Differential regulation of RGS proteins in the prefrontal cortex of short- and long-term human opiate abusers

Opiate addiction is characterized by drug tolerance and dependence which involve adaptive changes in μ-opioid receptors (MORs) signaling. Regulators of G-protein signaling RGS9, RGS4 and RGS10 proteins negatively regulate G(αi/o) protein activity modulating MOR function. An important role of RGS proteins in drug addiction has been described but the status of RGS proteins in human brain of opiate addicts remains unknown. The present study evaluated the immunoreactivity levels of RGS4, RGS9 and RGS10 proteins in prefrontal cortex of short- (n = 15) and long-term (n = 21) opiate abusers and in matched control subjects. RGS4 protein was not altered in short-term opiate abusers but, in long-term abusers it was significantly up-regulated (Δ = 29 ± 6%). RGS10 protein expression was significantly decreased in short-term (Δ = -42 ± 7%) but remained unaltered in long-term opiate abusers. RGS9 protein levels in opiate abusers did not differ from matched controls either in the short-term or in the long-term opiate abuser groups. RGS4, RGS9 and RGS10 levels were also studied in brains (frontal cortex) of rats submitted to acute and chronic morphine treatment and to spontaneous and naloxone-precipitated opiate withdrawal. Chronic morphine treatment in rats was associated with an increase in RGS4 protein immunoreactivity (Δ = 28 ± 7%), which persisted in spontaneous (Δ = 35 ± 8%) and naloxone-precipitated withdrawal (Δ = 30 ± 9%) without significant changes in RGS9 and RGS10 proteins. The specific modulation of RGS4 and RGS10 protein expression observed in the prefrontal cortex of opiate abusers might be relevant in the neurobiology of opiate tolerance, dependence and withdrawal. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.     

Nucleus Accumbens-Specific Interventions in RGS9-2 Activity Modulate Responses to Morphine

Regulator of G protein signalling 9-2 (Rgs9-2) modulates the actions of a wide range of CNS-acting drugs by controlling signal transduction of several GPCRs in the striatum. RGS9-2 acts via a complex mechanism that involves interactions with Gα subunits, the Gβ5 protein, and the adaptor protein R7BP. Our recent work identified Rgs9-2 complexes in the striatum associated with acute or chronic exposures to mu opioid receptor (MOR) agonists. In this study we use several new genetic tools that allow manipulations of Rgs9-2 activity in particular brain regions of adult mice in order to better understand the mechanism via which this protein modulates opiate addiction and analgesia. We used adeno-associated viruses (AAVs) to express forms of Rgs9-2 in the dorsal and ventral striatum (nucleus accumbens, NAc) in order to examine the influence of this protein in morphine actions. Consistent with earlier behavioural findings from constitutive Rgs9 knockout mice, we show that Rgs9-2 actions in the NAc modulate morphine reward and dependence. Notably, Rgs9-2 in the NAc affects the analgesic actions of morphine as well as the development of analgesic tolerance. Using optogenetics we demonstrate that activation of Channelrhodopsin2 in Rgs9-2-expressing neurons, or in D1 dopamine receptor (Drd1)-enriched medium spiny neurons, accelerates the development of morphine tolerance, whereas activation of D2 dopamine receptor (Drd2)-enriched neurons does not significantly affect the development of tolerance. Together, these data provide new information on the signal transduction mechanisms underlying opiate actions in the NAc.     

μ-Opioid receptors and regulators of G protein signaling (RGS) proteins: from a symposium on new concepts in mu-opioid pharmacology

Mu-opioid receptors (MOR) are the therapeutic target for opiate analgesic drugs and also mediate many of the side-effects and addiction liability of these compounds. MOR is a seven-transmembrane domain receptor that couples to intracellular signaling molecules by activating heterotrimeric G proteins. However, the receptor and G protein do not function in isolation but their activities are moderated by several accessory and scaffolding proteins. One important group of accessory proteins is the regulator of G protein signaling (RGS) protein family, a large family of more than thirty members which bind to the activated Gα subunit of the heterotrimeric G protein and serve to accelerate signal termination. This action negatively modulates receptor signaling and subsequent behavior. Several members of this family, in particular RGS4 and RGS9-2 have been demonstrated to influence MOR signaling and morphine-induced behaviors, including reward. Moreover, this interaction is not unidirectional since morphine has been demonstrated to modulate expression levels of RGS proteins, especially RGS4 and RGS9-2, in a tissue and time dependent manner. In this article, I will discuss our work on the regulation of MOR signaling by RGS protein activity in cultured cell systems in the context of other in vitro and behavioral studies. In addition I will consider implications of the bi-directional interaction between MOR receptor activation and RGS protein activity and whether RGS proteins might provide a suitable and novel target for medications to manage addictive behaviors.     

