Functional expression of human and mouse P2Y12 receptors in Saccharomyces cerevisiae

Pausch, Mark H.; Lai, M.O.; Tseng, Eugene; Paulsen, Janet; Bates, Brian; Kwak, Seung

doi:10.1016/j.bbrc.2004.09.034

Cited by 43 publications

(30 citation statements)

References 27 publications

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“…Several G i protein-coupled receptors of the P2Y 12 -like receptor group have been functionally expressed in sensitive and convenient yeast expression systems (48,49). Therefore, several GPR34 orthologs were cloned into the yeast expression plasmid p416GPD and expressed in the S. cerevisiae strain MPY578t5 (allowing coupling of G i protein-coupled receptors to the yeast mating pathway (50)).…”

Section: Lyso-ps Is Not An Agonist For Murine and Human Gpr34-mentioning

confidence: 99%

Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34

Liebscher¹,

Müller

Teupser

et al. 2011

Journal of Biological Chemistry

103

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The X-chromosomal GPR34 gene encodes an orphan G i protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-␣, GM-CSF, and IFN-␥ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges. G protein-coupled receptors (GPCR)2 form the largest gene family among transmembrane receptors, including more than 900 genes in humans and other mammals (1). A great number of stimuli, such as light, hormones, neurotransmitters, peptides, and nucleotides, activate the distinct receptors. Nonodorant receptors form about one-third of the GPCR repertoire. Although more than 200 non-odorant GPCR have been assigned to specific agonists and functions, about 155 socalled "orphan" GPCR (2) await identification of their physiological relevance. The importance of GPCR in controlling almost every physiological function makes this receptor family the most frequently used target for therapeutic drugs. Therefore, unveiling the function of orphan GPCR is a central issue in academic and industrial research.Among the five structurally different GPCR families (1, 3), the rhodopsin-like receptors form the largest in humans and other vertebrates. The rhodopsin-like family is divided further into subfamilies and groups. The P2Y 12 -like receptor group includes the ADP receptors P2Y 12 and P2Y 13 , the UDP-glucose receptor P2Y 14 , and the orphan receptors GPR87, GPR82, and GPR34 (4). Apart from the ADP receptor P2Y 12 , which has a central role in platelet aggregation and is the therapeutic target of clopidogrel (5, 6), very little is known about the function of the other members of this group.GPR34, an orphan receptor of the P2Y 12 -like receptor group, was first discovered by mining GenBank TM for novel GPCR sequences and homology cloning and has been assigned to the human X chromosome (7,8). Phylogenetic studies revealed that GPR34 has been highly conserved over the past 450 million years of vertebrate evolution, and no GPR34-deficient vertebrate has been identified yet (9). To date, there i...

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Section: Lyso-ps Is Not An Agonist For Murine and Human Gpr34-mentioning

confidence: 99%

Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34

Liebscher¹,

Müller

Teupser

et al. 2011

Journal of Biological Chemistry

103

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“…In previous experiments, we and others demonstrated that the human P2Y 12 is functionally expressed in the yeast system (Schulz and Schoneberg, 2003;Pausch et al, 2004), which lacks such problems. Numerous constitutively activating mutations have been described for GPCRs in natural or recombinant systems, but only a few have been reported for P2Y receptors (Ding et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Identification of Determinants Required for Agonistic and Inverse Agonistic Ligand Properties at the ADP Receptor P2Y₁₂

et al. 2012

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The ADP receptor P2Y 12 belongs to the superfamily of G protein-coupled receptors (GPCRs), and its activation triggers platelet aggregation. Therefore, potent antagonists, such as clopidogrel, are of high clinical relevance in prophylaxis and treatment of thromboembolic events. P2Y 12 displays an elevated basal activity in vitro, and as such, inverse agonists may be therapeutically beneficial compared with antagonists. Only a few inverse agonists of P2Y 12 have been described. To expand this limited chemical space and improve understanding of structural determinants of inverse agonist-receptor interaction, this study screened a purine compound library for lead structures using wild-type (WT) human P2Y 12

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“…P2RY12 is not expressed in splenic macrophages or in human alveolar macrophages and is expressed at negligible or very low levels in most other tissues (9,(31)(32)(33). Extracellular nucleotides such as ADP/ATP are released during tissue injury, inflammation, tumor growth, and aging (34)(35)(36)(37)(38).…”

Section: Introductionmentioning

confidence: 99%

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling

Su¹,

Floyd²,

Hughes³

et al. 2012

J. Clin. Invest.

104

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The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12 -/-OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12 -/-, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent α IIb β 3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β 3 gene knockout (Itgb3 -/-) OCs, but its effects were substantially blunted in P2ry12 -/-OCs. In vivo, P2ry12 -/-mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss. IntroductionOsteoclasts (OCs) are multinucleated myeloid lineage cells that are the principal source of bone resorptive activity (1). Enhanced OC activity, bone loss, and fractures are associated with rheumatoid arthritis, postmenopausal osteoporosis, and bone metastases (2). Modulation of osteoclastic bone resorption represents an attractive point of therapeutic intervention for the treatment of such conditions.Numerous purinergic G-protein-coupled nucleotide receptors are expressed in the bone microenvironment (3, 4). For example, uridine diphosphate-activated (UDP-activated) P2Y6 has been reported to increase NF-κB activation and OC survival (5), while P2Y2 (an ATP receptor) expression on osteoblasts (OBs) blocks bone mineralization (6, 7). Hoebertz et al. demonstrated that extracellular adenosine diphosphate (ADP) stimulates OC bone resorption in vitro, in part through the ADP receptor P2Y1 on OC (8); however, other ADP receptors, including purinergic receptor P2Y, G protein coupled, 12 (P2RY12), which is the target of the widely prescribed antiplatelet drug clopidogrel (Plavix), have not been evaluated for their roles in osteoclastic bone resorption.

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Functional expression of human and mouse P2Y12 receptors in Saccharomyces cerevisiae

Cited by 43 publications

References 27 publications

Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34

Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34

Identification of Determinants Required for Agonistic and Inverse Agonistic Ligand Properties at the ADP Receptor P2Y₁₂

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling

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Functional expression of human and mouse P2Y12 receptors in Saccharomyces cerevisiae

Cited by 43 publications

References 27 publications

Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34

Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34

Identification of Determinants Required for Agonistic and Inverse Agonistic Ligand Properties at the ADP Receptor P2Y12

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling

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Identification of Determinants Required for Agonistic and Inverse Agonistic Ligand Properties at the ADP Receptor P2Y₁₂