D2-Dopamine Receptors Target Regulator of G Protein Signaling 9-2 (RGS9-2) to Detergent-Resistant Membrane Fractions

Abstract

Detergent-resistant membranes (DRM) are thought to contain structures such as lipid rafts that are involved in compartmentalizing cell membranes. We report that the majority of D2-dopamine receptors (D2R) expressed endogenously in mouse striatum or expressed in immortalized cell-lines is found in DRM. In addition, exogenous co-expression of D2R in a cell line shifted the expression of regulator of G protein signaling 9-2 (RGS9-2) into DRM. RGS9-2 is a protein that is highly enriched in the striatum and specifically regulates striatal D2R. In the striatum, RGS9-2 is mostly associated with DRMs but when expressed in cell lines, RGS9-2 is present in the soluble cytoplasmic fraction. In contrast, the majority of mu opioid receptors (MOR) and delta opioid receptors (DOR) are found in detergent-soluble membrane and there was no shift of RGS9-2 into DRM after co-expression of MOR. These data suggest that the targeting of RGS9-2 to DRM in the striatum is mediated by D2R and that DRM is involved in the formation of a D2R signaling complex. D2R-mediated targeting of RGS9-2 to DRM was blocked by the deletion of the RGS9-2 DEP domain or by a point mutation that abolishes the GTPase accelerating protein function of RGS9-2.

Introduction

Dopamine receptors are members of the seven pass trans-membrane spanning G protein-coupled receptor (GPCR) superfamily (Missale et al. 1998) and can classified into two subfamilies, D1-like (D1 and D5) and D2-like (D2, D3 and D4) dopamine receptors (DRs). D2-like DR produce cellular effects via the activation of pertussis-toxin (PTX)-sensitive hetero-trimeric G_i/oG proteins. Of these receptors, the D2-dopamine receptor (D2R) appears to be the most widely distributed in the brain, as well as the most abundant in many brain areas (Missale et al. 1998; Defagot et al. 2000).

D2Rs are important clinically as they are the major targets of drugs used to alleviate symptoms of schizophrenia, Parkinson’s disease and depression (Missale et al. 1998; Neve et al. 2004; Bonci and Hopf 2005; Kehne et al. 2008). For example, receptor-based anti-Parkinsonian drugs work via stimulating D2R. Schizophrenia patients have up-regulated levels of a “high affinity” form of brain D2R (Seeman et al. 2006; Seeman et al. 2007) and a common property of all currently available anti-psychotic drugs is that they block D2Rs at therapeutic concentrations (Remington 2003). However, the cellular connection between activation or blockade of the receptor and the suppression of disease symptoms has not been elucidated. Thus, a more complete description of the cellular functioning of D2R could help to elucidate both the pathophysiology underlying several significant diseases and the mechanism of actions of clinically important drugs.

When an activated GPCR (e.g. dopamine-bound D2R) encounters a hetero-trimeric G protein, it catalyzes the exchange of guanosine-5′-triphosphate (GTP) for GDP at the G protein Gα subunit. Gα-GTP binding in turn leads to the dissociation of the GTP-bound Gα subunit from the Gβγ dimer. The activated GTP-bound Gα subunit and the freed Gβγ dimer regulate the activity of diverse cellular effector molecules. Signal termination is mediated by the intrinsic GTPase activity of the Gα, which hydrolyzes the bound GTP to GDP, allowing it to re-associate with the Gβγ dimer (Ross and Wilkie 2000). GTP hydrolysis catalyzed by the Gα subunits (i.e. GTPase activity) and consequent GPCR signal termination can be accelerated by the regulator of G-protein signaling (RGS) family of proteins (Willars 2006) via a conserved “RGS” domain.

RGS9-2 is an RGS protein that is specifically expressed in the striatum, a brain region involved in movement, motivation, mood and addiction (Anderson et al. 2009; Traynor et al. 2009). Dopaminergic neurons form major inputs to the striatum and a large number of studies have shown that RGS9-2 is a specific modulator of D2R function. RGS9-2 preferentially accelerates the termination of D2R signals (Kovoor et al. 2005) and specifically inhibits dopamine-induced D2R internalization (Celver et al. 2010). RGS9-2 is uniquely expressed in those medium-spiny striatal neurons that also express D2R and is involved in the specific functional compartmentalization of striatal D2R (Kovoor et al. 2005). Dialysis of striatal cholinergic neurons with RGS9 protein constructs reduced D2R-mediated inhibition of voltage-activated Ca²⁺ channels; M2-muscarinic acetylcholine receptor inhibition of the same Ca²⁺ channels was unaffected (Cabrera-Vera et al. 2004). Furthermore, viral-mediated overexpression of RGS9-2 in rat ventral striatum reduced loco motor responses to D2R but not to D1-dopamine receptor agonists (Rahman et al. 2003). D2R-RGS9-2 functional interactions have been implicated in schizophrenia etiology (Seeman et al. 2006; Seeman et al. 2007) and are involved in the development of loco motor side-effects of drugs used to treat schizophrenia and Parkinson’s disease (Kovoor et al. 2005; Gold et al. 2007). It is also likely that these functional interactions are involved in the striatal control of i) locomotion and ii) reward responses to psycho-stimulants (Rahman et al. 2003; Kovoor et al. 2005; Gold et al. 2007; Blundell et al. 2008).

The biochemical basis for the physiologically and clinically important D2R-RGS9-2 interactions described above is not understood. The specific regulation of D2R by RGS9-2 cannot be explained solely by the RGS9-2 GTPase accelerating protein (GAP) function, since medium spiny striatal neurons that express D2R also express other GPCRs that couple to G proteins that can be regulated by RGS9-2 (Cabrera-Vera et al. 2004; de Gortari and Mengod 2010).

It is thought that proteins associated with the plasma membrane are not homogeneously distributed, but are segregated into micro-domains (Lindner and Naim 2009). The targeting of receptors to separate plasma-membrane micro-domains may explain how signals from receptors with common signaling components can be compartmentalized and distinguished (Simons and Toomre 2000). Thus, the specific functional D2R-RGS9-2 interactions could be explained if the two proteins are shown to reside within the same membrane micro-domains.

In this study we provide the first pieces of evidence to support the above suggestion. We report here that the majority of D2R endogenously expressed in mouse brain, or exogenously expressed in immortalized cell-lines is found in membrane that is insoluble when treated with a cold solution of the non-ionic detergent, Triton X1-00 (TX100). Furthermore, we show that the localization of D2R in detergent-resistant membrane (DRM) has important physiological consequences. RGS9-2 when exogenously expressed in a mammalian cell lines is distributed throughout the cytoplasm and is extracted into a soluble biochemical fraction (Bouhamdan et al. 2004; Kovoor et al. 2005). In contrast, Mancuso and colleagues have reported, in a previous article in this journal (Mancuso et al. 2010), that in the striatum, only a small percentage of the endogenous RGS9-2 is present in the soluble cytoplasmic fraction. Most of the endogenously expressed RGS9-2 is instead associated with plasma-membrane structures that happen to be insoluble in non-ionic detergents. Mancuso and colleagues hypothesized that D2R is responsible for anchoring of RGS9-2 to DRM compartments within striatal neurons (Mancuso et al. 2010). In support of the above suggestion, we report that RGS9-2 is specifically recruited to DRMs upon co-expression of D2R. Thus the data presented here have identified a novel biochemical feature of D2R and suggest that the targeting of D2R to DRMs is physiologically important because it allows for the cellular compartmentalization of D2R and associated signal transducers.

