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David P Siderovski - One of the best experts on this subject based on the ideXlab platform.
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the effect of rgs12 on pdgfβ receptor signalling to p42 p44 mitogen activated protein kinase in mammalian cells
Cellular Signalling, 2006Co-Authors: Balwinder Sambi, David P Siderovski, Melinda D. Hains, Catherine M. Waters, Michelle Connell, Francis S. Willard, Adam J. Kimple, Susan Pyne, Nigel J. PyneAbstract:We have previously shown that the PDGFβ receptor uses a classical GPCR-mediated pathway in order to induce efficient activation of p42/p44 MAPK in response to PDGF. We therefore, considered the possibility that GTPase accelerating proteins (RGS proteins), which regulate GPCR signalling, modulate PDGFβ receptor-mediated signal transmission. Several lines of evidence were obtained to support functional interaction between the PDGFβ receptor and RGS12 in HEK 293 and airway smooth muscle cells. Firstly, the over-expression of the RGS12 PDZ/PTB domain N-terminus or RGS12 PTB domain reduced the PDGF-induced activation of p42/p44 MAPK. Secondly, the RGS12 PDZ/PTB domain N-terminus and RGS12 PDZ domain can form a complex with the PDGFβ receptor. Therefore, the results presented here provide the first evidence to support the concept that the PDZ/PTB domain N-terminus and/or the PTB domain of RGS12 may modulate PDGFβ receptor signalling. In airway smooth muscle cells, over-expressed recombinant RGS12 and the isolated PDZ/PTB domain N-terminus co-localised with PDGFβ receptor in cytoplasmic vesicles. To provide additional evidence for a role of the PDZ/PTB domain N-terminus, we used RGS14. RGS14 has the same C-terminal domain architecture of an RGS box, tandem Ras-binding domains (RBDs) and GoLoco motif as RGS12, but lacks the PDZ/PTB domain N-terminus. In this regard, RGS14 exhibited a different sub-cellular distribution compared with RGS12, being diffusely distributed in ASM cells. These findings suggest that RGS12 via its PDZ/PTB domain N-terminus may regulate trafficking of the PDGFβ receptor in ASM cells.
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The effect of RGS12 on PDGFβ receptor signalling to p42/p44 mitogen activated protein kinase in mammalian cells
Cellular signalling, 2005Co-Authors: Balwinder Sambi, David P Siderovski, Melinda D. Hains, Catherine M. Waters, Michelle Connell, Francis S. Willard, Adam J. Kimple, Susan Pyne, Nigel J. PyneAbstract:We have previously shown that the PDGFbeta receptor uses a classical GPCR-mediated pathway in order to induce efficient activation of p42/p44 MAPK in response to PDGF. We therefore, considered the possibility that GTPase accelerating proteins (RGS proteins), which regulate GPCR signalling, modulate PDGFbeta receptor-mediated signal transmission. Several lines of evidence were obtained to support functional interaction between the PDGFbeta receptor and RGS12 in HEK 293 and airway smooth muscle cells. Firstly, the over-expression of the RGS12 PDZ/PTB domain N-terminus or RGS12 PTB domain reduced the PDGF-induced activation of p42/p44 MAPK. Secondly, the RGS12 PDZ/PTB domain N-terminus and RGS12 PDZ domain can form a complex with the PDGFbeta receptor. Therefore, the results presented here provide the first evidence to support the concept that the PDZ/PTB domain N-terminus and/or the PTB domain of RGS12 may modulate PDGFbeta receptor signalling. In airway smooth muscle cells, over-expressed recombinant RGS12 and the isolated PDZ/PTB domain N-terminus co-localised with PDGFbeta receptor in cytoplasmic vesicles. To provide additional evidence for a role of the PDZ/PTB domain N-terminus, we used RGS14. RGS14 has the same C-terminal domain architecture of an RGS box, tandem Ras-binding domains (RBDs) and GoLoco motif as RGS12, but lacks the PDZ/PTB domain N-terminus. In this regard, RGS14 exhibited a different sub-cellular distribution compared with RGS12, being diffusely distributed in ASM cells. These findings suggest that RGS12 via its PDZ/PTB domain N-terminus may regulate trafficking of the PDGFbeta receptor in ASM cells.
