Pharmacological Sciences

Functional Assays For Deorphanization

The nature of the functional assay used to deorphanize 7TM proteins is crucial for the success rate of ligand identification. A number of expression systems

TABLE 7.2 Identified Functions of Orphan 7TM Proteins Since 2006

GPCRs

Identified Functions

Assay

Expression System Reference

GPR20 GPR22

GPR26 GPR78 GPR37

GPR50

GPR56

Constitutive inhibition of cAMP production through G^ Constitutive inhibition of cAMP production through Goi Protective role in response to hemodynamic stress Constitutive stimulation of cAMP production

Interacts with dopamine transporter and modulates its activity Heterodimerization with MTt inhibits melatonin binding and MTt signaling (G protein and P-arrestin recruitment) Regulator of energy metabolism Binds to tissue transglutaminase2 Inhibits tumor growth cAMP

cAMP

GPR22 deficient mice cAMP

Ligand binding, co-IP

GPR37 deficient mice BRET, co-IP, ligand binding, cAMP, GPR50 siRNA

GPR50 deficient mice Immunohistochemistry, radioimmunoprecipitation assay, proliferation

HEK293 (stable)

HEK293 (stable)

HEK293 (transient)

HEK293 (transient)

HEK293 (transient), hCMEC/D3 (endogenous)

HEK293, MC-1 cells (transient)

TABLE 7.2 (Continued)

GPCRs

Identified Functions

Assay

Expression System Reference

GPR81

GPR161

GPRC5A

EBI2 MrgE

LGR4

Constitutively couples to G; pathway

Influences brain size, behavior and vulnerability to schizophrenia Controls neurulation and lens development

Constitutive inhibition of Gas mRNA and cAMP production Increase the cell proliferation Constitutive inhibitions of cAMP production through G^ Heterodimerization with MrgD decreases internalization and increases ERK activation and intracellular [Ca2+] Role in selective pain behavioural responses Regulation of postnatal epididymal morphogenesis via maintenance of extracellular matrix

Gj/q chimera, inositol phosphate assay GPR81 transgenic mice GPR85 transgenic mice GPR85 deficient mice Vacuolated lens mouse model cAMP, real-time PCR, proliferation assay, GPRC5A siRNA cAMP, luciferase reporter

Internalization, ERK 1/2 phosphorylation, Ca2+

MrgE deficient mice LgrA hypomorphic mutant mice

HEK293 (transient)

Human thyroid follicular epithelial cells (endogenous) COS-7 (transient)

HEK293 (stable)

Co-IP, co-immunoprecipitation; hCMEC/D3, human endothelial cerebral cells.

including Xenopus oocytes, mammalian, and yeast cells combined with different functional readout systems contributed significantly to the deorphaniza-tion of orphan 7TM proteins in the past. These functional assays can be roughly divided into G protein-dependent assays, which have been extensively used in the past, and assays that rely on more recent strategies, namely receptor internalization and/or P-arrestin recruitment (Fig. 7.1). A recent review article provides an overview of the different assays with special emphasis on high - throughput screening formats [37] .