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.

We expressed the cloned μ‐opioid receptor (μR) in high abundance (5.5 × 106 sites/cell) with an amino‐terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope‐tagged receptor (EE‐μR) was similar to the untagged μR in 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 μ‐specific peptide agonist [d‐Ala2,MePhe4,Glyol5]enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist‐induced cellular redistribution of EE‐μR. Tracer binding studies suggested partial net internalization and a small degree of down‐regulation caused by DAMGO. EE‐μR‐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 ∼70 kDa with weaker bands at ∼65 kDa. EE‐μR was labeled with [γ‐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 ∼1.8‐fold with the addition of morphine. In conclusion, intracellular trafficking of the μR 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 in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-NЈ-(4-( (Snyder, 1976;Carlsson et al., 1999a). Unfortunately, antagonism of D 2 receptors also causes profound motor, endocrine, and cognitive side effects, which can 1 Current affiliation: TorreyPines Therapeutics,

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. Pretreatment tumor biopsies from 166 patients treated with anti-PD-1 across 10 academic cancer centers were fluorescently stained with multiple markers in discovery ( = 24) and validation ( = 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. 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 ( = 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 ( = 0.0096). These patients also experienced significantly improved progression-free survival (HR = 0.36; = 0.0004) and overall survival (HR = 0.39; = 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 ( = 0.000004). In contrast, PD-L1 expression was not predictive of survival. 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. .

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 [ 3 H] sulpiride-binding activity from the cell surface and by transfer of [ 3 H] 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.Keywords: desensitization; dopamine D2 receptor; G protein-coupled receptor kinase; internalization; sequestration.Guanine nucleotide-binding regulatory protein (G-protein)-coupled receptors are known to be sequestered or internalized when exposed to agonists, and thereby become inaccessible to extracellular ligand [1±5]. Sequestered receptors are recycled back into the plasma membrane, while a portion of sequestered receptors is thought to be transported to lysosomes and degraded. On the one hand, sequestration may reduce G-protein activation when the number of receptors is a limiting factor [6]. On the other hand, sequestration was suggested to be necessary for resensitization of desensitized receptors in the case of b2 adrenergic receptors (b receptors), where desensitized and resensitized receptors are assumed, although not proven, to correspond to phosphorylated and dephosphorylated receptor states, respectively [7±9].Tsuga et al. [10,11] have shown that sequestration of muscarinic receptor m2, m3, m4 and m5 subtypes (m2±m5 receptors) expressed in COS-7 cells is facilitated by coexpression of G-protein-coupled receptor kinase 2 (GRK2) and that sequestration of m2 and m4, but not m3 and m5, receptors is attenuated by coexpression of a dominant-negative mutant of GRK2 (DN-GRK2