Materials and Methods

Materials

Chemicals and reagents were purchased from Sigma Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburg, PA) or from the suppliers specifically identified below.

Complementary cDNA clones

The rat RGS9-2-V5, DEPless RGS9-2-V5, and RGS9-2 N364H-V5 (Bouhamdan et al. 2006; Celver et al. 2010), the Gβ5 short subunit (Kovoor et al. 2000), and the N-terminal FLAG epitope-tagged D2R long isoform, the N-terminal FLAG epitope-tagged delta opioid receptor (DOR) (Celver et al. 2001; Kearn et al. 2005), the human D2R short isoform (Kovoor et al. 2005), and the N-terminal FLAG epitope-tagged mu-opioid receptor (MOR) (Keith et al. 1996) constructs have been previously described. Enhanced green fluorescent protein (GFP) cDNA was from Clontech (Palo Alto, CA). The human Gα_i1 clone was obtained from Open Biosystems (Huntsville, AL) and a cDNA construct encoding for Gα_i1-tagged at the C-terminus with the V5 epitope was generated using standard PCR-based techniques.

Cell culture and transfection

Human embryonic kidney cells stably expressing the SV40 T-antigen (HEK293T, American Type Culture Collection, Manassas, VA) were grown in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, penicillin G (100 units/ml) and streptomycin sulfate (100 μg/ml). Mammalian expression plasmids containing the appropriate expression plasmids were transiently transfected using LTX transfection reagent (Invitrogen) according to the manufacturer’s instructions. Total transfected DNA was kept constant between groups using the empty plasmid vectors pcDNA6 or pcDNA3.1 (Invitrogen).

Isolation of Triton X-100 (TX100)-soluble and insoluble proteins from HEK293 cells

48-72 hr post-transfection cells were lysed in phosphate-buffered saline (PBS, in mmol/L: 137 NaCl, 2.7 KCl, 10 Na2HPO4, 2 KH2PO4, pH 7.4) containing 20 mL/L (2% v/v) of the non-ionic detergent, Triton X-100 (TX100), for 1 hr at 4 °C. The samples were centrifuged (10,000 g, 10 min, 4 °C) to pellet the insoluble proteins. The proteins in the supernatant (i.e. TX100-soluble proteins) were precipitated by the addition of trichloro-acetic acid (TCA, final concentration 100 g/L). Both the TX100 insoluble protein pellet and the pellet formed after TCA precipitation of the TX100-extracted, TX100-soluble proteins were resuspended in equal volumes of 1X SDS sample buffer (20 g/L SDS, 25 mL/L glycerol, 0.1 g/L bromophenol blue, in mmol/L: 350 dithiothreitol (DTT), 100 Tris–HCl, pH 6.8) containing 8 mol/L urea. The re-suspended pellet containing the TX100-insoluble material was passed through a QIAshredder column (Qiagen, Valencia, CA) to reduce sample viscosity prior to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 8 mol/L urea was included in the SDS-sample buffer to denature and ensure maximal extraction and dissolution of insoluble proteins from the precipitates. Equal volumes of the re-dissolved samples were then resolved by SDS-PAGE and the relative amounts of respective proteins were compared between TX100 soluble and insoluble fractions by immuno-blotting (see below).

Isolation of Triton X-100 (TX100)-soluble and insoluble biochemical fractions from mouse brain

All animal procedures with mice were performed according the Guidelines for the Care and Use of Laboratory Animals from the National Institutes of Health and with the approval of the University of Rhode Island’s Institutional Use and Care of Animals Committee (protocol #AN10-02-015). Brain samples were Dounce homogenized in PBS containing a cocktail of protease inhibitors (catalog #: S8830, Sigma-Aldrich, St. Louis, MO). Homogenates were mixed with an equal volume of PBS containing 40 mL/L TX100 and incubated on ice for 1 hr. Samples were centrifuged at 4 °C for 10 min at 10,000 g. The supernatant (TX100-soluble) and pellet fractions were processed as described above.

Protein immuno-blotting

Protein samples, prepared as described above, were resolved by SDS-PAGE and transferred, using wet electrophoretic elution buffer (in mmol/L: 25 Tris-base, 192 glycine, 100 mL/L methanol, 1 g/L SDS, pH~8.3), onto methanol-wetted polyvinylidene fluoride (PVDF) membranes. After blocking remaining protein-binding sites with 100 g/L non-fat milk powder in PBS, the proteins of interest were detected by probing the blots sequentially with the appropriate primary and horse-radish peroxidase (HRP)-conjugated secondary antibodies, diluted in PBS. The primary antibodies used were as follows: for D2R, polyclonal rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, catalog # sc-9113, 1:200 dilution); V5 epitope-tagged RGS9-2 constructs, purified mouse monoclonal antibody (Invitrogen, catalog # 46-0705, 1:5000 dilution); native RGS9-2, sheep polyclonal antibody, gift from Kirill Martemyanov, University of Minnesota (Martemyanov et al. 2005), FLAG-MOR and FLAG-DOR, mouse monoclonal anti-FLAG antibody (Sigma Aldrich, clone M2, catalog # F1804, 1:1000 dilution); green fluorescent protein (GFP), mouse monoclonal (Covance, Princeton, NJ, catalog # MMS-118P, 1:1000 dilution); Gβ5, rabbit polyclonal, CT215 (Watson et al. 1994), was a gift from Dr. Jason Chen and the mouse anti-transferrin (TFR) was obtained from Invitrogen (catalog# I36800, 1:1000 dilution). The blots were incubated with primary antibody (overnight at 4 °C), washed 3X in PBS and then incubated with the appropriate HRP-conjugated secondary antibody (1 hr at room temperature). Secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were diluted in PBS at concentrations recommended by the supplier HRP-catalyzed chemi-luminescent signals were generated using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce-Thermo Fisher Scientific Rockford, IL) and detected using a Chemidoc XRS Molecular Imager (Bio-Rad Laboratories, Hercules, CA).

