Identification of Signal Transduction Pathways Used by Orphan G Protein-Coupled Receptors

Abstract

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.

Neural Expression of G Protein-coupled Receptors GPR3, GPR6, and GPR12 Up-regulates Cyclic AMP Levels and Promotes Neurite Outgrowth

“…Transient expression of the GPR3, GPR6, or GPR12 in HEK-293 cells resulted in a significant increase in intracellular cAMP levels, which is consistent with previous reports (13,14,22,40). When the receptors were expressed in Neuro2a neuroblastoma cells, FIGURE 6.…”

Head Bobber: An Insertional Mutation Causes Inner Ear Defects, Hyperactive Circling, and Deafness

“…Gpr26 has been identified to be epigenetically silenced in human gliomas, but a function has not been ascribed (Boulay et al 2009). The GPCRs have diverse biological roles that transduce signals from a range of stimuli, including neurotransmitters (Bresnick et al 2003;Lee et al 2000;Lee et al 2001). Recently, a GPCR, sphingocine-1-phosphate receptor S1P2, was demonstrated to be essential for proper functioning of the auditory and vestibular systems.…”

G protein–coupled receptor 21 deletion improves insulin sensitivity in diet-induced obese mice

“…The orphan GPCR GPR21 was highly expressed in these temperature-sensing neurons, and we also noted that this receptor was highly expressed in macrophages. GPR21 couples to the Gq family of G proteins (16), but to our knowledge, there are no identified ligands, and its downstream signaling pathways remain to be determined.…”