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Differential expression of regulator of G-protein signaling R12 subfamily members during mouse development
Developmental dynamics : an official publication of the American Association of Anatomists, 2005Co-Authors: Luke Martin-mccaffrey, David P Siderovski, Melinda D. Hains, Grant A. Pritchard, Agnieszka Pajak, Lina Dagnino, Sudhir J.a. D'souzaAbstract:Regulators of G-protein Signaling (RGS proteins) are a multigene family of GTPase-accelerating proteins for the Galpha subunit of heterotrimeric G-proteins. The mammalian R12 RGS protein subfamily is composed of RGS12 and RGS14, two proteins characterized by their multidomain architecture of hallmark RGS domain, tandem Ras-binding domains (RBDs), and a second Galpha interacting domain, the GoLoco motif. The Rgs12 gene generates multiple splice variants, the largest of which encodes N-terminal PDZ and PTB domains in addition to the core RGS/RBD/GoLoco motifs. The Rgs14 gene encodes a protein similar to the non-PDZ/PTB domain RGS12 splice variants. The spatiotemporal expression patterns of RGS12 and RGS14 proteins were examined by immunohistochemistry in a developmental series of postimplantation mouse embryo. We report that RGS12 splice variants exhibit differential spatiotemporal patterns of expression during postimplantation embryogenesis, suggesting nonoverlapping roles. In contrast, RGS14 is found ubiquitously throughout the postimplantation period. We conclude that R12 subfamily RGS proteins likely play significant and different roles in specific tissues and periods of mouse embryogenesis.
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Return of the GDI: The GoLoco Motif in Cell Division
Annual review of biochemistry, 2004Co-Authors: Francis S. Willard, Randall J Kimple, David P SiderovskiAbstract:The GoLoco motif is a 19-amino-acid sequence with guanine nucleotide dissociation inhibitor activity against G-alpha subunits of the adenylyl-cyclase-inhibitory subclass. The GoLoco motif is present as an independent element within multidomain signaling regulators, such as Loco, RGS12, RGS14, and Rap1GAP, as well as in tandem arrays in proteins, such as AGS3, G18, LGN, Pcp-2/L7, and Partner of Inscuteable (Pins/Rapsynoid). Here we discuss the biochemical mechanisms of GoLoco motif action on G-alpha subunits in light of the recent crystal structure of G-alpha-i1 bound to the RGS14 GoLoco motif. Currently, there is sparse evidence for GoLoco motif regulation of canonical G-protein-coupled receptor signaling. Rather, studies of asymmetric cell division in Drosophila and Caenorhabditis elegans, as well as mammalian mitosis, implicate GoLoco proteins, such as Pins, GPR-1/GPR-2, LGN, and RGS14, in mitotic spindle organization and force generation. We discuss potential mechanisms by which GoLoco/Galpha complexes might modulate spindle dynamics.
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Purification and in vitro functional analyses of RGS12 and RGS14 GoLoco motif peptides
Methods in enzymology, 2004Co-Authors: Randall J Kimple, Francis S. Willard, David P SiderovskiAbstract:The GoLoco motif is a short polypeptide sequence that binds to heterotrimeric G-protein alpha subunits of the adenylyl cyclase-inhibitory (Galpha(i/o)) subclass in a nucleotide-dependent manner (i.e., solely to the GDP-bound ground state). This article describes methods used for the expression, purification, and in vitro evaluation of membrane-permeant tag fusion peptides derived from the GoLoco motif regions of "regulator of G-protein signaling" proteins type 12 (RGS12) and 14 (RGS14) and a consensus GoLoco sequence from the multiple GoLoco motif protein AGS3. Three different fluorescence-based assays are described for evaluating the in vitro function of these GoLoco peptides as guanine nucleotide dissociation inhibitors, including measurements of GTPgammaS binding and Galpha subunit activation by the planar ion aluminum tetrafluoride.
Neng-yao Shih - One of the best experts on this subject based on the ideXlab platform.