Immunostaining

HEK293T cells were grown on poly-D-lysine coated coverslips and transfected with the indicated cDNAs. 48 hours after transfection cells were fixed with fixed and permeabilized with cold acetone:methanol (1:1 v:v, 4 °C for 5 min). Cells were blocked with 5% (50 g/L) bovine serum albumin (BSA) in PBS and then incubated for 1 hr at room temperature with anti-FLAG antibody (1:1000 dilution) targeting the FLAG-tagged D2R construct, or anti-V5 antibody (1:500 dilution) targeting the V5-tagged RGS9-2 and Gαi1 constructs. Following 5 washes with PBS, cells were probed with anti-mouse Alexa Fluor 594 secondary (Invitrogen) for 30 min, washed 5 times with PBS and mounted on slides. Fluorescence was visualized and images captured using a Nikon microscope equipped with a Nikon 100X objective, epi-fluorescence filters and a digital data acquisition system (Nikon USA, Melville, NY).

Indirect detection of surface D2R and MOR in detergent-Insoluble and soluble fractions

HEK293T cells were transiently transfected with cDNA for FLAG-tagged D2R or MOR or with the corresponding empty vector, respectively. The intact cells were then incubated in cell-culture medium with antibody (Sigma Aldrich, clone M2, catalog # F1804, 1:500 dilution) directed against the extracellular FLAG-tag for 1 hr at 37 °C to label the respective surface receptors. The cells were then washed 5 times in ice-cold PBS (5 min incubation in PBS for each wash). TX100 soluble and insoluble proteins were isolated, resolved on SDS-PAGE and Western-blotted essentially as described above. The anti-FLAG antibody that labeled the surface receptors and fractionated into the TX100-soluble and insoluble fractions was visualized on the blot using a corresponding horse-radish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).

Data Analysis

Images were collected using exposure settings that did not saturate any of the charge-coupled device camera pixels. Signals from the target protein bands were quantified using ImageJ image processing and analysis software (National Institutes of Health, Bethesda MD, http://rsbweb.nih.gov/ij/). The signals resulting from detergent-soluble and insoluble preparations of a protein, respectively, were expressed as a fraction of the total signal and the Student’s t test was used to determine if the amounts of signal from the target protein bands in the different experimental groups were significantly different. Statistical significance for multiple comparisons was determined using ANOVA followed by Tukey’s multiple comparison test.

Results

D2R is expressed in a cell-membrane fraction that is resistant to solubilization by Triton X-100 (TX100)

Unless clearly specified, the longer alternatively spliced form of D2R (Missale et al. 1998) was used in this and subsequent experiments. We found that when D2R is exogenously expressed in HEK293T cells, the majority of D2R (76.7 ± 12.1%, mean ± S.E.M, n=8) is found in the cellular fraction that is insoluble in cold Triton X-100 (TX100) (Fig. 1A & E).

Fig. 1. Partitioning of cellular D2-dopamine receptor (D2R), the mu opioid receptor (MOR) and the delta opioid receptor (DOR) in Triton X-100 (TX100)-soluble and insoluble biochemical fractions.

D2R (A), DOR (B) and MOR (C) cDNA transfected HEK293T cells and mouse whole brain (Br., excluding cerebellum and brain stem) or mouse striatum (Str.) (D) were solubilized in TX100 detergent-containing buffer. Soluble and insoluble proteins were separated by centrifugation, resolved by SDS PAGE, and the D2R, DOR and MOR proteins in TX100 soluble (S) and insoluble (P, pellet) cell fractions were visualized by immuno-blotting, as described in the Materials and Methods section. CTRL refers to control HEK293T cell samples that were transfected with the corresponding empty plasmid vector. Anti-FLAG refers to the antibody used to visualize the recombinant FLAG epitope-tagged DOR and MOR proteins and anti-D2R refers to the antibody used to visualize D2R. E. Levels of the respective receptor proteins in A, B, C, and D, in TX100-soluble (closed black bars) and insoluble (open white bars) cellular fractions expressed as percentage of the total cellular signal for the respective receptor (mean ± SEM, n=4-8, *represents p<0.01, compared to TX100 insoluble signal for DOR or MOR).

G protein coupled mu-opioid receptors (MOR) are co-expressed with D2R in striatal neurons (de Gortari and Mengod 2010) and D2R, MOR and delta-opioid receptors (DOR) preferentially couple to the same pertussis toxin-sensitive (PTX) G proteins (Waldhoer et al. 2004). However, in contrast to what was observed for D2R, the majority of DOR and almost all of the detectable MOR, exogenously expressed in HEK293T cells, were found in the TX100-soluble fraction (Fig. 1B, C & E).

Western blots of TX100 soluble and insoluble fractions of mouse striatum and whole brain were probed with a D2R antibody. We found that i) the major band recognized by the D2R antibody in the TX100-insoluble brain and striatum fraction has a similar mobility as that of recombinant D2R expressed in HEK293T cells and ii) the vast majority of endogenous D2R expressed in mouse striatum and whole brain is localized to the TX100-insoluble fraction (Fig. 1D & E). These results suggest that the partitioning of D2R into the TX100-insoluble biochemical fraction was not an artifact of exogenous overexpression of the GPCR in HEK293T cells, but is a property of endogenously expressed D2R in the brain.

Co-expression of D2R translocates recombinantly expressed RGS9-2 to detergent-resistant membrane (DRM) fractions

Previous studies have showed that RGS9-2, when expressed in immortalized cell-lines, is distributed throughout the cytoplasm (Kovoor et al. 2005). In addition, most of the exogenously expressed RGS9-2 protein is readily extracted into solution using non-ionic detergents such as TX100 (Bouhamdan et al. 2004). In the striatum on the other hand, most of the endogenously expressed RGS9-2 is associated with plasma-membrane structures that are insoluble in non-ionic detergents (Mancuso et al. 2010). Prompted by these studies and by our finding that D2R was predominantly expressed in detergent-resistant membrane (DRM) (Fig. 1A, D & E) we investigated if D2R could re-localize RGS9-2 into the TX100-insoluble fraction.

We compared the relative amount of RGS9-2 that was extracted into the cold TX100 soluble cellular fraction or that remained in the insoluble fraction when RGS9-2 was expressed alone or when co-expressed with D2R in HEK293T cells. When expressed alone, greater than 75% of the total cellular RGS9-2 was detected in the TX100 soluble cell fraction but, upon D2R co-expression, the majority of the RGS9-2 signal was shifted into the TX100 insoluble fraction (Fig. 2A & B). In contrast, MOR co-expression did not increase the fraction of RGS9-2 that was detected in TX100-insoluble portion (Fig. 2A & B). Control experiments showed that D2R co-expression did not alter the TX100 solubility of either co-expressed cytoplasmic GFP or the endogenously expressed integral membrane transferrin receptor (TFR) protein (Fig. 2C-F).

Fig. 2. Effect of D2R or MOR co-expression of on TX100 solubility of RGS9-2 and effect of D2R co-expression on TX100 solubility of either the green fluorescent protein (GFP) or the transferrin receptors (TFR).