The GBeta5 subunit that associates with the R7 subfamily of RGS proteins regulates mu-opioid effects

The Gbeta5 protein, which is similar in sequence to other G-protein beta subunits, mainly associates with the G-protein gamma-like (GGL) domains of the R7 subfamily of regulators of G-protein signalling (RGS) proteins. This paper reports the presence of the Gbeta5 protein and its mRNA in all areas of mouse CNS, and also its involvement in the cellular signals initiated at mu- and delta-opioid receptors. The expression of Gbeta5 and RGS9-2 proteins (member of the R7 subfamily of RGS) was reduced by blocking their mRNAs with antisense oligodeoxynucleotides (ODN). Knock-down of these proteins enhanced the potency and duration of antinociception promoted by morphine and [D-Ala2, N-MePhe4,Gly-ol5]-enkephalin (DAMGO), agonists at mu opioid receptors. However, the activity of the selective agonist at delta opioid receptors, [D-Pen(2,5)]-encephalin (DPDPE), appeared to be reduced. 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 Gbeta5 or RGS9-2 knock-down animals. In a model of sustained morphine treatment, the impairment of Gbeta5 proteins facilitated the development of tolerance. This treatment did not alter the incidence of jumping behaviour precipitated by naloxone 3 days after commencing with chronic morphine. These results show differences in the signalling regulation of G-proteins when activated by mu or delta opioid agonists. For mu opioid receptors, acute tolerance, but probably not long-term tolerance, appears to depend on the function of Gbeta5 subunits and associated RGS proteins.     

R7BP Complexes With RGS9-2 and RGS7 in the Striatum Differentially Control Motor Learning and Locomotor Responses to Cocaine

In the striatum, signaling through G protein-coupled dopamine receptors mediates motor and reward behavior, and underlies the effects of addictive drugs. The extent of receptor responses is determined by RGS9-2/Gβ5 complexes, a striatally enriched regulator that limits the lifetime of activated G proteins. Recent studies suggest that the function of RGS9-2/Gβ5 is controlled by the association with an additional subunit, R7BP, making elucidation of its contribution to striatal signaling essential for understanding molecular mechanisms of behaviors mediated by the striatum. In this study, we report that elimination of R7BP in mice results in motor coordination deficits and greater locomotor response to morphine administration, consistent with the essential role of R7BP in maintaining RGS9-2 expression in the striatum. However, in contrast to previously reported observations with RGS9-2 knockouts, mice lacking R7BP do not show higher sensitivity to locomotor-stimulating effects of cocaine. Using a striatum-specific knockdown approach, we show that the sensitivity of motor stimulation to cocaine is instead dependent on RGS7, whose complex formation with R7BP is dictated by RGS9-2 expression. These results indicate that dopamine signaling in the striatum is controlled by concerted interplay between two RGS proteins, RGS7 and RGS9-2, which are balanced by a common subunit, R7BP.     

The R7 subfamily of RGS proteins assists tachyphylaxis and acute tolerance at mu-opioid receptors.

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.     

The ontogeny of opiate tolerance and withdrawal in infant rats

The acquisition of morphine analgesic tolerance was investigated in neonatal rats. Morphine was found to produce a potent analgesia, as measured by latency to retract a hindpaw from a 52 degree C hotplate, in rat pups as young as 1 day of age. Morphine analgesic tolerance, however, did not develop in rats until the third week of life. Rats given the same daily morphine regimen starting at 15 days of age or older showed rapid tolerance development. The data from four experiments indicate that experience with morphine prior to this age (Day 15) does not impact on the analgesic efficacy of the drug. Similarly, when morphine treatment was discontinued and the rats given a naloxone challenge, withdrawal symptoms were not observed in very young rats. Opiate withdrawal was first detected in rats that started their daily morphine treatment at 30 days of age and were then challenged with naloxone at 52 days of age. Therefore, two correlates of opiate addiction, tolerance and withdrawal, appear to be relatively late-developing phenomena in the rat.     

