Homologous desensitization of human histamine H3 receptors expressed in CHO-K1 cells

doi:10.1016/j.neuropharm.2013.09.011

Neuropharmacology

Volume 77, February 2014, Pages 387-397

https://doi.org/10.1016/j.neuropharm.2013.09.011 Get rights and content

Highlights

•
Histamine H₃ receptors (H₃Rs) modulate several nervous system functions.
•
Homologous desensitization was tested for human H₃Rs (hH₃Rs) expressed in CHO-K1 cells.
•
hH₃R binding and functional responses were markedly reduced by agonist pre-incubation.
•
hH₃Rs desensitize and internalize in a GRK2- and clathrin-dependent manner.
•
Desensitization is a relevant issue for pharmacological therapies aimed at H₃Rs.

Abstract

Histamine H₃ receptors (H₃Rs) modulate the function of the nervous system at the pre- and post-synaptic levels. In this work we aimed to determine whether, as other G protein-coupled receptors (GPCRs), H₃Rs desensitize in response to agonist exposure. By using CHO-K1 cells stably transfected with the human H₃R (hH₃R) we show that functional responses (inhibition of forskolin-induced cAMP accumulation in intact cells and stimulation of [³⁵S]-GTPγS binding to cell membranes) were markedly reduced after agonist exposure. For cAMP accumulation assays the effect was significant at 60 min with a maximum at 90 min. Agonist exposure resulted in decreased binding sites for the radioligand [³H]-N-methyl-histamine ([³H]-NMHA) to intact cells and modified the sub-cellular distribution of H₃Rs, as detected by sucrose density gradients and [³H]-NMHA binding to cell membranes, suggesting receptor internalization. The reduction in the inhibition of forskolin-stimulated cAMP formation observed after agonist pre-incubation was prevented by incubation in hypertonic medium or in ice-cold medium. Agonist-induced loss in binding sites was also prevented by hypertonic medium or incubation at 4 °C, but not by filipin III, indicating clathrin-dependent endocytosis. Immunodetection showed that CHO-K1 cells express GPCR kinases (GRKs) 2/3, and both the GRK general inhibitor ZnCl₂ and a small interfering RNA against GRK-2 reduced receptor desensitization. Taken together these results indicate that hH₃Rs experience homologous desensitization upon prolonged exposure to agonists, and that this process involves the action of GRK-2 and internalization via clathrin-coated vesicles.

Introduction

The primary function of the G protein-coupled receptors (GPCRs) in the nervous system is to act as mediators of the so-called slow chemical synaptic transmission (Gainetdinov et al., 2004), and in the mammalian brain histamine regulates several functions by interacting mainly with three (H₁, H₂ and H₃) of its four receptors cloned so far (H₁–H₄; Haas et al., 2008).

Histamine H₃ receptors (H₃Rs) are primarily located on nerve terminals where they control the release and synthesis of histamine as well as the release of several other neuroactive substances namely acetylcholine, noradrenaline, dopamine, γ-aminobutyric acid (GABA), glutamate, 5-hydroxytryptamine (5-HT) and substance P (Leurs et al., 2005, Feuerstein, 2008, Haas et al., 2008). However, there is also evidence for a post-synaptic expression of H₃Rs in some regions of the brain such as the striatum (projection neurons), cerebral cortex (layer V pyramidal neurons) and hippocampus (granule cells) (Pillot et al., 2002).

Through their coupling to Gαi/o proteins H₃Rs trigger several signaling pathways that include inhibition of adenylyl cyclase and thus of protein kinase A (PKA) activation, inhibition of voltage-operated Ca²⁺ channels thereby modulating neurotransmitter release, activation of phospholipase A₂ (PLA₂) leading to the release of arachidonic acid, modulation of the mitogen-activated protein kinase (MAPK) pathway and activation of the Akt/GSK-3β axis (Bongers et al., 2007a).

The functional response of GPCRs is regulated at the level of the receptor itself by two primary mechanisms: regulation of receptor density at cell surfaces and control of signaling efficiency (Hill, 2006). Homologous desensitization, an important regulatory mechanism that prevents GPCR overstimulation, implies a reduction in signaling upon agonist binding and receptor activation (Ferguson et al., 1996). The canonical mechanism involves phosphorylation of the occupied receptor by GPCR kinases (GRKs) resulting in increased affinity of the phosphorylated receptor for the proteins β-arrestins, which upon binding to the GPCR inhibit further coupling to G proteins and interact with clathrin and the β2-adaptin subunit of the clathrin adaptor AP-2 (adaptor protein complex-2) to target the receptor to clathrin-coated pits and the endocytic machinery, leading to receptor internalization (Gainetdinov et al., 2004). Endocyted receptors can be either re-inserted into the membrane or degraded in lysosomes, with the latter resulting in a reduction of total receptor number (down-regulation). The time-course of these processes spans from seconds (phosphorylation) to minutes (endocytosis) and hours (down-regulation) (Ferguson et al., 1996, Pierce et al., 2002).

