Mechanisms of drug action Advanced mass spectrometry methods enable monitoring tens thousands phosphorylation sites in proteins. This technology can potentially distinguish cellular signaling pathways that produce beneficial effects from those unwanted side effects. Liu et al. treated mice with various agonists the kappa opioid receptor (a G protein–coupled receptor) and monitored changes over time different brain regions. The patterns revealed distinct tissues, some which were associated...

The A118G SNP (single nucleotide polymorphism) of the hMOPR [human MOPR (μ opioid receptor)] gene OPRM1 results in an amino acid substitution (N40D). Subjects homozygous for 118G allele have been reported to require higher morphine doses achieve adequate analgesia, and is more prevalent among drug abusers. However, changes protein associated with this are unknown. Using a knockin mouse model (G/G mice; mice 112G MOPR) that possesses equivalent nucleotide/amino (A112G; N38D) gene, we...

We have investigated whether Ezrin-radixin-moesin (ERM)-binding phosphoprotein-50/Na+/H+exchanger regulatory factor (EBP50/NHERF), a PDZ domain-containing phosphoprotein, is associated with the human κ opioid receptor (hkor) and it regulates trafficking signaling of hkor. When expressed in CHO cells stably transfected FLAG-tagged hkor (FLAG-hkor), EBP50/NHERF co-immunoprecipitated FLAG-hkor, domain I, but not II, was involved interaction. Treatment agonist (−)-(trans)-3,4-...

Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated <i>in vitro</i> free Gβγ subunits negatively charged membrane phospholipids through pleckstrin homology (PH) domains. However, the molecular binding determinants physiological role for these ligands remain unclear. To address issues, we generated an array site-directed mutants within PH domain characterized interaction with...

We identified a truncated form (38–117) of GEC1 that interacts with the C-tail human κ opioid receptor (hKOR) by yeast two-hybrid screening. GEC1-(38–117) did not interact μ or δ receptors. GEC1, 117-amino acid protein (Pellerin, I., Vuillermoz, C., Jouvenot, M., Ordener, Royez, and Adessi, G. L. (1993) Mol. Cell Endocrinol. 90, R17–R21), is highly homologous to GABARAP, GATE-16, Apg8/aut7, all members microtubule associated (MAP) family. In pull-down assays, GST-GEC1 interacted directly...

Abstract Activation of κ opioid receptor (KOR) produces analgesia, antipruritic effect, sedation and dysphoria. To characterize neuroanatomy KOR at high resolutions circumvent issues specificity antibodies, we generated a knock-in mouse line expressing fused the C terminus with fluorescent protein tdTomato (KtdT). The selective agonist U50,488H caused anti-scratch effect hypolocomotion, indicating intact neuronal circuitries. Clearing brains CLARITY revealed three-dimensional (3-D) images...

Heterocyclic peptidomimetics are constrained compounds that mimic the biological efficacy of peptides while offering increased stability. We have previously generated a diazaheterocyclic peripherally selective, mixed-opioid agonist peptidomimetic produced synergistic antinociception with decreased side effects. Working from two earlier templates, we report here synthesis 15 new analogues. In vitro screening radioligand competition binding assays and [35S]GTPγS demonstrated variable affinity...

Abstract The opiates are well-established immunomodulatory factors, and recent evidence suggests that μ- δ-opioid receptor ligands alter chemokine-driven chemotactic responses through the process of heterologous desensitization. In present report, we sought to examine capacity δ-opioids modulate function chemokine receptors CCR5 CXCR4, two major human immunodeficiency virus (HIV) coreceptors. We found CCR1/5 ligand CCL5/regulated on activation, normal T expressed secreted, but not CXCR4...

Mutations within the "X1BBX2X3B" motif or its variants in junction of third intracellular (i3) loop and sixth transmembrane domain (TM6) have been shown to lead constitutive activation several G protein-coupled receptors (GPCRs). In this study, T6.34(279) at X3 locus rat μ opioid receptor was mutated Lys Asp, mutants were examined for binding signaling properties. The T6.34(279)K mutant poorly expressed, pretreatment with naloxone greatly enhanced expression. This construct exhibited...

