Discovery of Naturally Occurring Splice Variants of the Rat Histamine H3 Receptor That Act as Dominant-Negative Isoforms

• Published in: Molecular pharmacology 2006-04, Vol.69 (4), p.1194-1206
• Main Authors: Bakker, Remko A, Lozada, Adrian Flores, van Marle, André, Shenton, Fiona C, Drutel, Guillaume, Karlstedt, Kaj, Hoffmann, Marcel, Lintunen, Minnamaija, Yamamoto, Yumiko, van Rijn, Richard M, Chazot, Paul L, Panula, Pertti, Leurs, Rob
• Format: Article
• Language: English
• Subjects: Alternative Splicing; Amino Acid Sequence; Animals; Brain - metabolism; Cercopithecus aethiops; Cloning, Molecular; COS Cells; DNA Primers; DNA, Complementary; Fluorescence Resonance Energy Transfer; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Male; Molecular Sequence Data; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Histamine H3 - chemistry; Receptors, Histamine H3 - genetics; Receptors, Histamine H3 - metabolism; RNA, Messenger - genetics; Sequence Homology, Amino Acid
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.105.019299

We described previously the cDNA cloning of three functional rat histamine H 3 receptor (rH 3 R) isoforms as well as the differential brain expression patterns of their corresponding mRNAs and signaling properties of the resulting rH 3A , rH 3B , and rH 3C receptor isoforms ( Mol Pharmacol 59: 1–8). In the current report, we describe the cDNA cloning, mRNA localization in the rat central nervous system, and pharmacological characterization of three additional rH 3 R splice variants (rH 3D , rH 3E , and rH 3F ) that differ from the previously published isoforms in that they result from an additional alternative-splicing event. These new H 3 R isoforms lack the seventh transmembrane (TM) helix and contain an alternative, putatively extracellular, C terminus (6TM-rH 3 isoforms). After heterologous expression in COS-7 cells, radioligand binding or functional responses upon the application of various H 3 R ligands could not be detected for the 6TM-rH 3 isoforms. In contrast to the rH 3A receptor (rH 3A R), detection of the rH 3D isoform using hemagglutinin antibodies revealed that the rH 3D isoform remains mainly intracellular. The expression of the rH 3D-F splice variants, however, modulates the cell surface expression-levels and subsequent functional responses of the 7TM H 3 R isoforms. Coexpression of the rH 3A R and the rH 3D isoforms resulted in the intracellular retention of the rH 3A R and reduced rH 3A R functionality. Finally, we show that in rat brain, the H 3 R mRNA expression levels are modulated upon treatment with the convulsant pentylenetetrazole, suggesting that the rH 3 R isoforms described herein thus represent a novel physiological mechanism for controlling the activity of the histaminergic system.