Molecular cloning and pharmacological characterization of the guinea pig 5-HT1E receptor

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Abstract

The human 5-HT1E receptor gene was cloned more than a decade ago. Little is known about its function, and there have been no reports of its existence in the genome of small laboratory animals. In this study, attempts to clone the 5-HT1E gene from the rat and mouse were unsuccessful. In fact, a search of the mouse genome database revealed that the 5-HT1E receptor gene is missing from the mouse genome. However, the 5-HT1E gene was cloned from guinea pig genomic DNA and was characterized. The guinea pig 5-HT1E receptor gene encodes a protein of 365 amino acids. It shares 88% (nucleic acid) and 95% (amino acid) homology with the human receptor. The guinea pig 5-HT1E receptor showed similar pharmacology to the human 5-HT1E receptor in radioligand binding assays. Serotonin (5-hydroxytryptamine, 5-HT) dose-dependently stimulated [35S]GTPγS binding to the guinea pig 5-HT1E receptor with an EC50 of 13.6±1.92 nM, similar to that of the human 5-HT1E receptor (13.7±1.78 nM). Activation of the guinea pig 5-HT1E receptor was also achieved by ergonovine, α-methyl-5-HT, 1-naphthylpiperazine, methysergide, tryptamine, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Methiothepin exhibited antagonist activity. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that 5-HT1E mRNA was present in the guinea pig brain with the greatest abundance in the hippocampus, followed by the olfactory bulb. Lower levels were detected in the cortex, thalamus, pons, hypothalamus, midbrain, striatum, and cerebellum. Our current study marks the first identification of the 5-HT1E receptor gene in a commonly used laboratory animal species. This finding should allow the elucidation of the receptor's role(s) in the complex coordination of central serotonergic effects.

Introduction

To date, five subtypes of serotonin receptors have been identified in the 5-HT1 receptor family (5-HT1A, 1B, 1D, 1E, 1F) (Barnes and Sharp, 1999). The 5-HT1E receptor is a member of this family which represents a group of G protein-coupled receptors that activate the Gi/o types of G proteins. The 5-HT1E receptor was first identified by Leonhardt et al. (1989) in human cortical tissue using radioligand binding and demonstrated the characteristic pharmacologic feature of having high affinity for 5-HT and low affinity for 5-carboxyamidotryptamine (5-CT). Later, the 5-HT1E gene was cloned by several independent laboratories from human genomic libraries Levy et al., 1992, Zgombick et al., 1992 and a human cDNA library (McAllister et al., 1992). Further functional studies by these groups showed unanimously that the cloned receptors were able to mediate inhibition of adenylyl cyclase when activated by serotonin. Binding studies performed using transient or stable transfected cell lines revealed that the pharmacology of this newly cloned receptor was similar to that described by Leonhardt et al. (1989).

The human 5-HT1E receptor gene is intronless and encodes a protein of 365 amino acids McAllister et al., 1992, Zgombick et al., 1992, Gudermann et al., 1993. Sequence comparison demonstrates that it has the highest homology with the human 5-HT1F receptor, followed by the 5-HT1B and the 5-HT1D receptors McAllister et al., 1992, Zgombick et al., 1992, Barnes and Sharp, 1999. In situ hybridization of human metaphase chromosomes using the cloned human 5-HT1E gene shows that human 5-HT1E receptor gene is localized in human chromosome 6q14–q15 (Levy et al., 1994).