Effector antagonism by the regulators of G protein signalling (RGS) proteins causes desensitization of mu-opioid receptors in the CNS

Rationale In cell culture systems, agonists can promote the phosphorylation and internalization of receptors coupled to G proteins (GPCR), leading to their desensitization. However, in the CNS opioid agonists promote a profound desensitization of their analgesic effects without diminishing the presence of their receptors in the neuronal membrane. Recent studies have indicated that CNS proteins of the RGS family, specific regulators of G protein signalling, may be involved in mu-opioid receptor desensitization in vivo.Objective In this work we review the role played by RGS proteins in the intensity and duration of the effects of mu-opioid receptor agonists, and how they influence the delayed tolerance that develops in response to specific doses of opioids.Results RGS proteins are GTPase-activating proteins (GAP) that accelerate the hydrolysis of GGTP to terminate signalling at effectors. The GAP activity of RGS-R4 and RGS-Rz proteins restricts the amplitude of opioid analgesia, and the efficient deactivation of GzGTP subunits by RGS-Rz proteins prevents mu receptor desensitization. However, RGS-R7 proteins antagonize effectors by binding to and sequestering mu receptor-activated Gi/o/z subunits. Thus, they reduce the pool of receptor-regulated G proteins and hence, the effects of agonists. The delayed tolerance observed following morphine administration correlates with the transfer of G subunits from mu receptors to RGS-R7 proteins and the subsequent stabilization of this association.Conclusion In the CNS, the RGS proteins control the activity of mu opioid receptors through GAP-dependent (RGS-R4 and RGS-Rz) as well as by GAP-independent mechanisms (RGS-R7). As a result, they can both antagonize effectors and desensitize receptors under certain circumstances.     

G蛋白信号调节蛋白作为中枢神经系统药物新靶点研究进展

G蛋白偶联受体(G-prote in-coup led receptors,GPCR)是许多治疗药物的作用靶点。G蛋白信号调节蛋白(regu latorof G-prote in signaling,RGS)属一类新发现的蛋白家族,它们在GPCR信号传导中起重要作用。一般来说RGS可加速G蛋白失活进而终止GPCR信号传导,但也有些RGS同时具有效应分子和信号传递功能。兼具GPCR激动和RGS抑制功能的药物将大大增强信号传导,同时还能增加激动剂的区域特异性。由于RGS的多样性,组织分布特异性以及较强的调节活性,RGS很可能成为寻找新型中枢神经系统疾病治疗药物的新靶点。     

The role of formalin-induced pain in morphine tolerance, withdrawal, and reward

The effect of a commonly used experimental pain-induction procedure (formalin injection into a hindpaw site) on morphine tolerance, withdrawal, and reward was examined in rats. Results suggest that the effects of morphine are different in the organism that is experiencing pain at the time it receives the drug than in the organism that is pain free. The presence of pain at the time of each morphine administration decreased analgesic tolerance, decreased naloxone-precipitated withdrawal, and enhanced the rewarding effect of the opiate. These findings, together with those of previous studies, suggest that theories of opiate tolerance, withdrawal, and reward should incorporate the effects of pain.     

RGS-Rz and RGS9-2 proteins control mu-opioid receptor desensitisation in CNS: the role of activated Galphaz subunits

Two consecutive i.c.v. administrations of analgesic doses of mu-opioid receptor agonists lead to a profound desensitisation of the latter receptors; a third dose produced less than 20% of the effect obtained with the first administration. Desensitisation was still effective 24h later. Impairing the activity of Galphaz but not Galphai2 subunits prevented tolerance developing after the administration of three consecutive doses of morphine. Further, the i.c.v. injection of Galphai2 subunits potentiated morphine analgesia and abolished acute tolerance, whereas i.c.v.-administered Galphaz subunits produced a rapid and robust loss of the response to morphine. The RGSZ1 and RGSZ2 proteins selectively deactivate GalphazGTP subunits, and their knockdown increased the effects produced by the first dose of morphine. However, impairing their activity also accelerated tachyphylaxis following successive doses of morphine, and facilitated the development of acute morphine tolerance. In contrast, inhibiting the RGS9-2 proteins, which bind to GalphaoGTP and GalphaiGTP but only weakly deactivates them, preserved the effects of consecutive morphine doses and abolished the generation of acute tolerance. Therefore, desensitisation of mu-opioid receptors can be achieved by reducing the responsiveness of post-receptor elements (via the possible action of activated Galphaz subunits) and/or by depleting the pool of receptor-regulated G proteins that agonists need to propagate their effects, e.g., through the activity of RGS9-2 proteins.     

Opiate withdrawal behavior after focal brain stimulation

Electrical stimulation of the brainstem abolishes pain, while continued stimulation induces tolerance to the analgesic effect. Analgesic drugs producing tolerance also induce physical dependence, suggesting that the phenomenon of tolerance is associated with addiction. There is evidence that the neural mechanism for stimulation-produced analgesia is related to the release of opiate substances within the brain. We therefore propose that repeated or protracted brain stimulation elicits dependence upon the endorphins released by electrical stimulation of the neurons themselves. To investigate this possibility, rats were given repetitive bursts of analgesic electrical brain stimulation for two hours. Immediately thereafter, they were injected with the opiate antagonist, naloxone. Behaviors associated with low grade opiate withdrawal were observed. These data suggest that prolonged analgesic stimulation can result in naloxone-precipitated behaviors similar to the behaviors exhibited during opiate withdrawal.     

