How can rhythmic extracellular/environmental signals generate highly specific, rhythmic cellular responses at different time scales, while the repertoire of intracellular second messengers is limited?

FG Biochemie

Project members: Prof. Dr. Herberg, Prof. Dr. Mayer, Prof. Dr. Schaffrath, Prof. Dr. Stengl, PD Dr. Neupert, PD Dr. Popov

Clock cells can sense regular environmental rhythms at different time scales, such as the 24h light dark cycle or the superimposed ultradian rhythm in the change of light intensity at dusk and dawn. They transduce these environmental rhythms into respective intracellular rhythms of “second messengers” as well as in regular release of coupling factors such as neuropeptides. The rhythmically released neuropeptides in turn will generate intracellular changes of second messengers in all neuropeptide receptor expressing cells at the time course of the environmental cues. In the animal kingdom highly conserved neuropeptides such as pigment-dispersing factor (PDF) appear to serve similar physiological functions. Activation of neuropeptide receptors is translated into a limited repertoire of second messengers. Among these are nucleotide derivatives like cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), calcium (Ca2+), as well as signalling lipids including phosphatidylinositol phosphates (PIPs), or diacylglycerol (DAG). Also, the synthesis (adenylyl cyclases, phopholipases) and the degradation (phosphodiesterases, phosphatases) of second messengers is controlled by a limited repertoire of cellular proteins. Understanding intracellular signalling requires implementation of the concept of compartmentalization allowing the spatial/temporal control of the components involved. Signalling via defined subcellular compartments enables simultaneous as well as differential cellular responses at different frequencies from one second messenger species. In the present consortium multiscale timing processes in different model organisms are investigated and we will focus on the underlying molecular mechanisms. These include signalling processes via G-protein coupled receptors (GPCRs), such as the PDF-receptor, where all above-mentioned second messenger systems are involved [1]. Crosstalk between these signalling pathways is a critical featured to understand how molecular clocks work [2]. We have significant expertise and the tools to look at cyclic nucleotide derivatives to quantitatively describe their concentration and mode of action [3]. We also employ membrane permeable cyclic nucleotide analogues [4] and stapled peptides [5] as tools to selectively interfere with the targets of cAMP and cGMP. These compounds can not only be used for in vitro studies but also for cellular assays and in life tissue. We will extend our studies to Ca2+, where numerous intracellular sensors (FRET-based) are available, to study the effect of Ca2+ on PDF-induced cAMP-signalling and vice versa in the biological model systems of our graduate colleague. In Drosophila fluorescent based sensors simultaneously detect cAMP and Ca signalling [6]. Furthermore, for compartmentalized cAMP signalling, A-Kinase Anchoring Proteins (AKAPs) seem to be crucial in particular in higher eukaryotes [7]. For insects only a limited number of AKAPs has been described, yet. In Drosophila the AKAP-like scaffolding protein Nervy was reported to reduce PDF responses. With recombinant proteins of all isoforms and the respective structures of cAMP and cGMP-targets available (cAMP and cGMP dependent protein kinases from different organisms as well as EPAC and HCN-channels), we can easily combine biochemical, structural and cell-based assays to address attractive projects for prospective PhD-candidates.

Literature:

[1] Duvall LB and Taghert PH (2012) The Circadian Neuropeptide PDF Signals Preferentially through a Specific Adenylate Cyclase Isoform AC3 in M Pacemakers of Drosophila. PLoS Biol 10(6): e1001337.

[2] OT Shafer et al Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by real-time cyclic AMP imaging.Neuron, 58(2):223-37 (2008)

[3] Lorenz, R. et al. Mutations of PKA cyclic nucleotide binding domains reveal novel aspects of cyclic nucleotide selectivity. Biochem. J. 474(14), 2389-2403 (2017)

[4] Schwede F, et al. Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose- Stimulated Insulin Sekcretion Mol Endocrinol. Jul;29(7):988-1005 (2015)

[5] Bendzunas, N.G., et al. Investigating PKA-RII specificity using analogs of the PKA:AKAP peptide inhibitor STAD-2. Bioorg Med Chem.: S0968-0896(17)31514-6 (2018)

[6] Sprenger J.U. et al. Interactions of Calcium Fluctuations during Cardiomyocyte Contraction with Real-Time cAMP Dynamics Detected by FRET. PLoS OneDec 8;11(12):e0167974. (2016)

[7] Calebiro,D. and Maiellaro, I. cAMP signaling microdomains and their observation by optical methods Front. Cell. Neurosci. (2014)