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

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

G protein beta 5 is targeted to D2-dopamine receptor-containing biochemical compartments and blocks dopamine-dependent receptor internalization.

G beta 5 (Gbeta5, Gβ5) is a unique G protein β subunit that is thought to be expressed as an obligate heterodimer with R7 regulator of G protein signaling (RGS) proteins instead of with G gamma (Gγ) subunits. We found that D2-dopamine receptor (D2R) coexpression enhances the expression of Gβ5, but not that of the G beta 1 (Gβ1) subunit, in HEK293 cells, and that the enhancement of expression occurs through a stabilization of Gβ5 protein. We had previously demonstrated that the vast majority of D2R either expressed endogenously in the brain or exogenously in cell lines segregates into detergent-resistant biochemical fractions. We report that when expressed alone in HEK293 cells, Gβ5 is highly soluble, but is retargeted to the detergent-resistant fraction after D2R coexpression. Furthermore, an in-cell biotin transfer proximity assay indicated that D2R and Gβ5 segregating into the detergent-resistant fraction specifically interacted in intact living cell membranes. Dopamine-induced D2R internalization was blocked by coexpression of Gβ5, but not Gβ1. However, the same Gβ5 coexpression levels had no effect on agonist-induced internalization of the mu opioid receptor (MOR), cell surface D2R levels, dopamine-mediated recruitment of β-arrestin to D2R, the amplitude of D2R-G protein coupling, or the deactivation kinetics of D2R-activated G protein signals. The latter data suggest that the interactions between D2R and Gβ5 are not mediated by endogenously expressed R7 RGS proteins

Fluorescence-based, high-throughput assays for μ-opioid receptor activation using a membrane potential-sensitive dye

The development of new and improved opioid analgesics requires high-throughput screening (HTS) methods to identify potential therapeutics from large libraries of lead compounds. Here we describe two simple, real-time fluorescence-based assays of μ-opioid receptor activation that may be scaled up for HTS. In AtT-20 cells expressing the μ-opioid receptor (MOPr), opioids activate endogenous G protein gated inwardly rectifying K channels (GIRK channels), leading to membrane hyperpolarization. In Chinese hamster ovary cells expressing MOPr, adenylyl cyclase activation via forskolin results in membrane hyperpolarization, which is inhibited by opioids. Changes in membrane potential can be measured using a proprietary membrane potential-sensitive dye. In contrast to many HTS methods currently available, these assays reflect naturalistic coupling of the receptor to effector molecules.9 page(s

Coexpression of D2R enhances the expression of Gβ5 but not of Gβ1 and coexpression of MOR does not significantly alter expression of Gβ5.

<p><b>A.</b> Representative images from a Western blot depicting the relative levels of expression of Gβ5 (upper panels) and total cellular protein (lower panels) in HEK293 cells transfected with cDNA for the indicated proteins. <b>B.</b> Quantification of the relative cellular expression levels of Gβ5 after coexpression of MOR or D2R. The Gβ5 protein signal is expressed as a percent of the signal measured in cells expressing Gβ5 alone (mean ± SEM; n = 4, *p<0.05, ANOVA followed by Tukey’s post-hoc test, compared to the levels in cells expressing just Gβ5). <b>C.</b> Representative images from a Western blot depicting the relative levels of expression of Gβ1 (upper panels) or total cellular proteins (lower panels) in HEK293 cells transfected with cDNA for the indicated proteins. <b>D.</b> Quantification of the relative cellular expression levels of Gβ1 after coexpression of D2R. The Gβ1 protein signal is expressed as a percent of the signal measured in cells expressing Gβ1 alone (mean ± SEM; n = 3, *p<0.05, t-test, compared to the levels in cells expressing just Gβ5).</p

Effect of Gβ5 coexpression on arrestin recruitment to D2R-AP upon dopamine treatment as assessed by an in-cell “proximity biotin transfer assay”.