The β-adrenoceptors and, to a lesser extent, muscarinic receptors are to date the best studied regarding their desensitization and the mechanisms involved. For histamine receptors there is evidence that both H₁ and H₂ receptors undergo desensitization. While GRK-2 appears responsible for the desensitization of endogenous H₁ receptors expressed by uterine smooth-muscle cells (Self et al., 2005, Willets et al., 2008), in COS-7 cells desensitization of transfected H₂ receptors is mediated by GRK-2 and GRK-3 (Shayo et al., 2001).

Some pieces of evidence suggest that H₃Rs also experience homologous desensitization. In guinea-pig ileum where H₃R activation inhibits electrically-induced contraction most likely by reducing acetylcholine release from postganglionic cholinergic neurons, previous exposure to the agonist R-α-methylhistamine (RAMH) results in a reduction in both agonist potency and efficacy in a second agonist application (Perez-Garcia et al., 1998, Alguacil and Perez-Garcia, 2003). Further, we previously reported that the incubation of rat striatal slices with H₃R agonists results in a significant decrease in receptor density, as assessed by the binding of the labeled agonist [³H]-N-methyl-histamine ([³H]-NMHA) to membranes of treated slices. This effect was prevented, although partially, by incubation in a hypertonic medium suggesting the participation of clathrin-mediated endocytosis (Garduno-Torres and Arias-Montano, 2006).

The H₃R has become a promising drug target for the treatment of neuropathic pain, sleep-wake disorders and cognitive impairment associated with Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), schizophrenia and Parkinson's disease (Passani and Blandina, 2011). While H₃R antagonists/inverse agonists have attracted most attention, with pitolisant, GSK-189254 and JNJ-31001074 already in phase II-III clinical trials, H₃R agonists may be useful in the treatment of some types of insomnia (Passani et al., 2004), neuropathic pain (Cannon and Hough, 2005) and in the prevention of excitotoxic neuronal damage (Lintunen et al., 2005, Kukko-Lukjanov et al., 2006, Mariottini et al., 2009). One common drawback of otherwise effective pharmacological treatments is receptor desensitization, and in this work we therefore set out to study further whether H₃Rs experience homologous desensitization by using a heterologous expression system.

Section snippets

Cloning of the hH₃R

The hH₃R of 445 amino acids (hH₃R₄₄₅) was amplified by PCR from the human male PAC clone RP5-1005F21 and subsequently cloned into the pCIneo expression vector. DNA sequencing confirmed the identity of the cloned hH₃R with the published hH₃R₄₄₅ sequence (GenBank accession number NM_007232).

Construction of small-hairpin RNA (shRNA) expression vectors

In order to assess the effect of reducing endogenous GRK-2 (adrbk1) levels by RNA interference, shRNA-expression vectors were engineered to co-express shRNAs and the green fluorescent protein (GFP) which

Pharmacological characteristics of the hH₃R₄₄₅ stably expressed in CHO-K1cells

From saturation experiments specific [³H]-NMHA binding to cell membranes yielded maximum binding (B_max) 276 ± 26 fmol/mg protein (mean ± standard error, s.e.m.; 4 experiments, Fig. 1A) and equilibrium dissociation constant (K_d) 1.44 nM (pK_d 8.84 ± 0.08).

Specific [³H]-NMHA binding was inhibited in a concentration-dependent manner by the endogenous agonist histamine, the H₃R agonists immepip and (R)(−)-α-methylhistamine (RAMH) and the antagonist clobenpropit (Fig. 1B). Table 1 shows that

Discussion

Herein we show that the hH₃R₄₄₅ expressed in CHO-K1 cells experiences homologous desensitization, and provide information on the cellular mechanisms involved. These results are in accord with previous findings for native rodent H₃Rs (Perez-Garcia et al., 1998, Garduno-Torres and Arias-Montano, 2006) and with a preliminary report on the signaling properties of wild-type and EGFP-tagged hH₃Rs expressed in CHO-K1 cells (Powell and Hill, 2003) indicating that the tagged receptor was internalized

Conclusion

We have herein shown that the hH₃R₄₄₅ expressed in CHO-K1 cells experiences homologous desensitization, which is likely to involve the action of GRK-2 and clathrin-mediated receptor internalization. The H₃R has become a promising drug target for the treatment of several neurological alterations and H₃R agonists may be useful in the treatment of some types of insomnia such as those resulting from anxiety, stress or neuropathologies (Passani et al., 2004), neuropathic pain (Cannon and Hough, 2005

Conflicts of interest

The authors disclose no conflict of interest.

Acknowledgments

Supported by Cinvestav and Conacyt (grant 128205 to J.-A.A.-M.). We thank Raul Gonzalez-Pantoja for excellent technical assistance.

