• Corpus ID: 29676128

Histamine H 2 Receptor Trafficking : Role of Arrestin , Dynamin , and Clathrin in Histamine H 2 Receptor Internalization

@inproceedings{Fernndez2008HistamineH2,
  title={Histamine H 2 Receptor Trafficking : Role of Arrestin , Dynamin , and Clathrin in Histamine H 2 Receptor Internalization},
  author={Natalia Cristina Fern{\'a}ndez and Federico Monczor and Alberto Baldi and Carlos Alberto Davio and Carina Shayo},
  year={2008}
}

• N. Fernández, F. Monczor, +2 authors C. Shayo
• Published 2008
• Biology, Chemistry

Agonist-induced internalization of G protein-coupled receptors (GPCRs) has been implicated in receptor desensitization, resensitization, and down-regulation. In the present study, we sought to establish whether the histamine H2 receptor (H2r) agonist amthamine, besides promoting receptor desensitization, induced H2r internalization. We further studied the mechanisms involved and its potential role in receptor resensitization. In COS7 transfected cells, amthamine induced H2r time-dependent internalization, showing 70% of receptor endocytosis after 60-min exposure to amthamine. Agonist removal led to the rapid recovery of resensitized receptors to the cell surface. Similar results were obtained in the presence of cycloheximide, an inhibitor of protein synthesis. Treatment with okadaic acid, an inhibitor of the protein phosphatase 2A (PP2A) family of phosphatases, reduced the recovery of both H2r membrane sites and cAMP response. Arrestin 3 but not arrestin 2 overexpression reduced both H2r membrane sites and H2r-evoked cAMP response. Receptor cotransfection with dominant-negative mutants for arrestin, dynamin, Eps15 (a component of the clathrin-mediated endocytosis machinery), or RNA interference against arrestin 3 abolished both H2r internalization and resensitization. Similar results were obtained in U937 cells endogenously expressing H2r. Our findings suggest that amthamine-induced H2r internalization is crucial for H2r resensitization, processes independent of H2r de novo synthesis but dependent on PP2A-mediated dephosphorylation. Although we do not provide direct evidence for H2r interaction with -arrestin, dynamin, and/or clathrin, our results support their involvement in H2r endocytosis. The rapid receptor recycling to the cell surface and the specific involvement of arrestin 3 in receptor internalization further suggest that the H2r belongs to class A GPCRs. Histamine is a natural widely distributed body constituent that mediates numerous functions, especially in the central nervous system, mast cells, gastric mucosa, parietal cells, and basophils. Four distinct receptor subtypes (H1, H2, H3, and H4) belonging to the large family of G protein-coupled receptors (GPCRs) mediate histamine biological effects (Simons, 2004). The histamine H2 receptor (H2r) subserves hypotension, flushing, headache, increased gastric acid production, and enhanced vascular permeability (Sachs et al., 2002; Spitaler et al., 2002). By coupling to Gs, H2r triggers adenylyl cyclase activation and subsequent rapid cAMP accumulation (Hill, 1990). The activity of GPCRs results from a coordinated balance among the diverse mechanisms that govern receptor signaling at the different levels of signal propagation, and H2r-coupled signaling is not an exception. cAMP response to H2 agonists is attenuated within minutes of the onset of agonist stimulation by a process termed desensitization (Lemos Legnazzi et al., 2000). The desensitization of GPCRs is a physiologically adaptive mechanism triggered by continuous or repeated stimuli, which protects the cell from both shortand long-term receptor overstimulation. The underlying mechanisms are complex and involve receptor phosphorylation, uncoupling from G proteins, internalization, and ultimately receptor down-regulation (Zhang et al., 1997). These mechanisms have been extensively studied for the 2 adrenoreceptor, and based on these investigations, a general pathway for agonist-mediated receptor desensitization and internalization has been outlined (Zhang et al., 1997). In this canonical model, agonist-induced activation of This study was supported by grants from the Universidad de Buenos Aires (UBACyT B050) and the Agencia Nacional de Promoción Cientı́fica y Tecnológica (PICT 12164 and PICT 38318) and Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (CONICET) (PIP 6110). This work was made possible by CONICET fellowships. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.045336. ABBREVIATIONS: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; H2r, histamine H2 receptor; IBMX, isobutylmethylxanthine; amthamine, 2-amino-4-methylthiazole-5-ethanamine; RNAi, RNA interference; EGFP, enhanced green fluorescent protein; AP-2, activator protein 2. 