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Rationale: Clozapine is a unique antipsychotic, with efficacy against positive symptoms in treatment-resistant schizophrenic patients, and the ability to improve cognition and treat the negative symptoms characteristic of this disease. Despite its unique clinical actions, no specific molecular mechanism responsible for these actions has yet been described. Objectives and methods: To comprehensively profile a large library of neuropsychiatric drugs, including most antipsychotics, at human monoamine receptors using R-SAT, an in vitro functional assay. Results: Profiling revealed that N-desmethylclozapine (NDMC), the principal metabolite of clozapine, but not clozapine itself, is a potent and efficacious muscarinic receptor agonist, a molecular property not shared by any other antipsychotic. To further explore the role of NDMC muscarinic receptor agonist properties in mediating the physiological actions of clozapine, systemically administered NDMC was found to stimulate the phosphorylation of mitogen-activated protein kinase (MAP kinase) in mouse CA1 hippocampal neurons, an effect that was blocked by scopolamine, confirming central M1 muscarinic receptor agonist activity in vivo. Lastly, an analysis of clozapine and NDMC serum levels in schizophrenic patients indicated that high NDMC/clozapine ratios better predicted improvement in cognitive functioning and quality of life than the levels of either compound alone. Conclusions: The muscarinic receptor agonist activities of NDMC are unique among antipsychotics, and provide a possible molecular basis for the superior clinical effects of clozapine pharmacotherapy. [ABSTRACT FROM AUTHOR]

Abstract Background The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only δ opioid receptors. Activation profiles of most of these ligands at δ opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production. Results Naltrexone and nalorphine were classified as antagonists at δ opioid receptor. The other ligands studied were agonists at δ opioid receptors and demonstrated IC50 values of 0.1 nM to 2 μM, maximal inhibition of 39–77% and receptor binding affinities of 0.5 to 243 nM. The rank order of efficacy of the ligands tested was metazocine = xorphanol ≥ fentanyl = SKF 10047 = etorphine = hydromorphone = butorphanol = lofentanil > WIN 44,441 = Nalbuphine = cyclazocine ≥ met-enkephalin >> morphine = dezocine. For the first time these data describe and compare the function and relative efficacy of several ligands at δ opioid receptors. Conclusions The data produced from this study can lead to elucidation of the complete activation profiles of several opioid ligands, leading to clarification of the mechanisms involved in physiological effects of these ligands at δ opioid receptors. Furthermore, these data can be used as a basis for novel use of existing opioid ligands based on their pharmacology at δ opioid receptors.

Purpose: PD-1/L1 axis-directed therapies produce clinical responses in a subset of patients; therefore, biomarkers of response are needed. We hypothesized that quantifying key immunosuppression mechanisms within the tumor microenvironment by multiparameter algorithms would identify strong predictors of anti-PD-1 response. Experimental Design: Pretreatment tumor biopsies from 166 patients treated with anti-PD-1 across 10 academic cancer centers were fluorescently stained with multiple markers in discovery ( n = 24) and validation ( n = 142) cohorts. Biomarker-positive cells and their colocalization were spatially profiled in pathologist-selected tumor regions using novel Automated Quantitative Analysis algorithms. Selected biomarker signatures, PD-1/PD-L1 interaction score, and IDO-1/HLA-DR coexpression were evaluated for anti-PD-1 treatment outcomes. Results: In the discovery cohort, PD-1/PD-L1 interaction score and/or IDO-1/HLA-DR coexpression was strongly associated with anti-PD-1 response ( P = 0.0005). In contrast, individual biomarkers (PD-1, PD-L1, IDO-1, HLA-DR) were not associated with response or survival. This finding was replicated in an independent validation cohort: patients with high PD-1/PD-L1 and/or IDO-1/HLA-DR were more likely to respond ( P = 0.0096). These patients also experienced significantly improved progression-free survival (HR = 0.36; P = 0.0004) and overall survival (HR = 0.39; P = 0.0011). In the combined cohort, 80% of patients exhibiting higher levels of PD-1/PD-L1 interaction scores and IDO-1/HLA-DR responded to PD-1 blockers ( P = 0.000004). In contrast, PD-L1 expression was not predictive of survival. Conclusions: Quantitative spatial profiling of key tumor-immune suppression pathways by novel digital pathology algorithms could help more reliably select melanoma patients for PD-1 monotherapy. Clin Cancer Res; 24(21); 5250-60. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Current antipsychotic drug therapies for schizophrenia have limited efficacy and are notably ineffective at addressing the cognitive deficits associated with this disorder. The present study was designed to develop effective antipsychotic agents that would also ameliorate the cognitive deficits associated with this disease. In vitro studies comprised of binding and functional assays were utilized to identify compounds with the receptor profile that could provide both antipsychotic and pro-cognitive features. Antipsychotic and cognitive models assessing in vivo activity of these compounds included locomotor activity assays and novel object recognition assays. We developed a series of potential antipsychotic agents with a novel receptor activity profile comprised of muscarinic M(1) receptor agonism in addition to dopamine D(2) antagonism and serotonin 5-HT(2A) inverse agonism. Like other antipsychotic agents, these compounds reverse both amphetamine and dizocilpine-induced hyperactivity in animals. In addition, unlike other antipsychotic drugs, these compounds demonstrate pro-cognitive actions in the novel object recognition assay. The dual attributes of antipsychotic and pro-cognitive actions distinguish these compounds from other antipsychotic drugs and suggest that these compounds are prototype molecules in the development of novel pro-cognitive antipsychotic agents.

