Olfactory receptor localization and function: an emerging role for GPCR heterodimerization

Research on olfaction has been fraught with considerable frustration because none of the hundreds of olfactory receptors make it to the cell surface on their own when expressed in heterologous systems. Recent work indicates that the heterodimerization of olfactory receptors with beta2-adrenergic receptors results in surface expression of these G protein-coupled receptors. Similar conclusions--that heterodimerization is essential for surface expression of olfactory receptors--have been drawn from research in Drosophila utilizing completely different knockout and functional approaches. Together these findings may unlock the solution to a problem that has plagued the molecular study of olfaction since the cloning of the first olfactory G protein-coupled receptor over twelve years ago.     

Orexin-1 receptor expression after global ischemia in mice

Orexins are neuropeptides that have a range of physiological effects including the regulation of feeding behavior and the sleep-wakefulness cycle. Recently, we reported that level of orexin A in spinal fluid was decreased in the patients of some neurodegenerative diseases and it is considered that orexin A and the receptors might be related to central nervous system disorders. However, the expression and localization of orexin receptors is not elicited well. Therefore, the purpose of this study is to investigate the time-dependent changes and the cellular localization of orexin receptor focusing on orexin-1 receptor (OX1R) in the mouse brain after transient common carotid artery occlusion (tCCAO) model by using immunohistochemical techniques. OX1R immunoreactivity dramatically increased and peaked in the hippocampus and cortex 2 days after tCCAO, but remained unchanged in the hypothalamus. Using double-immunohistochemistry, the OX1R immunopositive cells at 2 days after tCCAO were co-localized not only with neuronal marker, NeuN-immunoreactivity but also with astroglial and oligodendroglial markers, GFAP- and CNPase-immunoreactivities, respectively. These results suggested that OX1R is induced other cells in addition to the neurons during stress such as ischemia and orexins and its receptor might play an important role for ischemic insult.     

The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization

One-third of the approximately 400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT(1) and MT(2) melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT(2) did not modify MT(2) function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT(1) protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT(1) without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT(1). Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs.     

Cytoplasmic localization of pregnane X receptor and ligand-dependent nuclear translocation in mouse liver

The pregnane X receptor (PXR) plays an important role in the response to xenobiotics and endogenous toxins. We have used a specific anti-PXR antibody in the Western blotting of mouse liver nuclear extracts to show that PXR is accumulated in the nucleus after treatment with 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN), followed by an increase in Cyp3a11 mRNA. Expression of wild type PXR and various mutants as green fluorescent fusion proteins in mouse livers showed that PXR was retained in the cytoplasm from where PCN treatment translocated PXR into the nucleus. Furthermore, the xenochemical response signal, the nuclear translocation signal, and the activation function 2 domain were all required for the nuclear translocation to occur. Immunoprecipitation experiments using the hsp90 antibody demonstrated the presence of PXR in a complex with the endogenous cytoplasmic constitutive active/androstane receptor retention protein (CCRP) in HepG2 cells. Fluorescence resonance energy transfer analysis of mouse liver sections after co-expression of cyan fluorescent protein-CCRP and yellow fluorescent protein-PXR also indicated that CCRP and PXR were closely associated in vivo. Overexpression of exogenous CCRP increased the cytoplasmic level of the PXR.CCRP.hsp90 complex, whereas a decrease in endogenous CCRP by treatment with small interfering RNA for CCRP repressed the PXR-mediated reporter activity in HepG2 cells. We conclude that the CCRP mediates the retention of PXR in the cytosol and modulates the activation of PXR in response to PCN treatment.     

