Frizzleds (FZDs) are classified as G‐protein‐coupling receptors, but how signals are initiated and specified through heterotrimeric G proteins is unknown. FZD ₆ regulates convergent extension movements, and its C‐terminal Arg511Cys mutation causes nail dysplasia in humans. We investigated the functional relationship between FZD ₆ , Disheveled (DVL), and heterotrimeric G proteins. Live cell imaging combined with fluorescence recovery after photobleaching (FRAP) revealed that inactive human FZD ₆ precouples to Gα _i1 and Gα _q but not to Gα _oA ,Gα _s , and Gα ₁₂ proteins. G‐protein coupling is measured as a 10–20% reduction in the mobile fraction of fluorescently tagged G proteins on chemical receptor surface cross‐linking. The FZD ₆ Arg511Cys mutation is incapable of G‐protein precoupling, even though it still binds DVL. Using both FRAP and Förster resonance energy transfer (FRET) technology, we showed that the FZD ₆ ‐Gα _i1 and FZD‐Gα _q complexes dissociate on WNT‐5A stimulation. Most important, G‐protein precoupling of FZD ₆ and WNT‐5A‐induced signaling to extracellular signal‐regulated kinase1/2 were impaired by DVL knockdown or overexpression, arguing for a strict dependence of FZD ₆ ‐G‐protein coupling on DVL levels and identifying DVL as a master regulator of FZD/G‐protein signaling. In summary, we propose a mechanistic connection between DVL and G proteins integrating WNT, FZD, G‐protein, and DVL function.—Kilander, M. B. C., Petersen, J., Andressen, K. W., Ganji, R. S. Levy, F. O., Schuster, J., Dahl N., Bryja, V., Schulte, G. Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6. FASEB J . 28, 2293–2305 (2014). www.fasebj.org

Current pharmacological treatment of congestive heart failure (CHF) addresses changes in neurohumoral stimulation or cardiac responsiveness to such stimulation. Yet, undiscovered neurohumoral changes, adaptive or maladaptive, may occur in CHF and suggest novel pharmacological treatment. Serotonin [5-hydroxytryptamine (5-HT)] enhances contractility and causes arrhythmias through 5-HT(4) receptors in human atrium and ventricle but not through rat ventricular 5-HT(4) receptors. We investigated whether CHF could induce ventricular responsiveness to serotonin. Postinfarction CHF was induced in male Wistar rats by coronary artery ligation. Contractility was measured in left ventricular papillary muscles 6 weeks after infarction. Messenger RNA was quantified by RT-PCR and cAMP by RIA. Serotonin caused positive inotropic (-logEC(50)=7.5) and lusitropic effects in CHF but not Sham papillary muscles. The inotropic effect of 10 muM serotonin in CHF (31.3+/-2.2%) was of similar size as the effect of 10 muM isoproterenol (34.0+/-1.7%). The effects of serotonin were antagonised by GR113808 (0.5-5 nM), consistent with mediation through 5-HT(4) receptors. This was further supported by positive inotropic effects of the 5-HT(4)-selective partial agonist RS67506. Carbachol blunted the serotonin responses and serotonin increased ventricular and cardiomyocyte cAMP, consistent with coupling to G(s) and adenylyl cyclase. Quantitative RT-PCR revealed fourfold increased 5-HT(4(b)) mRNA expression in CHF vs. Sham ventricles. Functional ventricular 5-HT(4) receptors are induced by myocardial infarction and CHF of the rat heart. We propose that they are a model for ventricular 5-HT(4) receptors of human failing heart and may play a pathophysiological role in heart failure.

Cardiac contractility is regulated by several neural, hormonal, paracrine, and autocrine factors. Amongst these, signaling through β-adrenergic and serotonin receptors generates the second messenger cyclic AMP (cAMP), whereas activation of natriuretic peptide receptors and soluble guanylyl cyclases generates cyclic GMP (cGMP). Both cyclic nucleotides regulate cardiac contractility through several mechanisms. Phosphodiesterases (PDEs) are enzymes that degrade cAMP and cGMP and therefore determine the dynamics of their downstream effects. In addition, the intracellular localization of the different PDEs may contribute to regulation of compartmented signaling of cAMP and cGMP. In this review, we will focus on the role of PDEs in regulating contractility and evaluate changes in heart failure.

Several FRET (fluorescence resonance energy transfer)-based biosensors for intracellular detection of cyclic nucleotides have been designed in the past decade. However, few such biosensors are available for cGMP, and even fewer that detect low nanomolar cGMP concentrations. Our aim was to develop a FRET-based cGMP biosensor with high affinity for cGMP as a tool for intracellular signaling studies. We used the carboxyl-terminal cyclic nucleotide binding domain of Plasmodium falciparum cGMP-dependent protein kinase (PKG) flanked by different FRET pairs to generate two cGMP biosensors (Yellow PfPKG and Red PfPKG). Here, we report that these cGMP biosensors display high affinity for cGMP (EC₅₀ of 23 ± 3 nM) and detect cGMP produced through soluble guanylyl cyclase and guanylyl cyclase A in stellate ganglion neurons and guanylyl cyclase B in cardiomyocytes. These biosensors are therefore optimal tools for real-time measurements of low concentrations of cGMP in living cells.