Galpha-subunits differentially alter the conformation and agonist affinity of kappa-opioid receptors

Biochemistry. 2008 Feb 12;47(6):1567-78. doi: 10.1021/bi701476b. Epub 2008 Jan 19.


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 Galpha-subunits (Galpha16 or Galphai2) 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 (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 Galpha16-overexpressed cells and Y3137.36, N3227.45, S3237.46, and L3297.52 in Galphai2-overexpressed cells. In addition, the degree of sensitivity for various TM7 residues was augmented, especially in Galphai2-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 Galpha-subunits differentially modulate the conformation and agonist affinity of a prototypical GPCR.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line
  • Humans
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Radioligand Assay
  • Receptors, Opioid, kappa / agonists*
  • Receptors, Opioid, kappa / chemistry*
  • Receptors, Opioid, kappa / genetics


  • Receptors, Opioid, kappa