Expression and pharmacological characterization of a canine 5-hydroxytryptamine1D receptor subtype.
RDC4 is a guanine nucleotide-binding protein-coupled receptor clone originally isolated from a canine thyroid cDNA library by Libert and colleagues [Science (Washington D. C.) 244:569-572 (1989)]. We have isolated the corresponding genomic clone for RDC4, have expressed this clone in murine LM (tk-) fibroblasts, and have determined that it encodes a serotonin 5-hydroxytryptamine1D (5-HT1D) receptor. RDC4 is an intronless gene encoding a protein of 377 amino acids, which exhibits greatest sequence identity (43%) to the 5-HT1A receptor and lower overall homology to other serotonergic and catecholaminergic receptors. Membranes prepared from murine LM (tk-) fibroblasts stably transfected with this clone were shown to bind [3H]5-HT in a saturable manner and displayed an apparently homogeneous population of high affinity (Kd = 3.6 nM, Bmax = 275 fmol/mg of protein) [3H]5-HT binding sites. High affinity [3H] 5-HT binding was unchanged using assay conditions [1 microM (+/- )-pindolol and 1 microM (R)-(+/- )-SCH 23390) to pharmacologically mask 5-HT1A, 5-HT1B, and 5-HT1C receptors. Serotonergic ligands displaced specific [3H]5-HT binding with a rank order of potency expected of a 5-HT1D receptor subtype, 5-carboxyamidotryptamine greater than 5-HT greater than yohimbine greater than 8-hydroxy-2-(di-n-propylamino)tetralin greater than ketanserin = spiperone greater than zacopride. Further, transfected cells responded to addition of 5-HT by decreasing the level of forskolin-stimulated cAMP accumulation. These data indicate that the gene RDC4 encodes a functional 5-HT1D receptor.
Zgombick
J M
JM
Neurogenetic Corporation, Paramus, New Jersey 07652.
Weinshank
R L
RL
Macchi
M
M
Schechter
L E
LE
Branchek
T A
TA
Hartig
P R
PR
D000067956:Adenylyl Cyclase Inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D004285:Dogs; D005347:Fibroblasts / Q000502:physiology; D015870:Gene Expression / Q000235:genetics; D007700:Kinetics; D051379:Mice; D011985:Receptors, Serotonin / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D012701:Serotonin / Q000378:metabolism; D014316:Tritium
D000067956:Adenylyl Cyclase Inhibitors; D011985:Receptors, Serotonin; D014316:Tritium; D012701:Serotonin