The critical role of carboxy-terminal amino acids in ligand-dependent and -independent transactivation of the constitutive androstane receptor

The mouse constitutive androstane receptor (CAR) is a unique member of the nuclear receptor superfamily, for which an inverse agonist, the testosterone metabolite 5alpha-androstan-3alpha-ol (androstanol), and an agonist, the xenobiotic 1,4-bis[2-(3, 5-dichloropyridyloxy)] benzene, are known. In this study the role of the transactivation domain 2 (AF-2) of CAR was investigated, which is formed by the seven most carboxy-terminal amino acids of the receptor. The AF-2 domain was shown to be critical for the constitutive activity by mediating a ligand-independent interaction of CAR with coactivator (CoA) proteins. In addition this domain increased and decreased contact with CoAs in the presence of agonist and inverse agonist, respectively. In analogy to classical endocrine nuclear receptors, in CAR the charge clamp between K187 (in helix 3) and E355 (within the AF-2 domain) was expected to be critical for its interaction with CoAs. However, the hydrophobic amino acids L352, L353, and I356 on the surface of the AF-2 domain were found to be more important for this protein-protein interaction. Moreover, these amino acids and C357 were shown to be involved in the response of CAR to androstanol. Interestingly, the cysteine at position 357 appears to block classical endocrine responsiveness of CAR to agonists, since mutagenesis of this amino acid both reduced CoA interaction in the absence of ligand and drastically increased inducibility by 1,4-bis[2-(3, 5-dichloropyridyloxy)] benzene. We showed that this blockade is not due to an intramolecular disulfide bridge, but is probably caused by an interaction between C357 and Y336.     

Differential requirements of arrestin-3 and clathrin for ligand-dependent and -independent internalization of human G protein-coupled receptor 40

G protein-coupled receptor 40 (GPR40) is believed to be an attractive target to enhance insulin secretion in patients with type 2 diabetes. GPR40 has been found to couple to Gq protein, leading to the activation of phospholipase C and subsequent increases in the intracellular Ca^2 + level. However, the underlying mechanisms that regulate the internalization and desensitization of GPR40 remain to be elucidated. In the present study, a construct of GPR40 fused with enhanced green fluorescent protein (EGFP) at its C-terminus was constructed for direct imaging of the localization and internalization of GPR40 by confocal microscopy. In stably transfected HEK-293 cells, GPR40 receptors underwent rapid agonist-induced internalization and constitutive ligand-independent internalization. Our data demonstrated that the agonist-mediated internalization of GPR40 was significantly blocked by hypertonic sucrose treatment and by siRNA mediated depletion of the heavy chain of clathrin. In contrast, constitutive GPR40 internalization was not affected by hypertonic sucrose or by knock-down of clathrin expression, but it was affected by treatment with methyl-β-cyclodextrin (MβCD) and nystatin. Furthermore, our results using an arrestin-3-EGFP redistribution assay and siRNA-mediated knock-down of arrestin-3 and GRK2 expression revealed that arrestin-3 and GRK2 play an essential role in the regulation of agonist-mediated GPR40 internalization, but are not involved in the regulation of constitutive GPR40 internalization. Additionally, our observation showed that upon activation by agonist, the internalized GPR40 receptors were rapidly recycled back to the plasma membrane via Rab4/Rab5 positive endosomes, whereas the constitutively internalized GPR40 receptors were recycled back to the cell surface through Rab5 positive endosomes. Because FFA receptors exhibit a high level of homology, our observations could be applicable to other members of this family.     

Impact of heterodimerization on intracellular localization of the ecdysteroid receptor (EcR)

Initially, nuclear import of the ecdysteroid receptor (EcR) in vertebrate cells (CHO-K1 and COS-7) does not afford a heterodimerization partner. Later on, EcR is retained in the nucleus only in the presence of a heterodimerization partner. Ultraspiracle (Usp) is more efficient compared to its vertebrate orthologue RXR and leads to an exclusively nuclear localization of EcR even in the absence of ligand. The DNA binding domain of the heterodimerization partner is important for retainment of EcR in the nucleus as shown by Usp4 (Usp(R130C)), which has lost its DNA binding capability. The C-terminal end of Usp (Usp(Delta205-508)) encompassing the C-terminal part of the D-domain and the E- and F-domains are essential for retainment of EcR in the nucleus. Nuclear localization is further influenced by cell-specific factors, since hormone and heterodimerization stabilizes the EcR protein in a cell-specific way.     

