Constitutive activation of the mu opioid receptor by mutation of D3.49(164), but not D3.32(147): D3.49(164) is critical for stabilization of the inactive form of the receptor and for its expression

The roles of conserved aspartates in the third transmembrane domain of the rat mu opioid receptor (RMOR) were explored with mutations of D3.32(147) an d D3.49(164). D3.49(164) in the highly conserved DRY motif was mutated to 1 3 amino acids. Except for the D3.49(164)E mutant, each mutant displayed lit tle or no detectable [H-3]diprenorphine binding, and pretreatment with nalo xone greatly enhanced binding. D3.49(164)H, -Q, -Y, -M, and -E mutants were further studied. D3.32(147) was substituted with A or N. All seven mutants exhibited similar binding affinities for the antagonist [H-3]diprenorphine as the wildtype. The D3.49(164)H, -Q, -Y, and -M mutants, but not the D3.4 9(164)E and D3.32(147) mutants, exhibited enhanced basal [S-35]GTP gammaS b inding which was comparable to the maximally activated level of the wild-ty pe and was related to expression levels. Naloxone, naltrexone, and naloxone methiodide significantly inhibited the basal [S-35]GTP gammaS binding of t he D3.49(164) mutants, indicating inverse agonist activities. Treatment of the D3.49(164)Y mutant with pertussis toxin greatly reduced the basal [S-35 ]GTP gammaS binding, demonstrating constitutive activation of G alpha (i)/a lpha (o). The D3.49(164)H, -Y, -M, and -Q mutants had higher affinities for DAMGO than the wild-type, which were not significantly lowered by GTP gamm aS. Thus, mutation of D3.49(164) to H, Y, M, or Q in RMOR resulted in recep tor assuming activated conformations. In contrast, the D3.49(164)E mutant d isplayed significantly lower basal [S-35]GTP,IS binding and reduced affinit y for DAMGO. Upon incubation of membranes at 37 degreesC, the constitutivel y active D3.49(164)Y mutant was structurally less stable, whereas the inact ivated D3.49(164)E mutant was more stable, than the wild-type. Computationa l simulations showed that the E3.49 side chain interacted strongly with the conserved R3.50 in the DRY motif and stabilized the inactive form of the r eceptor. Taken together, these results indicate that D3.49 plays an importa nt role in constraining the receptor in inactive conformations.