Dazap2 modulates transcription driven by the Wnt effector TCF-4.

A major outcome of the canonical Wnt/beta-catenin-signalling pathway is the transcriptional activation of a specific set of target genes. A typical feature of the transcriptional response induced by Wnt signalling is the involvement of Tcf/Lef factors that function in the nucleus as the principal mediators of signalling. Vertebrate Tcf/Lef proteins perform two well-characterized functions: in association with beta-catenin they activate gene expression, and in the absence of Wnt ligands they bind TLE/Groucho proteins to act as transcriptional repressors. Although the general characteristics of Tcf/Lef factors are well understood, the mechanisms that control their specific roles in various cellular backgrounds are much less defined. In this report we reveal that the evolutionary conserved Dazap2 protein functions as a TCF-4 interacting partner. We demonstrate that a short region proximal to the TCF-4 HMG box mediates the interaction and that all Tcf/Lef family members associate with Dazap2. Interestingly, knockdown of Dazap2 not only reduced the activity of Wnt signalling as measured by Tcf/beta-catenin reporters but additionally altered the expression of Wnt-signalling target genes. Finally, chromatin immunoprecipitation studies indicate that Dazap2 modulates the affinity of TCF-4 for its DNA-recognition motif.

The role of proline residues in the structure and function of human MT2 melatonin receptor.

Melatonin functions as an essential regulator of various physiological processes in all vertebrate species. In mammals, two G protein-coupled melatonin receptors (GPCR) mediate some melatonin's actions: MT1 and MT2. Transmembrane domains (TM) of most GPCRs contain a set of highly conserved proline residues that presumably play important structural and functional roles. As TM segments of MT2 receptor display several interesting differences in expression of specific proline residues compared to other rhodopsin-like receptors (rGPCRs), we investigated the role of proline residues in the structure and function of this receptor. All prolines in TM segments of MT2 receptor were individually replaced with alanine and/or glycine. In addition, the unusual NAxxY motif located in TM7 was mutated to generate highly conserved NPxxY motif found in the majority of rGPCR proteins. Following transient expression in CHO-K1 cells, binding properties of the mutant receptors and their ability to transduce signals were analyzed using (125)I-mel- and [(35)S]GTPgammaS-binding assays, respectively. The impact of the performed mutations on the receptor structure was assessed by molecular dynamic simulations of MT2 receptors embedded in the fully hydrated phospholipid bilayer. Our results indicate that residues P174, P212 and P266 are important for the ligand binding and/or signaling of the human MT2 receptor. We also show that changes within the unusual NAxxY sequence in the TM7 (mutations A305P and A305V) produce defective MT2 receptors indicating an important role of this motif in the function of melatonin receptors.

Circadian rhythmicity in AVP secretion and GABAergic synaptic transmission in the rat suprachiasmatic nucleus.

A variety of physiological and behavioral functions exhibit circadian changes and these circadian rhythms are driven by oscillatory expression of clock genes in the suprachiasmatic nuclei (SCN). It is still unknown how this molecular clockwork is controlled by extracellular neurohormones and neurotransmitters and which membrane receptors undergo circadian modulation. Circadian rhythm can be measured as a secretion of arginine vasopressin (AVP) in organotypic SCN culture for several weeks. Melatonin applied directly to the SCN late in the day induces a phase advance, when applied late at night or at the beginning of the day melatonin causes a phase delay. The time window for phase advance corresponds with the highest level of melatonin receptors in the SCN but the mechanism of melatonin-induced phase delay is unknown. The principal neurotransmitter on SCN synapses is gamma-aminobutyric acid (GABA), which acts at postsynaptic GABA(A) receptors. Spontaneous release of GABA from presynaptic nerve terminals, recorded as miniature inhibitory postsynaptic currents in the presence of TTX, does not change, but zinc sensitivity of exogenous GABA-induced currents varies during the day and night, possibly due to changes in subunit composition of GABA(A) receptors. We conclude that there is daily variation in the postsynaptic, but not presynaptic, function in the SCN.

