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  • Analysis of the thermosensory responses conferred


    Analysis of the thermosensory responses conferred by chimeric proteins suggests that both the ECD and ICD of AFD-rGCs contribute to their thermoresponsive properties. The presence of the GCY-18 or GCY-23 ICD and TMD in chimeric protein combinations generally corresponds to a higher or lower , respectively, suggesting that the may in part be regulated by these domains, possibly via interaction with proteins such as GCAPs (Duda et al., 1996, Laura et al., 1996). The absence of such activation mechanisms, or the presence of inhibitory factors, may account for the inability of GCY-8, or chimeras containing the GCY-8(ICD), to confer temperature responses upon misexpression. However, the ECDs of thermosensory rGCs are also necessary for temperature responses. Conformational changes upon ligand binding have been reported in transmembrane cyclases, resulting in allosteric activation of their enzymatic functions (Misono et al., 2005, Ogawa et al., 2004). Temperature responses by AFD-rGCs may require similar temperature-regulated conformational changes. Further analyses of thermosensory responses conferred by chimeric rGCs may allow us to identify residues in these proteins that contribute to thermosensation and plasticity. In addition to temperature-dependent regulation of rGC enzymatic activity, additional mechanisms contribute to the extraordinary thermosensitivity of AFD (Ramot et al., 2008). Similar to observations in mammalian photoreceptors, these mechanisms include the high density and compartmentalization of thermosensory molecules in the membranes of the complex AFD microvilli (Nguyen et al., 2014), as well as T-dependent Phosphatase Inhibitor Cocktail (2 Tubes, 100X) of that decreases gain and increases thermosensitivity (Biron et al., 2006, Ramot et al., 2008, Yu et al., 2014). The expression of highly thermosensitive AFD-rGCs together with neuron-specific amplification and adaptation mechanisms allows AFD, and hence C. elegans, to be exquisitely temperature sensitive across a wide temperature range. A thermosensory signaling cascade also has been proposed to amplify thermoresponses in Drosophila (Kwon et al., 2008). Intriguingly, recent work has shown that the guanylyl cyclase G rGC is both necessary and sufficient for sensing cool temperatures in the Grueneberg ganglion in the mouse nose (Chao et al., 2015). Identification of rGCs as possible thermosensitive proteins in rodents and C. elegans further diversifies the functions of these versatile signaling proteins and implies that thermosensory roles of these molecules may be conserved across phyla.
    Experimental Procedures Detailed protocols are provided in the Supplemental Experimental Procedures.
    Author Contributions
    Introduction The cyclic purine nucleotides cAMP and cGMP are established second messengers regulating numerous physiological processes, such as relaxation of smooth muscle cells, differentiation and neurotransmission [1], [2]. The existence of the cyclic pyrimidine nucleotides cCMP and cUMP in tissues had been postulated [3], [4]. Moreover, a specific cytidylyl cyclase and a cCMP-degrading PDE was claimed [5], [6]. However, previous methods used to demonstrate the occurrence and generation of cCMP lacked selectivity and sensitivity, resulting in controversial discussion [7]. As a result, very little research has been conducted in the cCMP and cUMP field over the past three decades. Recently, cCMP and cUMP have been shown to activate PKA with lower potency than, but similar efficacy as, cAMP [8]. cCMP and cUMP are less potent and effective activators of PKG than cGMP [8]. In contrast to cAMP, cGMP and cUMP, cCMP is not cleaved by several human recombinant PDEs [9], pointing to different roles of the various cNMPs in signal transduction. The membrane-permeable cCMP analog dibutyryl-cCMP induces vascular smooth muscle relaxation via PKG [10]. Moreover, cCMP and cUMP partially activate the ion channels HCN2 and 4 in recombinant cells and native cardiomyocytes [11]. Furthermore, by using radiometric-, HPLC- and MS approaches, purified bacterial adenylyl cyclase toxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis were shown to produce cCMP and cUMP [12]. Lastly, using a highly sensitive and specific HPLC–MS/MS method, purified sGC α1β1 has been shown to produce cCMP and cUMP NO-dependently [13]. Based on these data we developed the hypothesis that cCMP and cUMP play distinct roles as second messenger [14]. However, since cCMP- and cUMP formation by purified sGC occurred only in the presence of Mn2+, the physiological relevance of cyclic pyrimidine nucleotide formation by sGC remained unclear. Here, we show that sGC catalyzes cCMP- and cUMP formation also in intact cells.