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Histamine H2 Receptor Desensitization: Involvement of a Select Array of G Protein-Coupled Receptor Kinases

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Histamine H2 Receptor Desensitization: Involvement of a Select Array of G Protein-Coupled Receptor Kinases 0026-895X/01/6005-1049–1056$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 5 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 959/937817 Mol Pharmacol 60:1049–1056, 2001 Printed in U.S.A. Histamine H2 Receptor Desensitization: Involvement of a Select Array of G Protein-Coupled Receptor Kinases CARINA SHAYO, NATALIA FERNANDEZ, BIBIANA LEMOS LEGNAZZI, FEDERICO MONCZOR, ALEJANDRO MLADOVAN, ALBERTO BALDI, and CARLOS DAVIO School of Sciences (C.S.) and Radioisotopes Laboratory, School of Pharmacy and Biochemistry (N.F., B.L.L., F.M., C.D.), University of Buenos Aires, Buenos Aires, Argentina; Institute of Biology and Experimental Medicine, Buenos Aires, Argentina (C.S., N.F., A.M., A.B.); and National Research Council of Argentina, Buenos Aires, Argentina (F.M., A.B., C.D.) Received January 4, 2001; accepted July 27, 2001 This paper is available online at http://molpharm.aspetjournals.org ABSTRACT The histamine H2 receptor (H2r) belongs to the heptahelical (PKA and PKC) inhibitors but was remarkably reduced by Zn2ϩ, receptor family; upon agonist binding, members of this family an inhibitor of G protein-coupled receptor kinases (GRKs). Co- activate a G protein and the downstream effector adenylyl transfection experiments using tagged H2r and different GRKs cyclase. Like other G protein-coupled receptors, exposure of (2, 3, 5, or 6), demonstrated that GRK2 and GRK3 were the H2r to agonists produces a desensitization of the response. most potent in augmenting desensitization, causing a reduction The present study focused on the desensitization mechanism in the maximal response to amthamine and a decrease of the of this receptor. Using transiently transfected COS-7 cells ex- t1/2 for desensitization, whereas GRK5 and GRK6 did not affect pressing tagged-H2r, the desensitization induced by the signaling. Receptor phosphorylation correlates with desen- amthamine, characterized by decreased cAMP production, sitization for each GRK studied, whereas phosphorylation that was studied. Results show that the receptor was rapidly de- is dependent on protein kinases A and C seemed irrelevant in ϭ Ϯ sensitized with a t1/2 0.49 0.01 min. Because of the rapid receptor signal termination. These results indicate that in H2r- nature of H2r desensitization, receptor phosphorylation was transfected COS-7 cells, exposure to an agonist caused de- examined as a likely mechanism for signal attenuation. H2r sensitization controlled by H2r phosphorylation via GRK2 and desensitization was not affected by protein kinases A and C GRK3. Histamine is an intercellular signal molecule that exerts messenger-dependent kinases PKA and PKC, are responsible its effect through H1, H2, and H3 receptors (Leurs et al., for the homologous and heterologous desensitization, respec- 1995). Molecular biology studies indicate that H2r belongs to tively (Freedman and Lefkowitz, 1996). GRK-mediated phos- the large multigene family of G protein-coupled receptors phorylation of serine/threonine residues in the carboxyl tail (GPCRs) (Gantz et al., 1991). Structurally, these receptors and/or intracellular loops of GPCR increases the affinity for are characterized by seven transmembrane ␣-helices and arrestin-type proteins, and this binding prevents any further functionally by their ability to transmit signals to effector coupling between the receptor and G proteins. The complex molecules via G proteins (Dohlman et al., 1991). It is gener- formed by the phosphorylated GPCR and arrestin targets the ally accepted that the H2r mediates activation of adenylyl activated receptor to clathrin-coated pits for subsequent in- cyclase (AC) with subsequent increases in cAMP and protein ternalization (Pitcher et al., 1998). kinase A (PKA) activation (Nonaka et al., 1992). A charac- Recent studies have described that the H2r-mediated teristic feature of these GPCRs is that in the face of continu- cAMP response is rapidly desensitized in various cell types ing stimulation, signaling becomes attenuated or desensi- (Fukusima et al., 1993; Smit et al., 1994). However, in con- tized. For a large number of related GPCRs, rapid trast to other GPCR, the mechanism of H2r desensitization is desensitization seems to involve receptor phosphorylation. G protein-coupled receptor kinases (GRKs), and the second still not well understood. In the U937 promonocytic cell line, we described H1 and H2 histamine receptors (Davio et al., 1995 a), and we recently showed that the H2r was specifically This study was supported by grants from the University of Buenos Aires ϳ (JB52), the National Research Council of Argentina (PID 792/98), and the desensitized by an H2 agonist with a t1/2 of 20 min. This National Agency for Scientific and Technologic Promotion (PICT 1598). desensitization proved to be homologous and involved the ABBREVIATIONS: H2r, histamine H2 receptor; GPCR, G protein-coupled receptor; AC, adenylyl cyclase; PKA, protein kinase A; GRK, G protein-coupled receptor kinase; PKC, protein kinase C; IBMX, isobutylmethylxanthine; H9, N-(2-aminoethyl)-5-isoquinolinesulphonamide; HTMT dimaleate, 6[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptanecarboxamide; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; HA, hemagglutinin. 