The role of neuropeptide FF (NPFF) and its analogs in pain modulation is ambiguous. Although NPFF was first characterized as an antiopioid peptide, both antinociceptive and pronociceptive effects have been reported, depending on the route of administration. Currently, two NPFF receptors, termed FF1 and FF2, have been identified and cloned, but their roles in pain modulation remain elusive because of the lack of availability of selective compounds suitable for systemic administration in in vivo models. Ligand-binding studies confirm ubiquitous expression of both subtypes in brain, whereas only FF2 receptors are expressed spinally. This disparity in localization has served as the foundation of the hypothesis that FF1 receptors mediate the pronociceptive actions of NPFF. We have identified novel small molecule NPFF receptor agonists and antagonists with varying degrees of FF2/FF1 functional selectivity. Using these pharmacological tools in vivo has allowed us to define the roles of NPFF receptor subtypes as pertains to the modulation of nociception. We demonstrate that selective FF2 agonism does not modulate acute pain but instead ameliorates inflammatory and neuropathic pains. Treatment with a nonselective FF1/FF2 agonist potentiates allodynia in neuropathic rats and increases sensitivity to noxious thermal and to non-noxious mechanical stimuli in normal rats in an FF1 antagonist-reversible manner. Treatment with FF1 antagonists reversed established mechanical allodynia, indicating the possibility of increased NPFF tone through FF1 receptors. In conclusion, we provide evidence for the opposing roles of NPFF receptors and highlight selective FF2 agonism and/or selective FF1 antagonism as potential targets warranting further investigation.

The recent discovery of allosteric potentiators and agonists of the muscarinic M(1) receptor represents a significant advance in the muscarinic receptor pharmacology. In the current study we describe the receptor pharmacology and pro-cognitive action of the allosteric agonist AC-260584. Using in vitro cell-based assays with cell proliferation, phosphatidylinositol hydrolysis or calcium mobilization as endpoints, AC-260584 was found to be a potent (pEC(50) 7.6-7.7) and efficacious (90-98% of carbachol) muscarinic M(1) receptor agonist. Furthermore, as compared to orthosteric binding agonists, AC-260584 showed functional selectivity for the M(1) receptor over the M(2), M(3), M(4) and M(5) muscarinic receptor subtypes. Using GTPgammaS binding assays, its selectivity was found to be similar in native tissues expressing mAChRs to its profile in recombinant systems. In rodents, AC-260584 activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in the hippocampus, prefrontal cortex and perirhinal cortex. The ERK1/2 activation was dependent upon muscarinic M(1) receptor activation since it was not observed in M(1) knockout mice. AC-260584 also improved the cognitive performance of mice in the novel object recognition assay and its action is blocked by the muscarinic receptor antagonist pirenzepine. Taken together these results indicate for the first time that a M(1) receptor agonist selective over the other mAChR subtypes can have a symptomatically pro-cognitive action. In addition, AC-260584 was found to be orally bioavailable in rodents. Therefore, AC-260584 may serve as a lead compound in the development of M(1) selective drugs for the treatment of cognitive impairment associated with schizophrenia and Alzheimer's disease., (2009 Elsevier Ltd. All rights reserved.)