Expression of neural RGS-R7 and Gbeta5 Proteins in Response to Acute and Chronic Morphine.

The R7 subfamily of regulators of G-protein signaling (RGS) proteins (RGS6, RGS7, RGS9-2, and RGS11), and its binding protein Gbeta5, are found in neural structures of mouse brain. A single intracerebroventricular priming dose of 10 nmol morphine gave rise to acute tolerance to the analgesic effects of successive identical test doses of the opioid. At 2 h after administering the acute opioid, RGS7 mRNA levels in the striatum plus those of RGS9-2 in the striatum and thalamus were increased, whereas RGS9-2 and RGS11 mRNA were reduced in the cortex. Similar but attenuated RGS-R7 mRNA changes persisted 24 h after acute morphine administration. No changes in Gbeta5 mRNA levels were observed. At 2 days after commencing sustained morphine treatment, the levels of mRNA for RGS7, RGS9-2, RGS11, and Gbeta5 increased in most of the brain structures studied (striatum, thalamus, periaqueductal gray matter (PAG), and cortex). In these morphine tolerant-dependent mice, the greater changes were found for RGS9-2 in the thalamus (>500%) and PAG (>200%). In post-dependent mice, the increases in RGS-R7 and Gbeta5 mRNA still persisted in the PAG and striatum at 8 and 16 days after starting the chronic opioid treatment. The raised mRNA levels promoted by chronic, but not by acute, morphine, were accompanied by increases in the encoded proteins. This is probably a result of the costabilization of the RGS-R7 and Gbeta5 proteins forming heterodimers. Opioid-induced adaptations of RGS-R7 and Gbeta5 genes may regulate the severity of morphine-induced tolerance/dependence and the duration of the post-dependent period, helping to recover the normal response.     

Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins

G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization processes and GPCR up- and downregulation. GPCR function can also be regulated by several proteins that directly interact with the receptor and thereby modulate receptor activity. An additional mechanism by which receptor signalling is regulated involves an emerging class of proteins, the so-called regulators of G protein signalling (RGS). In this review we will describe some of these control mechanisms in more detail with some specific examples in the cardiovascular system. In addition, we will provide an overview on RGS proteins and the involvement of RGS proteins in cardiovascular function.     

RGS6 as a Novel Therapeutic Target in CNS Diseases and Cancer

Regulator of G protein signaling (RGS) proteins are gatekeepers regulating the cellular responses induced by G protein-coupled receptor (GPCR)-mediated activation of heterotrimeric G proteins. Specifically, RGS proteins determine the magnitude and duration of GPCR signaling by acting as a GTPase-activating protein for Gα subunits, an activity facilitated by their semiconserved RGS domain. The R7 subfamily of RGS proteins is distinguished by two unique domains, DEP/DHEX and GGL, which mediate membrane targeting and stability of these proteins. RGS6, a member of the R7 subfamily, has been shown to specifically modulate Gα_i/o protein activity which is critically important in the central nervous system (CNS) for neuronal responses to a wide array of neurotransmitters. As such, RGS6 has been implicated in several CNS pathologies associated with altered neurotransmission, including the following: alcoholism, anxiety/depression, and Parkinson’s disease. In addition, unlike other members of the R7 subfamily, RGS6 has been shown to regulate G protein-independent signaling mechanisms which appear to promote both apoptotic and growth-suppressive pathways that are important in its tumor suppressor function in breast and possibly other tissues. Further highlighting the importance of RGS6 as a target in cancer, RGS6 mediates the chemotherapeutic actions of doxorubicin and blocks reticular activating system (Ras)-induced cellular transformation by promoting degradation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) to prevent its silencing of pro-apoptotic and tumor suppressor genes. Together, these findings demonstrate the critical role of RGS6 in regulating both G protein-dependent CNS pathology and G protein-independent cancer pathology implicating RGS6 as a novel therapeutic target.     

Inhibition of calcium/calmodulin-dependent protein kinase II in rat hippocampus attenuates morphine tolerance and dependence.

