Identification of signal transduction pathways used by orphan g protein-coupled receptors

Assay Drug Dev Technol. 2003 Apr;1(2):239-49. doi: 10.1089/15406580360545053.


The superfamily of GPCRs have diverse biological roles, transducing signals from a range of stimuli, from photon recognition by opsins to neurotransmitter regulation of neuronal function. Of the many identified genes encoding GPCRs, >130 are orphan receptors ( i.e., their endogenous ligands are unknown), and this subset represents putative novel therapeutic targets for pharmaceutical intervention in a variety of diseases. As an initial step toward drug discovery, determining a biological function for these newly identified receptors is of vital importance, and thus identification of a natural ligand(s) is a primary aim. There are several established methods for doing this, but many have drawbacks and usually require some in-depth knowledge about how the receptor functions. The technique described here utilizes a transcription-based reporter assay in live cells. This allows the determination of the signal transduction pathway any given oGPCR uses, without any prior knowledge of the endogenous ligand. This can therefore reduce the redundancy of effort involved in screening ligands at a given receptor in multiple formats (i.e., Galpha(s), Galpha(i/0), and Galpha(q) assays), as well as ensuring that the receptor targeted is capable of signaling if appropriately activated. Such knowledge is often laboriously obtained, and for almost all oGPCRs, this kind of information is not yet available. This technology can also be used to develop inverse agonist as well as agonist sensitive high throughput assays for oGPCRs. The veracity of this approach is demonstrated, using a number of known GPCRs. The likely signaling pathways of the GPR3, GPR12, GPR19, GPR21, and HG55 oGPCRs are shown, and a high throughput assay for GPR26 receptors developed. The methods outlined here for elucidation of the signal transduction pathways for oGPCRs and development of functional assays should speed up the process of identification of ligands for this potentially therapeutically useful group of receptors.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Antineoplastic Combined Chemotherapy Protocols
  • Calcium Signaling / physiology
  • Cell Line
  • Cricetinae
  • Cyclophosphamide
  • Doxorubicin
  • Drug Industry / methods
  • Fluorescence
  • Genes, Reporter
  • Green Fluorescent Proteins
  • Kidney / cytology
  • Luminescent Proteins
  • Membrane Proteins / metabolism
  • Nerve Tissue Proteins
  • Receptor, Serotonin, 5-HT2A / physiology
  • Receptors, Dopamine D2
  • Receptors, G-Protein-Coupled / chemistry
  • Receptors, G-Protein-Coupled / drug effects
  • Receptors, G-Protein-Coupled / metabolism
  • Receptors, G-Protein-Coupled / physiology*
  • Receptors, Leukotriene / metabolism
  • Receptors, Neurotransmitter / metabolism
  • Receptors, Serotonin / physiology
  • Signal Transduction / physiology*
  • Transcription, Genetic / physiology
  • Transfection / methods
  • Vincristine


  • GPR19 protein, human
  • GPR21 protein, human
  • GPR26 protein, human
  • GPR3 protein, human
  • Luminescent Proteins
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Receptor, Serotonin, 5-HT2A
  • Receptors, Dopamine D2
  • Receptors, G-Protein-Coupled
  • Receptors, Leukotriene
  • Receptors, Neurotransmitter
  • Receptors, Serotonin
  • serotonin 7 receptor
  • Green Fluorescent Proteins
  • Vincristine
  • Doxorubicin
  • Cyclophosphamide
  • leukotriene D4 receptor

Supplementary concepts

  • CAV protocol