Classical Assays of GPCR Deorphanization

Whenever high-affinity radioligands are available, these pharmacological tools are an integral part of the ligand screening process. Chemical libraries or tissue extracts are typically screened for compounds that compete with the radioli-gand for the same binding site.

Binding of agonists to GPCRs induces a conformational change and promotes the activation of heterotrimeric G proteins. Their activation can be most easily and directly detected by monitoring the binding of nonhydrolyzable guanosine 5'-O-(3-thiotriphosphate) (GTP) analogs such as [35S]GTPyS to the G protein. These [35S]GTPyS binding assays are widely used in pharmaceutical industry and 1536-well scintillation proximity assays have been developed [38].

Measuring second messengers such as cyclic adenosine monophosphate (cAMP) constitutes another widely used functional readout system (Table 7.1). Activation or inhibition of the cAMP pathway can be either measured directly on the second messenger level or in a reporter gene assay based on the activation of a cAMP response element located in a promoter that drives a luciferase reporter gene. These assays are specifically designed for those receptors that either activate (Gs- coupled) or inactivate (Gi- coupled) adenylyl cyclases. Natural cell systems such as Xenopus laevis melanophores offer additional options for ligand screening of orphan 7TM proteins [39]. The aggregation or dispersion of intracellular organelles termed melanosomes can be controlled by GPCR signaling mainly, but not exclusively, by the adenylyl cyclase pathway. This strategy has been recently used to deorphanize GPRC6A which binds several L -a - amino acids (Table 7.1 ) [30] .

Many GPCRs physiologically regulate changes in intracellular Ca2+ either through an IP3-mediated mechanism involving changes in stored Ca2+ or through the regulation of voltage-sensitive ion channels, which modulate Ca2+ influx. Indeed, this strategy has been applied to the ligand screening of about 40 orphan 7TM proteins (Table 7.1). Two of the most commonly employed instruments used to measure changes in intracellular calcium concentration are the Fluorescence Imaging Plate Reader (FLIPRâ„¢ ; Molecular Devices, Sunnyvale, CA) and the functional drug screening system (FDSS, Hamamatsu Corporation, Hamamatsu City, Japan) which use calcium indicators such as Fluo-3 or Fura-2 [40,41]. Another Ca2+-sensitive approach is based on aequo-rin, an induced Ca2+-sensitive luciferase [42]. The assay was further optimized by the addition of promiscuous Ga proteins (Ga15 and Ga16) or chimeric Ga subunits (Gaq/i5) to facilitate the receptor-G protein coupling [43, 44].

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