STRUCTURE, CHARACTERIZATION, AND EXPRESSION OF THE GENE ENCODING THE MOUSE MEL(1A) MELATONIN RECEPTOR

Recently, a distinct family of G protein-coupled receptors has been cl oned that mediates the biological effects of melatonin. Of two subtype s cloned from mammals (Mel(1a) and Mel(1b)), the Mel(1a) receptor appe ars to mediate the circadian and reproductive effects of the hormone. We now report the cloning, characterization, and expression of the gen e encoding the Mel(1a) receptor in mice. The receptor gene is composed of two exons, separated by an intron of greater than 13 kilobases. Ex on 1 encodes the entire 5'-untranslated region and the coding region t hrough the first cytoplasmic loop. Exon 2 encodes the rest of the codi ng region and the entire 3'-untranslated region. 5'-Rapid amplificatio n of complementary DNA ends and ribonuclease protection analyses show that the major transcription start site is 103 nucleotides upstream of the translation start codon. Sequence analysis of 1.1 kilobases of th e 5'-flanking region reveals that it does not contain TATA or CAAT box es. The 5'-flanking region drives luciferase expression 114-fold over basal levels in a murine retinal cell line that endogenously expresses the Mel(1a) receptor. The mouse receptor binds 2-[(125)]iodomelatonin with high affinity (K-d = 55.6 pM) when expressed transiently in COS- 7 cells. In situ hybridization studies establish that Mel(1a) receptor messenger RNA is expressed in the hypothalamic suprachiasmatic nuclei and hypophyseal pars tuberalis, presumed sites of the circadian and s ome of reproductive actions of melatonin, respectively. These results provide information on Mel(1a) receptor gene structure essential for d esigning transgenic and gene knock-out studies and analyzing the trans criptional regulation of receptor gene expression.