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Diagnosing the RGS11 Lung Cancer Biomarker: The Integration of Competitive Immunoassay and Isothermal Nucleic Acid Exponential Amplification Reaction.
Analytical chemistry, 2019Co-Authors: Ying-feng Chang, Yi-qi Huang, Amily Fang-ju Jou, Neng-yao ShihAbstract:Lung cancer is the primary cause of cancer-associated mortality worldwide, which makes the identification of reliable lung cancer biomarkers a pressing need for early diagnosis and prognosis. RGS11, which is a regulator of G-protein signaling and also a lung cancer biomarker, plays an important role in cancer-related metastasis. However, trace levels of RGS11 (in the range of pg/mL) in serum samples make it difficult to quantify using currently available enzyme-linked immunosorbent assay (ELISA) kits and, therefore, this hinders progress in the discovery of new approaches for treating lung cancer. The aim of this study is to develop a rapid, sensitive, and reliable platform for the detection of RGS11 lung cancer biomarker based on a suspension immunoassay coupled with an isothermal exponential amplification strategy. Our study was initiated by the functionalization of magnetic beads with anti-RGS11 antibodies (Ab-MB) by EDC (1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide)/NHS (N-hydroxysulfosuccinimide)...
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Diagnosing the RGS11 Lung Cancer Biomarker: The Integration of Competitive Immunoassay and Isothermal Nucleic Acid Exponential Amplification Reaction
2019Co-Authors: Ying-feng Chang, Yi-qi Huang, Amily Fang-ju Jou, Neng-yao ShihAbstract:Lung cancer is the primary cause of cancer-associated mortality worldwide, which makes the identification of reliable lung cancer biomarkers a pressing need for early diagnosis and prognosis. RGS11, which is a regulator of G-protein signaling and also a lung cancer biomarker, plays an important role in cancer-related metastasis. However, trace levels of RGS11 (in the range of pg/mL) in serum samples make it difficult to quantify using currently available enzyme-linked immunosorbent assay (ELISA) kits and, therefore, this hinders progress in the discovery of new approaches for treating lung cancer. The aim of this study is to develop a rapid, sensitive, and reliable platform for the detection of RGS11 lung cancer biomarker based on a suspension immunoassay coupled with an isothermal exponential amplification strategy. Our study was initiated by the functionalization of magnetic beads with anti-RGS11 antibodies (Ab-MB) by EDC (1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide)/NHS (N-hydroxysulfosuccinimide) activation. Ab-MB served as a sensing probe for the competitive immunorecognitions between known concentrations of His-tag RGS11 and unknown concentrations of target RGS11 in serum. The reporter anti-His antibodies, which were modified with primers that induced an isothermal exponential amplification reaction, were subsequently introduced to the reaction mixture that resulted in the formation of immunosandwich complexes. The exponentially amplified DNA duplex that was intercalated with SYBR Green was designated as a signal reporter for the assessment of RGS11 in an inversely proportional relationship. The sensing platform was excellent for the determination of RGS11 with an exceptional detection limit of 148 fg/mL and a linear dynamic range of 0.1–10 pg/mL using a minimal sample volume (20 μL) and with a reaction time of 1.5 h. In addition, we challenged the sensing platform with RGS11-spiked samples (in 2× diluted serum), and an acceptable recovery rate (>90%) was observed. Finally, 24 clinical samples acquired from patients with advanced lung cancer (C), inflammation (I), and heart failure (H) were analyzed by this newly developed sensing platform and a commercial ELISA kit for validation. This sensing platform has potential in biomedical applications for clinically diagnosing liquid biopsy samples for patients with lung cancer. Moreover, the universal design of our proposed system is easily adapted to detect any other protein if a His-tag recombinant protein is available
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Overexpression of regulator of G protein signaling 11 promotes cell migration and associates with advanced stages and aggressiveness of lung adenocarcinoma.