Background: Activation of muscarinic M1 receptors is mediated via interaction of orthosteric agonists with the acetylcholine binding site or via interaction of allosteric agonists with different site(s) on the receptor. The focus of the present study was to determine if M1 receptors activated by allosteric agonists undergo the same regulatory fate as M1 receptors activated by orthosteric agonists., Results: The orthosteric agonists carbachol, oxotremorine-M and pilocarpine were compared to the allosteric agonists AC-42, AC-260584, N-desmethylclozapine and xanomeline. All ligands activated M1 receptors and stimulated interaction of the receptors with beta-arrestin-1. All ligands reduced cell surface binding and induced the loss of total receptor binding. Receptor internalization was blocked by treatment with hypertonic sucrose indicating that all ligands induced formation of clathrin coated vesicles. However, internalized receptors recycled to the cell surface following removal of orthosteric, but not allosteric agonists. Whereas all ligands induced loss of cell surface receptor binding, no intracellular vesicles could be observed after treatment with AC-260584 or xanomeline. Brief stimulation of M1 receptors with AC-260584 or xanomeline resulted in persistent activation of M1 receptors, suggesting that continual receptor signaling might impede or delay receptor endocytosis into intracellular vesicles., Conclusion: These results indicate that allosteric agonists differ from orthosteric ligands and among each other in their ability to induce different regulatory pathways. Thus, signaling and regulatory pathways induced by different allosteric ligands are ligand specific.

We report the discovery and initial characterization of a novel class of selective NPFF2 agonists. HTS screening using R-SAT, a whole cell based functional assay, identified a class of aryliminoguanidines as NPFF1 and NPFF2 ligands. Subsequent optimization led to molecules exhibiting selective NPFF2 agonistic activity. Systemic administration showed that selective NPFF2 agonists (1 and 3) are active in various pain models in vivo, whereas administration of a nonselective NPFF1 and NPFF2 agonist (9) increases sensitivity to noxious and non-noxious stimuli.

We report the first small-molecule protease-activated receptor (PAR) 2 agonists, AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide], each representing a distinct chemical series. AC-55541 and AC-264613 each activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca(2+) mobilization assays, with potencies ranging from 200 to 1000 nM for AC-55541 and 30 to 100 nM for AC-264613. In comparison, the PAR2-activating peptide 2-furoyl-LIGRLO-NH(2) had similar potency, whereas SLIGRL-NH(2) was 30 to 300 times less potent. Neither AC-55541 nor AC-264613 had activity at any of the other PAR receptor subtypes, nor did they have any significant affinity for over 30 other molecular targets involved in nociception. Visualization of EYFP-tagged PAR2 receptors showed that each compound stimulated internalization of PAR2 receptors. AC-55541 and AC-264613 were well absorbed when administered intraperitoneally to rats, each reaching micromolar peak plasma concentrations. AC-55541 and AC-264613 were each stable to metabolism by liver microsomes and maintained sustained exposure in rats, with elimination half-lives of 6.1 and 2.5 h, respectively. Intrapaw administration of AC-55541 or AC-264613 elicited robust and persistent thermal hyperalgesia and edema. Coadministration of either a tachykinin 1 (neurokinin 1) receptor antagonist or a transient receptor potential vanilloid (TRPV) 1 antagonist completely blocked these effects. Systemic administration of either AC-55541 or AC-264613 produced a similar degree of hyperalgesia as was observed when the compounds were administered locally. These compounds represent novel small-molecule PAR2 agonists that will be useful in probing the physiological functions of PAR2 receptors.

Peptides with agonist activity at the vasopressin V(2) receptor are used clinically to treat fluid homeostasis disorders such as polyuria and central diabetes insipidus. Of these peptides, the most commonly used is desmopressin, which displays poor bioavailability as well as potent activity at the V(1b) receptor, with possible stress-related adverse effects. Thus, there is a strong need for the development of small molecule chemistries with selective V(2) receptor agonist activity. Using the functional cell-based assay Receptor Selection and Amplification Technology (R-SAT((R))), a screening effort identified three small molecule chemotypes (AC-94544, AC-88324, and AC-110484) with selective agonist activity at the V(2) receptor. One of these compounds, AC-94544, displayed over 180-fold selectivity at the V(2) receptor compared to related vasopressin and oxytocin receptors and no activity at 28 other G protein-coupled receptors (GPCRs). All three compounds also showed partial agonist activity at the V(2) receptor in a cAMP accumulation assay. In addition, in a rat model of central diabetes insipidus, AC-94544 was able to significantly reduce urine output in a dose-dependent manner. Thus, AC-94544, AC-88324, and AC-110484 represent novel opportunities for the treatment of disorders associated with V(2) receptor agonist deficiency.

We assessed a simple, noninvasive method of monitoring transcutaneous partial pressure of CO2 (Ptcco2) in mice to determine whether it would provide an accurate and reproducible method to assess ventilatory depression in mice. To this end, Ptcco2 and Paco2 (partial pressure of arterial CO2) measurements were performed on isoflurane-anesthetized male C57Bl/6 mice breathing differing percentages of CO2 or fentanyl, a known ventilatory depressive drug. All doses of fentanyl produced a sharp increase in Ptcco2 values within 20 min with difference in Ptcco2 values between saline and all fentanyl groups being statistically significant (P < 0.0001). A good correlation between Paco2 and Ptcco2 values was established (r2 = 0.91). A Bland-Altman analysis likewise found that Ptcco2 measurements in the mice reliably and accurately reflected their Paco2 values. Therefore, under controlled conditions, Ptcco2 measurements were found to reliably reflect Paco2 values in mice. Consequently, the Ptcco2 method can be used as a means to rapidly and quantitatively assess the ventilatory depressive properties of a wide spectrum of drugs, under varying conditions in numerous mouse models.

The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)(2A) receptor inverse agonist ACP-103 competitively antagonized the binding of [(3)H]ketanserin to heterologously expressed human 5-HT(2A) receptors with a mean pK(i) of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC(50) of 8.7. ACP-103 demonstrated lesser affinity (mean pK(i) of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC(50) 7.1 in R-SAT) at human 5-HT(2C) receptors, and lacked affinity and functional activity at 5-HT(2B) receptors, dopamine D(2) receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT(2A) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT(2A) receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT(2A) receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.

Background: The aim of the present study was to describe the activity of a set of opioid drugs, including partial agonists, in a human embryonic kidney cell system stably expressing only the mouse kappa-opioid receptors. Receptor activation was assessed by measuring the inhibition of cyclic adenosine mono phosphate (cAMP) production stimulated by 5 microM forskolin. Intrinsic activities and potencies of these ligands were determined relative to the endogenous ligand dynorphin and the kappa agonist with the highest intrinsic activity that was identified in this study, fentanyl., Results: Among the ligands studied naltrexone, WIN 44,441 and dezocine, were classified as antagonists, while the remaining ligands were agonists. Intrinsic activity of agonists was assessed by determining the extent of inhibition of forskolin-stimulated cAMP production. The absolute levels of inhibition of cAMP production by each ligand was used to describe the rank order of intrinsic activity of the agonists; fentanyl = lofentanil > or = hydromorphone = morphine = nalorphine > or = etorphine > or = xorphanol > or = metazocine > or = SKF 10047 = cyclazocine > or = butorphanol > nalbuphine. The rank order of affinity of these ligands was; cyclazocine > naltrexone > or = SKF 10047 > or = xorphanol > or = WIN 44,441 > nalorphine > butorphanol > nalbuphine > or = lofentanil > dezocine > or = metazocine > or = morphine > hydromorphone > fentanyl., Conclusion: These results elucidate the relative activities of a set of opioid ligands at kappa-opioid receptor and can serve as the initial step in a systematic study leading to understanding of the mode of action of these opioid ligands at this receptor.