Lipid rafts are microdomains of plasma membranes enriched in cholesterol and sphingolipids the outer layer. We determined whether κ opioid receptors (KOR) human placenta FLAG (DYKDDDDK)-tagged KOR (FLAG-hKOR) expressed Chinese hamster ovary (CHO) cells localized lipid changes contents affect hKOR properties signaling. were prepared from CHO expressing FLAG-hKOR using Na₂CO₃ method fractionation through a sucrose density gradient. The majority cells, by...

Binding of β-funaltrexamine (β-FNA) to the cloned rat μ opioid receptor expressed in COS-1 cells or Chinese hamster ovary was examined. β-FNA bound with high affinity. Irreversible binding [3H]β-FNA defined as that could not be dissociated by trichloroacetic acid. Na+ greatly enhanced specific irreversible receptor, which concentration- and time-dependent. Specific potently inhibited CTAP (a ligand), but ICI174,864 δ ligand) U50,488H κ ligand). These results indicate binds irreversibly...

To investigate the interaction between opioid receptor and its ligands, we compared binding of μ-selective ligands to two μ/κ chimeric receptors μ k receptors. The chimeras were constructed from cloned rat μ, in which a segment middle third intracellular loop C terminus was exchanged. When this portion replaced by that receptor, affinities selective agonists, DAMGO (Tyr-D-Ala-Gly-NMePhe-Gly-ol), PL017 (Tyr-Pro-NMePhe-D-Pro-NH2), sufentanil, morphine, greatly increased as those for receptor....

The roles of conserved aspartates in the third transmembrane domain rat mu opioid receptor (RMOR) were explored with mutations D3.32(147) and D3.49(164). D3.49(164) highly DRY motif was mutated to 13 amino acids. Except for D3.49(164)E mutant, each mutant displayed little or no detectable [(3)H]diprenorphine binding, pretreatment naloxone greatly enhanced binding. D3.49(164)H, -Q, -Y, -M, -E mutants further studied. substituted A N. All seven exhibited similar binding affinities antagonist...

We examined glycosylation of FLAG-hKOR expressed in CHO cells and determined its functional significance. was resolved as a broad diffuse 55-kDa band less 45-kDa by immunoblotting, indicating that the receptor is glycosylated. Endoglycosidase H cleaved to ∼38 kDa but did not change band, demonstrating N-glycosylated with high-mannose or hybrid-type glycan. Peptide−N-glycosidase F digestion solubilized hKOR incubation tunicamycin resulted two species 43 38 kDa, suggesting 43-kDa...

The agonist (−)(<i>trans</i>)-3,4-dichloro-<i>N</i>-methyl-<i>N</i>-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide [(−)U50,488H] caused desensitization of the human κ-opioid receptor (hkor) and Flag-tagged hkor (Flag-hkor) but not rat (rkor) rkor (Flag-rkor) stably expressed in CHO cells as assessed by guanosine 5′-<i>O</i>-(3-[³⁵S]thiotriphosphate) binding. In addition, (−)U50,488H stimulation enhanced phosphorylation Flag-hkor, Flag-rkor. (−)U50,488H-induced Flag-hkor was...

Two peptide agonists, eight nonpeptide and five antagonists were evaluated for their capacity to regulate FLAG (DYKDDDDK)-tagged human κ opioid receptors (hKORs) stably expressed in Chinese hamster ovary cells after incubation 4 h with a ligand at concentration ∼1000-fold of its EC₅₀ (agonist) or <i>K</i>_i (antagonist) value. Dynorphins A B decreased the fully glycosylated mature form (55-kDa) FLAG-hKOR by 70%, whereas full agonists...

We demonstrated previously that GEC1, a member of the microtubule-associated protein (MAP) family, bound to human κ opioid receptor (hKOPR) and promoted hKOPR cell surface expression by facilitating its trafficking along secretory pathway. GABA(A) receptor-associated (GABARAP), GEC1 analog, also enhanced KOPR expression, but lesser extent. The MAP family proteins undergo cleavage their C-terminal residue(s), exposed conserved glycine forms conjugates with phosphatidylethanolamine, which...