We found that zaprinast, a well-known cyclic guanosine monophosphate-specific phosphodiesterase inhibitor, acted as an agonist for a G protein-coupled receptor, GPR35. In our intracellular calcium mobilization assay, zaprinast activated rat GPR35 strongly (geometric mean EC 50 value of 16 nM), whereas it activated human GPR35 moderately (geometric mean EC 50 value of 840 nM). We also demonstrated that GPR35 acted as a Ga i/o -and Ga 16 -coupled receptor for zaprinast when heterologously expressed in human embryonic kidney 293 (HEK 293) cells. These findings will facilitate the research on GPR35 and the drug discovery of the GPR35 modulators.

In the cyanobacterium Synechococcus elongatus PCC 7942, circadian timing is transmitted from the KaiABC-based central oscillator to the transcription factor RpaA via the KaiC-interacting histidine kinase SasA to activate transcription, thereby generating rhythmic circadian gene expression. However, KaiC can also repress circadian gene expression, including its own. The mechanism and significance of this negative feedback regulation have been unclear. Here, we report a novel gene, labA (low-amplitude and bright), that is required for negative feedback regulation of KaiC. Disruption of labA abolished transcriptional repression caused by overexpression of KaiC and elevated the trough levels of circadian gene expression, resulting in a low-amplitude phenotype. In contrast, overexpression of labA significantly lowered circadian gene expression. Furthermore, genetic analysis indicated that labA and sasA function in parallel pathways to regulate kaiBC expression, whereas rpaA functions downstream from labA for kaiBC expression. These results suggest that temporal information from the KaiABC-based oscillator diverges into a LabA-dependent negative pathway and a SasA-dependent positive pathway, and then converges onto RpaA to generate robust circadian gene expression. It is likely that quantitative information of KaiC is transmitted to RpaA through LabA, whereas SasA mediates the state of the KaiABC-based oscillator.[Keywords: Circadian clock; cyanobacteria; KaiC; labA; RpaA; SasA] Supplemental material is available at http://www.genesdev.org. The circadian clock is an endogenous timing system that controls various biological activities with a period of ∼24 h. Most organisms use self-sustained oscillation to coordinate with and adapt to daily environmental changes. KaiA, KaiB, and KaiC have been identified as essential components for circadian oscillation in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus). KaiC, an autokinase and autophosphatase, is the central component of the cyanobacterial circadian clock and interacts with KaiA and KaiB Iwasaki et al. 1999;Nishiwaki et al. 2000;Taniguchi et al. 2001). KaiA enhances the autokinase activity of KaiC (Iwasaki et al. 2002;Williams et al. 2002;Uzumaki et al. 2004) and/or inhibits its autophosphatase activity (Kitayama et al. 2003;Xu et al. 2003), while KaiB attenuates the activity of KaiA (Williams et al. 2002;Kitayama et al. 2003;Xu et al. 2003). KaiC phosphorylation oscillates with a period of ∼24 h when the three recombinant Kai proteins are incubated in vitro in the presence of ATP . This oscillation period is refractory to changes in temperature, an important feature of circadian rhythms ). Thus, Kai-based chemical oscillation is thought to be the basic circadian timing loop in Synechococcus Tomita et al. 2005).KaiC also interacts with a sensory histidine kinase, SasA . Autophosphorylation of SasA is enhanced in response to KaiC binding (Smith and Williams 2006;Takai et al. 2006), and this phosphate group is then transferred to the putati...

Circadian kaiBC expression in the cyanobacterium Synechococcus elongatus PCC 7942 is generated by temporal information transmission from the KaiABC-based circadian oscillator to RpaA, a putative transcriptional factor, via the SasA-dependent positive pathway and the LabA-dependent negative pathway which is responsible for feedback regulation of KaiC. However, the labA/sasA double mutant has a circadian kaiBC expression rhythm, suggesting that there is an additional circadian output pathway. Here we describe a third circadian output pathway, which is CikA-dependent. The cikA mutation attenuates KaiC overexpression-induced kaiBC repression and exacerbates the low-amplitude phenotype of the labA mutant, suggesting that cikA acts as a negative regulator of kaiBC expression independent of the LabA-dependent pathway. In the labA/sasA/cikA triple mutant, kaiBC promoter activity becomes almost arrhythmic, despite preservation of the circadian KaiC phosphorylation rhythm, suggesting that CikA largely accounts for the residual kaiBC expression rhythm observed in the labA/sasA double mutant. These results also strongly suggest that transcriptional regulation in the labA/sasA/cikA triple mutant is insulated from the circadian signals of the KaiABC-based oscillator. Based on these observations, we propose a model in which temporal information from the KaiABC-based circadian oscillator is transmitted to gene expression through three separate output pathways.CikA | circadian clock | LabA | SasA | transcriptional feedback

kaiABC, a gene cluster, encodes KaiA, KaiB and KaiC proteins that are essential to circadian rhythms in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. Kai proteins can interact with each other in all possible combinations. This study identified two KaiA-binding domains (C KABD1 and C KABD2 ) in KaiC at corresponding regions of its duplicated structure. Clock mutations on the two domains and kaiA altered the strength of C KABD^K aiA interactions assayed by the yeast two-hybrid system. Thus, interaction between KaiA and KaiC through C KABD1 and C KABD2 is likely important for circadian timing in the cyanobacterium. ß

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