Optimized Binding of [35S]GTPγS to Gq-Like Proteins Stimulated with Dopamine D1-Like Receptor Agonists

Subtypes of dopamine D₁-like receptors are coupled through the G proteins Gs or Gq to stimulate either adenylate cyclase or phospholipase C signaling cascades. In the present study, we have uncovered the marked enhancement by sodium deoxycholate of D1-like agonist-stimulated [³⁵S]GTPS binding to Gq-like G proteins in brain membranes, and determined the optimal experimental conditions for assessing agonist effects on [³⁵S]GTPS binding in the presence of the detergent. Factors and their optimal levels that were found to significantly enhance the sensitivity and robustness of the agonist-stimulated [³⁵S]GTPS binding reaction include protein concentration at 40 g/ml, cationic concentrations of 120 mM Na⁺, 1.8 mM K⁺, and 20 mM Mg²⁺, a molar guanine nucleotide ratio of 100,000 GDP to [³⁵S]GTPS, the presence of 1 mM deoxycholate, and an overall incubation duration of 30–120 min. Under the optimized conditions, the D₁-like agonist SKF38393 induced potent and highly efficacious (up to 1000%) stimulation of [³⁵S]GTPS binding in membrane preparations from the striatum and other rat brain regions. In striatal membranes incubated with drug for 2 h, immunoprecipitation of the [³⁵S]GTPS-bound proteins with specific G antibodies showed that at least 70% of SKF38393-stimulated [³⁵S]GTPS binding was to Gq. The present reaction parameters are consistent with conditions previously found to support dopaminergic stimulation of phospholipase C-mediated signaling in brain slice preparations. These results imply that different but equally physiologically relevant conditions can be obtained under which subtypes of dopaminergic receptors may couple preferentially to Gs and the adenylate cyclase pathway or to Gq and the phospholipase C pathway.     

Ca(2+)/calmodulin-mediated regulation of the desensitizing process in G(q) protein-coupled histamine H(1) receptor-mediated Ca(2+) responses in human U373 MG astrocytoma cells

We investigated Ca(2+)/calmodulin (CaM)-mediated regulation of the desensitizing process of the histamine H(1) receptor-mediated increase in intracellular Ca(2+) concentration in human U373 MG astrocytoma cells. The desensitizing process was evaluated by measuring the histamine-induced Ca(2+) responses in cells pretreated with histamine for 15 s-30 min under various conditions. Under normal physiological conditions, desensitization developed with three successive phases : a fast desensitization within 15 s, a transient resensitization at 45 s, and a prompt and sustained redesensitization from 1 to 30 min. Similar processes of desensitization/resensitization occurred even under hypertonic conditions, where histamine-mediated internalization of the histamine H(1) receptor is inhibited. The transient resensitization phase was selectively prevented by deprivation of extracellular Ca(2+) and, even more strikingly, by the presence of W-7 (a CaM antagonist). FK506 and cyclosporin A, Ca(2+)/CaM-dependent protein phosphatase (PP2B) inhibitors, mimicked such effects. In the presence of KN-62, a Ca(2+)/CaM-dependent protein kinase II (CaM kinase II) inhibitor, the early development of desensitization disappeared, allowing a slow and simple development of desensitization. The early processes of desensitization and resensitization were unaffected by W-5, okadaic acid, and KN-04 (less potent inhibitors against CaM, PP2B, and CaM kinase II, respectively) or by GF109203X and chelerythrine (protein kinase C inhibitors). The high-affinity site for histamine was converted to a lower-affinity site by histamine treatment, which also showed a transient restoration phase at 45 s in a manner sensitive to KN-62 and FK506. These results provide the first evidence that Ca(2+)/CaM plays a crucial role in determining the early phase of the desensitizing process via activation of CaM kinase II and PP2B, by regulating agonist affinity for histamine H(1) receptors.     