Oncotarget, 2016Co-Authors: Sheng-huei Yang, Pei-yi Chu, Li-tzong Chen, Wan-wen Chen, Chia-hung Han, Jr-hau Lung, Neng-yao ShihAbstract:Regulator of G protein signaling 11 (RGS11), a member of the R7 subfamily of RGS proteins, is a well-characterized GTPase-accelerating protein that is involved in the heterotrimeric G protein regulation of the amplitude and kinetics of receptor-promoted signaling in retinal bipolar and nerve cells. However, the role of RGS11 in cancer is completely unclear. Using subtractive hybridization analysis, we found that RGS11 was highly expressed in the lymph-node metastatic tissues and bone-metastatic tumors obtained from patients with lung adenocarcinoma. Characterization of the clinicopathological features of 91 patients showed that around 57.1% of the tumor samples displayed RGS11 overexpression that was associated with primary tumor status, nodal metastasis and increased disease stages. Its high expression was an independent predictive factor for poor prognosis of these patients. Cotransfection of guanine nucleotide-binding protein beta-5 (GNB5) markedly increased RGS11 expression. Enhancement or attenuation of RGS11 expression pinpointed its specific role in cell migration, but not in cell invasion and proliferation. Signaling events initiated by the RGS11-GNB5 coexpression activated the c-Raf/ERK/FAK-mediated pathway through upregulation of the Rac1 activity. Consistently, increasing the cell invasiveness of the transfectants by additional cotransfection of the exogenous urokinase-plasminogen activator gene caused a significant promotion in cell invasion in vitro and in vivo, confirming that RGS11 functions in cell migration, but requires additional proteolytic activity for cell and tissue invasion. Collectively, overexpression of RGS11 promotes cell migration, participates in tumor metastasis, and correlates the clinicopathological conditions of patients with lung adenocarcinoma.
Key-sun Kim - One of the best experts on this subject based on the ideXlab platform.
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RGS11 interacts preferentially with r7bp over gαoa characterization of gβ5 free RGS11
Biochemical and Biophysical Research Communications, 2009Co-Authors: Yasar Saleem, Key-sun KimAbstract:Abstract Regulator of G protein signaling 11 (RGS11) is the least characterized member of the R7 family of Gγ-like GGL domain-containing RGS proteins. All R7-RGS proteins of a variety of cell types are found in Gβ5-containing complexes that exhibit a number of unique functional properties. However, presence of Gβ5 reduced the affinity of R7-RGS7 for Gα subunits, also only RGS7 bound to Muscarinic M3-Receptor, but the Gβ5-RGS7 dimer did not, making it difficult to study differential interaction of R7-RGS proteins. Here, we report the successful purification of functionally intact, Gβ5-free recombinant RGS11 (rRGS11), obtained by expressing N- and C-terminally truncated form of RGS11 in Escherichia coli BL 21 (DE3), that differentially interact with R7BP and Gαoa. rRGS11 was capable of interacting with Gαoa and R7BP (RGS7 family binding protein) with equilibrium dissociation constants (KD) of 904 (±208) nM, and 308 (±97) nM, respectively. It also induced several-fold increase in the GTPase activity of Gαoa. The binding of rRGS11 was differential with a binding preference for R7BP over Gαoa implying extended roles of R7BP. In addition, we identified a novel interaction between Gαoa and R7BP with a KD of 592 (±150) nM. The production of stable and functional rRGS11 would provide chances to discover more functions of RGS11 yet to be identified.
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RGS11 interacts preferentially with R7BP over Gαoa – Characterization of Gβ5-free RGS11
Biochemical and biophysical research communications, 2009Co-Authors: Yasar Saleem, Key-sun KimAbstract:Abstract Regulator of G protein signaling 11 (RGS11) is the least characterized member of the R7 family of Gγ-like GGL domain-containing RGS proteins. All R7-RGS proteins of a variety of cell types are found in Gβ5-containing complexes that exhibit a number of unique functional properties. However, presence of Gβ5 reduced the affinity of R7-RGS7 for Gα subunits, also only RGS7 bound to Muscarinic M3-Receptor, but the Gβ5-RGS7 dimer did not, making it difficult to study differential interaction of R7-RGS proteins. Here, we report the successful purification of functionally intact, Gβ5-free recombinant RGS11 (rRGS11), obtained by expressing N- and C-terminally truncated form of RGS11 in Escherichia coli BL 21 (DE3), that differentially interact with R7BP and Gαoa. rRGS11 was capable of interacting with Gαoa and R7BP (RGS7 family binding protein) with equilibrium dissociation constants (KD) of 904 (±208) nM, and 308 (±97) nM, respectively. It also induced several-fold increase in the GTPase activity of Gαoa. The binding of rRGS11 was differential with a binding preference for R7BP over Gαoa implying extended roles of R7BP. In addition, we identified a novel interaction between Gαoa and R7BP with a KD of 592 (±150) nM. The production of stable and functional rRGS11 would provide chances to discover more functions of RGS11 yet to be identified.
