The end of a myth: cloning and characterization of the ovine melatonin MT(2) receptor.
OBJECTIVE
For many years, it was suspected that sheep expressed only one melatonin receptor (closely resembling MT(1) from other mammal species). Here we report the cloning of another melatonin receptor, MT(2), from sheep.


METHODS
Using a thermo-resistant reverse transcriptase and polymerase chain reaction primer set homologous to the bovine MT(2) mRNA sequence, we have cloned and characterized MT(2) receptors from sheep retina.


RESULTS
The ovine MT(2) receptor presents 96%, 72% and 67% identity with cattle, human and rat respectively. This MT(2) receptor stably expressed in CHO-K1 cells showed high-affinity 2[(125)I]-iodomelatonin binding (K(D)= 0.04 nM). The rank order of inhibition of 2[(125)I]-iodomelatonin binding by melatonin, 4-phenyl-2-propionamidotetralin and luzindole was similar to that exhibited by MT(2) receptors of other species (melatonin > 4-phenyl-2-propionamidotetralin > luzindole). However, its pharmacological profile was closer to that of rat, rather than human MT(2) receptors. Functionally, the ovine MT(2) receptors were coupled to G(i) proteins leading to inhibition of adenylyl cyclase, as the other melatonin receptors. In sheep brain, MT(2) mRNA was expressed in pars tuberalis, choroid plexus and retina, and moderately in mammillary bodies. Real-time polymerase chain reaction showed that in sheep pars tuberalis, premammillary hypothalamus and mammillary bodies, the temporal pattern of expression of MT(1) and MT(2) mRNA was not parallel in the three tissues.


CONCLUSIONS
Co-expression of MT(1) and MT(2) receptors in all analysed sheep brain tissues suggests that MT(2) receptors may participate in melatonin regulation of seasonal anovulatory activity in ewes by modulating MT(1) receptor action.
Cogé
F
F
Pharmacologie Moléculaire et Cellulaire, Institut de Recherches SERVIER, Suresnes, France.
Guenin
S P
SP
Fery
I
I
Migaud
M
M
Devavry
S
S
Slugocki
C
C
Legros
C
C
Ouvry
C
C
Cohen
W
W
Renault
N
N
Nosjean
O
O
Malpaux
B
B
Delagrange
P
P
Boutin
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002417:Cattle; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009928:Organ Specificity; D012333:RNA, Messenger / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D044122:Receptor, Melatonin, MT1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D044123:Receptor, Melatonin, MT2 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013764:Tetrahydronaphthalenes / Q000494:pharmacology; D014363:Tryptamines / Q000494:pharmacology
C114459:4-phenyl-2-propionamidotetraline; D012333:RNA, Messenger; D044122:Receptor, Melatonin, MT1; D044123:Receptor, Melatonin, MT2; D011994:Recombinant Proteins; D013764:Tetrahydronaphthalenes; D014363:Tryptamines; C057154:luzindole; D019204:GTP-Binding Proteins