Regulator of G protein signaling 9-2 (RGS9-2) mRNA is up regulated during neuronal differentiation of mouse embryonic stem cells

In this study we demonstrate up-regulation of mRNA for Regulator of G protein Signaling (RGS) 6, 7, 9 and 11, R7 family RGS binding protein (R7BP) and RGS9 anchor protein (R9AP) during neuronal differentiation of mouse embryonic stem cells (mESCs). This expression pattern was most robust for RGS9 whose transcript level was low in undifferentiated mESCs but increased over 125 fold when differentiating mESCs began to exhibit a neuronal precursor cell (NPC) phenotype. In addition, we demonstrate that RGS9 mRNA is expressed in neuronal stem cells isolated from embryonic mouse cortex. The expression of RGS9 in two distinct populations of NPCs suggests that RGS9 and its accessory proteins may play an important role in neuron development

Plasma Membrane Compartmentalization of D2 Dopamine Receptors

Plasma membrane microcompartments could allow different signaling pathways to operate more efficiently and prevent cross-talk. We utilized a novel in-cell biotin transfer assay to demonstrate that the majority of plasma membrane-expressed D2 dopamine receptor (D2R) is microcompartmentalized within detergent-resistant structures. Conversely, a minority of D2R existed in a detergent-soluble form and interacted in a relatively unrestricted manner with other cellular proteins. The microcompartmentalization of D2R had functional consequences because dopamine-induced internalization of D2R was largely restricted to the compartmentalized receptor. The D2R-containing microcompartments did not correspond to putative detergent-resistant lipid raft structures. First, the detergent-insoluble D2R structures were significantly denser than detergent-resistant membrane fragments containing flotillin, a widely utilized lipid raft marker protein. Second, the detergent solubility of D2R was unaffected by treatment of cells with the cholesterol chelating agent, methyl-β-cyclodextrin, that is thought to disrupt lipid rafts. Finally, the in-cell biotinylation assay did not provide any evidence for the membrane compartmentalization of peptide motifs thought to target to lipid rafts. Thus, our observations form one of the first demonstrations, in living cells, of plasma membrane microcompartments defined by the ability of the compartment structure to broadly restrict the interaction of resident molecules with other cellular proteins

Membrane Anchor R9AP Potentiates GTPase-accelerating Protein Activity of RGS11·Gβ\u3csub\u3e5\u3c/sub\u3e Complex and Accelerates Inactivation of the mGluR6-G\u3csub\u3e0\u3c/sub\u3e Signaling

The R7 subfamily of RGS proteins critically regulates neuronal G protein-signaling pathways that are essential for vision, nociception, motor coordination, and reward processing. A member of the R7 RGS family, RGS11, is a GTPase-accelerating protein specifically expressed in retinal ON-bipolar cells where it forms complexes with the atypical G protein β subunit, Gβ5, and transmembrane protein R9AP. Association with R9AP has been shown to be critical for the proteolytic stability of the complex in the retina. In this study we report that R9AP can in addition stimulate the GTPase-accelerating protein activity of the RGS11·Gβ5 complex at Gαo. Single turnover GTPase assays reveal that R9AP co-localizes RGS11·Gβ5 and Gαo on the membrane and allosterically potentiates the GTPase-accelerating function of RGS11·Gβ5. Reconstitution of mGluR6-Gαo signaling in Xenopus oocytes indicates that RGS11·Gβ5-mediated GTPase acceleration in this system requires co-expression of R9AP. The results provide new insight into the regulation of mGluR6-Gαo signaling by the RGS11·Gβ5·R9AP complex and establish R9AP as a general GTPase-accelerating protein activity regulator of R7 RGS complexes

Removal of Giα1 Constraints on Adenylyl Cyclase in the Hippocampus Enhances LTP and Impairs Memory Formation

