ACCELERATED COMMUNICATION Discovery of a Novel Member of the Histamine Receptor Family

We report the discovery, tissue distribution and pharmacological characterization of a novel receptor, which we have named H4. Like the three histamine receptors reported previously (H1, H2, and H3), the H4 receptor is a G protein-coupled receptor and is most closely related to the H3 receptor, sharing 58% identity in the transmembrane regions. The gene encoding the H4 receptor was discovered initially in a search of the GenBank databases as sequence fragments retrieved in a partially sequenced human genomic contig mapped to chromosome 18. These sequences were used to retrieve a partial cDNA clone and, in combination with genomic fragments, were used to determine the full-length open reading frame of 390 amino acids. Northern analysis revealed a 3.0-kb transcript in rat testis and intestine. Radioligand binding studies indicated that the H4 receptor has a unique pharmacology and binds [H]histamine (Kd 5 44 nM) and [ H]pyrilamine (Kd 5 32 nM) and several psychoactive compounds (amitriptyline, chlorpromazine, cyproheptadine, mianserin) with moderate affinity (Ki range of 33–750 nM). Additionally, histamine induced a rapid internalization of HAtagged H4 receptors in transfected human embryonic kidney 293 cells. Histamine is a monoamine neurotransmitter thus far known to activate three G protein-coupled receptors (GPCRs), the H1, H2, and H3 receptors (Hill et al., 1997). Although molecular cloning has made possible the identification, isolation, and characterization of the majority of known GPCRs, the histamine receptor subtypes have proven more difficult to identify. The first two histamine receptor genes cloned were H1 (Yamashita et al., 1991) and H2 (Gantz et al., 1991). The identification of the H3 receptor came nearly a decade later (Lovenberg et al., 1999). Collectively, the H1, H2, and H3 receptors share less than 35% identity with one another and each has greater sequence identities with other aminergic receptors. Thus, the histamine receptor gene family is significantly divergent and may have evolved from different ancestral genes (Leurs et al., 2000). In addition, there is evidence that multiple subtypes of the H3 receptor may exist. Pharmacological studies performed on membranes extracted from rat brain tissue revealed two classes of H3 binding sites (West et al., 1990; Leurs et al., 1996). However, efforts to clone a second H3 receptor subtype in the brain have thus far been unsuccessful. Through molecular cloning techniques, we have identified numerous novel GPCRs, including many subtypes not suspected to exist on the basis of pharmacology. Over the past decade, our cloning efforts have identified a number of GPCR genes, including genes encoding such receptors as the cysteinyl leukotriene CysLT2 (Heise et al., 2000), galanin GalR2 and GalR3 (Kolakowski et al., 1998), thyrotropin-releasing hormone TRH-R2 (O’Dowd et al., 2000), uridine nucleotide receptor UNR (Nguyen et al., 1995), as well as a large cohort of orphan GPCRs for which the endogenous ligands remain to be elucidated (Marchese et al., 1999; Lee et al., 2001). In a This research was funded by the Canadian Institutes of Health Research (B.F.O. and S.R.G.), the National Institute on Drug Abuse (B.F.O. and S.R.G.), and in part by K02-MH01366 and N01–80005 to B.L.R. T.N. and D.A.S. contributed equally to this work. ABBREVIATIONS: GPCR(s), G protein-coupled receptor(s); HTGS, high-throughput genomic sequence; TM, transmembrane; NCBI, National Center for Biotechnology Information; PCR, polymerase chain reaction; HA, hemagglutinin; HEK, human embryonic kidney cells; kb, kilobase pair. 0026-895X/01/5903-427–433$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 3 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 617/892595 Mol Pharmacol 59:427–433, 2001 Printed in U.S.A. 427 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from scan of the GenBank high-throughput genomic sequence (HTGS) database, we identified a small DNA fragment that had greatest homology to the known histamine receptor genes. Here we report the discovery, tissue distribution, and pharmacological characterization of human DNA encoding a receptor, H4. H4 receptor mRNA had discrete and limited expression in rat testis and intestine. Although the H4 receptor was most closely related to the H3 receptor [58% identity in the transmembrane (TM) regions], it seemed to possess a unique pharmacology, with highest affinity for psychoactive drugs (amitriptyline, chlorpromazine) with a tricyclic structure. Materials and Methods Database Searching. A customized search was used to query the GenBank HTGS database of high-throughput genomic sequences maintained by the National Center for Biotechnology Information (NCBI) with the sequences of various GPCRs, using the FAST_PAN program (Retief et al., 1999). Screening and Construction of the H4 Receptor Gene. Partial sequences encoding the H4 receptor were used to design primers to PCR-amplify H4-encoding fragments from human genomic DNA. A fragment encoding the start methionine to TM2 was amplified using primers P1 (59-ATGCCAGATACTAATAGCACAATC-39) and P2 (59-CACAAAGAAGTCAGAGATGG-39) and another fragment encoding from TM5 to TM6 using primers P3 (59-TGGTACATCCTTGCCATC-39) and P4 (59-TATGGAGCCCAGCAAACAG-39). PCR products were extracted with phenol/chloroform, precipitated with ethanol, and electrophoresed on a low-melting agarose gel. DNA in the expected size range were excised from the gel, ligated into the EcoRV site of the pBluescript vector (Stratagene, La Jolla, CA), and then the sequence was determined. H4 receptor-encoding fragments were used to screen human and rat genomic libraries (CLONTECH, Palo Alto, CA) and a human testis cDNA library (CLONTECH), as described previously (Marchese et al., 1994). Isolated phages from the human and rat genomic libraries were purified and subcloned, and the sequence was determined as described previously (Marchese et al., 1994). Isolated phages from the human cDNA library were subjected to PCR amplification using primers specific for regions flanking the insert of the lgt10 library vector, subcloned, and se-