Cloning and characterization of the monkey histamine H₃ receptor isoforms

Abstract

We have recently identified three splice isoforms of the histamine H₃ receptor in multiple brain regions of cynomolgus monkey (Macaca fascicularis). Two of the novel isoforms displayed a deletion in the third intracellular loop (H₃(413) and H₃(410)), the third isoform H₃(335) displayed a deletion in the i3 intracellular loop and a complete deletion of the putative fifth transmembrane domain TM5. We have confirmed by RT-PCR the expression of full-length H₃(445) mRNA as well as H₃(413), H₃(410), and H₃(335) splice isoform mRNA in multiple monkey brain regions including the frontal, parietal and occipital cortex, parahippocampal gyrus, hippocampus, amygdala, caudate nucleus, putamen, thalamus, hypothalamus, and cerebellum. The full-length isoform H₃(445) was predominant in all of the regions tested, followed by H₃(335), with the H₃(413) and H₃(410) being of low abundance. When expressed in C6 cells, H₃(445), H₃(413), and H₃(410) exhibit high affinity binding to the agonist ligand [³H]-(N)-α-methylhistamine with respective pK_D values of 9.7, 9.7, and 9.6. As expected, the H₃(335) isoform did not display any saturable binding with [³H]-(N)-α-methylhistamine. The histamine H₃ receptor agonists histamine, (R)-α-methylhistamine, imetit and proxyfan were able to activate calcium mobilization responses through H₃(445), H₃(413) and H₃(410) receptors when they were co-expressed with the chimeric Gα_qi5-protein in HEK293 cells, while no response was elicited in cells expressing the H₃(335) isoform. The existence of multiple H₃ receptor splice isoforms across species raises the possibility that isoform specific properties including ligand affinity, signal transduction coupling, and brain localization may differentially contribute to observed in vivo effects of histamine H₃ receptor antagonists.

Introduction

The histamine H₃ receptors belong to the class of G-protein coupled receptors, and like all such receptors, they contain 7 putative transmembrane domains with extracellular N-terminus and intracellular C-terminus. Histamine H₃ receptors are expressed almost exclusively in the central nervous system (CNS) (Esbenshade et al., 2006, Wijtmans et al., 2007) where they control release of several important neurotransmitters, including acetylcholine, dopamine, norepinephrine, serotonin, and histamine itself. Involvement of these neurotransmitters in the cognitive process has made H₃ receptors an important target of both academic and pharmaceutical research. In the past decade significant efforts were dedicated to cloning and pharmacological characterization of histamine receptors with Lovenberg reporting cloning of the human histamine H₃ receptor in 1999 (Lovenberg et al., 1999). Soon thereafter, cloning of mouse, rat, and guinea pig histamine H₃ receptors has followed (Chen et al., 2003, Lovenberg et al., 2000, Tardivel-Lacombe et al., 2000). Subsequent research revealed significant functional and structural heterogeneity among the histamine H₃ receptors from different species. Distinct ligand binding properties have been observed for human and rat histamine H₃ receptors despite the fact that they share 93% identity of their amino acid sequences (Arrang et al., 1987, Ireland-Denny et al., 2001, Lovenberg et al., 1999, Lovenberg et al., 2000, Yao et al., 2003b). Using a human/rat chimeric histamine H₃ receptor and site directed mutagenesis, two amino acids at positions 119 (T for human and A for rat) and 122 (A for human and V for rat) have been identified to be mainly responsible for the pharmacological differences between rat and human species (Ligneau et al., 2000, Yao et al., 2003b). In addition to that, human, rat, mouse, and guinea pig histamine H₃ receptors were shown to have multiple isoforms (Cogé et al., 2001, Drutel et al., 2001, Lovenberg et al., 2000, Morisset et al., 2000, Morisset et al., 2001, Rouleau et al., 2004, Tardivel-Lacombe et al., 2000, Wellendorph et al., 2002). For most part, these isoforms contain deletions of various sizes typically starting at a common position in the third intracellular loop between transmembrane domains 5 and 6. The nucleotide sequences surrounding the deleted regions resemble those of a splice junction, suggesting that these isoforms are derived from alternative splicing of the long form of the histamine H₃ receptor.

In 2003 we reported cloning of the monkey histamine H₃ receptor (Yao et al., 2003a). However, unlike the human or rat, at that time we found no evidence for additional splice isoforms of the monkey histamine H₃ receptor gene. It has been shown that splice isoforms of histamine H₃ receptor may possess ligand binding and functional properties that are not identical to those of the full-length receptor (Bongers et al., 2007, Drutel et al., 2001). The monkey serves as a useful primate model in drug discovery research, particularly in studies of compounds that affect learning and memory (Buccafusco et al., 1995, Decker et al., 1997). Histamine H₃ receptor ligands are currently under investigation for a number of indications including cognition and attention, and understanding the pharmacological characteristics of the monkey histamine H₃ receptor could provide critical information for evaluation of histamine H₃ receptor ligands in monkey behavioral models. Taking these considerations into account we decided to further investigate the issue of additional splicing events in the monkey histamine H₃ gene. In this report we describe cloning of additional monkey histamine H₃ receptor splice isoforms and their pharmacological characterization.