title: Galpha-subunits differentially alter the conformation and agonist affinity of kappa-opioid receptors.
abstract: 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.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Yan
authors.forename: Philip D
authors.initials: PD
authors.lastname: Mosier
authors.forename: Richard B
authors.initials: RB
authors.lastname: Westkaemper
authors.forename: Bryan L
authors.initials: BL
authors.lastname: Roth
mesh_terms: D002460:Cell Line; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011869:Radioligand Assay; D017473:Receptors, Opioid, kappa / Q000819:agonists* / Q000737:chemistry* / Q000235:genetics
chemical_list: D017473:Receptors, Opioid, kappa