A. HEK293T cells were transfected with RGS9-2-V5-tagged cDNA (RGS9) alone or with RGS9 and D2R cDNA (RGS9+D2R) or RGS9 and MOR cDNA (RGS9+MOR) as indicated. CTRL refers to a control group of cells transfected with empty plasmid vector. Cells were solubilized in TX100 detergent-containing buffer. Soluble and insoluble proteins were separated by centrifugation, resolved by SDS PAGE, and the RGS9-2 protein in TX100 soluble (S) and insoluble (P, pellet) cell fractions were visualized by immunoblotting using an anti-V5 epitope tag antibody, as described in the Materials and Methods section. B. Levels of RGS9-2 protein in TX100-soluble (closed black bars) and insoluble (open white bars) cellular fractions visualized in A and expressed as percentage of the total cellular RGS9-2 protein (mean ± SEM, n=4-5, *represents p<0.01 compared to RGS9-2 cDNA only transfected group). C. HEK293T cells were transfected with GFP cDNA alone or with GFP and D2R cDNA as indicated. CTRL refers to a control group of cells transfected with empty plasmid vector. Cells were solubilized in TX100 detergent-containing buffer. Soluble and insoluble proteins were separated by centrifugation, resolved by SDS PAGE, and the GFP protein in TX100 soluble (S) and insoluble (P, pellet) cell fractions were visualized by immuno-blotting using an anti-GFP antibody, as described in the Materials and Methods section. D. Levels of GFP protein in TX100-soluble (closed black bars) and insoluble (open bars) cellular fractions visualized in C and expressed as percentage of the total cellular GFP protein (mean ± SD, n=2). E. HEK293T cells were transfected with either empty vector control (CTRL) or D2R cDNA as indicated. Soluble and insoluble proteins were separated and resolved by SDS PAGE as described above and the endogenously expressed TFR in TX100 soluble (S) and insoluble (P, pellet) cell fractions were visualized by immuno-blotting using an anti-TFR antibody, as described in the Materials and Methods. F. Levels of TFR protein in TX100-soluble (closed black bars) and insoluble (open bars) cellular fractions visualized in E and expressed as percentage of the total cellular TFR protein (mean ± SEM, n=4).

D2R-mediated targeting of RGS9-2 to TX100-insoluble membrane compartments is observed after co-expression of the G protein subunit, Gβ5

RGS9-2 is a member of the R7 RGS protein subfamily (Anderson et al. 2009) whose members are defined by the presence of a Gγ-like domain (GGL) that binds Gβ5, an outlying member of the G protein beta subunit family. Therefore we asked if co-expression of D2R could mediate translocation of the Gβ5 complex into DRM. The shorter alternatively spliced version of Gβ5 is the Gβ5 isoform that is expressed in the brain and this isoform was utilized in the following studies described here (Watson et al. 1996).

In the absence of D2R expression both RGS9-2 and Gβ5 are predominately TX100-soluble (Fig. 3A-D). After D2R co-expression both RGS9-2 and co-expressed Gβ5 were relocated from the TX100-soluble to the TX100-insoluble fraction (Fig. 3A-D). Gβ5 co-expression also significantly increased the amount of RGS9-2 that partitioned into the TX100-insoluble fraction upon D2R co-transfection (Fig. 2B compared to 3B, p<0.01). It has been suggested that, in vivo, Gβ5 is an obligate partner of R7 RGS proteins (Chen et al. 2003; Anderson et al. 2009), and our data suggest that the RGS9-2/Gβ5 complex is more efficiently recruited to D2R in DRM than RGS9-2 alone.

Fig. 3. Effect of D2R co-expression on the TX100 solubility of RGS9-2 and Gβ5 co-expressed in HEK293T cells and effect of dopamine (DA)-treatment on D2R-mediated translocation of RGS9-2 to TX100-insoluble cellular fractions.

A. HEK293T cells were transfected with cDNA for RGS9-2-V5-tagged (RGS9) and Gβ5 or with cDNA for RGS9, Gβ5 and D2R. Western blot (WB) of RGS9-2-V5 (RGS9) in TX100 detergent soluble (S) and insoluble (P, pellet) cell fractions probed with an anti-V5 epitope-tagged antibody, as described in the Materials and Methods. B. Levels of RGS9-2 protein in TX100-soluble (closed black bars) and insoluble (open bars) cellular fractions visualized in A and expressed as percentage of the total expressed RGS9-2 protein (mean ± S.D., n=4, *represents p<0.01 compared to RGS9-2 cDNA/Gβ5 cDNA transfected group). C. Western blot (WB) of Gβ5 in TX100 detergent soluble (S) and insoluble (P, pellet) cell fractions described in A. D. Levels of Gβ5 protein in TX100-soluble (closed black bars) and insoluble (open bars) cellular fractions visualized in C and expressed as percentage of the total expressed Gβ5 protein (mean ± SEM, n=4, *represents p<0.01 compared to RGS9-2 cDNA/Gβ5 cDNA transfected group). E. HEK293T cells transfected with expression plasmids for D2R, Gβ5 and a RGS9-2 were treated for 10 or 30 min with 1 μM dopamine (DA). RGS9-2 proteins in TX100 detergent soluble (S) and insoluble (P, pellet) fractions from cells treated with DA as indicated and visualized by immunoblotting. F. Levels of RGS9-2 protein in TX100-soluble (closed black bars) and insoluble (open bars) cellular fractions visualized in E and expressed as percentage of the total cellular RGS9-2 protein (mean ± SEM, n=4).

As previously described (Celver et al. 2010; Masuho et al. 2011), we saw no effect of Gβ5 co-expression on RGS9-2 protein expression levels when the two proteins were co-expressed in HEK293T cells (data not shown). Although, it has been demonstrated that association with Gβ5 can protect R7 RGS proteins from proteolysis (Chen et al. 2003; Witherow and Slepak 2003; Anderson et al. 2009) these data and previous studies (Celver et al. 2010; Masuho et al. 2011) indicate that HEK293 cells do not efficiently degrade RGS9-2 protein that is not associated with Gβ5.

In addition, D2R co-expression did not significantly alter protein expression levels of either component of the RGS9-2/Gβ5 complex (RGS9-2: 77 ± 15.1%, p=0.25; Gβ5: 146 ± 27%, p=0.11, with D2R compared to without D2R co-expression, mean ± SEM).

Activation of D2R with dopamine (10 μmol/L for 10 or 30 min) did not alter RGS9/Gβ5 translocation to DRM (Fig. 3E & F). In addition, the major fraction of D2R expressed in HEK293T cells remained in the DRM fraction and was not significantly altered after either agonist-treatment (dopamine, 10 μmol/L, 60 min), or after co-expression of RGS9-2 (data not shown).

Biochemical fractionation of brain RGS9-2 and Gβ5

We found that majority of the endogenous RGS9-2, which is largely localized to the striatum, is extracted into the TX100-insoluble biochemical fraction (Fig. 4A & B).

Fig. 4. TX100 solubility of RGS9-2 and Gβ5 in mouse brain.