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RGS11 interacts preferentially with R7BP over Gαoa – Characterization of Gβ5-free RGS11
Biochemical and biophysical research communications, 2009Co-Authors: Yasar Saleem, Key-sun KimAbstract:Regulator of G protein signaling 11 (RGS11) is the least characterized member of the R7 family of Ggamma-like GGL domain-containing RGS proteins. All R7-RGS proteins of a variety of cell types are found in Gbeta5-containing complexes that exhibit a number of unique functional properties. However, presence of Gbeta5 reduced the affinity of R7-RGS7 for Galpha subunits, also only RGS7 bound to Muscarinic M3-Receptor, but the Gbeta5-RGS7 dimer did not, making it difficult to study differential interaction of R7-RGS proteins. Here, we report the successful purification of functionally intact, Gbeta5-free recombinant RGS11 (rRGS11), obtained by expressing N- and C-terminally truncated form of RGS11 in Escherichia coli BL 21 (DE3), that differentially interact with R7BP and Galpha(oa). rRGS11 was capable of interacting with Galpha(oa) and R7BP (RGS7 family binding protein) with equilibrium dissociation constants (K(D)) of 904 (+/- 208) nM, and 308 (+/- 97) nM, respectively. It also induced several-fold increase in the GTPase activity of Galpha(oa). The binding of rRGS11 was differential with a binding preference for R7BP over Galpha(oa) implying extended roles of R7BP. In addition, we identified a novel interaction between Galpha(oa) and R7BP with a K(D) of 592 (+/- 150) nM. The production of stable and functional rRGS11 would provide chances to discover more functions of RGS11 yet to be identified.
Pilar Sánchez-blázquez - One of the best experts on this subject based on the ideXlab platform.
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The RGSZ2 Protein Exists in a Complex with μ-Opioid Receptors and Regulates the Desensitizing Capacity of Gz Proteins
Neuropsychopharmacology, 2005Co-Authors: Javier Garzon, Almudena López-fando, María Rodríguez-muñoz, Pilar Sánchez-blázquezAbstract:The regulator of G-protein signaling RGS17(Z2) is a member of the RGS-Rz subfamily of GTPase-activating proteins (GAP) that efficiently deactivate G α zGTP subunits. We have found that in the central nervous system (CNS), the levels of RGSZ2 mRNA and protein are elevated in the hypothalamus, midbrain, and pons-medulla, and that RGSZ2 is glycosylated in synaptosomal membranes isolated from CNS tissue. In analyzing the function of RGSZ2 in the CNS, we found that when the expression of RGSZ2 was impaired, the antinociceptive response to morphine and [ D -Ala^2, N -MePhe^4, Gly-ol^5]-enkephalin (DAMGO) augmented. This potentiation involved μ -opioid receptors and increased tolerance to further doses of these agonists administered 24 h later. High doses of morphine promoted agonist desensitization even within the analgesia time-course, a phenomenon that appears to be related to the great capacity of morphine to activate Gz proteins. In contrast, the knockdown of RGSZ2 proteins did not affect the activity of δ receptor agonists, [ D -Pen^2,5]-enkephalin (DPDPE), and [ D -Ala^2] deltorphin II. In membranes from periaqueductal gray matter (PAG), both RGSZ2 and the related RGS20(Z1) co-precipitated with μ -opioid receptors. While a morphine challenge reduced the association of Gi/o/z with μ receptors, it increased their association with the RGSZ2 and RGSZ1 proteins. However, only G α z subunits co-precipitated with RGSZ2. Doses of morphine that produced acute tolerance maintained the association of G α subunits with RGSZ proteins even after the analgesic effects had ceased. These results indicate that both RGSZ1 and RGSZ2 proteins influence μ receptor signaling by sequestering G α subunits, therefore behaving as effector antagonists.