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There are seven GRK subtypes classified in three subfamilies based on sequence and functional similarity: GRK1/7, GRK2/3 and GRK4/5/6 (Gainetdinov et al., 2004). GRK2/3 and GRK5/6 are widely expressed in the brain (Premont and Gainetdinov, 2007), and we previously reported that the hH3R445 stably expressed in CHO-K1 cells experienced homologous desensitization that involved the actions of GRK2/3 and receptor internalization (Osorio-Espinoza et al., 2014). The hH3R365 lacks 80 residues in the third intracellular loop (IL3), an important region for receptor coupling to G proteins (Oldham and Hamm, 2007; Wess, 1997).
Histamine H₃ receptors (H₃Rs) signal through Gα_i/o proteins and are found in neuronal cells as auto- and hetero-receptors. Alternative splicing of the human H₃R (hH₃R) originates 20 isoforms, and the mRNAs of two receptors of 445 and 365 amino acids (hH₃R₄₄₅ and hH₃R₃₆₅) are widely expressed in the human brain. We previously showed that the hH₃R₄₄₅ stably expressed in CHO-K1 cells experiences homologous desensitization. The hH₃R₃₆₅ lacks 80 residues in the third intracellular loop, and in this work we therefore studied whether this isoform also experiences homologous desensitization and the possible differences with the hH₃R₄₄₅. In clones of CHO-K1 cells stably expressing similar receptor levels (211 ± 12 and 199 ± 16 fmol/mg protein for hH₃R₄₄₅ and hH₃R₃₆₅, respectively), there were no differences in receptor affinity for selective H₃R ligands or for agonist-induced [³⁵S]-GTPγS binding to membranes and inhibition of forskolin-stimulated cAMP accumulation in intact cells. For both cell clones, pre-incubation with the H₃R agonist RAMH (1 μM) resulted in functional receptor desensitization, as indicated by cAMP accumulation assays, and loss of receptors from the cell surface and reduced affinity for the agonist immepip in cell membranes, evaluated by radioligand binding. However, functional desensitization differed in the maximal extent (96 ± 15% and 58 ± 8% for hH₃R₄₄₅ and hH₃R₃₆₅, respectively) and the length of pre-exposure required to reach the maximum desensitization (60 and 30 min, respectively). Furthermore, the isoforms differed in their recovery from desensitization. These results indicate that the hH₃R₃₆₅ experiences homologous desensitization, but that the process differs between the isoforms in time-course, magnitude and re-sensitization.
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Homologous desensitization of human histamine H3 receptors expressed in CHO-K1 cells

Highlights

Abstract

Introduction

Section snippets

Cloning of the hH3R

Construction of small-hairpin RNA (shRNA) expression vectors

Pharmacological characteristics of the hH3R445 stably expressed in CHO-K1cells

Discussion

Conclusion

Conflicts of interest

Acknowledgments

Trends Pharmacol. Sci.

J. Biol. Chem.

Biochem. Pharmacol.

J. Pain

Mol. Cell Neurosci.

Jpn. J. Pharmacol.

Mol. Cell

Biochem. Pharmacol.

Neurobiol. Dis.

Biochem. Biophys. Res. Commun.

Trends Pharmacol. Sci.

Trends Pharmacol. Sci.

Exp. Cell Res.

Eur. J. Pharmacol.

Neuroscience

J. Biol. Chem.

Bioorg. Med. Chem.

Mol. Cell Proteomics

Histamine H3 receptor: a potential drug target for the treatment of central nervous system disorders

Curr. Drug Targets CNS Neurol. Disord.

An 80-amino acid deletion in the third intracellular loop of a naturally occurring human histamine H3 isoform confers pharmacological differences and constitutive activity

J. Pharmacol. Exp. Ther.

Genomic organization and characterization of splice variants of the human histamine H3 receptor

Biochem. J.

Subtype-specific kinetics of inhibitory adenosine receptor internalization are determined by sensitivity to phosphorylation by G protein-coupled receptor kinases

Mol. Pharmacol.

Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling

Pharmacol. Rev.

G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins

Can. J. Physiol. Pharmacol.

Presynaptic receptors for dopamine, histamine, and serotonin

Handb. Exp. Pharmacol.

A single-point mutation (Ala280Val) in the third intracellular loop alters the signalling properties of the human histamine H3 receptor stably expressed in CHO-K1 cells

Br. J. Pharmacol.

Desensitization of G protein-coupled receptors and neuronal functions

Annu. Rev. Neurosci.

Pharmacological analysis of dopamine stimulation of [35S]-GTPγS binding via human D2short and D2long dopamine receptors expressed in recombinant cells

Br. J. Pharmacol.

Homologous down-regulation of histamine H3 receptors in rat striatal slices

Synapse

Homologous desensitization of human histamine H₃ receptors expressed in CHO-K1 cells

Cloning of the hH₃R

Pharmacological characteristics of the hH₃R₄₄₅ stably expressed in CHO-K1cells

Histamine H₃ receptor: a potential drug target for the treatment of central nervous system disorders

An 80-amino acid deletion in the third intracellular loop of a naturally occurring human histamine H₃ isoform confers pharmacological differences and constitutive activity

Genomic organization and characterization of splice variants of the human histamine H₃ receptor

A single-point mutation (Ala280Val) in the third intracellular loop alters the signalling properties of the human histamine H₃ receptor stably expressed in CHO-K1 cells

Pharmacological analysis of dopamine stimulation of [³⁵S]-GTPγS binding via human D_2short and D_2long dopamine receptors expressed in recombinant cells

Homologous down-regulation of histamine H₃ receptors in rat striatal slices