A. Western blot (WB) of RGS9-2 in TX100 soluble (S) and insoluble (P, pellet) cell fractions from mouse cortex and striatum. B. Levels of RGS9-2 protein in TX100-soluble (closed black bars) and insoluble (open bars) striatal fractions visualized in A and expressed as percentage of the total striatal RGS9-2 signal (mean ± SEM, n=4, *represents p<0.01 compared to RGS9-2 protein signal in the TX100 soluble fraction). C. Western blot (WB) of Gβ5 in TX100 soluble (S) and insoluble (P, pellet) cell fractions from mouse cortex and striatum. D. Levels of Gβ5 protein in TX100-soluble (closed black bars) and insoluble (open bars) cellular fractions visualized in C and expressed as percentage of the total detected Gβ5 protein signal detected, respectively, in mouse cortex and striatum (mean ± SEM, n=4, *represents p<0.01 compared to Gβ5 protein signal in the TX100 insoluble fraction from the cortex).

In addition, in the striatum where both RGS9-2 and D2R are highly expressed, more than 40% the endogenous Gβ5 was detected in the TX100-insoluble fraction (4C & D). In contrast, in the cortex, where RGS9-2 protein levels are almost undetectable (Fig. 4A), more than 90% of the endogenous Gβ5 was, instead, extracted into the TX100-soluble biochemical fraction (4C & D).

D2R does not mediate DRM-translocation of RGS9-2 constructs with either i) a deleted DEP domain or ii) a point mutation in the RGS domain that abolishes GTPase accelerating protein (GAP) function

An RGS9-2 construct in which the DEP domain was deleted (DEPless RGS9-2) and expressed in HEK293T cells was mostly soluble in cold TX100 (>70%, Fig. 5A & B). Co-expression of D2R did not significantly alter the TX100 solubility of the DEPless protein construct (Fig. 5A & B). The above results indicate that the DEP domain is required for the D2R-mediated translocation of RGS9-2 into the TX100 insoluble compartment. Note that while wild-type RGS9-2 appears to run as a single band, the DEPless RGS9-2 construct resolves as a doublet on the SDS-PAGE gel and could reflect increased proteolysis or other post-translational modifications such as phosphorylation that occur as a result of the DEP domain deletion.

Fig. 5. The effect of D2R co-expression on TX100 solubility of mutant RGS9-2 constructs.

HEK293T cells were transfected with cDNAs for Gβ5 and a DEPless RGS9-2 construct (DEPless) or with cDNAs for Gβ5 and RGS9-2 N364H (N364H). Both RGS9-2 constructs were V5-tagged. Two more experimental groups were transfected in addition, with cDNA for D2R (+D2R). A. RGS9-2 mutant proteins in TX100 detergent soluble (S) and insoluble (P, pellet) cell fractions visualized by immuno-blotting, as described in the Materials and Methods, RGS9-2 N364H (left panels), DEPless RGS9-2 construct (right panels). The distribution of the mutant RGS9-2 constructs in TX100-soluble and insoluble cellular fractions after D2R co-expression (+D2R) is shown in the lower panel. B. Levels of RGS9-2 mutant proteins in TX100-soluble (closed black bars) and insoluble (open bars) cellular fractions visualized in A and expressed as percentage of the respective total cellular mutant RGS9-2 protein (mean ± SEM, n=4-5).

The canonical function that has been described for RGS proteins is to accelerate the GTPase activity of Gα G proteins (GAP function). Therefore, we tested if a mutant RGS9-2 construct that does not interact with Gα G proteins, can be relocated into the TX100-insoluble fraction by D2R co-expression. N364 in RGS9-2 is an asparagine residue that is conserved within the RGS domain of RGS proteins and mutation of this residue to histidine has been shown in RGS family members, including RGS9-2, to block Gα G protein binding and/or abolish GAP function (Natochin et al. 1998; Srinivasa et al. 1998; Posner et al. 1999; Celver et al. 2010). We found that like wild-type RGS9-2, the RGS9-2 N364H construct was soluble in TX100 when expressed alone in HEK293T cells, but unlike wild-type RGS9-2, RGS9-2 N364H remained soluble upon co-expression of D2R (Fig. 5A & B). In both sets of the experiments D2R co-expression was confirmed by immuno-blotting.

RGS9-2, RGS9-2 N364H and Gα_i1 are targeted to the plasma membrane upon D2R co-expression

We had previously demonstrated that the RGS9 DEP domain was both necessary and sufficient for mediating the intracellular co-localization of RGS9-2 and D2R (Kovoor et al. 2005) and were intrigued by the failure of the RGS9-2 N364H mutant to translocate to DRM after D2R co-expression. Therefore, we examined if the RGS9-2 N364H mutant could co-localize spatially with D2R when the two proteins were co-expressed in HEK293T cells. The labeling for D2R is seen predominantly along the cell boundary as would be expected from a receptor protein that is targeted to the plasma membrane (Fig. 6A). RGS9-2 and the N364H mutant can be seen distributed largely through the cytoplasm when expressed with Gβ5 (Fig. 6B & C, left panels). Upon D2R co-expression, however, both wild-type RGS9-2 and the RGS9-2 N364H mutant appeared to move to the cell boundary with a cellular localization that was similar to that observed for D2R (Fig. 6B & C, right panels).

Fig.6. Effect of D2R co-expression on the cellular distribution of RGS9-2, RGS9-2 N364H, and Gα_i1 in transfected HEK293T cells.

A. D2R distribution in HEK293T cells that were transfected with cDNA for D2R alone. B. RGS9-2 distribution in cells that were transfected with cDNA for the RGS9-2-V5-tag (RGS9) and Gβ5 (Gβ5) (left panel) and in cells that were transfected, in addition, with cDNA for D2R (right panel). C. RGS9-2 N364E distribution in cells that were transfected with cDNA for the RGS9-2 N364E-V5-tag (RGS9 N364E) and Gβ5 (Gβ5) (left panel) and in cells that were transfected in addition with cDNA for D2R (right panel). D. Gα_i1 distribution in cells that were transfected with cDNA for Gα_i1 alone (left panel) or with cDNA for D2R (right panel). E. Western blot (WB) of Gα_i1 in TX100 detergent soluble (S) and insoluble (P, pellet) HEK293T cell fractions visualized by immuno-blotting, as described in the Materials and Methods,

Similar results were obtained Gα_i1, a G protein isoform that can couple to D2R and can functionally interact with RGS9-2, which also appeared to re-localize to the plasma membrane after D2R co-expression (Fig. 6D).

A biochemical analysis indicated that almost all of the Gα_i1 was detected in TX100-insoluble membrane fraction (Fig. 6E) which is consistent with previous reports demonstrating that Gαi G proteins target to detergent-resistant membranes (Oh and Schnitzer 2001; Patel et al. 2008). These data together suggest that the DEP domain is likely sufficient to target RGS9-2 to D2R but an additional interaction with G protein may be required for stabilizing the complex during cold detergent extraction.