Homo- and heterodimerization of somatostatin receptor subtypes. Inactivation of sst(3) receptor function by heterodimerization with sst(2A)

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst(2A) and sst(3) exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and sst(3). The sst(2A)-sst(3) heterodimer exhibited high affinity binding to somatostatin-14 and the sst(2)-selective ligand L-779,976 but not to the sst(3)-selective ligand L-796,778. Like the sst(2A) homodimer, the sst(2A)-sst(3) heterodimer stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding, inhibition of adenylyl cyclase, and activation of extracellular signal-regulated kinases after exposure to the sst(2)-selective ligand L-779,976. However, unlike the sst(3) homodimer, the sst(2A)-sst(3) heterodimer did not promote GTPgammaS binding, adenylyl cyclase inhibition, or extracellular signal-regulated kinase activation in the presence of the sst(3)-selective ligand L-796,778. Interestingly, during prolonged somatostatin-14 exposure, the sst(2A)-sst(3) heterodimer desensitized at a slower rate than the sst(2A) and sst(3) homodimers. Both sst(2A) and sst(3) homodimers underwent agonist-induced endocytosis in the presence of somatostatin-14. In contrast, the sst(2A)-sst(3) heterodimer separated at the plasma membrane, and only sst(2A) but not sst(3) underwent agonist-induced endocytosis after exposure to somatostatin-14. Together, heterodimerization of sst(2A) and sst(3) results in a new receptor with a pharmacological and functional profile resembling that of the sst(2A) receptor, however with a greater resistance to agonist-induced desensitization. Thus, inactivation of sst(3) receptor function by heterodimerization with sst(2A) or possibly other G protein-coupled receptors may explain some of the difficulties in detecting sst(3)-specific binding and signaling in mammalian tissues.     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.     

Quantitative analysis of muscarinic acetylcholine receptor homo- and heterodimerization in live cells: regulation of receptor down-regulation by heterodimerization

Although previous pharmacological and biochemical data support the notion that muscarinic acetylcholine receptors (mAChR) form homo- and heterodimers, the existence of mAChR oligomers in live cells is still a matter of controversy. Here we used bioluminescence resonance energy transfer to demonstrate that M(1), M(2), and M(3) mAChR can form constitutive homo- and heterodimers in living HEK 293 cells. Quantitative bioluminescence resonance energy transfer analysis has revealed that the cell receptor population in cells expressing a single subtype of M(1), M(2), or M(3) mAChR is predominantly composed of high affinity homodimers. Saturation curve analysis of cells expressing two receptor subtypes demonstrates the existence of high affinity M(1)/M(2), M(2)/M(3), and M(1)/M(3) mAChR heterodimers, although the relative affinity values were slightly lower than those for mAChR homodimers. Short term agonist treatment did not modify the oligomeric status of homo- and heterodimers. When expressed in JEG-3 cells, the M(2) receptor exhibits much higher susceptibility than the M(3) receptor to agonist-induced down-regulation. Coexpression of M(3) mAChR with increasing amounts of the M(2) subtype in JEG-3 cells resulted in an increased agonist-induced down-regulation of M(3), suggesting a novel role of heterodimerization in the mechanism of mAChR long term regulation.     

