regulation (14Smit M.J. Timmerman H. Hijzelendoorn J.C. Fukui H. Leurs R. Br. J. Pharmacol. 1996; 117: 1071-1080Crossref PubMed Scopus (60) Google Scholar, 18Pype J.L. Mak J.C. Dupont L.J. Verleden G.M. Barnes P.J. Br. J. Pharmacol. 1998; 125: 1477-1484Crossref PubMed Scopus (18) Google Scholar). However, beyond a basic appreciation that the H1HR desensitizes and internalizes, little is known regarding the mechanisms by which these processes are mediated. In the present study, we examined the roles of PKC and GRKs in agonist-specific H1HR desensitization, and assessed the relative contribution of distinct functional domains within GRK2 responsible for terminating H1HR signaling. Materials—A pcDNA3-H1HR expression construct encoding the human H1 histamine receptor was provided by Dr. R. Leurs (Vrije University, Amsterdam, The Netherlands). Gq binding-defective GRK2 mutants (GRK2-D110A, GRK2-R106A) were provided by Dr. R. Sterne-Marr (Siena College, Loudonville, NY). Human embryonic kidney (HEK293) cells were purchased from the American Type Culture Collection (Manassas, VA). FuGENE-6 was from Roche Applied Science. Monoclonal and polyclonal antibodies for the hemagglutinin (HA) epitope were purchased from Covance Research Products (Berkeley, CA). Alexa 594 conjugate anti-HA monoclonal antibody was from Molecular Probes (Eugene, OR). myo-[3H]Inositol and [32P]orthophosphate were purchased from PerkinElmer Life Sciences, whereas inositol-free DMEM and sodium phosphate-free DMEM were from Life Technologies, Inc. GRK-specific and scrambled (SC) siRNAs were purchased from Dharmacon. Plasmid Construction—To eliminate the promoter sequence, the 5′-terminal region of H1HR in pcDNA3-H1HR was amplified by PCR using two oligos, 5′-CGGGGGTACCCGGGCACCATG AGCCTCCCCAATTCCTCC-3′ and 5′-TAACATCTGATCCTCTGATATCTCGC-3′, and cloned back into the KpnI sites of the original pcDNA3-H1HR construct. The open reading frame of pcDNA3-H1HR was also subcloned in-frame into pcDNA3 containing a 5′-HA epitope tag cassette (20Parent J.L. Labrecque P. Orsini M.J. Benovic J.L. J. Biol. Chem. 1999; 274: 8941-8948Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) to generate HA-H1HR. Constructs encoding GRK2, GRK2-K220R, GRK2-(45–178) (GRK2-RGS), GRK2-(468–689) (GRK2-CT), GRK2-R106A, GRK2-D110A, GRK5, and GRK6 have been described previously (21Carman 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 (284) Google Scholar, 22Sterne-Marr R. Tesmer J.J.G. Day P.W. Stracquatanio R.P. Cilente J-A.E. 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 (101) Google Scholar, 23Pronin A.N. Carman C.V. Benovic J.L. J. Biol. Chem. 1998; 273: 31510-31518Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). GRK5-K215R (24Tiruppathi C. Yan W. Sandoval R. Naqvi T. Pronin A.N. Benovic J.L. Malik A.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7440-7445Crossref PubMed Scopus (83) Google Scholar) was subcloned into pcDNA3 by PCR cloning. All constructs were sequenced to confirm the correct orientation and to ensure that no spurious mutations were introduced. Transient Transfection and H1HR Radioligand Binding—HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 10 IU/ml penicillin, and 10 μg/ml streptomycin. Cells grown in 100-mm dishes to subconfluence were transfected with 5 μg of wild type or HA-tagged pcDNA3-H1HR using FuGENE-6 according to the manufacturer's protocol. Cells expressing H1HRs were harvested by scraping into ice-cold 50 mm phosphate buffer (pH 7.5) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 5 mm benzamidine, 10 μg/ml leupeptin, and 10 μg/ml pepstatin) and recovered by a 10-min centrifugation at 500 × g. The cells were homogenized using a Polytron homogenizer, and the homogenate (0.1–0.3 mg of protein/assay tube) was incubated for 1 h at 37°C in 1 ml of ice-cold 50 mm phosphate buffer (pH 7.5) containing protease inhibitors and 0.5–16 nm [3H]pyrilamine (25Hill S.J. Young J.M. Marrian D.H. Nature. 1977; 270: 361-363Crossref PubMed Scopus (99) Google Scholar). The binding reaction was terminated by the addition of 3 ml of ice-cold 50 mm phosphate buffer (pH 7.5) followed by rapid filtration through What-man GF/C filters in a cell harvester followed by three washes with 3 ml of ice-cold buffer. Nonspecific binding of [3H]pyrilamine was determined as binding in the presence of 20 μm doxepin, a specific antagonist for H1HR. Triplicate samples were assayed for each point. Protein concentrations were determined using a Bradford protein assay kit. Transfection of GRK siRNAs—siRNA transfection of the GRK2, GRK5, and SC RNA duplexes (Dharmacon, Lafayette, CO) was performed in HEK293 cells at ∼90% confluence. The cells were