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] .

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