Topmouth culter melanocortin-3 receptor: regulation by two isoforms of melanocortin-2 receptor accessory protein 2

Melanocortin-3 receptor (MC3R) is a regulator of energy homeostasis, and interaction of MC3R and melanocortin-2 receptor accessory protein 2 (MRAP2) plays a critical role in MC3R signaling of mammals. However, the physiological roles of MC3R in teleosts are not well understood. In this study, qRT-PCR was used to measure gene expression. Radioligand binding assay was used to study the binding properties of topmouth culter MC3R (caMC3R). Intracellular cAMP generation was determined by radioimmunoassay and caMC3R expression was quantified with flow cytometry. We showed that culter mc3r had higher expression in the central nervous system. All agonists could bind and stimulate caMC3R to increase dose-dependently intracellular cAMP accumulation. Compared to hMC3R, culter MC3R showed higher constitutive activity, higher efficacies and Rmax to α-MSH, des-α-MSH, and ACTH. Both caMRAP2a and caMRAP2b markedly decreased caMC3R basal cAMP production. However, only caMRAP2a significantly decreased cell surface expression, Bmax and Rmax of caMC3R. Expression analysis suggested that MRAP2a and MRAP2b might be more important in regulating MC3R/MC4R signaling during larval period, and reduced mc3r, mc4r, and pomc expression might be primarily involved in modulation of MC3R/MC4R in adults. These data indicated that the cloned caMC3R was a functional receptor. MRAP2a and MRAP2a had different effects on expression and signaling of caMC3R. In addition, expression analysis suggested that MRAP2s, receptors, and hormone might play different roles in regulating culter development and growth.

The IgG3 Subclass of β1-adrenergic receptor autoantibody is an endogenous biaser of β1AR signaling

ABSTRACTAutoantibodies recognizing human β1ARs generated due to dysregulation in autoimmune response are generally associated with deleterious cardiac outcomes. However, cellular studies show that isolates of β1AR autoantibody from patients differentially modulate β1AR function. β1AR autoantibodies belong to the IgG class of immunoglobulins, however it is not known whether the IgG sub-classes mediate variability in β1AR responses. To determine whether the IgG3 subclass of β1AR autoantibodies uniquely modulate β1AR function, HEK293 cells stably expressing human β1ARs were utilized. Treatment of cells with IgG3(-) serum resulted in significant increase of cAMP compared to IgG3(+) serum. Pre-treatment of cells with IgG3(+) serum impaired dobutamine-mediated Adenylate Cyclase (AC) activity and cAMP generation whereas, it surprisingly increased AC activity and cAMP generation with β-blocker metoprolol. Consistently, purified IgG3(+) β1AR autoantibodies impaired dobutamine-mediated cAMP while elevating metoprolol-mediated AC activity and cAMP. Despite IgG3(+) autoantibodies reducing cAMP response to dobutamine, they mediate significant ERK activation upon dobutamine. IgG3(+) β1AR autoantibodies did not alter β2AR function, reflecting their specificity. The study shows that IgG3(+) β1AR autoantibody impairs agonist-mediated G-protein coupling while preferentially mediating G-protein-independent ERK activation. Furthermore, it uniquely biases β-blocker towards G-protein coupling. This unique biasing capabilities of IgG3(+) β1AR autoantibodies may underlie the beneficial outcomes in patients.

Allosteric Inhibition of Adenylyl Cyclase Type 5 by G-Protein: A Molecular Dynamics Study

Adenylyl cyclases (ACs) have a crucial role in many signal transduction pathways, in particular in the intricate control of cyclic AMP (cAMP) generation from adenosine triphosphate (ATP). Using homology models developed from existing structural data and docking experiments, we have carried out all-atom, microsecond-scale molecular dynamics simulations on the AC5 isoform of adenylyl cyclase bound to the inhibitory G-protein subunit Gαi in the presence and in the absence of ATP. The results show that Gαi has significant effects on the structure and flexibility of adenylyl cyclase, as observed earlier for the binding of ATP and Gsα. New data on Gαi bound to the C1 domain of AC5 help explain how Gαi inhibits enzyme activity and obtain insight on its regulation. Simulations also suggest a crucial role of ATP in the regulation of the stimulation and inhibition of AC5.

Gq/11-dependent regulation of endosomal cAMP generation by parathyroid hormone class B GPCR

cAMP production upon activation of Gs by G protein-coupled receptors has classically been considered to be plasma membrane-delimited, but a shift in this paradigm has occurred in recent years with the identification of several receptors that continue to signal from early endosomes after internalization. The molecular mechanisms regulating this aspect of signaling remain incompletely understood. Here, we investigated the role of Gq/11 activation by the parathyroid hormone (PTH) type 1 receptor (PTHR) in mediating endosomal cAMP responses. Inhibition of Gq/11 signaling by FR900359 markedly reduced the duration of PTH-induced cAMP production, and this effect was mimicked in cells lacking endogenous Gαq/11. We determined that modulation of cAMP generation by Gq/11 occurs at the level of the heterotrimeric G protein via liberation of cell surface Gβγ subunits, which, in turn, act in a phosphoinositide-3 kinase-dependent manner to promote the assembly of PTHR–βarrestin–Gβγ signaling complexes that mediate endosomal cAMP responses. These results unveil insights into the spatiotemporal regulation of Gs-dependent cAMP signaling.

The Importance of Oxidative Stress in Determining the Functionality of Mammalian Spermatozoa: A Two-Edged Sword

This article addresses the importance of oxidative processes in both the generation of functional gametes and the aetiology of defective sperm function. Functionally, sperm capacitation is recognized as a redox-regulated process, wherein a low level of reactive oxygen species (ROS) generation is intimately involved in driving such events as the stimulation of tyrosine phosphorylation, the facilitation of cholesterol efflux and the promotion of cAMP generation. However, the continuous generation of ROS ultimately creates problems for spermatozoa because their unique physical architecture and unusual biochemical composition means that they are vulnerable to oxidative stress. As a consequence, they are heavily dependent on the antioxidant protection afforded by the fluids in the male and female reproductive tracts and, during the precarious process of insemination, seminal plasma. If this antioxidant protection should be compromised for any reason, then the spermatozoa experience pathological oxidative damage. In addition, situations may prevail that cause the spermatozoa to become exposed to high levels of ROS emanating either from other cells in the immediate vicinity (particularly neutrophils) or from the spermatozoa themselves. The environmental and lifestyle factors that promote ROS generation by the spermatozoa are reviewed in this article, as are the techniques that might be used in a diagnostic context to identify patients whose reproductive capacity is under oxidative threat. Understanding the strengths and weaknesses of ROS-monitoring methodologies is critical if we are to effectively identify those patients for whom treatment with antioxidants might be considered a rational management strategy.

Gene-signatures predict biologically relevant dose-response potencies in phenotypic assays

Multiplexed gene-signature-based phenotypic assays are increasingly used for the identification and profiling of small molecule-tool compounds and drugs. Here we introduce a method (provided as R-package) for the quantification of the dose-response potency of a gene-signature as EC50 and IC50 values.Two signaling pathways were used as models to validate our methods: beta-adrenergic agonistic activity on cAMP generation (dedicated dataset generated for this study) and EGFR inhibitory effect on cancer cell viability. In both cases, potencies derived from multi-gene expression data were highly correlated with orthogonal potencies derived from cAMP and cell growth readouts, and superior to potencies derived from single individual genes.Our results show that gene-signature potencies are a novel valid alternative to conventional readouts for compound potency quantification, in particular in scenarios where no other established readouts are available.