Learning and memory have been suggested to be important in the development of opiate addiction. Based on the recent findings that calcium/calmodulin-dependent protein kinase II (CaMKII) is essential in learning and memory processes, and morphine treatment increases CaMKII activity in hippocampus, the present study was undertaken to examine whether inhibition of hippocampal CaMKII prevents morphine tolerance and dependence. Here, we report that inhibition of CaMKII by intrahippocampal dentate gyrus administration of the specific inhibitors KN-62 and KN-93 to rats significantly attenuated the tolerance to the analgesic effect of morphine and the abstinence syndrome precipitated by opiate antagonist naloxone. In contrast, both KN-04 and KN-92, the inactive structural analogs of KN-62 and KN-93, failed to attenuate morphine tolerance and dependence, indicating that the observed effects of KN-62 and KN-93 are mediated through inhibition of CaMKII. Furthermore, administration of CaMKII antisense oligonucleotide into rat hippocampal dentate gyrus, which decreased the expression of CaMKII specifically, also attenuated morphine tolerance and dependence, while the corresponding sense oligonucleotide of CaMKII did not exhibit such inhibitory effect. Moreover, the KN-62 treatment abolished the rewarding properties of morphine as measured by the conditioned place preference. These results suggest that hippocampal CaMKII is critically involved in the development of morphine tolerance and dependence, and inhibition of this kinase may have some therapeutic benefit in the treatment of opiate tolerance and dependence.     

Morphine alters the selective association between mu-opioid receptors and specific RGS proteins in mouse periaqueductal gray matter

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 G alpha i/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.     

Tolerance and withdrawal to chronic morphine treatment in the week-old rat pup.

Chronic treatment of adult animals with morphine results in tolerance but there are fewer reports on the effects of chronic opiates during ontogeny. The present experiments assessed the development of morphine-induced tolerance and withdrawal in infant rats. Pups were injected with morphine twice daily from ages 1-7 days and then tested on day 7 for morphine-induced analgesia in a hot-water immersion test, and separation-induced ultrasonic vocalizations in response to isolation from the dam and littermates at 7 and 10 days of age. Tolerance occurred to the analgesic effects of morphine but not to its suppression of ultrasonic vocalizations. Separation-induced vocalizations were greatly increased in chronic morphine-treated pups following naltrexone-precipitated withdrawal at 7 days of age. The increase in ultrasonic vocalizations following naltrexone treatment in morphine exposed pups may be a developmentally unique sign of opiate withdrawal.     

Effects of (-)-N6-(R-phenylisopropyl)-adenosine (PIA) and caffeine on nociception and morphine-induced analgesia, tolerance and dependence in mice

(-)-N6-(R-phenylisopropyl)-adenosine (PIA) was shown to possess analgesic activity in both the tail flick and acetic acid writhing assays. The analgesic actions of PIA were antagonized by caffeine in a dose-dependent manner. An apparent pA2 analysis in vivo suggested that the antagonism by caffeine was not competitive. Subanalgesic doses of PIA potentiated morphine-induced analgesia, tolerance and dependence. Caffeine antagonized these effects of morphine. PIA attenuated while caffeine exacerbated opiate withdrawal. While a low dose of caffeine antagonized PIA effects on withdrawal, a low dose of PIA did not antagonize the effects of caffeine. These results indicate that PIA can facilitate, and caffeine can antagonize the actions of morphine and that caffeine may be exerting some of its actions independent of adenosine receptor antagonism.     

Impact of fusion to Gα(i2) and co-expression with RGS proteins on pharmacological properties of human cannabinoid receptors CB₁R and CB₂R

Objectives G protein coupled receptor (GPCR)‐Gα fusion proteins are often employed to investigate receptor/G protein interaction. In this study, the impact of Gα fusion proteins on pharmacology of CBRs, both mediating signals through Gα_i proteins, were investigated. Gα_i2 was fused to the C‐terminus of the CBRs or co‐expressed with non‐fused Gα_i2 in Sf9 cells, always together with Gβ₁γ₂. Furthermore, the impact of RGS proteins on CBR signaling in combination with the CBR fusion approach was examined, using RGS4 and RGS19 as paradigms. Methods CBR ligands were characterized in the steady‐state GTPase assay and pharmacological properties of ligands in the different test systems were correlated. Key findings Fusion of CBRs to Gα_i2 enhanced the maximal stimulatory effects of ligands compared to the co‐expression system, especially for CB₂R. RGS4, but not RGS19, behaved as a GTPase‐activating protein at CBRs in the Gα_i2 co‐expression and fusion system. Fusion of GPCR, most prominently CB₂R, to Gα_i2, and co‐expression with RGS4 altered the pharmacological properties of ligands. Conclusions Our data suggest that fusion of CB₂R to Gα_i2 and co‐expression with RGS4 impedes with conformational changes. Moreover, our results support the concept of ligand‐specific receptor conformations. Finally, this paper describes the most sensitive CBR test system currently available.