CB1-independent inhibition of dopamine transporter activity by cannabinoids in mouse dorsal striatum

Cannabinoid drugs are known to affect dopaminergic neurotransmission in the basal ganglia circuitry. In this study, we used in vitro and in vivo techniques to investigate whether cannabinoid agonists and antagonist could affect dopaminergic transmission in the striatum by acting at the dopamine transporter. Incubation of striatal synaptosomes with the cannabinoid agonists WIN55,212-2 or methanandamide decreased dopamine uptake (IC(50) = 2.0 micromol/L and 3.1 micromol/L, respectively). A similar inhibitory effect was observed after application of the inactive WIN55,212-2 isomer, S(-)WIN55,212-3. The CB(1) antagonist AM251 did not reverse WIN55,212-2 effect but rather mimicked it. WIN55,212-2 and AM251 partially displaced the binding of the cocaine analog [(3)H]WIN35,428, thus acting as dopamine transporter pseudo-substrates in the high micromolar range. High-speed chronoamperometry measurements showed that WIN55,212-2 (4 mg/kg, i.p.) caused significant release of endogenous dopamine via activation of CB(1) receptors, followed by a reduction of dopamine clearance. This reduction was CB(1)-independent, as it was mimicked by S(-)WIN55,212-3. Administration of AM251 (1 and 4 mg/kg, i.p.) increased the signal amplitude and reduced the clearance of dopamine pressure ejected into the striatum. These results indicate that both cannabinoid agonists and antagonists inhibit dopamine transporter activity via molecular targets other than CB(1) receptors.     

Agonist efficacy and receptor efficiency in heterozygous CB1 knockout mice: relationship of reduced CB1 receptor density to G-protein activation

Heterozygous CB1 receptor knockout mice were used to examine the effect of reduced CB1 receptor density on G-protein activation in membranes prepared from four brain regions: cerebellum, hippocampus, striatum/globus pallidus (striatum/GP) and cingulate cortex. Results showed that CB1 receptor levels were approximately 50% lower in heterozygous mice in all regions examined. However, maximal stimulation of [(35)S]guanosine-5'-(gamma-O-thio) triphosphate ([(35)S]GTPgammaS) binding by the high efficacy agonist WIN 55,212-2 was reduced by only 20-25% in most brain regions, with the exception of striatum/GP where the decrease in stimulation was as predicted (approximately 50%). Furthermore, although the efficacies of the cannabinoid partial agonists, methanandamide and (9)-tetrahydrocannabinol, were similarly lower in heterozygous mice, their relative efficacies compared with WIN 55,212-2 were generally unchanged. Saturation analysis of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding showed that decreased stimulation by WIN 55,212-2 in striatum/GP of heterozygous mice was caused by a decrease in the apparent affinity of net-stimulated [(35)S]GTPgammaS binding. The apparent maximal number of binding sites (B(max)) values of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding were unchanged in cerebellum and striatum/GP of heterozygous mice, but decreased in cingulate cortex, with a similar trend in hippocampus. Moreover, in every region except cingulate cortex, the maximal number of net-stimulated [(35)S]GTPgammaS binding sites per receptor was significantly increased in heterozygous mice. These results indicate region-dependent increases in the apparent efficiency of CB1 receptor-mediated G-protein activation in heterozygous CB1 knockout mice.     

下丘脑腹内侧核Orexin-1及其受体对大鼠胃酸分泌的影响及其机制

目的:探讨下丘脑腹内侧核Orexin-1及其受体对大鼠胃酸分泌的影响及其机制。方法:大鼠麻醉后侧脑室及VMH置管,大鼠分组后分别VMH注射orexin-A、[Pro~(34)]-酪酪肽、[c PP1-7、NPY~(19-23)、Ala~(31)、Aib~(32)、Gln~(34)]胰多肽;腹腔注射SB-334867;皮下注射阿托品;侧脑室微量注射GR-231118、CGP-71683。给药结束后使用幽门结扎模型检测大鼠的胃酸分泌。结果:OXA能够促进胃酸分泌,且呈量效依赖关系。腹腔注射SB-334867能够抑制胃酸分泌,且呈量效依赖关系;SB-334867能够抑制orexin-A对胃酸分泌的促进作用;阿托品不但能够抑制胃酸分泌并且还能够完全阻断OXA的促胃酸分泌作用。侧脑室微量注射GR-231118或CGP-71683胃酸及胃液量减少,呈量效依赖关系,并且能够完全阻断OXA的促胃酸分泌作用。VMH内微量注射[cPP~(1-7),NPY~(19-23),Ala~(31),Aib~(32),Gln~(34)]胰多肽胃酸分泌增多,且呈量效依赖关系。结论:Orexin-A能够作用于下丘脑VMH促进胃酸分泌,orexin受体、Y1和Y5受体以及迷走神经系统均参与该过程。     

Heteromultimerization of cannabinoid CB(1) receptor and orexin OX(1) receptor generates a unique complex in which both protomers are regulated by orexin A

Agonist-induced internalization was observed for both inducible and constitutively expressed forms of the cannabinoid CB(1) receptor. These were also internalized by the peptide orexin A, which has no direct affinity for the cannabinoid CB(1) receptor, but only when the orexin OX(1) receptor was co-expressed along with the cannabinoid CB(1) receptor. This effect of orexin A was concentration-dependent and blocked by OX(1) receptor antagonists. Moreover, the ability of orexin A to internalize the CB(1) receptor was also blocked by CB(1) receptor antagonists. Remarkably, orexin A was substantially more potent in producing internalization of the CB(1) receptor than in causing internalization of the bulk OX(1) receptor population, and this was true in cells in which the CB(1) receptor was maintained at a constant level, whereas levels of OX(1) could be varied and vice versa. Both co-immunoprecipitation and cell surface, homogenous time-resolved fluorescence resonance energy transfer based on covalent labeling of N-terminal "SNAP" and "CLIP" tags present in the extracellular N-terminal domain of the receptors confirmed the capacity of these two receptors to heteromultimerize. These studies confirm the capacity of the CB(1) and OX(1) receptors to interact directly and demonstrate that this complex has unique regulatory characteristics. The higher potency of the agonist orexin A to regulate the CB(1)-OX(1) heteromer compared with the OX(1)-OX(1) homomer present in the same cells and the effects of CB(1) receptor antagonists on the function of orexin A suggest an interplay between these two systems that may modulate appetite, feeding, and wakefulness.     

Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function

The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.     

CB1 receptor signaling regulates social anxiety and memory

The endocannabinoid (eCB) system regulates emotion, stress, memory and cognition through the cannabinoid type 1 (CB₁) receptor. To test the role of CB₁ signaling in social anxiety and memory, we utilized a genetic knockout (KO) and a pharmacological approach. Specifically, we assessed the effects of a constitutive KO of CB₁ receptors (CB₁KOs) and systemic administration of a CB₁ antagonist (AM251; 5 mg/kg) on social anxiety in a social investigation paradigm and social memory in a social discrimination test. Results showed that when compared with wild‐type (WT) and vehicle‐treated animals, CB₁KOs and WT animals that received an acute dose of AM251 displayed anxiety‐like behaviors toward a novel male conspecific. When compared with WT animals, KOs showed both active and passive defensive coping behaviors, i.e. elevated avoidance, freezing and risk‐assessment behaviors, all consistent with an anxiety‐like profile. Animals that received acute doses of AM251 also showed an anxiety‐like profile when compared with vehicle‐treated animals, yet did not show an active coping strategy, i.e. changes in risk‐assessment behaviors. In the social discrimination test, CB₁KOs and animals that received the CB₁ antagonist showed enhanced levels of social memory relative to their respective controls. These results clearly implicate CB₁ receptors in the regulation of social anxiety, memory and arousal. The elevated arousal/anxiety resulting from either total CB₁ deletion or an acute CB₁ blockade may promote enhanced social discrimination/memory. These findings may emphasize the role of the eCB system in anxiety and memory to affect social behavior .     

Constitutive activity of the cannabinoid CB1 receptor regulates the function of co-expressed Mu opioid receptors

The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.