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RGSZ1 and GAIP Regulate μ- but Not δ-Opioid Receptors in Mouse CNS: Role in Tachyphylaxis and Acute Tolerance
Neuropsychopharmacology, 2004Co-Authors: Javier Garzon, Almudena López-fando, María Rodríguez-muñoz, Antonio Garcia-españa, Pilar Sánchez-blázquezAbstract:In the CNS, the regulators of G-protein signaling (RGS) proteins belonging to the Rz subfamily, RGS19 (G α interacting protein (GAIP)) and RGS20 (Z1), control the activity of opioid agonists at μ but not at δ receptors. Rz proteins show high selectivity in deactivating G α z-GTP subunits. After reducing the expression of RGSZ1 with antisense oligodeoxynucleotides (ODN), the supraspinal antinociception produced by morphine, heroin, DAMGO ([D-Ala^2, N -MePhe^4,Gly-ol^5]-enkephalin), and endomorphin-1 was notably increased. No change was observed in the effect of endomorphin-2. This agrees with the proposed existence of different μ receptors for the endomorphins. The activities of DPDPE ([D-Pen^2,5]-enkephalin) and [D-Ala^2] deltorphin II, agonists at δ receptors, were also unchanged. Knockdown of GAIP and of the GAIP interacting protein C-terminus (GIPC) led to changes in agonist effects at μ but not at δ receptors. The impairment of RGSZ1 extended the duration of morphine analgesia by at least 1 h beyond that observed in control animals. CTOP (Cys^2, Tyr^3, Orn^5, Pen^7-amide) antagonized morphine analgesia when given during the period in which the effect of morphine was enhanced by RGSZ1 knockdown. Thus, in naive mice, morphine tachyphylaxis originated in the presence of the opioid agonist and during the analgesia time course. The knockdown of RGSZ1 facilitated the development of tolerance to a single dose of morphine and accelerated tolerance to continuous delivery of the opioid. These results indicate that μ but not δ receptors are linked to Rz regulation. The μ receptor-mediated activation of Gz proteins is effective at recruiting the adaptive mechanisms leading to the development of opioid desensitization.
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The R7 Subfamily of RGS Proteins Assists Tachyphylaxis and Acute Tolerance at μ -Opioid Receptors
Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, 2003Co-Authors: Javier Garzon, Almudena López-fando, Pilar Sánchez-blázquezAbstract:Members of the R7 subfamily of regulators of G-protein signaling (RGS) proteins (RGS6, RGS7, RGS9-2, and RGS11) are found in the mouse CNS. The expression of these proteins was effectively reduced in different neural structures by blocking their mRNA with antisense oligodeoxynucleotides (ODNs). This was achieved without noticeable changes in the binding characteristics of labeled beta-endorphin to opioid receptors. Knockdown of R7 proteins enhanced the potency of antinociception promoted by morphine and [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO)-both agonists at mu-opioid receptors. The duration of morphine analgesia was greatly increased in RGS9-2 and in RGS11 knockdown mice. The impairment of R7 proteins brought about different changes in the analgesic activity of selective delta agonists. Knockdown of RGS11 reduced [D-Ala(2)]deltorphin II analgesic effects. Those of RGS6 and RGS9-2 proteins caused [D-Ala(2)]deltorphin II to produce a smoothened time-course curve-the peak effect blunted and analgesia extended during the declining phase. RGS9-2 impairment also promoted a similar pattern of change for [D-Pen(2,5)]-enkephalin (DPDPE). RGS7-deficient mice showed an increased response to both [D-Ala(2)]deltorphin II and DPDPE analgesic effects. A single intracerebroventricular (i.c.v.) ED(80) analgesic dose of morphine gave rise to acute tolerance in control mice, but did not promote tolerance in RGS6, RGS7, RGS9-2, or RGS11 knockdown animals. Thus, R7 proteins play a critical role in agonist tachyphylaxis and acute tolerance at mu-opioid receptors, and show differences in their modulation of delta-opioid receptors.
