Functional Role of a Conserved Motif in TM6 of the Rat μ Opioid Receptor:  Constitutively Active and Inactive Receptors Result from Substitutions of Thr6.34(279) with Lys and Asp

Abstract

Mutations within the "X1BBX2X3B" motif or its variants in the junction of the third intracellular (i3) loop and the sixth transmembrane domain (TM6) have been shown to lead to constitutive activation of several G protein-coupled receptors (GPCRs). In this study, T6.34(279) at the X3 locus of the rat mu opioid receptor was mutated to Lys and Asp, and the mutants were examined for binding and signaling properties. The T6.34(279)K mutant was poorly expressed, and pretreatment with naloxone greatly enhanced its expression. This construct exhibited properties identified previously with constitutive activation: (1) compared with the wild type, it produced much higher agonist-independent [35S]GTPgammaS binding, which was abolished by pertussis toxin treatment; (2) it displayed an enhanced affinity for the agonist DAMGO similar to that of the high-affinity state of the wild type, which was not altered by GTPgammaS, while having unchanged affinity for the antagonist diprenorphine. The T6.34(279)K mutant displayed a higher intracellular receptor pool than the wild type. Naloxone inhibited the basal [35S]GTPgammaS binding of the T6.34(279)K mutant, demonstrating inverse agonist activity at this mutant receptor. In contrast, the T6.34(279)D substitution did not increase basal [35S]GTPgammaS binding, greatly reduced agonist-promoted [35S]GTPgammaS binding, and markedly decreased affinity for DAMGO. Thus, the T6.34(279)D mutant adopts conformations corresponding to inactive states of the receptor. The results were interpreted in the structural context of a model for the mu opioid receptor that incorporates the information from the crystal structure of rhodopsin. The interaction of T6.34(279) with R3.50(165) in the mu opioid receptor is considered to stabilize the inactive conformations. The T6.34(279)K substitution would then disrupt this interaction and support agonist-free activation, while T6.34(279)D mutation should strengthen this interaction which keeps the receptor in inactive states. T6.34(279) may, in addition, interact with the neighboring R6.35(280) to help constrain the receptor in inactive states, and T6.34(279)K and T6.34(279)D mutations would affect this interaction by disrupting or strengthening it, respectively. To the best of our knowledge, the results presented here represent the first structurally rationalized demonstration that mutations of this locus can lead to dramatically different properties of a GPCR.

Agonist treatment did not affect association of mu opioid receptors with lipid rafts and cholesterol reduction had opposite effects on the receptor-mediated signaling in rat brain and CHO cells

“…clonal CHO cell line stably expressing HA-rMOR was established as described previously (Huang et al, 2001b), with a B max value of 1.8 pmol/mg membrane protein. CHO-HA-rMOR cells were cultured in Dulbecco's modified Eagle's medium F12 HAM supplemented with 10% fetal calf serum, 0.1 mg/ml geneticin, 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere consisting of 5% CO 2 and 95% air at 37 °C.…”

“…Intracellular receptors were assessed according to a modification of a previously described procedure (Huang et al, 2001b). CHO-HA-rMOR cells were cultured with or without 1 mM etorphine or 10 mM morphine, washed three times with 1× PBS buffer, and harvested.…”

The Crystallographic Model of Rhodopsin and Its Use in Studies of Other G Protein–Coupled Receptors

“…Residues D3.25 [122] and D3.29 [126] in helix III and F6. 51[261] and H6.54 [264] in helix VI decreased the binding of the melanocyte-stimulating hormone. In a study of the mouse receptor (45), several residues involved in agonist and antagonist binding were identified using a model built before the crystallographic structure became available as well as one built using the crystal structure.…”

“…Groups have investigated this motif in rat μ opioid receptor (51,62), β 2 -adrenergic receptor (7), α 1b -adrenergic receptor (41), lutropin/choriogonadotropin receptor (4), and 5-hydroxytryptamine 2A serotonin receptor (100). Mutagenesis of D3.49 in the opioid receptor results in activation, and neutralization of the charge on E6.30 in the adrenergic receptors does likewise.…”

Brain region-specific N-glycosylation and lipid rafts association of the rat mu opioid receptor

“…Biotinylated anti-μC: biotinylation of the anti-μC by sulfo-NHS-LC-Biotin was carried out according to the instructions (Pierce). The clonal CHO cell line stably expressing HA-rMOR (CHO-HA-rMOR) was established and cultured as described previously [15], with a B max value of 1.8 pmol/mg membrane protein. MOR-knockout (K/O) mice were originally developed in the lab of Dr. John Pintar by disruption of exon-1 of the MOR-1 gene through homologous recombination [16].…”