The 5-HT1A receptor is a critical mediator of serotonergic (5-HT) function. We have identified 13 potential single nucleotide polymorphisms resulting in amino acid changes throughout the human 5-HT1A receptor. The pharmacological profiles of these 13 polymorphic variants were then characterized using a high-throughput assay based on ligand-dependent transformation of NIH/3T3 cells. The majority of the polymorphic variants displayed wild-type pharmacological profiles in response to a panel of well-established agonists at the 5-HT1A receptor. However, the A50V polymorphic variant, which had an alanine to valine substitution in transmembrane 1, exhibited a loss of detectable response to 5-HT. Interestingly, all other agonists tested, including buspirone, lisuride, and (+)8-OH-DPAT, exhibited efficacies similar to that of the wild-type receptor. The competitive antagonist, methiothepin, also displayed a 19-fold decrease in potency at the A50V variant receptor. However, both 5-HT and methiothepin were able to compete for [3H]WAY-100635 binding to the A50V variant with affinities similar to the wild-type receptor. Moreover, the Bmax of [3H]WAY-100635 binding was 14-fold lower for the A50V variant than for the wild-type receptor. Thus, the A50V receptor variant exhibited ligand-specific functional alterations in addition to lower expression levels. These data suggest a previously unappreciated role for transmembrane 1 in mediating 5-HT response at the 5-HT1A receptor. Furthermore, individuals that potentially harbor the A50V polymorphism might display aberrant affective behaviors and altered responses to drugs targeting the 5-HT1A receptor.

Background: The aim of the present study was to describe the activity of a set of opioid drugs, including partial agonists, in a cell system expressing only mu opioid receptors. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cyclic adenosine mono phosphate (cAMP) production. Efficacies and potencies of these ligands were determined relative to the endogenous ligand beta-endorphin and the common mu agonist, morphine., Results: Among the ligands studied naltrexone, WIN 44,441 and SKF 10047, were classified as antagonists, while the remaining ligands were agonists. Agonist efficacy was assessed by determining the extent of inhibition of forskolin-stimulated cAMP production. The rank order of efficacy of the agonists was fentanyl = hydromorphone = beta-endorphin > etorphine = lofentanil = butorphanol = morphine = nalbuphine = nalorphine > cyclazocine = dezocine = metazocine >or= xorphanol. The rank order of potency of these ligands was different from that of their efficacies; etorphine > hydromorphone > dezocine > xorphanol = nalorphine = butorphanol = lofentanil > metazocine > nalbuphine > cyclazocine > fentanyl > morphine >>>> beta-endorphin., Conclusion: These results elucidate the relative activities of a set of opioid ligands at mu opioid receptor and can serve as the initial step in a systematic study leading to understanding of the mode of action of opioid ligands at this receptor. Furthermore, these results can assist in understanding the physiological effect of many opioid ligands acting through mu opioid receptors.

Background: The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only delta opioid receptors. Activation profiles of most of these ligands at delta opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production., Results: Naltrexone and nalorphine were classified as antagonists at delta opioid receptor. The other ligands studied were agonists at delta opioid receptors and demonstrated IC50 values of 0.1 nM to 2 microM, maximal inhibition of 39-77% and receptor binding affinities of 0.5 to 243 nM. The rank order of efficacy of the ligands tested was metazocine = xorphanol > or = fentanyl = SKF 10047 = etorphine = hydromorphone = butorphanol = lofentanil > WIN 44,441 = Nalbuphine = cyclazocine > or = met-enkephalin >> morphine = dezocine. For the first time these data describe and compare the function and relative efficacy of several ligands at delta opioid receptors., Conclusions: The data produced from this study can lead to elucidation of the complete activation profiles of several opioid ligands, leading to clarification of the mechanisms involved in physiological effects of these ligands at delta opioid receptors. Furthermore, these data can be used as a basis for novel use of existing opioid ligands based on their pharmacology at delta opioid receptors.

