Delineation of ligand binding and receptor signaling activities of purified P2Y receptors reconstituted with heterotrimeric G proteins.

Autor: Bodor ET; Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA., Waldo GL, Blaesius R, Harden TK
Jazyk: angličtina
Zdroj: Purinergic signalling [Purinergic Signal] 2004 Dec; Vol. 1 (1), pp. 43-9.
DOI: 10.1007/s11302-004-4748-1
Abstrakt: P2Y receptors are G protein coupled receptors that respond to extracellular nucleotides to promote a multitude of signaling events. Our laboratory has purified several P2Y receptors with the goal of providing molecular insight into their: (1) ligand binding properties, (2) G protein signaling selectivities, and (3) regulation by RGS proteins and other signaling cohorts. The human P2Y(1) receptor and the human P2Y(12) receptor, both of which are intimately involved in ADP-mediated platelet aggregation, were purified to near homogeneity and studied in detail. After high-level expression from recombinant baculovirus infection of Sf9 insect cells, approximately 50% of the receptors were successfully extracted with digitonin. Purification of nearly homogeneous epitope-tagged P2Y receptor was achieved using metal-affinity chromatography followed by other traditional chromatographic steps. Yields of purified P2Y receptors range from 10 to 100 mug/l of infected cells. Once purified, the receptors were reconstituted in model lipid vesicles along with their cognate G proteins to assess receptor function. Agonist-promoted increases in steady-state GTPase assays demonstrated the functional activity of the reconstituted purified receptor. We have utilized this reconstitution system to assess the action of various nucleotide agonists and antagonists, the relative G protein selectivity, and the influence of other proteins, such as phospholipase C, on P2Y receptor-promoted signaling. Furthermore, we have identified the RGS expression profile of platelets and have begun to assess the action of these RGS proteins in a reconstituted P2Y receptor/G protein platelet model.
Databáze: MEDLINE
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