Mammalian melatonin receptors: molecular biology and signal transduction

The pineal hormone, melatonin, is an important regulator of seasonal reproduction and circadian rhythms. Its effects are mediated via high-affinity melatonin receptors, located on cells of the pituitary pars tuberalis (PT) and suprachiasmatic nucleus (SCN), respectively. Two subtypes of mammalian melatonin receptors have been cloned and characterized, the MT1 (Mel(1a)) and the MT2 (Mel(1b)) melatonin receptor subtypes. Both subtypes are members of the seven-transmembrane G protein-coupled receptor family. By using recombinant melatonin receptors it has been shown that the MT1 melatonin receptor is coupled to different G proteins that mediate adenylyl cyclase inhibition and phospholipase C beta activation. The MT2 receptor is also coupled to inhibition of adenylyl cyclase and additionally it inhibits the soluble guanylyl cyclase pathway. In mice with a targeted deletion of the MT1 receptor, the acute inhibitory effects of melatonin on SCN multiunit activity are completely abolished, while the phase-shifting responses to melatonin (given in physiological concentrations) appear normal. Furthermore, melatonin inhibits the phosphorylation of the transcription factor cyclic AMP response element binding protein, induced by the pituitary adenylate cyclase-activating polypeptide in SCN cells predominantly via the MT1 receptor. However, a functional MT2 receptor in the rodent SCN is partially able to compensate for the absence of the MT1 receptor in MT1 receptor-deficient mice. These findings indicate redundant and non-redundant roles of the receptor subtypes in regulating SCN function. In the PT, a functional MT1 receptor is essential for the rhythmic synthesis of the clock gene product mPER1. Melatonin produces a long-lasting sensitization of adenylyl cyclase and thus amplifies cyclic AMP signaling when melatonin levels decline at dawn. This action of melatonin amplifies gene expression rhythms in the PT and provides a mechanism for reinforcing rhythmicity in peripheral tissues which themselves lack the capacity for self-sustained oscillation. Mice with targeted deletion of melatonin receptor subtypes provide an excellent model to understand cellular mechanisms through which melatonin modulates circadian and photoperiodic rhythmicity

Rhythms in clock proteins in the mouse pars tuberalis depend on MT1 melatonin receptor signalling

Melatonin provides a rhythmic neuroendocrine output, driven by a central circadian clock that encodes information about phase and length of the night. In the hypophyseal pars tuberalis (PT), melatonin is crucial for rhythmic expression of the clock genes mPer1 and mCry1, and melatonin acting in the PT influences prolactin secretion from the pars distalis. To examine further the possibility of a circadian clockwork functioning in the PT, and the impact of melatonin on this tissue, we assessed circadian clock proteins by immunohistochemistry and compared the diurnal expression in the PT of wild type (WT), and MT1 melatonin receptor-deficient (MT1-/-) mice. While in the PT of WT mice mPER1, mPER2, and mCRY1 showed a pronounced rhythm, mCRY2, CLOCK, and BMAL1 were constitutively present. Despite reported differences in maximal levels and timing of mCry1, mPer1, and mPer2 RNAs, the corresponding protein levels peaked simultaneously during late day, suggesting a codependency for their stabilization and/or nuclear entry. MT1-/- mice had reduced levels of mPER1, mCRY1, CLOCK and BMAL1, consistent with the earlier reported reduction in mRNA expression of these clock genes. Surprisingly, mPER2-immunoreaction was constitutively low, although mPer2 was rhythmically expressed in the PT of MT1-/- mice. This suggests that mPER2 is degraded due to the reduced levels of its stabilizing interaction partners mPER1 and mCRY1. The results show that melatonin, acting through the MT1, determines availability of the circadian proteins mPER1, mPER2 and mCRY1 and thus plays a crucial role in regulating rhythmicity in PT cells

Melatonin plays a crucial role in the regulation of rhythmic clock gene expression in the mouse pars tuberalis

Circadian rhythms in physiology and behavior are driven by a central clock residing within the hypothalamic suprachiasmatic nucleus (SCN). Molecularly, the biological clock is based on the transcriptional/translational feedback loop of clock genes (mPer, mCry, Clock, and Bmal1). Circadian expression of clock genes is not limited to the SCN, but is found in many peripheral tissues. Peripheral rhythms depend on neuroendocrine/neuronal output from the SCN. Melatonin, the hormone of darkness, represents an important neuroendocrine output of the circadian clock. The hypophyseal pars tuberalis (PT) is one of the main target regions for melatonin. The aim of the study was to test whether mPer, mCry, Clock, and Bmal1 are rhythmically expressed in the mouse PT and how the absence of melatonin receptors affects clock gene expression. We analyzed clock gene expression by in situ hybridization and compared wild-type (WT), melatonin 1 receptor knockout (MT1 ko), and melatonin 2 receptor knockout (MT2 ko) mice. mPer1, mCry1, Clock, and Bmal1, but not mPer2 and mCry2, were rhythmically expressed in the PT of WT and MT2 ko mice. In the PT of MT1 ko mice, expression of mPer1, mCry1, Clock, and Bmal1 was dramatically reduced. We conclude that melatonin, acting through the MT1 receptor, is an important regulator of rhythmic clock gene expression in the mouse PT

Targeted Disruption of the Mouse Mel(1b) Melatonin Receptor

Two high-affinity, G protein-coupled melatonin receptor subtypes have been identified in mammals. Targeted disruption of the Mel(1a) melatonin receptor prevents some, but not all, responses to the hormone, suggesting functional redundancy among receptor subtypes (Liu et al., Neuron 19:91-102, 1997). In the present work, the mouse Mel(1b) melatonin receptor cDNA was isolated and characterized, and the gene has been disrupted. The cDNA encodes a receptor with high affinity for melatonin and a pharmacological profile consistent with its assignment as encoding a melatonin receptor. Mice with targeted disruption of the Mel(1b) receptor have no obvious circadian phenotype. Melatonin suppressed multiunit electrical activity in the suprachiasmatic nucleus (SCN) in Mel(1b) receptor-deficient mice as effectively as in wild-type controls. The neuropeptide, pituitary adenylyl cyclase activating peptide, increases the level of phosphorylated cyclic AMP response element binding protein (CREB) in SCN slices, and melatonin reduces this effect. The Mel(1a) receptor subtype mediates this inhibitory response at moderate ligand concentrations (1 nM). A residual response apparent in Mel(1a) receptor-deficient C3H mice at higher melatonin concentrations (100 nM) is absent in Mel(1a)-Mel(1b) double-mutant mice, indicating that the Mel(1b) receptor mediates this effect of melatonin. These data indicate that there is a limited functional redundancy between the receptor subtypes in the SCN. Mice with targeted disruption of melatonin receptor subtypes will allow molecular dissection of other melatonin receptor-mediated responses

Rhythmic gene expression in pituitary depends on heterologous sensitization by the neurohormone melatonin

In mammals, many daily cycles are driven by a central circadian clock, which is based on the cell-autonomous rhythmic expression of clock genes. It is not clear, however, how peripheral cells are able to interpret the rhythmic signals disseminated from this central oscillator. Here we show that cycling expression of the clock gene Period1 in rodent pituitary cells depends on the heterologous sensitization of the adenosine A2b receptor, which occurs through the nocturnal activation of melatonin mt1 receptors. Eliminating the impact of the neurohormone melatonin simultaneously suppresses the expression of Period1 and evokes an increase in the release of pituitary prolactin. Our findings expose a mechanism by which two convergent signals interact within a temporal dimension to establish high-amplitude, precise and robust cycles of gene expression