Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores.
Using an expression cloning strategy, a high-affinity melatonin receptor cDNA has been isolated from Xenopus laevis dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in high-affinity 2-[125I]-iodomelatonin binding (Kd = 6.3 +/- 0.3 x 10(-11) M). In addition, six ligands exhibited a rank order of inhibition of specific 2-[125I]iodomelatonin binding that was identical to that reported for endogenous high-affinity receptors. Functional studies of CHO cells stably expressing the receptor cDNA showed that melatonin acting through the cloned receptor inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner. Northern blot analysis showed that melatonin receptor transcripts are moderately expressed in Xenopus dermal melanophores. The cDNA encodes a protein of 420 amino acids, which contains seven hydrophobic segments. Structural analysis revealed that the receptor protein is a newly discovered member of the guanine nucleotide binding protein-coupled receptor family.
Ebisawa
T
T
Laboratory of Developmental Chronobiology, Children's Service, Massachusetts General Hospital, Boston.
Karne
S
S
Lerner
M R
MR
Reppert
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D007668:Kidney; D007700:Kinetics; D008547:Melanophores / Q000378:metabolism*; D008550:Melatonin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011061:Poly A / Q000032:analysis / Q000096:biosynthesis; D017433:Protein Structure, Secondary; D012313:RNA / Q000032:analysis / Q000096:biosynthesis; D012333:RNA, Messenger; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D044094:Receptors, Melatonin; D012867:Skin / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014982:Xenopus laevis
D018076:DNA, Complementary; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D044094:Receptors, Melatonin; D005576:Colforsin; D011061:Poly A; D012313:RNA; D004247:DNA; D000242:Cyclic AMP; D008550:Melatonin