Functional expression of human and mouse P2Y12 receptors in Saccharomyces cerevisiae.
DNA sequences encoding the murine ortholog of the human P2Y12 receptor were cloned. The human and mouse P2Y12 receptors were expressed in a yeast cell-based GPCR expression technology containing chimeric yeast Galpha protein (Gpa1) constructs in which the 5 C-terminal amino acids were identical to corresponding sequences from mammalian Galphai/o proteins. LacZ reporter gene assays of agonist-induced activation of the G protein-coupled mating signal transduction pathway revealed murine P2Y12 functional pharmacological properties that closely resembled those exhibited by the human P2Y12 receptor. In NIH3T3 cells, the mouse P2Y12 stimulated calcium uptake monitored in FLIPR via coupling to a Galphaq/i3 chimeric protein. Murine P2Y12 mRNA was expressed at high levels in the brain and at lower levels in a variety of peripheral tissues. In situ hybridization analysis indicated glia-specific expression within the brain.
Discovery Neuroscience, Wyeth Research, Princeton, NJ 08543-8000, USA. pauschm@wyeth.com <pauschm@wyeth.com>
Mark H
MH
Pausch
Margaret
M
Lai
Eugene
E
Tseng
Janet
J
Paulsen
Brian
B
Bates
Seung
S
Kwak
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D017403:In Situ Hybridization; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058925:Receptors, Purinergic P2Y12; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D008565:Membrane Proteins; C548576:P2RY12 protein, human; C548577:P2ry12 protein, mouse; C553058:P2ry12 protein, rat; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D011993:Recombinant Fusion Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium