Purification of the human P2Y(12) receptor: Ligand binding, G protein coupling, and regulation by RGS proteins.

摘要

The human P2Y12 receptor is a member of the G protein coupled P2Y receptor family intimately involved in platelet physiology. We describe here the purification and functional characterization of recombinant P2Y12 receptor following high-level expression from a baculovirus in Sf9 insect cells with the goal of providing molecular insight into the receptor's: (i) ligand binding properties, (ii) G protein signaling selectivities, and (iii) regulation by RGS proteins. Following expression in Sf9 insect cells and extraction with digitonin, nearly homogeneous P2Y 12 receptor purification was achieved using metal-affinity chromatography and other traditional chromatographic steps. Yields of purified P2Y12 receptor ranged from 10--100 mug per liter of infected cells. Once purified, the receptor was reconstituted in model lipid vesicles along with heterotrimeric G proteins to assess receptor function. Agonist-promoted increases in steady-state GTPase activity demonstrated the functional activity of the reconstituted purified receptor. This reconstitution system was utilized to assess the action of various nucleotide agonists and antagonists as well as the relative G protein selectivity. Furthermore, the relative expression of RGS proteins in human platelets was determined and the action of these RGS proteins was assessed in reconstitution studies utilizing either receptor-expressing membranes or purified receptors. RGS16 but not other RGS proteins enhanced carbachol-promoted guanine nucleotide exchange by Gi in both M2 receptor-expressing membranes and model lipid vesicles containing purified M2 receptor. P2Y 12 receptor-expressing membranes were rendered inactive following treatment with urea and could not be tested in this system. However, in proteoliposomes containing purified P2Y12 receptor and Galphai2beta 1gamma2, RGS4, and possibly RGS16, appeared to enhance exchange by the G protein. These results suggest a potential scaffolding role for R4 RGS proteins, allowing for efficient receptor/G protein coupling.