Discovery of naturally occurring splice variants of the rat histamine H3 receptor that act as dominant-negative isoforms.
We described previously the cDNA cloning of three functional rat histamine H3 receptor (rH3R) isoforms as well as the differential brain expression patterns of their corresponding mRNAs and signaling properties of the resulting rH3A, rH3B, and rH3C 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 rH3R splice variants (rH3D, rH3E, and rH3F) that differ from the previously published isoforms in that they result from an additional alternative-splicing event. These new H3R isoforms lack the seventh transmembrane (TM) helix and contain an alternative, putatively extracellular, C terminus (6TM-rH3 isoforms). After heterologous expression in COS-7 cells, radioligand binding or functional responses upon the application of various H3R ligands could not be detected for the 6TM-rH3 isoforms. In contrast to the rH3A receptor (rH3AR), detection of the rH3D isoform using hemagglutinin antibodies revealed that the rH3D isoform remains mainly intracellular. The expression of the rH3D-F splice variants, however, modulates the cell surface expression-levels and subsequent functional responses of the 7TM H3R isoforms. Coexpression of the rH3AR and the rH3D isoforms resulted in the intracellular retention of the rH3AR and reduced rH3AR functionality. Finally, we show that in rat brain, the H3R mRNA expression levels are modulated upon treatment with the convulsant pentylenetetrazole, suggesting that the rH3R isoforms described herein thus represent a novel physiological mechanism for controlling the activity of the histaminergic system.
Bakker
Remko A
RA
The Leiden/Amsterdam Center for Drug Research, Department of Medicinal Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands.
Lozada
Adrian Flores
AF
van Marle
André
A
Shenton
Fiona C
FC
Drutel
Guillaume
G
Karlstedt
Kaj
K
Hoffmann
Marcel
M
Lintunen
Minnamaija
M
Yamamoto
Yumiko
Y
van Rijn
Richard M
RM
Chazot
Paul L
PL
Panula
Pertti
P
Leurs
Rob
R
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D031541:Fluorescence Resonance Energy Transfer; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D018100:Receptors, Histamine H3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D020033:Protein Isoforms; D012333:RNA, Messenger; D018100:Receptors, Histamine H3; D016244:Guanosine 5'-O-(3-Thiotriphosphate)