AMINO-ACIDS IN THE CLONED MOUSE KAPPA-RECEPTOR THAT ARE NECESSARY FORHIGH-AFFINITY AGONIST BINDING BUT NOT ANTAGONIST BINDING

Opioids exert their biological effects through membrane-bound receptor s. We have been interested in examining the domains and specific resid ues of the opioid receptors involved in ligand binding. In this report we demonstrate the critical roles of two highly conserved aspartate r esidues in the second and third transmembrane domains (TM2 and TM3) of the kappa receptor in ligand binding. Two mutant receptors were gener ated in which the negatively charged aspartate residues residing in TM 2 and TM3 were independently mutated to neutrally charged asparagines (D105N and D138N). These aspartate residues are absolutely required fo r high affinity kappa-selective agonist binding, in addition, we excha nged the region encompassing TM4 and the second extracellular loop bet ween the kappa and delta opioid receptors. One of these chimeras, the kappa receptor with TM4 and the delta second extracellular loop (kappa /delta-2eloop), was unable to bind kappa-selective agonists but bound antagonists and nonselective opioids in a manner identical to wild-typ e kappa receptor. The ability of kappa-selective agonists to inhibit f orskolin-stimulated cAMP accumulation in COS-7 cells transfected with the kappa/delta-2eloop chimera was completely abolished, consistent wi th the loss of binding observed. These results indicate that TM4 and/o r the second extracellular loop are involved in kappa-selective agonis t binding.