1049 1050 Shayo et al. ␮ ␮ GRK family. Binding experiments showed that receptor in- 20 M staurosporine, or 200 M ZnCl2 for 20 min at 37°C, followed ternalization began just after 2 h, when total desensitization by desensitization assays carried out in the presence of inhibitors. ϩ took place (Lemos Legnazzi et al., 2000). Zn2 showed a maximal inhibition on desensitization response in the In an attempt to better understand the H2r desensitization range of 100 to 400 ␮M. Higher concentrations became toxic for the mechanism, a transfection system that allowed the overex- cells. cAMP production is expressed as the stimulus relative to basal levels. pression of the different GRKs was used. Results demon- Radioligand Binding Assay. Triplicate assays were performed strate that in COS-7 transfected cells, agonist-dependent in COS-7 transfected cells in 24 multiwell plates. For saturation H2r phosphorylation is coincident with receptor desensitiza- studies, increasing concentrations of [3H]tiotidine were incubated in tion and involves GRK2 and GRK3 in both processes. A the absence or presence of 1 ␮M tiotidine, in a total volume of 200 ␮l better knowledge of the molecular regulation of the H2r of 50 mM Tris-HCl, pH 7.4. After 40 min at 4°C, incubation was should contribute to the understanding and manipulation of stopped by dilution with 3 ml of ice-cold 50 mM Tris-HCl, pH 7.4, the processes they regulate. followed by washes with ice-cold buffer. Experiments on intact cells were carried out at 4°C to avoid internalization of the ligand. Kinetic Experimental Procedures studies showed that equilibrium was reached after 30 min and persisted for 4 h (data not shown). Materials. Restriction enzymes were purchased from New En- Western Blots. COS-7 cells were lysed in sample buffer (50 mM gland Biolabs, Inc. (Beverly, MA), and Taq polymerase from Invitro- Tris-HCl, pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol, 10% glycerol, gen (Carlsbad, CA). Isobutylmethylxanthine (IBMX), cAMP, famoti- and 0.05% bromphenol blue), and sonicated to shear DNA. The dine, pyrilamine, protease inhibitors, and phosphatase inhibitors anti-HA immunoprecipitates were also analyzed. Samples were were obtained from Sigma Chemical Company (St. Louis, MO). boiled for 5 min, and aliquots were electrophoresed in 12% SDS- Amthamine, N-(2-aminoethyl)-5-isoquinolinesulphonamide (H9), polyacrylamide gels and transferred to nitrocellulose membranes. and 6[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)hep- The residual binding sites were blocked with 5% nonfat powdered tanecarboxamide (HTMT dimaleate), were from Tocris Cookson Inc. milk in PBS-Tween-20 (PBS containing 0.05% Tween-20), and mem- (Ballwin, MO). [3H]cAMP and [3H]tiotidine were purchased from branes were incubated with 1 ␮g/ml of anti-GRK2, 3, 5, 6 or anti-HA New England Nuclear (Boston, MA), and [32P]orthophosphate from (for the immunoprecipitates) rabbit antibody (Santa Cruz Biotech- Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, nology, Santa Cruz, CA), in PBS-Tween-20. All subsequent washes UK). All other chemicals were of analytical grade. were performed with the same buffer. Reactivity was developed Expression Plasmid Preparation. Oligonucleotides were syn- using an anti-rabbit polyclonal antibody linked to horseradish per- thesized using the known cDNA sequence for human H2r, and a oxidase and enhanced chemiluminescence reagents, according to the full-length nucleotide sequence was amplified from the human cell manufacturer’s instructions (Amersham Pharmacia Biotech). line U937 cDNA by polymerase chain reaction. The cDNA was then Phosphorylation Assays. Transfected cells were preincubated cloned into the SpeI and EcoRV sites of the eukaryotic expression 1hat37°C in phosphate-free DMEM, and labeled 3 h with 100 ␮ 32 vector pCEFL (pCEFL-H2r) (Teramoto et al., 1996) or into the BglII Ci/ml of Pi at 37°C in fresh medium. H2 agonist was applied as and XbaI sites of pCEFL-HA (pCEFL-HA-H2r), a modified pCEFL indicated in figure legends. To assay kinase inhibitors, cells were ϩ expression plasmid encoding the HA nonapeptide epitope. GRK2, -3, pretreated for 20 min with 20 ␮MH9or200␮MZn2 at 37°C, and -5, and -6 cDNAs were subcloned into the pCEFL vector (pCEFL- agonist stimulation was carried out in the
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