Protein kinase C (PKC)ζ-mediated Gαq stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts

Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gα(q)-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gα(q) acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gα(q)/PKCζ/ERK5 signaling axis.     

Sweet taste receptor interacting protein CIB1 is a general inhibitor of InsP3-dependent Ca2+ release in vivo

In a search for sweet taste receptor interacting proteins, we have identified the calcium- and integrin-binding protein 1 (CIB1) as specific binding partner of the intracellular carboxyterminal domain of the rat sweet taste receptor subunit Tas1r2. In heterologous human embryonic kidney 293 (HEK293) cells, the G protein chimeras Gα_16gust44 and Gα_15i3 link the sweet taste receptor dimer TAS1R2/TAS1R3 to an inositol 1,4,5-trisphosphate (InsP₃)-dependent Ca²⁺ release pathway. To demonstrate the influence of CIB1 on the cytosolic Ca²⁺ concentration, we used sweet and umami compounds as well as other InsP₃-generating ligands in FURA-2-based Ca²⁺ assays in wild-type HEK293 cells and HEK293 cells expressing functional human sweet and umami taste receptor dimers. Stable and transient depletion of CIB1 by short-hairpin RNA increased the Ca²⁺ response of HEK293 cells to the InsP₃-generating ligands ATP, UTP and carbachol. Over-expression of CIB1 had the opposite effect as shown for the sweet ligand saccharin, the umami receptor ligand monosodium glutamate and UTP. The CIB1 effect was dependent on the thapsigargin-sensitive Ca²⁺ store of the endoplasmic reticulum (ER) and independent of extracellular Ca²⁺. The function of CIB1 on InsP₃-evoked Ca²⁺ release from the ER is most likely mediated by its interaction with the InsP₃ receptor. Thus, CIB1 seems to be an inhibitor of InsP₃-dependent Ca²⁺ release in vivo .     

Ca2+ responses to thyrotropin-releasing hormone and angiotensin II: the role of plasma membrane integrity and effect of G11alpha protein overexpression on homologous and heterologous desensitization

The molecular mechanisms involved in GPCR-initiated signaling cascades where the two receptors share the same signaling cascade, such as thyrotropin-releasing hormone (TRH) and angiotensin II (ANG II), are still far from being understood. Here, we analyzed hormone-induced Ca(2+) responses and the process of desensitization in HEK-293 cells, which express endogenous ANG II receptors. These cells were transfected to express exogenously high levels of TRH receptors (clone E2) or both TRH receptors and G(11)alpha protein (clone E2M11). We observed that the characteristics of the Ca(2+) response, as well as the process of desensitization, were both strongly dependent on receptor number and G(11)alpha protein level. Whereas treatment of E2 cells with TRH or ANG II led to significant desensitization of the Ca(2+) response to subsequent addition of either hormone, the response was not desensitized in E2M11 cells expressing high levels of G(11)alpha. In addition, stimulation of both cell lines with THR elicited a clear heterologous desensitization to subsequent stimulation with ANG II. On the other hand, ANG II did not affect a subsequent response to TRH. ANG II-mediated signal transduction was strongly dependent on plasma membrane integrity modified by cholesterol depletion, but signaling through TRH receptors was altered only slightly under these conditions. It may be concluded that the level of expression of G-protein-coupled receptors and their cognate G-proteins strongly influences not only the magnitude of the Ca(2+) response but also the process of desensitization and resistance to subsequent hormone addition.     

Volume transmission signalling via astrocytes

The influence of astrocytes on synaptic function has been increasingly studied, owing to the discovery of both gliotransmission and morphological ensheathment of synapses. While astrocytes exhibit at best modest membrane potential fluctuations, activation of G-protein coupled receptors (GPCRs) leads to a prominent elevation of intracellular calcium which has been reported to correlate with gliotransmission. In this review, the possible role of astrocytic GPCR activation is discussed as a trigger to promote synaptic plasticity, by affecting synaptic receptors through gliotransmitters. Moreover, we suggest that volume transmission of neuromodulators could be a biological mechanism to activate astrocytic GPCRs and thereby to switch synaptic networks to the plastic mode during states of attention in cerebral cortical structures.     