The short isoform of the D2-dopamine receptor (D2Rsh) is expressed in a TX100 insoluble fraction and recruits RGS9-2 to this fraction

D2R short (D2Rsh) is an alternatively spliced form of D2R lacking a stretch of 29 amino acids found in the third cytoplasmic loop of the longer isoform (Missale et al. 1998). We found that a slightly but significantly larger percentage of D2Rsh is found in the cold TX100-insoluble fraction when compared to D2R (p<0.05, comparing Fig. 7C & 1E). The slightly differing T100-isolubility of D2R and D2Rsh could be indicative of the involvement of the third cytoplasmic loop in targeting D2R to DRM. Co-expression of D2Rsh also alters the biochemical localization of the RGS9-2/Gβ5 complex so that the majority of the complex is detected in the TX100 insoluble biochemical fraction (Fig. 7B & C).

Fig. 7. Effect of co-expression of D2R short isoform (D2Rsh) on the TX100 solubility of the RGS9-2/Gβ5 complex.

HEK293T cells were transfected with cDNA for the D2R short isoform (D2Rsh) alone, or with cDNA for RGS9-2-V5-tagged (RGS9), Gβ5 and D2Rsh. A. D2Rsh protein in TX100 detergent soluble (S) and insoluble (P, pellet) cell fractions visualized by immuno-blotting, using an anti-D2R antibody, as described in the Materials and Methods section. CTRL refers to a control group of cells transfected with empty plasmid vector. B. RGS9-2 (RGS9) protein, co-expressed with D2Rsh, in TX100 detergent soluble (S) and insoluble (P, pellet) cell fractions visualized by immuno-blotting, using an anti-V5 epitope-tag antibody, as described in the Materials and Methods section. C. Levels of D2Rsh and RGS9-2 protein in TX100-soluble (closed black bar) and insoluble (open bar) cellular fractions visualized in B and expressed as percentage of the respective total cellular protein (mean ± SEM, n=3-5).

D2R expressing detergent-resistant membrane is present at the cell surface

The crude cell fraction insoluble in cold TX100 is a heterogeneous mixture of detergent-insoluble membrane structures and detergent-insoluble proteins from within the cell. Therefore we asked if any D2R that was isolated in the TX100-insoluble pellet originated from the plasma membrane at the cell surface. As described in the Materials and Methods section, intact cells, expressing either D2R or MOR, were treated with an anti-FLAG antibody targeting an extracellular FLAG-tag epitope fused to the respective receptors. The anti-FLAG antibody that is detected in the TX100-insoluble (pellet) biochemical fraction is targeted into this fraction as a result of binding to surface receptor that partitioned into the TX100-insoluble pellet fraction. In cells expressing D2R, greater than 50% of the antibody that bound surface receptors is detected in the DRM pellet fraction (Fig. 8). In contrast, less than 10% of the anti-FLAG antibody targeting surface MOR was detected in the DRM pellet fraction (Fig. 8). Treatment of the intact cells with anti-FLAG antibody did not alter the amounts of D2R in detergent-resistant and soluble fractions (data not shown).

Fig. 8. Detection of plasma membrane receptors expressed at the cell surface in the detergent-resistant membrane (DRM) fraction.

HEK293T cells were transfected with cDNA for the N-terminal FLAG-tagged D2R or MOR or empty vector (control) and 48 hr post-transfections were incubated for 1 hr at 37 °C with an anti-FLAG antibody targeting the extracellular FLAG epitope on the respective receptors. The cells were subsequently washed three times in ice cold PBS, treated for 1 hr with 2% v/v (20mL/L) TX100 at 4 °C and the TX100-insoluble and soluble fractions were separated by centrifugation as outlined in the Materials and Methods section. A. The receptor-targeting antibody in TX100 detergent soluble (S) and insoluble (P, pellet) cell fractions visualized by immuno-blotting, using appropriate secondary antibodies, as described in the Materials and Methods sections. “control” refers to a control group of cells transfected with empty plasmid vector and then incubated with anti-FLAG antibody. B. Levels of extracellular epitope targeting FLAG antibody in TX100-soluble (closed black bars) and insoluble (open bar) cellular fractions visualized in A and expressed as percentage of the total antibody detected in both the TX100 soluble and insoluble fractions (mean±SEM, n=5, *p<0.02).

If it is assumed that D2R expressed in TX100-soluble and insoluble plasma membrane compartments were equally accessible to the antibody and bound the antibody with equal affinity, than the results depicted in Fig 8 suggest that the majority of D2R expressed at the cell surface (i.e. D2R inserted into the plasma membrane) is present in plasma membrane compartments that are resistant to solubilization in TX100.

It is important to note, however, that the ratio of antibody partitioning into TX100-insoluble versus TX100-soluble biochemical fractions, does not accurately represent the ratio of surface D2R partitioning into these fractions, and likely underestimates that ratio, for the following reason. Both ionic and non-ionic detergents can significantly reduce antibody-antigen binding. For example, Qualtiere and colleagues (Qualtiere et al. 1977) showed that TX100 concentrations as low as 0.1% v/v inhibited a specific antibody-antigen interaction, that they investigated, by approximately 8-10%. Since most antibody-antigen interactions are reversible, treatment with TX100 is likely to cause some of the anti-FLAG antibody, bound to the plasma-membrane receptors, to dissociate and partition into the TX100-containing solution, leading to an underestimation of the proportion of antibody that had bound surface D2R in TX100-insoluble membrane and an overestimation of antibody bound to TX100-soluble D2R.

Discussion

D2R is expressed in detergent-resistant membrane (DRM) compartments and can direct the biochemical co-compartmentalization of associated signal transducers

Detergent-resistant membrane (DRM) fractions are thought to contain structures such as lipid rafts (Simons and Toomre 2000; Lingwood and Simons 2010) that are hypothesized to micro-compartmentalize cellular processes by specific inclusion or exclusion of cellular components. Here we have provided evidence to show that D2R, either endogenously expressed in the brain or exogenously expressed in HEK293T cells, is expressed in detergent-resistant biochemical compartments. The importance of the above finding is highlighted by our observations that, in contrast, the majority of the delta opioid receptor (DOR) and almost all the detectable mu opioid receptors (MOR) are expressed in the detergent, soluble membrane fractions (Fig. 1). Thus, our data provide an explanation for how D2R-elicited signals can be segregated from other GPCR pathways and compartmentalized within cells.

Support for the above suggestion and an important physiologically relevant consequence of the expression of D2R in DRM is provided by the subsequent demonstration that, D2R can mediate the re-localization of RGS9-2 and, even more efficiently, the RGS9-2/Gβ5 complex into D2R-expressing DRM fractions when the proteins are co-expressed in HEK293T cells (Fig. 2). RGS9-2 is a striatally enriched protein that has been previously shown to specifically regulate D2R and is expressed in striatal DRM fractions (Mancuso et al. 2010). When expressed in HEK293T cells, however, the majority of RGS9-2 is detergent-soluble but upon D2R co-expression a large proportion of the recombinantly expressed RGS9-2 is translocated into DRM. We believe that this is the first report of the involvement of a GPCR in directing the compartmentalization of an associated signal transducer into detergent-resistant micro-compartments, possibly allowing for the specific functional interactions between the two proteins.