0026-895X/08/7404-1109–1118$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 4 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 45336/3385390 Mol Pharmacol 74:1109–1118, 2008 Printed in U.S.A. 1109 at A PE T Jornals on Jne 7, 2017 m oharm .aspeurnals.org D ow nladed from GPCRs leads to receptor phosphorylation by second messenger-dependent kinases and/or specific G protein-coupled receptor kinases (GRKs) (Pitcher et al., 1998). In turn, GRKmediated phosphorylation facilitates the binding of the third intracellular loop and the carboxyl-terminal tail of the receptor to cytoplasmic accessory proteins called arrestins, which physically uncouple the receptor from the G protein. In addition, the interaction of GPCRs with arrestins targets the phosphorylated GPCR to clathrin-coated pits and initiates internalization by the interaction of the carboxyl-terminal of arrestin with both the clathrin heavy chain and the 2adaptin subunit of AP-2 complex (Laporte et al., 1999). After pinching off the vesicles from plasma membrane by dynamin (Sever, 2002), the receptor is sequestered into intracellular vesicular compartments (endosomes) (von Zastrow, 2003). Based on their aptitude to bind -arrestin, GPCRs are divided into A and B classes. Class A GPCRs are dephosphorylated in the endosomal compartment after internalization and are recycled rapidly to the cell surface (rapid resensitization). Class B GPCRs are retained in the endosomal compartment and slowly recycled to the plasma membrane (slow resensitization) or targeted to lysosomes for degradation (down-regulation) (Oakley et al., 2000; Prossnitz, 2004). However, other mechanisms for GPCR internalization have also been described. Some GPCRs are internalized by clathrin-independent endocytic mechanisms via caveola and lipid rafts (Ginés et al., 2001). The latter are small clathrinfree microdomains of the cell membrane enriched in cholesterol and sphingolipids that can present the addition of members of the caveolin family of scaffolding proteins (Anderson and Jacobson, 2002). Clathrin-independent internalization has been described in some cell types for 2-adrenoreceptors (Raposo et al., 1989). The internalization of AT1A angiotensin receptor and M2 muscarinic receptor is mediated by a dynamin-independent mechanism (Pals-Rylaarsdam et al., 1997; Zhang et al., 1997), whereas that of the 5-hydroxytryptamine-2A receptor is mediated by an arrestin-independent pathway (Bhatnagar et al., 2001). These findings clearly indicate that the mechanisms underlying receptor internalization is by no means universal for all GPCRs, thus supporting the complexity of GPCR signaling, desensitization, and internalization. This spatial and temporal control determines the specificity of receptor-mediated signal transduction among the distinct downstream effectors and the ultimate cellular response. Despite the wide therapeutic use of H2 ligands for gastric ulcers, their cardioprotective effects in patients with chronic heart failure (Asanuma et al., 2006; Kim et al., 2006), and their implication in HL-60 and U937 leukemic cell maturation (Tasaka et al., 1994; Fernández et al., 2002), little is known about H2r regulation. H2r internalization was first reported in human embryonic kidney 293 cells in which the authors showed that histamine treatment induces loss of H2r membrane immunoreactivity (Smit et al., 1995). However, the molecular mechanism underlying H2r desensitization, internalization, and H2r fate after endocytosis still remain uncertain. The purpose of the present study was to investigate agonist-induced H2r internalization in COS7 and U937 leukemic cells and to determine the role of -arrestins, dynamin, and clathrin in this process as well as in H2r response resensitization. Our findings show that agonistinduced H2r internalization is crucial for the rapid recovery of H2r-mediated cAMP response, which is independent of de novo H2r synthesis. Furthermore, arrestin 3, dynamin, and clathrin are involved in both the internalization and resensitization of the H2r. Materials and Methods Materials. U937 and COS7 cells were obtained from the American Type Culture Collection (Manassas, VA). Cell culture medium, antibiotics, isobutylmethylxanthine (IBMX), cAMP, cycloheximide, okadaic acid, G418, and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO). Amthamine and tiotidine were from Tocris Cookson Inc. (Ballwin, MO). [H]cAMP ( 31 Ci/mmol) and [H]tiotidine ( 75 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). Other chemicals used were of analytical grade. pcDNA31arrestin (arrestin 2), pcDNA32arrestin (arrestin 3), pcDNA3-HA-dynaminK44A, and pcDNA31-arrestin(319–418) were generous gifts from Dr. J. Benovic (Thomas Jefferson University, Microbiology and Immunology Department, Kimmel Cancer Center, Philadelphia, PA). pEGFP-C2Eps15 EH29, pEGFP-C2-Eps15 DIII, and pEGFP-C2-Eps15 D3 2 constructs were generous gifts from Dr. Benmerah (Université Paris 5, Institut Cochin, Departement de Maladies Infectieuses, Paris, France). The RNAi sequences targeting arrestin 2 (5 -CCAAUCUCAUAGAACUUGACACAAA-3 ) or arrestin 3 (5 -GGGUCUUCAAGAAGUCGAGCCCUAA-3 and 5 -GCCACAGAUGAUGACAUUGUGUUUG-3 ) and the RNAi control duplexes were Stealth RNAi purchased from Invitrogen (Carlsbad, CA). Cell Culture and Transfection. COS7 cells were cultured in a humidified atmosphere of 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 50 g/ml gentamicin. For transient transfection, COS7 cells were grown to 80 to 90% confluence. cDNA constructs were tr