AbstractStimulation of adenylyl cyclase in the hippocampus is critical for memory formation. However, generation of cAMP signals within an optimal range for memory may require a balance between stimulatory and inhibitory mechanisms. The role of adenylyl cyclase inhibitory mechanisms for memory has not been addressed. One of the mechanisms for inhibition of adenylyl cyclase is through activation of Gi-coupled receptors, a mechanism that could serve as a constraint on memory formation. Here we report that ablation of Giα1 by gene disruption increases hippocampal adenylyl cyclase activity and enhances LTP in area CA1. Furthermore, gene ablation of Giα1 or antisense oligonucleotide-mediated depletion of Giα1 disrupted hippocampus-dependent memory. We conclude that Giα1 provides a critical mechanism for tonic inhibition of adenylyl cyclase activity in the hippocampus. We hypothesize that loss of Giα1 amplifies the responsiveness of CA1 postsynaptic neurons to stimuli that strengthen synaptic efficacy, thereby diminishing synapse-specific plasticity required for new memory formation

Distinct domains of the -opioid receptor control uncoupling and internalization.

ABSTRACT Homologous desensitization of the opioid receptor (OR) can be resolved into distinct processes that include the uncoupling of the OR from its G-protein effectors and internalization of cell surface receptors. Using electrophysiological recordings of OR activation of G-protein-coupled K ϩ channels (K ir 3) in Xenopus laevis oocytes and AtT20 cells, confocal microscopy of receptor localization, and radioligand binding of cell surface receptors, we resolved these desensitization mechanisms to determine the domain of OR important for receptor uncoupling. Activation of OR by saturating concentrations of [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO), methadone, or fentanyl, but not morphine, produced robust internalization of a green fluorescent protein-tagged OR. A subsaturating concentration of DAMGO (100 nM) did not cause receptor internalization but markedly reduced the subsequent responsiveness of K ir 3 by uncoupling OR. OR desensitization in AtT20 cells was confirmed to be homologous, because desensitization by 100 nM DAMGO was blocked by dominant-negative forms of either G protein-coupled receptor kinase (GRK) or arrestin, and pretreatment with DAMGO did not affect the K ir 3 response to somatostatin receptor activation. Alanine substitution of a single threonine in the second cytoplasmic loop of the OR (Threonine 180) blocked agonist-dependent receptor uncoupling without affecting receptor internalization. These results suggest that GRK-dependent phosphorylation of OR required threonine 180 for uncoupling but that a different GRK and arrestin-dependent mechanism controlled OR internalization in AtT20 cells

A Polymorphic Microdeletion in the RGS9 Gene Suppresses PTB Binding and Associates with Obesity

Objective: RGS9 is a member of the family of Regulators of G-Protein Signaling (RGS) proteins defined by the presence of an RGS domain which can accelerate the GTPase-activity of G protein Gα subunits. An insertion/deletion (I/D) polymorphism of the nucleotide sequence TTTCT (rs3215227) has been identified in the human RGS9 gene, which matches the consensus high affinity binding motif for the ubiquitously expressed RNA binding Polypyrimidine Tract Binding Protein (PTB). In this study, we evaluate the genetic association and functional relevance of this polymorphism in type 2 diabetes and obesity. Subjects and methods: We genotyped a larger population of 9272 Chinese and Malaysian individuals for the RGS9 I/D polymorphism using TaqMan allelic discrimination protocols. We found that the D allele of the RGS9 polymorphism was associated with a decreased prevalence of obesity in women (P=0.003, OR=0.753 95%CI 0.625-0.906) and girls (P=0.002, OR=0.604 95%CI 0.437-0.835). The association was moderate in boys (P=0.038, OR=0.724 95%CI 0.533-0.983) and not significant in men. Furthermore, we found that the transcript deletion variant exhibited a 10-fold reduction in PTB binding in vitro and that the splicing of the deletion variant was less affected by PTB co-expression. Conclusions: We provide genetic and biochemical data to support a genetic role of RGS9 in obesity but unlikely in T2D. The RGS9 I/D polymorphism influence the post-transcriptional processing of the gene through an altered affinity for the splicing factor PTB and are associated with obesity

Lack of Antinociceptive Cross-Tolerance With Co-Administration of Morphine and Fentanyl Into the Periaqueductal Gray of Male Sprague-Dawley Rats