Recovery of Ins(1,4,5)-trisphosphate-dependent calcium signaling in neonatal gonadotrophs.

Pituitary gonadotrophs express non-desensitizing gonadotropin-releasing hormone (GnRH) receptors and their activations leads to inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ mobilization. When added in physiological concentration range GnRH induces baseline Ca2+ oscillations, whereas in higher concentrations it induces a prolonged spike response accompanied with non-oscillatory or oscillatory plateau response. Here, we studied the recovery of calcium signaling during repetitive stimulation with short (10-30 s) GnRH pulses and variable interpulse intervals in neonatal gonadotrophs perfused with Ca2+/Na+ -containing, Ca2+ -deficient/Na+ -containing, and Ca2+ -containing/Na+ -deficient media. In Ca2+/Na+ -containing medium, baseline Ca2+ oscillations recovered without refractory period and with a time constant of approximately 20 s, whereas the recovery of spike response occurred after 25-35 s refractory period and with a time constant of approximately 30 s. During repetitive GnRH stimulation, removal of Ca2+ had only a minor effect on baseline oscillations but abolished spike response, whereas removal of Na+ slightly extended duration of baseline oscillations and considerably prolonged spike response. These results indicate that two calcium handling mechanisms are operative in gonadotrophs: redistribution of calcium within InsP3-sensitive and -insensitive pools and a sodium-dependent calcium efflux followed by calcium influx. Redistribution of Ca2+ within the cell leads to rapid recovery of InsP3-dependent pool, whereas the Na+ -dependent Ca2+ efflux pathway is activated by spike response and limits the time of exposure to elevated cytosolic Ca2+ concentrations.

Ligand binding to the human MT2 melatonin receptor: the role of residues in transmembrane domains 3, 6, and 7.

To better understand the mechanism of interactions between G-protein-coupled melatonin receptors and their ligands, our previously reported homology model of human MT2 receptor with docked 2-iodomelatonin was further refined and used to select residues within TM3, TM6, and TM7 potentially important for receptor-ligand interactions. Selected residues were mutated and radioligand-binding assay was used to test the binding affinities of hMT2 receptors transiently expressed in HEK293 cells. Our data demonstrate that residues N268 and A275 in TM6 as well as residues V291 and L295 in TM7 are essential for 2-iodomelatonin binding to the hMT2 receptor, while TM3 residues M120, G121, V124, and I125 may participate in binding of other receptor agonists and/or antagonists. Presented data also hint at possible specific interaction between the side-chain of Y188 in second extracellular loop and N-acetyl group of 2-iodomelatonin.

Molecular modeling of human MT2 melatonin receptor: the role of Val204, Leu272 and Tyr298 in ligand binding.

A model of the helical part of the human MT2 melatonin (hMT2) receptor, a member of the G protein-coupled receptors superfamily has been generated, based on the structure of bovine rhodopsin. Modeling has been combined with site-directed mutagenesis to investigate the role of the specific amino acid residues within the transmembrane domains (TM) numbers V, VI and VII of hMT2 receptor in the interaction with 2-iodomelatonin. Saturation binding assays with 2-iodomelatonin demonstrated that the substitution V204A (TMV) resulted in total loss of binding while the mutation V205A had no effect. The replacement of F209 with alanine led to a significant decrease in the Bmax value of receptor binding while mutations V205A and F209A also within TM V did not significantly change binding properties of the hMT2 receptor. In the case of TM VI, the substitution G271T caused substantial decrease in 2-iodomelatonin binding to the hMT2 receptor. The change L272A (TM VI) as well as mutation Y298A within TM VII completely abolished ligand binding to the receptor. These data suggest that several new amino acid residues within TM V, VI and VII are involved in ligand-MT2 receptor interaction.