Pilar Sanchezblazquez - One of the best experts on this subject based on the ideXlab platform.
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morphine alters the selective association between mu opioid receptors and specific rgs proteins in mouse periaqueductal gray matter
Neuropharmacology, 2005Co-Authors: Maria Rodriguezmunoz, Pilar SanchezblazquezAbstract:Abstract In the CNS, several regulators of G-protein signalling (RGS) modulate the activity of mu-opioid receptors. In pull-down assays performed on membranes from mouse periaqueductal gray matter (PAG), mu-opioid receptors co-precipitated with delta-opioid receptors, Gi/o/z/q proteins, and the regulators of G-protein signalling RGS4, RGS9-2, RGS14, RGSZ1 and RGSZ2. No RGS2, RGS7, RGS10 and RGS11 proteins were associated with the mu receptors in these PAG membranes. In mice, an intracerebroventricular dose of 10 nmol morphine produced acute tolerance at mu receptors but did not disrupt the co-precipitation of mu-delta receptor complexes. However, this opioid reduced by more than 50% the co-precipitation of Gαi/o/z subunits with mu receptors, and altered their association with some of the RGS proteins at 30 min, 3 h and 24 h after its administration. The association of RGS9-2 with mu receptors diminished by 30–40% 24 h after the administration of morphine, while that of RGSZ2 and of RGSZ1 increased. Morphine treatment recruited RGS4 to the PAG membranes, and 30 min and 3 h after the opioid challenge its association with mu receptors had increased. However, 24 h after morphine administration, the co-precipitation of RGS4 had decreased by about 30%. The opioid produced no change in the membrane levels of RGS9-2, RGS14, RGSZ1 and RGSZ2. Thus, in PAG synaptosomal membranes, a dynamic and selective link exists between, mu-opioid receptors, Gi/o/z proteins and certain RGS proteins.
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the r7 subfamily of rgs proteins assists tachyphylaxis and acute tolerance at μ opioid receptors
Neuropsychopharmacology, 2003Co-Authors: Almudena Lopezfando, Pilar SanchezblazquezAbstract:Members of the R7 subfamily of regulators of G-protein signaling (RGS) proteins (RGS6, RGS7, RGS9-2, and RGS11) are found in the mouse CNS. The expression of these proteins was effectively reduced in different neural structures by blocking their mRNA with antisense oligodeoxynucleotides (ODNs). This was achieved without noticeable changes in the binding characteristics of labeled β-endorphin to opioid receptors. Knockdown of R7 proteins enhanced the potency of antinociception promoted by morphine and [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin (DAMGO)—both agonists at μ-opioid receptors. The duration of morphine analgesia was greatly increased in RGS9-2 and in RGS11 knockdown mice. The impairment of R7 proteins brought about different changes in the analgesic activity of selective δ agonists. Knockdown of RGS11 reduced [D-Ala2]deltorphin II analgesic effects. Those of RGS6 and RGS9-2 proteins caused [D-Ala2]deltorphin II to produce a smoothened time-course curve—the peak effect blunted and analgesia extended during the declining phase. RGS9-2 impairment also promoted a similar pattern of change for [D-Pen2,5]-enkephalin (DPDPE). RGS7-deficient mice showed an increased response to both [D-Ala2]deltorphin II and DPDPE analgesic effects. A single intracerebroventricular (i.c.v.) ED80 analgesic dose of morphine gave rise to acute tolerance in control mice, but did not promote tolerance in RGS6, RGS7, RGS9-2, or RGS11 knockdown animals. Thus, R7 proteins play a critical role in agonist tachyphylaxis and acute tolerance at μ-opioid receptors, and show differences in their modulation of δ-opioid receptors.