Novel screening assay for the selective detection of G-protein-coupled receptor heteromer signaling

J Pharmacol Exp Ther. 2013 Jan;344(1):179-88. doi: 10.1124/jpet.112.198655. Epub 2012 Oct 24.


Drugs targeting G-protein-coupled receptors (GPCRs) make up more than 25% of all prescribed medicines. The ability of GPCRs to form heteromers with unique signaling properties suggests an entirely new and unexplored pool of drug targets. However, current in vitro assays are ill equipped to detect heteromer-selective compounds. We have successfully adapted an approach, using fusion proteins of GPCRs and chimeric G proteins, to create an in vitro screening assay (in human embryonic kidney cells) in which only activated heteromers are detectable. Here we show that this assay can demonstrate heteromer-selective G-protein bias as well as measure transinhibition. Using this assay, we reveal that the δ-opioid receptor agonist ADL5859, which is currently in clinical trials, has a 10-fold higher potency against δ-opioid receptor homomers than δ/μ-opioid receptor heteromers (pEC(50) = 6.7 ± 0.1 versus 5.8 ± 0.2). The assay enables the screening of large compound libraries to identify heteromer-selective compounds that could then be used in vivo to determine the functional role of heteromers and develop potential therapeutic agents.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Benzamides / pharmacology
  • Calcium / metabolism
  • Calcium Signaling
  • Cloning, Molecular
  • Dose-Response Relationship, Drug
  • Drug Evaluation, Preclinical
  • Enkephalin, Leucine / pharmacology
  • Enzyme-Linked Immunosorbent Assay
  • GTP-Binding Protein alpha Subunits, Gi-Go / drug effects
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism
  • GTP-Binding Protein alpha Subunits, Gs / drug effects
  • GTP-Binding Protein alpha Subunits, Gs / metabolism
  • HEK293 Cells
  • Humans
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Mutant Chimeric Proteins / chemistry
  • Mutant Chimeric Proteins / genetics
  • Piperazines / pharmacology
  • Polymerase Chain Reaction
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / physiology*
  • Receptors, Opioid, delta / drug effects
  • Receptors, Opioid, delta / genetics
  • Recombinant Fusion Proteins
  • Signal Transduction / drug effects*


  • Benzamides
  • Mutant Chimeric Proteins
  • Piperazines
  • Receptors, G-Protein-Coupled
  • Receptors, Opioid, delta
  • Recombinant Fusion Proteins
  • 4-(alpha-(4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl)-N,N-diethylbenzamide
  • Enkephalin, Leucine
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • GTP-Binding Protein alpha Subunits, Gs
  • Calcium