MUTAGENESIS OF THE MOUSE DELTA-OPIOID RECEPTOR CONVERTS (-)-BUPRENORPHINE FROM A PARTIAL AGONIST TO AN ANTAGONIST

An aspartic acid at residue 95 (Asp(95)) in the delta receptor has pre viously been shown to be critical for the binding affinity oi selectiv e delta agonists. To gain a better understanding of the functional con sequence of agonist action at the delta receptor, the Asp(95) residue was mutated to an asparagine (D95N) and opioids were tested for bindin g and functional activation of the wild-type and mutant delta receptor s. Selective agonists such as [D-Ser(2), D-Leu(5)]enkephalin-Thr(6) (D SLET) and [D-Ala(2), D-Leu(5)]enkephalin (DADLE) had greatly reduced a ffinity for the D95N mutant receptor but still inhibited cAMP accumula tion, which indicated that the mutant receptor was still functionally coupled to adenylyl cyclase. Antagonist binding was not affected by th e Asp(95) mutation. Similarly, the partial agonist buprenorphine bound with equally high affinity to the D95N mutant and the wild-type delta receptor, which indicated that Asp(95) in not essential for the bindi ng affinity of this opioid. Buprenorphine did not affect cAMP accumula tion in HEK 293 cells expressing the D95N mutant, and it blocked the a bility of DSLET and bremazocine to inhibit cAMP accumulation via the D 95N mutant, which indicated that buprenorphine acts as an antagonist a t the D95N mutant. These findings confirm the essential role of Asp(95 ) in the activation of the delta receptor by agonists and reveal a mol ecular basis of the unique property of buprenorphine.