For G protein-coupled receptors, limited information is available on the role of agonist binding or of the second-messenger products of receptor signaling on receptor endocytosis. We explored this problem using the opossum PTH/PTH-related protein (PTHrP) receptor, a prototypical Class II G protein-coupled receptor, as a model. In one approach, we evaluated the endocytic properties of mutated forms of the opossum PTH/PTHrP receptor that we had previously shown to be impaired in their ability to initiate agonist-induced signaling when expressed in COS-7 cells. A point mutation in the third cytoplasmic loop (K382A) that severely impairs PTH/PTHrP receptor signaling significantly reduced internalization, whereas two mutant receptors that displayed only partial defects in signaling were internalized normally. To explore more directly the role of second-messenger pathways, we used a cleavable biotinylation method to assess endocytosis of the wild-type receptor stably expressed in human embryonic kidney (HEK) 293 cells. A low rate of constitutive internalization was detected (<5% over a 30-min incubation at 37 C); the rate of receptor internalization was enhanced about 10-fold by the receptor agonists PTH(1-34) or PTHrP(1-34), whereas the receptor antagonist PTH(7-34) had no effect. Forskolin treatment produced a minimal increase in constitutive receptor endocytosis, and the protein kinase (PK)-A inhibitor H-89 failed to block agonist-stimulated endocytosis. Similarly, activation of PK-C, by treatment with phorbol 12-myristate 13-acetate, elicited only a minimal increase in constitutive receptor endocytosis; and blockade of the PK-C pathway, by treatment with a bisindolylmaleimide, failed to inhibit agonist-induced receptor endocytosis. Immunofluorescence confocal microscopic studies of PTH/PTHrP receptor internalization confirmed the results using receptor biotinylation. These findings suggest that: 1) agonist binding is required for the efficient endocytosis of the PTH/PTHrP receptor; 2) receptor activation (agonist-induced receptor conformational change) and/or coupling to G proteins plays a critical role in receptor internalization; and 3) activation of PK-A and PK-C is neither necessary nor sufficient for agonist-stimulated receptor internalization.

We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride-binding activity from the cell surface and by transfer of [3H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway.

We have studied the effect of an antibody against the epitope EYMPME on the internalization of the human muscarinic cholinergic receptor hm1 tagged with the epitope at the amino terminus. The antibody to the tag induces internalization of the hm1 receptor within minutes after exposure of human embryonic kidney 293 cells transfected with the tagged receptor. This antibody-induced internalization is reversible following removal of the antibody. In contrast to hm1 internalization induced by the agonist carbachol, internalization induced by antibody is not blocked by the muscarinic antagonist atropine. The mechanism of antibody-mediated internalization does not appear to involve receptor dimerization by the antibody, as Fab fragments derived from the antibody also induce internalization. The pathway of antibody-induced internalization, similar to the agonist-induced process, is mediated by clathrin-coated vesicles. Furthermore, antibody treatment does not result in any second messenger production, as measured by phosphoinositide accumulation. Our data show that internalization of a G protein-coupled receptor can be triggered by interaction of the amino terminus of the receptor with an exogenous ligand and can occur independently of second messenger production. This result suggests that the receptor can exist in multiple conformations, each mediating distinct downstream events.

