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 G alpha-subunits (G alpha(16) or G alpha(i2)) with the kappa-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 G alpha(16)-overexpressed cells and Y313(7.36), N322(7.45), S323(7.46), and L329(7.12) in G alpha(i2)-overexpressed cells. In addition, the degree of sensitivity for various TM7 residues was augmented, especially in G alpha(i2)-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 G alpha-subunits differentially modulate the conformation and agonist affinity of a prototypical GPCR.