Constitutive receptor systems for drug discovery

Chen, Grace; Jayawickreme, Channa; Way, James M.; Armour, Susan; Queen, Ken; Watson, Chris; Ignar, Diane M.; Chen, Wen Ji; Kenakin, Terrence Peter

doi:10.1016/s1056-8719(00)00075-7

Cited by 40 publications

(29 citation statements)

References 13 publications

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“…The ligand-independent activation of GPR20 was observed not only in HEK293 cells but also in the rat hepatoma cell line RH7777 (data not shown). Although the constitutive activation of the exogenously expressed GPCR was sometimes observed because of an excess of the receptor in cultured cells (24), as shown in Fig. 4A, we demonstrated that at least GPR20 had a higher basal activity than hBLT1.…”

Section: Discussionsupporting

confidence: 49%

Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates Gi Proteins

Hase

Yokomizo

Shimizu

et al. 2008

Journal of Biological Chemistry

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GPR20 was isolated as an orphan G protein-coupled receptor from genomic DNA by PCR amplification. Although GPR20 was closely related to nucleotide or lipid receptors, the functional role of this receptor, as well as its endogenous ligand, remains unclear. Here we demonstrate that GPR20 is constitutively active in the absence of ligand, leading to continuous activation of its coupled G proteins. When GPR20 was exogenously expressed in HEK293 cells, both the basal level and the prostaglandin E 2 -induced production of cAMP were significantly decreased. A remarkable increase in [35 S]guanosine 5-(␥-thio)triphosphate (GTP␥S) binding to membrane preparations was also observed in GPR20-expressing cells. These effects of GPR20 overexpression were diminished in cells treated with pertussis toxin, suggesting that the expression of GPR20 results in the activation of G i/o proteins. Involvement of GPR20 in the activation of G i/o proteins was also supported by evidence that the disruption of a conserved DRY motif in GPR20 attenuated both [35 S]GTP␥S incorporation and inhibition of the prostaglandin E 2 -induced cAMP production. Knockdown of GPR20 in PC12h cells resulted in an elevation of the basal cAMP level, suggesting that the endogenous GPR20 achieves a constitutively or spontaneously active conformation. Furthermore, enhancement of [ 3 H]thymidine incorporation was also observed in the GPR20-silencing cells, implying that the GPR20 expression seems to attenuate PC12h cell growth. Taken together, these data indicate that GPR20 constitutively activates G i proteins without ligand stimulation. The receptor may be involved in cellular processes, including control of intracellular cAMP levels and mitogenic signaling. Mammalian G protein-coupled receptors (GPCRs)2 are a diverse superfamily of proteins with hundreds of members, and these receptors regulate many processes in vivo via interactions with a variety of ligands, including small organic molecules, lipids, protons, hormones, short and large polypeptides, glycoproteins, and even photons. Despite the vast and longstanding efforts of academic and industrial researchers to pair GPCRs with potential ligands, more than 150 nonsensory GPCRs still remain orphan receptors, for which the cognate ligands have not yet been identified (1). Because GPCRs have proven particularly amenable to modulation by small molecules and are the targets of approximately half of currently marketed prescription drugs, the nonsensory GPCRs are clearly important therapeutic targets (2). Consequently, the orphan GPCRs constitute a vast reservoir of potential drug targets for therapeutic development. Indeed, numerous research groups, including ours, work on characterizing these receptors.GPR20 is one of the orphan GPCRs that has been identified from human genomic DNA by PCR amplification using primers based on the sequences of the opioid/somatostatin-related receptors, GPR7 and GPR8 (3). The expression of human GPR20 has been detected in several brain regions, including the caudate nuclei, putamen, a...

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Section: Discussionsupporting

confidence: 49%

Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates Gi Proteins

Hase

Yokomizo

Shimizu

et al. 2008

Journal of Biological Chemistry

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“…GPCRs can convert spontaneously from inactive to active conformations (30,53,54). We found that basal activity of CCR5 was reversed to different extents by TAK779 and MVC, thus identifying both molecules as inverse agonists for the receptor.…”

Section: Tak779 and Mvc Are Inverse Agonists For Ccr5 Which Stabilizmentioning

confidence: 81%

New Insights into the Mechanisms whereby Low Molecular Weight CCR5 Ligands Inhibit HIV-1 Infection

García-Pérez

Rueda

Staropoli

et al. 2011

Journal of Biological Chemistry

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“…In the double-stable HEK293T cells expressing the rat TSHR cDNA and a CRE-luciferase reporter construct, TSH stimulation promoted luciferase expression due to the CRE element upstream of the luciferase cDNA. Luciferase value is known to correlate with cAMP levels (Chen et al 1999). In the same cell line, we have also monitored [Ca 2C ] i in response to TSH or recombinant proteins.…”

Section: Resultsmentioning

confidence: 99%

Differential expression of the thyrostimulin subunits, glycoprotein 2 and 5 in the rat pituitary

Nagasaki

Wang

Jackson

et al. 2006

Journal of Molecular Endocrinology

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Two glycoprotein hormone subunits, (glycoprotein hormone a2-subunit GPA2) and (glycoprotein hormone b5-subunit GPB5) have been recently discovered which, when expressed in vitro, heterodimerize to form a new hormone called thyrostimulin. Thyrostimulin activates the thyroid-stimulating hormone receptor (TSHR) and has thyrotropic activity. Immunological studies have indicated that both subunits co-localize in pituitary cells. To explore the function of thyrostimulin in the rat, we have cloned rat GPA2 and GPB5, reconstituted the heterodimers in vitro, and confirmed that rat thyrostimulin activates TSHR with an affinity similar to that of TSH. In situ hybridization of the pituitary showed that while GPA2 is expressed in the anterior lobe, GPB5 is not detected in any of the lobes. A quantitative analysis showed that the co-localization of GPA2 and GPB5 is restricted in the rat to the eye and the testis. We found that GPB5 can be detected in the pituitary by quantitative-PCR, but at extremely low levels, 2000-fold lower than TSH b-subunit (GPBtsh). Furthermore, the levels of GPB5 remain constant during the estrus cycle, while those of GPA2 vary. Finally, we found that none of the thyrostimulin subunits was induced by TRH in pituitary cell culture. These data point at the thyrostimulin system as being functionally different to the TSH system.

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Constitutive receptor systems for drug discovery

Cited by 40 publications

References 13 publications

Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates Gi Proteins

Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates Gi Proteins

New Insights into the Mechanisms whereby Low Molecular Weight CCR5 Ligands Inhibit HIV-1 Infection

Differential expression of the thyrostimulin subunits, glycoprotein 2 and 5 in the rat pituitary

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