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation, whereas certain other receptor types recycle constitutively, e.g., the transferrin receptor. To investigate structural domains involved in mu-opioid receptor internalization, we constructed two truncation mutants bracketing a Ser/Thr-rich domain (354ThrSerSerThrIleGluGlnGlnAsn362) unique to the C-terminus of the mu-opioid receptor (mutants Trunc354 and Trunc363). Ligand binding did not differ substantially, and G protein coupling was slightly lower for these mu-receptor constructs, in particular for Trunc363. To permit localization of the receptor by immunocytochemistry, an epitope tag was added to the N-terminus of the wild-type and mutant receptors. Both the wild-type mu-opioid receptor and Trunc363 resided largely at the plasma membrane and internalized into vesicles upon stimulation with the agonist [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin. Internalization occurred into vesicles that contain transferrin receptors, as shown previously, as well as clathrin, but not caveolin. In contrast, even without any agonist present, Trunc354 colocalized in intracellular vesicles with clathrin and transferrin receptors, but not caveolin. On blocking internalization by hyperosmolar sucrose or acid treatment, Trunc354 translocated to the plasma membrane, indicating that the mutant internalized into clathrin-coated vesicles and recycled constitutively. Despite agonist-independent internalization of Trunc354, basal G protein coupling was not elevated, suggesting distinct mechanisms for coupling and internalization. Furthermore, a portion of the C-terminus, particularly the Ser/Thr domain, appears to suppress mu-receptor internalization, which can be overcome by agonist stimulation. These results demonstrate that a mutant GPCR can be constructed such that internalization, normally an agonist-dependent process, can occur spontaneously without concomitant G protein activation.

Human m1 muscarinic acetylcholine receptor mutants were screened to determine receptor domains and cellular pathways relevant to down-regulation. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for receptor activation or internalization had been previously demonstrated in HEK293 cells, were selected for this study. To assess receptor down-regulation, the m1 receptor mutants were transfected into Chinese hamster ovary cells. Because receptor internalization is expected to precede down-regulation, mutants displaying intact internalization were selected to permit interpretation of mutational effects on down-regulation alone. Four mutations were identified that specifically impaired down-regulation without altering receptor internalization: V127A, I211A, E360A, and K362A. The results define new receptor domains in the second intracellular loop and the junctions of the third intracellular loop that are involved in down-regulation. These same four mutants were also defective in signaling via the phospholipase C and the adenylyl cyclase pathways and in G protein activation, as measured by [35S]GTP gamma S binding. However, the level of second messenger stimulation correlated poorly with the extent of down-regulation. In summary, several mutations of the m1 receptor selectively affect down-regulation, demonstrating that internalization and down-regulation represent distinct events driven by different cellular mechanisms.

We expressed the cloned mu-opioid receptor (muR) in high abundance (5.5 x 10(6) sites/cell) with an amino-terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope-tagged receptor (EE-muR) was similar to the untagged mu R ligand binding and agonist-dependent inhibition of cyclic AMP accumulation. By confocal microscopy, the labeled receptor was shown to be largely confined to the plasma membrane. Pretreatment with morphine failed to affect the cellular distribution of the receptor as judged by immunofluorescence and tracer binding studies. In contrast, exposure to the mu-specific peptide agonist [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist-induced cellular redistribution of EE-muR. Tracer binding studies suggested partial net internalization and a small degree of down-regulation caused by DAMGO. EE-muR-containing membranes were solubilized in detergent [3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate] and immunoprecipitated by an anti-epitope monoclonal antibody. Immunoblotting revealed a prominent band at approximately 70 kDa with weaker bands at approximately 65 kDa. EE-muR was labeled with [gamma-32P]ATP in permeabilized cells, immunoprecipitated, and analyzed by polyacrylamide gel electrophoresis autoradiography. A prominent band at 65-70 kDa indicated the presence of basal receptor phosphorylation occurring in the absence of agonist, which was enhanced approximately 1.8-fold with the addition of morphine. In conclusion, intracellular trafficking of the muR appears to depend on the agonist, with morphine and DAMGO having markedly different effects. Unlike other G protein-coupled receptors, basal phosphorylation is substantial, even in the absence of agonist.