transfected with siRNA duplexes (600 pmol of siRNA in a 10-cm dish, final concentration of 40 nm) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's instructions and were analyzed for GRK expression and calcium flux 3 days after transfection. In some experiments, cells were transfected with siRNA, split after 6 h, transfected a second time after 24 h, and then analyzed 4 days after the initial transfection. All siRNAs were 21-nucleotide duplexes and had the following sequences: siRNA-GRK2, 5′-GAU CUU CGA CUC AUA CAU CdTdT-3′; siRNA-GRK5, 5′-GAU CCU CUG CGG CUU AGA AdTdT-3′; and siRNA-SC, 5′-GCG CGC UUU GUA GGA UUC GdTdT-3′. Receptor Phosphorylation—HEK293 cells grown in 100-mm dishes were transfected with 5 μg of pcDNA3-HA-H1HR and 5 μg of pcDNA3-GRK2, GRK5, or GRK6 or vector control. The following day, the cells were seeded into two 10-cm dishes for phosphorylation analysis and one 6-cm dish for radioligand binding (to confirm equivalent expression). Forty-eight h after transfection, the cells were washed twice in serum-free and sodium phosphate-free DMEM followed by incubation in the same medium for 1 h. The cells were subsequently labeled with 0.5 mCi of [32P]orthophosphate for 2 h and then incubated with or without 100 μm histamine for 10 min. The medium was removed, and the cells were washed three times with buffer (25 mm Tris-HCl, pH 7.5, 137 mm NaCl, 5 mm KCl, 0.9 mm CaCl2, 0.5 mm MgCl2, 0.7 mm Na2HPO4) and then scraped into 0.8 ml of ice-cold lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, 1% (v/v) Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mm sodium fluoride, 10 mm sodium pyrophosphate, 1 mm phenylmethylsulfonyl fluoride, 5 mm benzamidine, 10 μg/ml pepstatin, 10 μg/ml leupeptin, 2 μg/ml aprotinin). All subsequent steps were performed at 4 °C. The lysate was solubilized for 1 h on a rocker and then centrifuged at 100,000 × g for 20 min. The resultant supernatant was isolated and precleared by the addition of 50 μl of an ∼50% slurry of protein A-agarose beads with gentle rocking for 50 min. Sixteen μl of anti-HA polyclonal antibody was then added to the precleared supernatant and incubated for 1 h; 50 μl of protein A-agarose beads were added, and the suspension was incubated on a rocker overnight. Immune complexes were collected by centrifugation the following day and washed 4 times with ice-cold lysis buffer. The beads were resuspended in 50 μl of SDS sample buffer, and the immunoprecipitates were resolved on 10% polyacrylamide gels. The gels were stained with Coomassie Blue, destained, dried, and subjected to autoradiography at -80 °C. Inositol Phosphate Production—Measurement of inositol phosphate (IP) production in cells was as described previously (21Carman 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 (284) Google Scholar). Briefly, sub-confluent HEK293 cells grown in 100-mm dishes were transfected with pcDNA3-HA-H1HR and GRK constructs using FuGENE-6. The total amount of transfected plasmids was adjusted to 10 μg by the addition of pcDNA3 vector. The following day, the cells were seeded onto a 24-well dish and labeled with 1 μCi/ml myo-[3H]inositol for 17–22 h in 0.5% bovine serum albumin in DMEM. The cells were washed two times and incubated with inositol-free DMEM containing 5 mm LiCl for 30 min at 37 °C and then stimulated with various concentrations of histamine for 30 min. The medium was removed, and the cells were lysed with 1 ml of 20 mm formic acid for 30 min at 4 °C and then neutralized with 130 μl of 3% ammonium hydroxide. The inositol fractions were separated using Dowex AGX (100–200 mesh) columns, counted, and data reported as described previously (21Carman 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 (284) Google Scholar). Measurement of Ca2+ Flux—HEK293 cells transfected with SC, GRK2, or GRK5 siRNAs were harvested with Cellstripper (Mediatech, Herndon, VA), washed twice with phosphate-buffered saline, and resuspended at ∼5 × 106 cells/ml in Hanks' balanced salt solution (140 mm NaCl, 5 mm KCl, 10 mm HEPES, pH 7.4, 1 mm CaCl2, 1 mm MgCl2, 1 mg/ml glucose) containing 0.025% bovine serum albumin. The cells were then loaded with 3 μm Fura-2 acetoxymethyl ester derivative (Fura-2/AM) (Molecular Probes, Eugene, OR) for 30 min at 37 °C. The cells were washed once in Hanks' solution, resuspended in Hanks', incubated at room temperature for 15 min, washed twice in Hanks' solution, and then resuspended in Hanks' at a concentration of ∼3 × 107 cells/ml. A typical experiment contained 1.5 × 106 cells/1.6 ml in a quartz cuvette and stimulation with 1–1000 μm histamine. Receptor specificity studies were done by measuring the calcium flux stimulated by 100 μm histamine