The protein distribution of 5-HT1E receptor in the brain is inconclusive because no selective ligands or antibodies are available to perform either radioligand autoradiographic-based or immunohistochemical-based mapping. Most distribution studies using [3H]5-HT in the presence of 5-CT and mesulergine (to mask 5-HT1A, 5-HT1B, 5-HT1D and 5-HT2C sites ) depict a combined localization of 5-HT1E/1F sites. The combined 5-HT1E/1F sites have been reported in human, guinea pig, rat, and mouse brains Leonhardt et al., 1989, Miller and Teitler, 1992, Barone et al., 1994, Palacios et al., 1996, Fugelli et al., 1997. In human, these 5-HT1E/1F sites are detected in the cortex, caudate putamen, claustrum, hippocampus, and amygdala Leonhardt et al., 1989, Miller and Teitler, 1992, Barone et al., 1994, Palacios et al., 1996, Fugelli et al., 1997. It is now known that the 5-HT1E binding site has lower affinity for sumatriptan, a characteristic that makes it distinguishable from the 5-HT1F binding sites. However, there is no report on the brain distribution of pure 5-HT1E receptor after masking the 5-HT1F binding sites. Nevertheless, the distributions of 5-HT1E mRNA in both human and monkey brains were determined by in situ hybridization analysis, showing the presence of 5-HT1E mRNA in the brain areas of caudate, putamen, hypothalamus, and cortex (Bruinvels et al., 1994).

While the 5-HT1E receptor gene has been cloned from the human genome for more than a decade, there has been no report on the isolation of this gene from any commonly used laboratory species. The unknown nature of the presence of the 5-HT1E receptor in species other than the human makes it difficult to establish an ideal experimental animal model to study the physiological function of this receptor. In the present study, the 5-HT1E receptor gene was cloned from genomic DNA isolated from guinea pig. The cloned guinea pig 5-HT1E receptor was then pharmacologically and functionally characterized. The results of this study revealed that the guinea pig 5-HT1E receptor gene has a great homology with the human 5-HT1E receptor. It also shared similar pharmacologic profiles as the human 5-HT1E receptor when expressed in the same cell line. These findings represent the first identification of the 5-HT1E receptor in a commonly used laboratory animal species.

Section snippets

Animal tissues, chemicals and radioactive ligands

Brain tissue from monkey, dog, rabbit, guinea pig, chicken, gerbil, and hamster and choriod plexus from pig were obtained from the Neuroscience and Toxicology Divisions of Lilly Research Laboratories (Indianapolis, IN). All animal use procedures were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals. All efforts were made to reduce the number of animals used and to minimize animal suffering. Protocols were approved by the Lilly Research

Molecular cloning

PCR analysis using primers designed to correspond to the N-terminal and the C-terminal sequences of the human 5-HT1E receptor gene yielded a product of ∼1.1 kb from human genomic DNA. Genomic fragments of the same size were also found in the monkey, pig, rabbit, and guinea pig, but not in the dog, chicken, gerbil, rat, mouse, or hamster (Fig. 1). Although both the rabbit and the guinea pig are relatively small laboratory animals, subsequent work focused only on the guinea pig receptor. The

Discussion

In the present study, the 5-HT1E receptor gene was successfully cloned from guinea pig genomic DNA. An AV-12 cell line, which stably expresses the cloned guinea pig 5-HT1E receptor, was established and tested for pharmacology and function of the cloned receptor. In addition, the relative quantity of the 5-HT1E mRNA was determined in various guinea pig brain regions. This represents the first identification, isolation, and characterization of the 5-HT1E gene in a commonly used small laboratory

Acknowledgments

The authors would like to thank David B Wainscott and Virginia L Lucaites for the technical and scientific input to this work. We would also like to thank Tony Gardner and Dr. Don McClure for the large-scale production of the guinea pig and human 5-HT1E cell lines used in this study.

References (26)

  • S. Swillens

    How to estimate the total receptor concentration when the specific radioactivity of the ligand is unknown

    Trends Pharmacol. Sci.

    (1992)
  • D.B. Wainscott et al.

    Human 5-HT1F receptor-stimulated [35S]GTPgammaS binding: correlation with inhibition of guinea pig dural plasma protein extravasation

    Eur. J. Pharmacol.

    (1998)
  • Y. Cheng et al.

    Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction

    Biochem. Pharmacol.

    (1973)
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