<p><b>A.</b> Schematic of the Arr-BL construct, a fusion of arrestin and BirA, an <i>E. coli</i> derived biotin ligase (BL) enzyme that specifically biotinylates a unique acceptor peptide (AP) sequence. N and C refer to the N- and C-terminus, respectively, of the fusion protein. <b>B.</b> Representative images from a Western blot depicting total cellular biotinylated D2R-AP by coexpressed Arr-BL (top panels) and coexpressed Gβ5 (bottom panels). The top left panel represents samples prepared from cells that were untransfected with either D2R-AP (−) or Arr-BL (−) and treated with 10 µM biotin for 2 min (biotin +, left) or samples that were transfected with both D2R-AP (+) and Arr-BL (+) and not treated with biotin (biotin −, right). The top center panel represents samples prepared from cells which were transfected with both D2R-AP (+) and Arr-BL (+) and treated with vehicle (dopamine −, left) or 10 µM dopamine for 30 min (dopamine +, right) and treated with biotin. The top right panel represents samples prepared from cells which were transfected with both D2R-AP (+), Arr-BL (+), and Gβ5 (+) and treated with vehicle (dopamine −, left) or 10 µM dopamine for 30 minutes (dopamine +, right) and treated with biotin (+).D2R-AP biotinylated by Arr-BL was detected by probing the blots with streptavidin. The middle panels represent corresponding western blots from samples in the upper panel probed for Gβ5. The bottom panels represent corresponding western blots from samples in the upper panel stained for total protein. <b>C.</b> Quantification of the relative levels of D2R-AP biotinylated by Arr-BL in response to dopamine treatment (10 µM for 30 min) in cells expressing only D2R-AP and Arr-BL or cells expressing D2R-AP, Arr-BL, and Gβ5. The biotinylated D2R-AP signal is expressed as a percentage of biotinylated D2R-AP signal from cells that were not treated with dopamine (mean ± SEM; n = 4, *p<0.05, t-test, compared to biotinyalted D2R-AP signal from corresponding cells not treated with dopamine).</p

Effect of Gβ5 on dopamine-mediated activation of D2R-coupled G protein signaling as measured by a fast kinetic BRET assay.

<p><b>A.</b> Average BRET dose-repsonse curve elicited by the application of 10 pM - 10 µM concentrations of dopamine to cells expressing only D2R (black trace) and cells transiently coexpressing low (dark grey) and high (light grey) levels of Gβ5. D2R stimulation by dopamine application leads to the dissociation of the G protein heterotrimer into Gβγ-Venus and GTP-bound Gαo subunits. Free Gβγ-Venus interacts with masGRK3ct-NanoLuc to produce a BRET signal. The black trace is from HEK293 cells that did not coexpress Gβ5 and the dark and light grey traces are from HEK293 cells transiently coexpressing two different levels (l, for low and h, for high) of Gβ5 (mean ± SEM; n = 4). <b>B.</b> Quantification of the maximal amplitude (E_max) of the BRET signal elicited by the application of dopamine in the cells described above. The E_max only significantly differed between D2R and D2R + Gβ5 (h) (*p<0.01, ANOVA followed by Tukey’s post-hoc test). <b>C.</b> Quantification of the average EC₅₀ derived for the dopamine mediated activation of D2R in the cells described in D. The response at each dose is expressed as a percent of the average maximal response (n = 4). The EC₅₀ (in nM) for D2R (black bar), D2R + Gβ5 (l, dark grey bar), and D2R + Gβ5 (h, light grey bar) was 19.8±0.825, 21.3±0.863, and 25.4±0.431 (mean ± SEM), respectively. The EC₅₀ only significantly differed between D2R and D2R + Gβ5 (h) (*p<0.01, ANOVA followed by Tukey’s post-hoc test). <b>D.</b> Averaged traces (± SEM) of changes in the BRET signal (ΔBRET or the BRET response) over time obtained from HEK293 cells transfected with cDNA for D2R, Gαo, Venus-Gβγ, masGRK3ct-NanoLuc and treated sequentially with dopamine (10 nM) and haloperidol (100 µM). <b>E.</b> Quantification of the deactivation kinetics of the dopamine-elicited BRET response after application of the D2R antagonist, haloperidol (100 µM).</p

Coexpression of D2R enhances the stability of Gβ5.

<p><b>A.</b> Representative image of a Western blot which depicts Gβ5 cellular expression levels levels (upper panel) or total cellular protein (lower panel), from HEK293 cells transiently expressing either Gβ5 alone or Gβ5 coexpressed with D2R, at times, t = 0, 3, or 6 hr after treatment with cycloheximide (100 µM). <b>B.</b> Quantification of the reduction in cellular Gβ5 levels after treatment of cells with cycloheximide. The Gβ5 levels at times 3 and 6 hr after cycloheximide treatment are expressed as a percentage of the levels of Gβ5 measured in cells that were not treated with cycloheximide (mean ± SEM; n = 4, *p<0.05, t-test, comparing to the cells that did not coexpress D2R).</p