Tolerance to the antinociceptive effect of mu-opioid receptor (MOPr) agonists, such as morphine and fentanyl, greatly limits their effectiveness for long-term use to treat pain. Clinical studies have shown that combination therapy and opioid rotation can be used to enhance opioid-induced antinociception once tolerance has developed. The mechanism and brain regions involved in these processes are unknown. The purpose of this study was to evaluate the contribution of the ventrolateral periaqueductal gray (vlPAG) to antinociceptive tolerance and cross-tolerance between administration and co- administration of morphine and fentanyl. Tolerance was induced by pretreating rats with morphine or fentanyl or low-dose combination of morphine and fentanyl into the vlPAG followed by assessment of cross-tolerance to the other opioid. In addition, tolerance to the combined treatment was assessed. Cross-tolerance did not develop between repeated vlPAG microinjections of morphine and fentanyl. Likewise, there was no evidence of cross-tolerance from morphine or fentanyl to co-administration of morphine and fentanyl. Co-administration did not cause cross-tolerance to fentanyl. Cross- tolerance was only evident to morphine or morphine and fentanyl combined in rats pretreated with co-administration of low-doses of morphine and fentanyl. In conclusion, cross-tolerance does not develop between morphine and fentanyl within the vlPAG. This finding is consistent with the functionally selective signaling that has been reported for antinociception and tolerance following morphine and fentanyl binding to the MOPr. This research supports the notion that combination therapy and opioid rotation may be useful clinical practices to reduce opioid tolerance and other side effects. Perspective: This preclinical study shows that there is a reduction in cross tolerance between morphine and fentanyl within the periaqueductal gray which is key brain region in opioid antinociception and tolerance

Association between Regulator of G Protein Signaling 9–2 and Body Weight

Regulator of G protein signaling 9–2 (RGS9–2) is a protein that is highly enriched in the striatum, a brain region that mediates motivation, movement and reward responses. We identified a naturally occurring 5 nucleotide deletion polymorphism in the human RGS9 gene and found that the mean body mass index (BMI) of individuals with the deletion was significantly higher than those without. A splicing reporter minigene assay demonstrated that the deletion had the potential to significantly decrease the levels of correctly spliced RGS9 gene product. We measured the weights of rats after virally transduced overexpression of RGS9–2 or the structurally related RGS proteins, RGS7, or RGS11, in the nucleus accumbens (NAc) and observed a reduction in body weight after overexpression of RGS9–2 but not RGS7 or 11. Conversely, we found that the RGS9 knockout mice were heavier than their wild-type littermates and had significantly higher percentages of abdominal fat. The constituent adipocytes were found to have a mean cross-sectional area that was more than double that of corresponding cells from wild-type mice. However, food intake and locomotion were not significantly different between the two strains. These studies with humans, rats and mice implicate RGS9–2 as a factor in regulating body weight.National Institute of Mental Health (U.S.) (R41MH78570 award)National Center for Research Resources (U.S.) (Rhode Island IDeA Network of Biomedical Research Excellence (RI-INBRE) Award P20RR016457-10

Association between Regulator of G Protein Signaling 9-2 and Body Weight

Abstract Regulator of G protein signaling 9-2 (RGS9-2) is a protein that is highly enriched in the striatum, a brain region that mediates motivation, movement and reward responses. We identified a naturally occurring 5 nucleotide deletion polymorphism in the human RGS9 gene and found that the mean body mass index (BMI) of individuals with the deletion was significantly higher than those without. A splicing reporter minigene assay demonstrated that the deletion had the potential to significantly decrease the levels of correctly spliced RGS9 gene product. We measured the weights of rats after virally transduced overexpression of RGS9-2 or the structurally related RGS proteins, RGS7, or RGS11, in the nucleus accumbens (NAc) and observed a reduction in body weight after overexpression of RGS9-2 but not RGS7 or 11. Conversely, we found that the RGS9 knockout mice were heavier than their wild-type littermates and had significantly higher percentages of abdominal fat. The constituent adipocytes were found to have a mean cross-sectional area that was more than double that of corresponding cells from wild-type mice. However, food intake and locomotion were not significantly different between the two strains. These studies with humans, rats and mice implicate RGS9-2 as a factor in regulating body weight