EXPRESSION AND PHARMACOLOGICAL CHARACTERIZATION OF A CANINE 5-HYDROXYTRYPTAMINE1D RECEPTOR SUBTYPE

RDC4 is a guanine nucleotide-binding protein-coupled receptor clone or iginally isolated from a canine thyroid cDNA library by Libert and col leagues [Science (Washington D. C.) 244:569-572 (1989)]. We have isola ted the corresponding genomic clone for RDC4, have expressed this clon e in murine LM (tk-) fibroblasts, and have determined that it encodes a serotonin 5-hydroxytryptamine1D (5-HT1D) receptor. RDC4 is an intron less gene encoding a protein of 377 amino acids, which exhibits greate st sequence identity (43%) to the 5-HT1A receptor and lower overall ho mology to other serotonergic and catecholaminergic receptors. Membrane s prepared from murine LM (tk-) fibroblasts stably transfected with th is clone were shown to bind [H-3]5-HT in a saturable manner and displa yed an apparently homogeneous population of high affinity (K(d) = 3.6 nM, B(max) = 275 fmol/mg of protein) [H-3]5-HT binding sites. High aff inity [H-3] 5-HT binding was unchanged using assay conditions [1-mu-M (+/-)-pindolol and 1-mu-M (R)-(+/-)-SCH 23390) to pharmacologically ma sk 5-HT1A, 5-HT1B, and 5-HT1C receptors. Serotonergic ligands displace d specific [H-3]5-HT binding with a rank order of potency expected of a 5-HT, 1D receptor subtype, 5-carboxyamidotryptamine > 5-HT > yohimbi ne > 8-hydroxy-2-(di-n-propylamino)tetralin > ketanserin = spiperone > zacopride. Further, transfected cells responded to addition of 5-HT b y decreasing the level of forskolin-stimulated cAMP accumulation. Thes e data indicate that the gene RDC4 encodes a functional 5-HT1D recepto r.