For example, the GTPase accelerating function of RGS9-2 is specifically targeted to D2R-coupled G proteins even though medium spiny neurons that express D2R also express other GPCRs that couple to G proteins that can be regulated by RGS9-2 (Cabrera-Vera et al. 2004; de Gortari and Mengod 2010). Our data provide an explanation for how this specific regulation could occur. The expression of D2R in DRM micro-compartments and the D2R-mediated translocation of RGS9-2 to these membrane compartments positions RGS9-2 close to the D2R-coupled Gα subunits, before dopamine-mediated activation of D2R. After the Gα subunits are activated by dopamine-bound D2R, the proximity of RGS9-2 to D2R allows RGS9-2 to readily inactivate these nearby G protein subunits. Such a model is consistent with our data indicating that D2R, RGS9-2 and coupled G proteins are pre-assembled in DRM even before agonist treatment and the targeting of both D2R and RGS9-2 to DRM is unaffected by agonist treatment.

The specificity resulting from such translocation is likely amplified in neurons, which have a complex morphology and regions such as axon terminals and dendrites that are spatially distant from each other.

The finding that the majority of brain D2R is expressed DRM fractions has exposed a major limitation of common approaches for identifying components of the D2R signaling complex in the brain. Due to the physiological and clinical importance of D2R, a large amount of such effort has been invested in identifying the different protein interactions that comprise the D2R “signalplex” (Kabbani and Levenson 2007; Shioda et al. 2010). However, the common approach to identifying D2R-interacting proteins involves the solubilization of D2R in solutions of mild detergents, such as TX100, followed by the identification of co-immunoprecipitating proteins (Kabbani and Levenson 2007); the use of mild detergents for solubilization of D2R is necessitated because stronger detergents will likely disrupt protein-protein interactions. Unfortunately, our data indicates that the detergent-soluble fraction represents an insignificant percentage of the total brain D2R (Fig. 1D). Thus, the vast majority of D2R-interacting proteins is likely found in the insoluble pellet, with D2R, and thus remains unidentified.

These results also suggest a possible explanation for why a physical or biochemical interaction between endogenous D2R and RGS9-2 has not been observed, even though there are multiple reports of physiologically important and specific D2R-RGS9-2 functional interactions (reviewed in Introduction). For example proteomic and biochemical approaches undertaken to detect brain RGS9-2 binding partners have previously identified multiple neuronal proteins, including R7BP (Martemyanov et al. 2005; Posokhova et al.), but D2R has not been reported as present in the RGS9-2-containing complexes. The studies used Triton X-100 (TX100) extracts of striatal tissue and thus, the fraction containing both RGS9-2 and D2R, being TX100-insoluble, was excluded.

The targeting of D2R and RGS9-2 to DRM is physiological and specific

We have identified a new biochemical property of native D2R expressed in the brain and the striatum, namely targeting to DRM. In addition, we have shown that the biochemical partitioning of over-expressed recombinant D2R protein in HEK293T in DRM mirrors the distribution of endogenous D2R from the striatum. These results suggest that the partitioning of D2R into the TX100-insoluble biochemical fraction was not an artifact of exogenous overexpression of the GPCR in HEK293T cells, but is a property of endogenously expressed D2R in the brain. They also suggest that the targeting of D2R to DRM is either due to some intrinsic physical property of D2R or is due to an interaction with detergent-resistant cellular components expressed in both the striatum and in HEK cells.

On the other hand, the D2R-medated translocation of RGS9-2 from detergent soluble biochemical fraction to DRM fractions in HEK293T cells indicates that DRM localization of striatal RGS9-2 is not an intrinsic property of the native RGS9-2 protein but may be mediated by direct or indirect interactions with D2R.

Furthermore, we found that, in the striatum, where D2R (Missale et al. 1998) and RGS9-2 (Gold et al. 1997; Rahman et al. 1999; Zhang et al. 1999) concentrations are known to be high, the majority of Gβ5 is found in the TX100 insoluble fraction. In contrast, in the cortex where RGS9-2 levels are barely detectable (Fig. 4A), the majority of Gβ5 is found in the TX100 soluble fraction. These data further suggest that the D2R-mediated translocation of RGS9-2/Gβ5 complex into detergent resistant membrane (DRM) fractions in HEK cells is a physiologically relevant phenomenon.

Evidence for specificity of the D2R-RGS9-2 interaction is provided by our observation that cytoplasmic GFP and the endogenously expressed, integral membrane transferrin receptor (TFR) protein was not re-localized to DRMs by D2R co-expression (Fig. 2C-F) and that co-expression of MOR did not relocate RGS9-2 to DRMs (Fig. 2A & B). In addition, an RGS9-2 construct lacking the DEP domain or a point mutant RGS9-2 construct that lacked GTPase accelerating protein (GAP) function failed to re-localize to DRMs upon D2R co-expression (Fig. 4)

Structural features involved in D2R-mediated RGS9-2 translocation to DRM

It has been demonstrated that specific DEP domains can mediate interactions with specific GPCRs (Ballon et al. 2006; Chen and Hamm 2006; Anderson et al. 2009). We show here that the RGS9-2 DEP domain was necessary but not a sufficient structural determinant for the D2R-mediated RGS9-2 re-localization into DRM: a point mutation within the RGS domain (RGS9-2 N364H), which was previously shown to block interactions with Gα G protein subunits, also blocked RGS9-2 re-localization (Fig. 5).

The latter data suggests that, in addition to the interactions mediated by the DEP domain, interactions with G proteins could be important for the partitioning of RGS9-2 into DRMs. It is known that members of the family of Gα G proteins that are activated by D2R activation are associated with lipid rafts (Patel et al. 2008) and RGS proteins bind with high affinity to the transition state conformation of the Gα G protein, necessary for GTP hydrolysis (Tesmer et al. 1997). However, DRM translocation of RGS9-2 cannot be simply explained via the binding of RGS9-2 to DRM-associated Gα G proteins after receptor-mediated GDP-GTP exchange at Gα. First, DRM translocation of RGS9-2 requires the DEP domain (Fig. 5), but the DEP domain is not required for G protein binding (Tesmer et al. 1997). Secondly, activation of D2R is not necessary for DRM translocation (Fig. 2A & B) but high-affinity RGS binding to Gα requires receptor-activation for catalyzing GDP-GTP exchange at Gα. Finally, co-expression of MOR, which activates the same G proteins as does D2R, does not produce RGS9-2 DRM translocation (Fig. 2A & B).

