Molecular and pharmacological characterization of the mouse histamine H3 receptor

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Abstract

Human, guinea pig and rat histamine H3 receptors have been investigated at both pharmacological and molecular levels in recent years. Here we report the cloning, molecular, and pharmacological characterization of the mouse histamine H3 receptor. The amino acid sequence of the mouse histamine H3 receptor exhibits high homology to rat, guinea pig and human histamine H3 receptors with 98%, 95%, 94% identities, respectively. The distribution of the mRNA encoding the mouse histamine H3 receptor was predominant in the brain as detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and RNase protection assay. Although several splice forms have been reported for human, guinea pig and rat histamine H3 receptor mRNAs, we did not detect equivalent isoforms in the mouse in several tissues by either RNase protection assay or robust Polymerase Chain Reaction (PCR) amplifications. Human embryonic kidney (HEK)-293 cells transiently transfected with mouse histamine H3 receptor cDNA and Gqi5 exhibited increases of intracellular Ca2+ in response to histamine and several histamine H3 receptor agonists. COS-7 (African green monkey kidney) cells transfected with mouse histamine H3 receptor cDNA showed high affinity binding for histamine H3 receptor ligands in competition binding assays. The pharmacological comparison of human, guinea pig, rat and mouse histamine H3 receptors indicated that, as expected, the mouse histamine H3 receptor exhibited a more similar pharmacological profile to the rat histamine H3 receptor than to either the human or the guinea pig histamine H3 receptor. Taken together, these findings allow a further appreciation of the histamine H3 receptor at the molecular level and provide an additional species to assist in the pharmacological assessment of histamine H3 receptor function.

Introduction

Histamine plays an important role in a number of physiological functions, including inflammation, gastric acid secretion, neurotransmission and immune regulation through its H1, H2, H3 and H4 receptors, respectively. Since the first cDNA cloning of the human histamine H3 receptor (Lovenberg et al., 1999), molecular characterization of histamine H3 receptors of human, guinea pig and rat have been extensively investigated Lovenberg et al., 1999, Lovenberg et al., 2000, Tardivel-lacombe et al., 2000, Drutel et al., 2000, Liu et al., 2000. These Gi-coupled presynaptic receptors from different species share high amino acid homology, yet exhibit some differences in pharmacology (Lovenberg et al., 2000). Since the advent of gene-knockout technology, the mouse has become a more important species for the evaluation of gene expressed in the brain. Our interest in the histamine H3 receptor has led us to explore the histamine H3 receptor pharmacology in the mouse.

In addition to known pharmacological variations, the existence of splice variants has been reported for rat and guinea pig Drutel et al., 2000, Tardivel-lacombe et al., 2000. Also, controversial results have also been reported for the existence of the human histamine H3 receptor splice variants Liu et al., 2000, Coge et al., 2001, Wiedemann et al., 2002, Wellendorph et al., 2002. These isoforms have been demonstrated to have distinct pharmacological properties and in some cases different tissue distribution from their corresponding “original” or “long” forms.

We have previously identified a fragment of the mouse histamine H3 receptor cDNA which was used to knockout the histamine H3 receptor mRNA expression in mice (Toyota et al., 2002). In the present study, we cloned the full length of mouse histamine H3 receptor to characterize it at both molecular and pharmacological levels and investigate whether the alternative splice variants of mouse histamine H3 receptor exist.

Section snippets

Cloning of the mouse histamine H3 receptor cDNA coding region

A 1 kb mouse histamine H3 receptor cDNA fragment was Polymerase Chain Reaction (PCR)-amplified from mouse brain cDNA (Clontech, Palo Alto, CA) using two primers [forward primer: 5′ GGC AAC GCG CTG GTC ATG CTC GCC TTC GTG 3′ (rat histamine H3 receptor coding region 151–180) and reverse primer: 5′ GGG CCC AGC AGA GCC CAA AGA TGC TCA CGA TGA 3′ (rat histamine H3 receptor coding region 1085–1118)] which were designed according to the conserved regions between the human and rat histamine H3 receptor

Comparison of the sequences of human, guinea pig, rat and mouse histamine H3 receptors

A 1 kb DNA fragment was PCR-amplified from mouse brain cDNA using two primers designed from the conserved region between the human and rat histamine H3 receptor cDNA sequences. The DNA sequence is unique and shares greater than 90% sequence identity to the human and rat histamine H3 receptor sequences, suggesting that it is a mouse histamine H3 receptor cDNA fragment. The 5′ end and 3′ end of the mouse histamine H3 receptor cDNA were PCR-amplified by RACE using mouse histamine H3 receptor

Discussion

In this study, the cloning and molecular and pharmacological characterization of the mouse histamine H3 receptor are described. The open reading frame of the mouse histamine H3 receptor mRNA encodes a 445-amino acid protein that has high homology and similar pharmacology to the rat, guinea pig and human histamine H3 receptors. The amino acid sequence analysis revealed that the seven transmembrane domains of the mouse histamine H3 receptor are identical to those of the rat histamine H3 receptor.

Acknowledgments

All of the experimental protocols comply with European Community guidelines for the use of experimental animals and have been approved by Johnson & Johnson Pharmaceutical Research and Development L.L.C IACUC.

References (14)

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