Gα-Subunits Differentially Alter the Conformation and Agonist Affinity of κ-Opioid Receptors†

Although ligand-induced conformational changes in G protein-coupled receptors (GPCRs) are well-documented, there is little direct evidence for G protein-induced changes in GPCR conformation. To investigate this possibility, the effects of overexpressing Gα-subunits (Gα 16 or Gα ι2 ) with the κ-opioid receptor (KOR) were examined. The changes in KOR conformation were subequently examined via the substituted cysteine accessibility method (SCAM) in transmembrane domains 6 (TM6) and 7 (TM7) and extracellular loop 2 (EL2). Significant conformational changes were observed on TM7, the extracellular portion of TM6, and EL2. Seven SCAM-sensitive residues (S310 7.33 , F314 7.37 , and I316 7.39 to Y320 7.43 ) on TM7 presented a cluster pattern when the KOR was exposed to baseline amounts of G protein, and additional residues became sensitive upon overexpression of various G proteins. In TM7, S311 7.34 and N326 7.49 were found to be sensitive in Gα 16 -overexpressed cells and Y313 7.36 , N322 7.45 , S323 7.46 , and L329 7.52 in Gα ι2 -overexpressed cells. In addition, the degree of sensitivity for various TM7 residues was augmented, especially in Gα ι2 -overexpressed cells. A similar phenomenon was also observed for residues in TM6 and EL2. In addition to an enhanced sensitivity of certain residues, our findings also indicated that a slight rotation was predicted to occur in the upper part of TM7 upon G protein overexpression. These relatively modest conformational changes engendered by G protein overexpression had both profound and differential effects on the abilities of agonists to bind to KOR. These data are significant because they demonstrate that Ga-subunits differentially modulate the conformation and agonist affinity of a prototypical GPCR.