Post-activation-mediated changes in opioid receptors detected by N-terminal antibodies

J Biol Chem. 2008 Apr 18;283(16):10735-44. doi: 10.1074/jbc.M709454200. Epub 2008 Feb 6.

Abstract

The majority of studies examining activity-induced conformational changes in G protein-coupled receptors have focused on transmembrane helices or intracellular regions. Relatively few studies have examined the involvement of the extracellular region in general and the N-terminal region in particular in this process. To begin to address this, we generated a series of antibodies to the N-terminal region of opioid receptors. Characterization of these antibodies revealed that they differentially recognize activated receptors. Recently, we generated monoclonal antibodies that recognize regions proximal to glycosylation sites in the receptor N terminus. Characterization of these antibodies revealed that agonist treatment leads to a decrease in epitope recognition by the antibody presumably because of a movement of the region of the N terminus proximal to glycosylation sites. The time course of the decrease in antibody recognition suggested that it could be due to a post-activation-mediated event. Examination of the involvement of receptor residues in the C-tail and beta-arrestin binding using site-directed mutagenesis and cells or tissues lacking beta-arrestin 2 suggests a role for these desensitization-related mechanisms in governing antibody binding to the receptor. Thus, these N-terminally directed antibodies can differentially recognize post-activation-mediated changes in the C-terminal (intracellular) region of the receptor. Therefore, these conformation-sensitive antibodies represent powerful reagents to probe receptor activation states and provide a potential tool for identifying and characterizing new compounds of therapeutic interest.

Publication types

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

MeSH terms

  • Animals
  • Antibodies / chemistry*
  • Antibodies, Monoclonal / chemistry
  • Arrestins / chemistry*
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Dose-Response Relationship, Drug
  • Glycosylation
  • Ligands
  • Phosphorylation
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Receptors, Opioid / chemistry*
  • beta-Arrestins

Substances

  • Antibodies
  • Antibodies, Monoclonal
  • Arrestins
  • Ligands
  • Receptors, Opioid
  • beta-Arrestins