Protein PERIOD2 Research Articles

Expression of circadian rhythm genes in gonadotropin-releasing hormone-secreting GT1-7 neurons.

The center for circadian rhythms in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus, composed of single cell circadian oscillators driven by a transcriptional/translational feedback loop where clock proteins drive clock gene expression. These genes are expressed in peripheral tissues and several brain areas outside the SCN. It is likely that some peripheral oscillators are synchronized by the SCN. The pineal hormone melatonin plays an important role in the entrainment of circadian rhythms through feedback to the SCN. Melatonin also plays a role in reproduction, including direct effects on GnRH-secreting GT1-7 neurons. The intrinsic rhythmicity of GnRH neurons suggests that these neurons may express the components of the circadian oscillator. Using the GT1-7 cell line, we demonstrate expression of the circadian rhythm genes, clock, BMAL1,timeless (tim), period1,period2, cryptochrome1, andcryptochrome2. Furthermore, semiquantitative RT-PCR demonstrates that BMAL1, period1, andperiod2 as well as GnRH mRNAs are expressed with a circadian-like rhythm after synchronization over 54 h. With available antibodies, we demonstrated CLOCK, BMAL1, and PERIOD1 protein expression in these cells, with BMAL1 protein levels showing a rhythmic expression pattern. In addition, receptors for melatonin, mt1 and MT2, also show a circadian expression pattern in the GT1-7 cells, and their expression is down-regulated by melatonin treatment. These findings suggest that the components of the clock machinery in mammals may play a role in GnRH neuronal function.

Nuclear entry mechanism of rat PER2 (rPER2): role of rPER2 in nuclear localization of CRY protein.

Mammalian PERIOD2 protein (PER2) is the product of a clock gene that controls circadian rhythms, because PER2-deficient mice have an arrhythmic phenotype. The nuclear entry regulation of clock gene products is a key step in proper circadian rhythm formation in both Drosophila and mammals, because the periodic transcription of clock genes is controlled by an intracellular, oscillating, negative feedback loop. The present study used deletion mutants of rat PER2 (rPER2) to identify the functional nuclear localization signal (NLS) in rPER2. The elimination of putative NLS (residues 778 to 794) from the rPER2 fragment resulted in the loss of nuclear entry activity. Adding the NLS to the cytosolic protein (bacterial alkaline phosphatase) translocates the fusion protein to the nuclei. The data indicate the presence of a functional NLS in rPER2. Furthermore, intact rPER2 was preferentially translocated from the cytoplasm to the nucleus when coexpressed with human CRY1 (hCRY1). However, rPER2 mutants lacking a carboxyl-terminal domain could not enter the nucleus even in the presence of hCRY1. In addition, coexpression of the nuclear localization domain (residues 512 to 794) lacking rPER2 and CRY1 changed the subcellular localization of CRY1 from the nucleus to the cytoplasm. In vitro protein interaction studies demonstrated that the carboxyl-terminal domain of rPER2 is essential for binding to CRY1. The data suggested that both the rPER2 NLS and carboxyl-terminal CRY binding domain are essential for nuclear entry of the rPER2-CRY1 complex.