The ligand binding pocket of many G protein-coupled receptors is thought to be located within the core formed by their seven transmembrane domains (TMDs). Previous results suggested that muscarinic antagonists bind to a pocket located toward the extracellular region of the TMDs, primarily at TMDs 2, 3, 6, and 7. Tyrosine-82 (Y82) is located in TMD2 only one helical turn from the presumed membrane surface of Hm1, whereas a phenylalanine (F124) is found in the equivalent position of the closely related Hm3. In order to determine the contribution of Y82 to Hm1 ligand binding and selectivity versus Hm3, we constructed the point mutation Y82 F of Hm1 and measured binding affinities of various ligands, with 3H-N-methylscopolamine (3H-NMS) as the tracer. The Hm1 wild-type receptor and the Y82F mutant were transfected into human embryonic kidney U293 cells. Whereas the affinities of NMS, carbachol, and atropine were either unchanged (carbachol) or enhanced by less than twofold (atropine and NMS), the affinity of the Hm1-selective pirenzepine was reduced threefold by the Y82F mutation. These changes parallel affinity differences of Hm1 and Hm3, indicating that the Y82 F mutation affects the binding pocket and that Y82 contributes to the binding selectivity among closely related muscarinic receptors.

The clonal human neuroblastoma cell line SK-N-SH-SY5Y was previously shown to express mu-opioid and alpha 2-adrenoceptors which are both negatively coupled to adenylyl cyclase. Because of the potential use of alpha 2-agonists in the treatment of narcotic dependence, we tested the interactions among he alpha 2-agonists, clonidine and norepinephrine, and morphine on AC in SH-SY5Y cells. Pretreatment with retinoic acid resulting in partial neuronal differentiation greatly enhanced the cells' sensitivity towards adenylyl cyclase stimulation by prostaglandin E1, and its inhibition by morphine and alpha 2-agonists. Norepinephrine (EC50 = 69 nM) maximally inhibited prostaglandin E1-stimulated cAMP accumulation (by approximately 83%), and the alpha 2-agonist yohimbine reversed these effects. Clonidine (EC50 = 32 nM) was a partial agonist, with 50 to 60% maximal inhibition. The combined effects of morphine (maximum approximately 70% inhibition) and norepinephrine exceeded the effect of either agent alone, yielding more than 90% inhibition of prostaglandin E1-stimulated cAMP accumulation. As previously reported for morphine, only a partial tolerance was observed for adenylyl cyclase inhibition by norepinephrine. Further, no cross-tolerance was observed between clonidine and morphine. The combined results indicate that mu-opioid receptors and an alpha 2-adrenoceptor subtype are colocalized on the same cells in SH-SY5Y culture, which hence serves as a model to study opioid-alpha 2-adrenergic interactions.

We have isolated a portion of the canine gene encoding the orphan receptor RDC1 [1]. The complete coding sequence is contained in a single exon, and an intron divides the 5' untranslated region of RDC1 mRNA. The RDC1 protein is 94% homologous to the gene product of GPRN1, which has been proposed to serve as a VIP receptor when expressed in CHO-K1 and COS-7 cells (Sreedharan, S.P. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 4986-4990). Northern analysis indicates that CHO-K1 cells endogenously express a 2.1 kb RDC1 mRNA. However, while CHO-K1 cells possess detectable low affinity [125I]VIP binding sites, VIP binding is not altered in membranes of CHO-K1 cells expressing varying amounts of the RDC1 gene construct. Further, endogenous VIP binding is not increased by transient expression of RDC1 in COS-7 cells. Taken together, the data suggest that RDC1 is not a canine homolog of the proposed VIP receptor.

The mouse neuroblastoma x rat glioma hybrid NG108-15 was previously shown to express delta opioid receptors. Because neuroblastoma cells display different phenotypes and cloned cell lines are heterogenous, we studied the characteristics and distribution of human 125I-beta-endorphin (125I-beta E) binding sites in cultures of NG108-15 cells with the use of micro-autoradiography and light microscopy. 125I-beta E labeled delta sites in NG108-15 in the presence of the non-opioid blocking peptide, beta-endorphin (6-31) (beta E (6-31)). Silver grains resulting from 125I-beta E binding to the opioid sites occurred in diffuse patches over several cells, with preferential location in dense cell patches. Pretreatment of NG108-15 with the delta agonist DADLE, previously shown to decrease beta E binding to delta sites on intact cells, also reduced silver grain density; however, some cells located in dense cell clusters were resistant to substantial agonist induced loss of labeling. These results suggest that delta opioid binding has a heterogenous cellular distribution in NG108.