Interestingly, our results also suggest a role for D2R-coupled Gα G proteins in stabilizing the D2R-mediated translocation of RGS9-2 to DRM. A point mutation N364H, which disrupts RGS9-2-Gα G protein binding, does not block D2R-mediated targeting of RGS9-2 to the plasma membrane (Fig. 6), but completely disrupts D2R-mediated targeting of RGS9-2 to DRM (Fig. 5). A role of Gα protein in the stability of the D2R/RGS9/Gb5 complex is also supported by the finding that D2R is likely pre-coupled to G proteins as D2R significantly increased the membrane localization of a Gα_iG protein isoform at the plasma membrane when the proteins were co-expressed in HEK293T cells (Fig. 6D).

One model that explains all the available data is that the RGS9 DEP domain is both necessary and sufficient for targeting RGS9-2/Gβ5 to D2R at the plasma membrane. Additional interactions with D2R-associated G proteins may be required for targeting of RGS9-2 to D2R in DRM micro-compartments and may help to stabilize RGS9-2 in the DRM complex during detergent extraction. The N364H mutation abolishes RGS9-2 G protein interactions and the mutant RGS9-2 N364H construct consequently partitions into the TX100-soluble biochemical fraction.

Previously, we showed that RGS9-2 specifically blocks the agonist-mediated internalization of D2R when the two proteins are expressed together in HEK293T cells (Celver et al. 2010). Interestingly, both the RGS9-2-mediated inhibition of D2R internalization, described previously (Celver et al. 2010), and the D2R-mediated translocation of RGS9-2 into DRM, described here, were blocked by the deletion of the RGS9-2 DEP domain or by the introduction of the RGS9-2 N364H mutation (Fig. 5). These data suggest that inhibition of D2R internalization by RGS9-2 involves the targeting of RGS9-2 to D2R-containing DRM. Gβ5 co-expression was also necessary for the RGS9-2-mediated inhibition of D2R internalization (Celver et al. 2010). Our finding that RGS9-2 targeting to D2R is enhanced by Gβ5 expression may explain the requirement of Gβ5 for RGS9-2 mediated inhibition D2R internalization.

The DEP domain of RGS9-2 interacts with R7BP, a small membrane-associated protein, and RGS9-2 can be targeted to the plasma membrane via this interaction (Martemyanov et al. 2005; Anderson et al. 2009). R7BP is also important for controlling the proteolytic stability of RGS9 2 (Anderson et al. 2007) and dramatic reductions in the levels of striatal RGS9-2 are observed in mice after elimination of R7BP expression. However, we cannot necessarily conclude from the above studies that all of the RGS9-2 associated with the cell membrane is targeted to the membrane via an interaction with R7BP or that there is no cellular RGS9-2 that is not bound to R7BP. For example, the RGS9-2/Gβ5/R7BP complex could help to stabilize total cellular RGS9-2, including the proportion of free RGS9-2.

In fact, Mancuso and colleagues found that the majority of RGS9-2 endogenously expressed in the striatum is TX100 insoluble while R7BP is TX100 soluble (Mancuso et al. 2010). They concluded that the TX100-insolublity of striatal RGS9-2 must result “through an interaction other than or in addition to binding palmitoylated R7BP.” The data we present in this manuscript suggests that an interaction “other than” binding R7BP, that is mediated by D2R, is likely responsible for the TX100-insolubility of striatal RGS9-2. We were unable to detect R7BP transcripts in HEK293T cells using reverse transcriptase PCR (see Supporting Information) indicating that endogenous expression of R7BP is not influencing the interpretation of the results reported here.

D2R expressed in the plasma membrane at the cell surface is found in detergent-resistant structures

The resistance of D2R to solubilization by cold TX100 is not purely a result of the non-physiological aggregation of the recombinantly expressed protein. Mis-folded or non-physiologically aggregated plasma membrane-targeted proteins usually remain in the endoplasmic reticulum (ER) where they are sorted for ER-associated degradation (Shimizu and Hendershot 2007; Maattanen et al. 2010) but our results indicate that a significant fraction of D2R in DRM resides at the cell surface. First, in a previous publication we estimated that slightly more than 50% of the exogenously expressed D2R in HEK293T cells is inserted into the plasma membrane and the rest is present in intracellular compartments (Celver et al. 2010). Based on this estimated cellular percentage of D2R at the plasma membrane, and our observation showing that greater than 70% of the total cellular D2R is found in DRM (Fig. 1E), it can be easily concluded that DRM-containing D2R is present both at the plasma membrane and within the cell. The intracellular TX100-insoluble D2R fraction could represent mis-folded D2R or the physiologically relevant insertion of D2R into DRM within intracellular compartments such as the endoplasmic reticulum and Golgi.

Furthermore, we have confirmed the presence of D2R in DRM at the cell surface using antibodies targeting the extracellular FLAG-epitope fused to D2R to show that the majority of antibody that bound D2R-expressing intact cells was fractionated into the DRM pellet. A similar experiment using antibodies to specifically bind surface-expressed MOR found a drastically smaller percentage of the MOR-labeling antibodies in the DRM fraction (Fig. 8).

Lipid rafts represent a set of hypothesized plasma membrane micro-domains that are thought to consist of dynamic assemblies of cholesterol, sphingolipids and associated proteins (Lindner and Naim 2009; Lingwood and Simons 2010). An early definitive biochemical feature that was attributed to these membrane structures is resistance to extraction in cold non-ionic detergents (Simons and Toomre 2000; Pike 2004; Brown 2006; Lindner and Naim 2009; Lingwood and Simons 2010) but this early definition of biological rafts is thought to be simplistic (Munro 2003). For example, detergent-insolubility of D2R could arise from an association with detergent insoluble cytoskeletal protein. Future experiments will investigate the molecular basis for the targeting of D2R to DRM and attempt to identify whether D2R is targeted to lipid-rafts or other detergent-insoluble cellular structures.

Our study shows that D2R is targeted to DRM structures and a direct or indirect interaction with D2R could be responsible for the targeting of endogenous RGS9-2 into DRM. Consequently, this study is an important step in the development of a biochemical model for explaining many of the previously described and physiologically important D2R-RGS9-2 functional interactions (reviewed in Introduction) and the description of the D2R ‘signalplex” in the brain.

Abbreviations used

D2R

D2-dopamine receptor long isoform

D2Rsh

D2-dopamine receptor short isoform

DEP

dishevelled

EGL-10

pleckstrin homology

DRM

detergent-resistant membrane

DOR

delta opioid receptor

FLAG

octa-peptide with amino acid sequence of DYKDDDDK

GAP

GTPase accelerating protein

Gβ5

G protein G beta 5 short isoform

GFP

green fluorescent protein

GPCR

G protein-coupled receptor

GTP

guanosine-5′-triphosphate

HEK293T

human embryonic kidney 23 cells stably expressing the SV40 T-antigen

MOR

mu opioid receptor

PTX

pertussis-toxin

DTT

dithiothreitol

HRP

horse-radish peroxidase

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

PVDF

polyvinylidene fluoride

RGS

regulator of G protein signaling

RGS9-2

regulator of G protein signaling 9-2

SDS

sodium dodecyl sulfate

TCA

trichloro-acetic acid

TFR

transferrin receptor

TX100

Triton X-100