Epithelial microRNA‐9a regulates dendrite growth through Fmi‐Gq signaling in Drosophila sensory neurons

microRNA‐9 (miR‐9) is highly expressed in the nervous system across species and plays essential roles in neurogenesis and axon growth; however, little is known about the mechanisms that link miR‐9 with dendrite growth. Using an in vivo model of Drosophila class I dendrite arborization (da) neurons, we show that miR‐9a, a Drosophila homolog of mammalian miR‐9, downregulates the cadherin protein Flamingo (Fmi) thereby attenuating dendrite development in a non‐cell autonomous manner. In miR‐9a knockout mutants, the dendrite length of a sensory neuron ddaE was significantly increased. Intriguingly, miR‐9a is specifically expressed in epithelial cells but not in neurons, thus the expression of epithelial but not neuronal Fmi is greatly elevated in miR‐9a mutants. In contrast, overexpression of Fmi in the neuron resulted in a reduction in dendrite growth, suggesting that neuronal Fmi plays a suppressive role in dendrite growth, and that increased epithelial Fmi might promote dendrite growth by competitively binding to neuronal Fmi. Fmi has been proposed as a G protein‐coupled receptor (GPCR), we find that neuronal G protein Gαq (Gq), but not Go, may function downstream of Fmi to negatively regulate dendrite growth. Taken together, our results reveal a novel function of miR‐9a in dendrite morphogenesis. Moreover, we suggest that Gq might mediate the intercellular signal of Fmi in neurons to suppress dendrite growth. Our findings provide novel insights into the complex regulatory mechanisms of microRNAs in dendrite development, and further reveal the interplay between the different components of Fmi, functioning in cadherin adhesion and GPCR signalling. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 225–237, 2016     

The effect of detergents on trimeric G-protein activity in isolated plasma membranes from rat brain cortex: Correlation with studies of DPH and Laurdan fluorescence

The effect of non-ionic detergents on baclofen (GABA_B-R agonist)-stimulated G-protein activity was measured as a [³⁵S]GTPγS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic — a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABA_B-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58 > Triton X-100 > Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (r_DPH) incorporated into the hydrophobic PM interior. Decrease of r_DPH, in the order of Brij58 > Triton X-100 > Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time ? paralleled by an increase of diffusion wobbling constant D_w (analysis by time-resolved fluorescence according to “wobble-in-cone” model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58 > Triton X-100 > Digitonin.     

Brain region-specific alterations of 5-HT2A and 5-HT2C receptors in serotonin transporter knockout mice

The aim of the present studies was to determine the effects of reduced or absent serotonin (5-HT) transporters (5-HTTs) on 5-HT2A and 5-HT2C receptors. The density of 5-HT2C receptors was significantly increased in the amygdala and choroid plexus of 5-HTT knockout mice. On the other hand, the density of 5-HT2A receptors was significantly increased in the hypothalamus and septum, but reduced in the striatum, of 5-HTT knockout mice. However, 5-HT2A mRNA was not changed in any brain region measured. 5-HT2C mRNA was significantly reduced in the choroid plexus and lateral habenula nucleus of these mice. The function of 5-HT2A receptors was evaluated by hormonal responses to (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Oxytocin, but not adrenocorticotrophic hormone or corticosterone, responses to DOI were significantly greater in 5-HTT knockout mice. In addition, Gq and G11 proteins were not significantly changed in any brain region measured. The present results suggest that the constitutive alteration in the function of 5-HTTs changes the density of 5-HT2A and 5-HT2C receptors in a brain region-specific manner. These changes may not be mediated by alterations in their gene expression or in the level of Gq/11 proteins. The alterations in these receptors may be related to the altered behaviors of 5-HTT knockout mice.