in the presence of H1-, H2-, or H3-specific antagonists. Calcium flux was measured using excitation at 340 and 380 nm in a fluorescence spectrometer (LS55, PerkinElmer Life Sciences). Calibration was performed using 0.1% Triton X-100 for total fluorophore release and 10 mm EGTA to chelate free Ca2+. Intracellular Ca2+ concentrations were calculated using a fluorescence spectrometer measurement program as described previously (26Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Pharmacological and Functional Properties of HA-tagged H1HRs—Our initial series of studies focused on characterizing H1HRs transiently expressed in HEK293 cells. [3H]Pyrilamine binding assays demonstrated high affinity binding of recombinant H1HRs expressed in HEK293 cells (Kd = 1.5 ± 0.8 nm, Bmax = 4.0 ± 1.3 pmol/mg protein; n = 3), similar to previously reported values (27Wieland K. Laak A.M. Smit M.J. Kuhne R. Timmerman H. Leurs R. J. Biol. Chem. 1999; 274: 29994-30000Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Kd and Bmax values obtained for HA-H1HR (Kd = 0.8 ± 0.1 nm, Bmax = 5.5 ± 0.5 pmol/mg protein; n = 3) were similar to those for untagged H1HR. Histamine-stimulated IP production in HEK293 cells expressing H1HR (EC50 = 69 ± 13 nm, fold basal increase = 5.2 ± 1.3) and HA-H1HR (EC50 = 78 ± 16 nm, fold basal increase = 3.9 ± 0.3) was also comparable. Thus, the HA tag at the N terminus of the H1HR does not appear to affect H1HR expression, ligand binding, or signaling properties. Role of GRKs in H1HR Phosphorylation—To assess the ability of the H1HR to undergo agonist-promoted phosphorylation, HEK293 cells were transfected with or without HA-H1HR and loaded with [32P]orthophosphate as described under "Experimental Procedures." The HA-H1HR migrated as a broad band of 70–100 kDa, as assessed by Western blotting, using an anti-HA antibody (Fig. 1A, left panel). The H1HR also appeared to undergo agonist-promoted phosphorylation, as treatment with 100 μm histamine for 10 min resulted in increased phosphorylation of an ∼70–90-kDa protein in immunoprecipitates from HA-H1HR transfected cells (Fig. 1A, right panel). This protein was not present in mock-transfected cells, verifying that it was the HA-tagged H1HR (Fig. 1A, right panel). Note that the low level of agonist-independent phosphorylation appears to be mediated by endogenous PKC, because it was inhibited by treating the cells with the selective PKC inhibitor bisindolylmaleimide I (data not shown). To assess whether GRKs might contribute to H1HR phosphorylation, HEK293 cells were co-transfected with HA-H1HR and either vector, GRK2, GRK5, or GRK6. Agonist-induced phosphorylation of the H1HR was significantly enhanced by co-expression of GRK2 (180% increase), whereas co-expression of GRK5 or GRK6 promoted a significantly lesser (50 and 80% increase, respectively) degree of agonist-induced phosphorylation (Fig. 1B). Interestingly, all three GRKs also led to a mobility shift in the diffuse H1HR band observed in the absence of GRK co-expression to a much sharper band of ∼90 kDa (Fig. 1B). Similar agonist-promoted mobility shifts have been observed with other GPCRs (28Stadel J.M. Nambi P. Shorr R.G. Sawyer D.F. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3173-3177Crossref PubMed Scopus (159) Google Scholar, 29Tiberi M. Nash S.R. Bertrand L. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1996; 271: 3771-3778Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 30Oppermann M. Mack M. Proudfoot A.E. Olbrich H. J. Biol. Chem. 1999; 274: 8875-8885Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and this likely reflects enhanced phosphorylation of specific residues on the H1HR. GRK Function in Quenching H1HR-mediated Inositol Phosphate Production—We next examined the capacity and functional properties of GRKs in quenching H1HR signaling. Co-expression of GRK2 with HA-H1HR completely inhibited H1HR-mediated IP production (Fig. 2A), consistent with previous studies demonstrating the ability of GRK2 to effect rapid agonist-specific desensitization of numerous other GPCRs (1Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar, 2Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar, 3Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (452) Google Scholar). Interestingly, catalytically inactive GRK2 (GRK2-K220R) (31Kong G. Penn R. Benovic J.L. J. Biol. Chem. 1994; 269: 13084-13087Abstract Full Text PDF PubMed Google Scholar), previously shown as being capable of acting in a dominant negative fashion to reverse desensitization of some GPCRs (1Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar, 2Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar, 3Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (452) Google Scholar), also significantly attenuated histamine-stimulated IP production, suggesting that the kinase activity of GRK2 is partially dispensable in its ability to quench H1HR signaling. Subsequent experiments were performed to explore how a catalytically inactive GRK2 quenches H1HR signaling and to clarify the role of discrete GRK2 functional domains in regulating H1HR signaling. Carman et al. (21Carman 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 (284) Google Scholar) previously demonstrated that an N-terminal polypeptide containing residues 45–178 of GRK2 (GRK2-RGS) associates strongly with both the transition and activated states of Gαq and functions as an RGS protein to inhibit Gαq-stimulated PLC activity. We therefore tested the capacity of this N-terminal functional domain to inhibit H1HR-mediated IP production. Co-expression of GRK2-RGS and H1HR resulted in ∼60% inhibition of histamine-stimulated IP production (Fig. 2B), demonstrating that the RGS function of GRK2 can effectively inhibit H1HR signaling. Conversely, co-expression of GRK2-CT, a C-terminal pleckstrin homology domain construct of GRK2 known to sequester Gβγ subunits necessary for activation of endogenous GRK2 (32Koch 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, 33Carman C.V. Barak L.S. Chen C. Liu-Chen L-Y. Onorato J.J. Kennedy S.P. Caron M.G. Benovic J.L. J. Biol. Chem. 2000; 275: 10443-10452Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), significantly increased histamine-stimulated IP production. This result is similar to recent findings on the metabotropic glutamate 1 receptor (34Dhami G.K. Anborgh P.H. Dale L.B. Sterne-Marr R. Ferguson S.S. J. Biol. Chem. 2002; 277: 25266-25272Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and supports a role for endogenous GRK2 in the regulation of H1HR signaling in HEK293 cells. The involvement of the RGS domain of GRK2 in H1HR regulation was subsequently examined by employing a GRK2 mutant (GRK2-D110A) that lacks the ability to interact with Gαq (22Sterne-Marr R. Tesmer J.J.G. Day P.W. Stracquatanio R.P. Cilente J-A.E. 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 (101) Google Scholar). Co-expression of GRK2-D110A significantly inhibited H1HR-mediated IP production (>80%; Fig. 2C) to an extent even greater than that observed for GRK2-RGS (Fig. 2B), suggesting that the RGS function of GRK is at least partially dispensable in the ability of GRK2 to inhibit H1HR signaling. Co-expression of GRK2-R106A, which also lacks the ability to interact with Gαq (22Sterne-Marr R. Tesmer J.J.G. Day P.W. Stracquatanio R.P. Cilente J-A.E. 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 (101) Google Scholar), inhibited H1HR-mediated IP production in a manner identical to that affected by GRK2-D110A (data not shown). To assess the requirement for kinase and RGS function in GRK2-mediated desensitization of the H1HR, we introduced the D110A mutation into catalytically inactive GRK2 to generate a construct lacking both kinase and RGS function (GRK2-K220R/D110A). Co-expression of GRK2-K220R/D110A with H1HR had a relatively weak inhibitory effect on IP production at low histamine concentrations but approached 40–50% at saturating concentrations (Fig. 2C). A possible explanation for this residual efficacy of GRK2-K220R/D110A is the steric hindrance of receptor-Gαq coupling caused by GRK2-K220R/D110A binding to H1HR. By binding to the activated receptor, exogenous GRK mutants not only block access of endogenous GRK to the receptor, but potentially block receptor-Gα interactions. Because this latter function might be considered experimentally artifactual, we reduced the levels of co-expressed GRK constructs by reducing the concentration of their respective plasmid DNAs in our transfection protocol. Under these conditions, both GRK2 and GRK2-K220R largely retained their ability to inhibit H1HR-mediated IP production (Fig. 2D). However, lower levels of GRK2-K220R/D110A expression reduced the inhibitory effect of this construct to ∼20% (Fig. 2D). These data suggest that steric hindrance of H1HR-Gαq coupling probably contributes to inhibition of H1HR signaling under experimental conditions, although under physiologic conditions, GRK2 likely regulates H1HR signaling via its kinase and RGS activities. We next examined the capacity of other kinases to effect H1HR desensitization. We first examined the effects of wild type GRK5 and GRK6, both shown to weakly phosphorylate H1HR (Fig. 1). Co-expression of GRK6 with H1HR had a modest effect on histamine-stimulated IP production, whe

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