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

• Published in: Biochemistry (Easton) 2008-02, Vol.47 (6), p.1567-1578
• Main Authors: Yan, Feng, Mosier, Philip D, Westkaemper, Richard B, Roth, Bryan L
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi701476b

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 (Gα16 or Gαi2) 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 (S3107.33, F3147.37, and I3167.39 to Y3207.43) 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, S3117.34 and N3267.49 were found to be sensitive in Gα16-overexpressed cells and Y3137.36, N3227.45, S3237.46, and L3297.52 in Gαi2-overexpressed cells. In addition, the degree of sensitivity for various TM7 residues was augmented, especially in Gαi2-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 Gα-subunits differentially modulate the conformation and agonist affinity of a prototypical GPCR.