Functional role of a conserved motif in TM6 of the rat mu opioid receptor:Constitutively active and inactive receptors result from substitutions of Thr6.34(279) with Lys and Asp

Mutations within the "X1BBX2X3B" motif or its variants in the junction of t he third intracellular (i3) loop and the sixth transmembrane domain (TM6) h ave been shown to lead to constitutive activation of several G protein-coup led receptors (GPCRs). In this study, T6.34(279) at the X-3 locus of the ra t mu opioid receptor was mutated to Lys and Asp, and the mutants were exami ned 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 constitutiv e activation: (1) compared with the wild type, it produced much higher agon ist-independent [S-35]GTP gammaS binding, which was abolished by pertussis toxin treatment; (2) it displayed an enhanced affinity for the agonist DAMG O similar to that of the high-affinity state of the wild type, which was no t altered by GTP gammaS, while having unchanged affinity for the antagonist diprenorphine. The T6.34(279)K mutant displayed a higher intracellular rec eptor pool than the wild type. Naloxone inhibited the basal [S-35]GTP gamma S 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 [S-35]GTP gammaS binding, greatly reduced agonist-promoted [S-35]GTP gammaS binding, and markedly decreased affinity for DAMGO. Thus, the T6.34(279)D mutant adopts conformations corresponding to inactive state s of the receptor. The results were interpreted in the structural context o f 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 c onformations. The T6.34(279)K substitution would then disrupt this interact ion 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 rep resent the first structurally rationalized demonstration that mutations of this locus can lead to dramatically different properties of a GPCR.