Delineation of ligand binding and receptor signaling activities of purified P2Y receptors reconstituted with heterotrimeric G proteins
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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 P2Y1 receptor and the human P2Y12 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 μg/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.
Key wordsG protein coupled receptor nucleotide(s) P2Y receptor platelets receptor purification RGS proteins
GTPase activating protein
guanine nucleotide dissociation inhibitor
guanine nucleotide exchange factor
G protein coupled receptor
regulator of G protein signaling
G protein-coupled receptors (GPCRs) represent one of the largest targets for therapeutic development. The P2Y receptor family of GPCRs are heptahelical transmembrane proteins that allow extracellular signals, in the form of nucleotides, to initiate intracellular signaling cascades that result in a wide range of physiological responses [1, 2, 3]. P2Y receptors have been subdivided into two groups based on sequence homology and G protein selectivity. Subtypes of the P2Y1-like family, comprised of the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors, all couple to Gαq to activate phospholipase C (PLC). These five receptors were the first P2Y receptors cloned and were illustrated to be activated by ADP (P2Y1), ATP and UTP (P2Y2), UTP (P2Y4), UDP (P2Y6), and ATP (P2Y11). Existence of a Gi/adenylyl cyclase-coupled receptor for ADP was initially reported by Cooper and Rodbell  in 1979, but molecular identification of this Gi-coupled P2Y receptor remained elusive for two decades. However, the P2Y12 receptor was ultimately cloned in 2001 and a P2Y12-like subfamily of P2Y receptors exists that includes the ADP-activated P2Y12 receptor, the ADP-activated P2Y13 receptor, and the nucleotide-sugar-activated P2Y14 receptor [5, 6, 7, 8, 9, 10].
Unambiguous delineation of the ligand selectivity of P2Y receptors is difficult due to the nature of nucleotide-promoted signaling. All cell systems used to study P2Y receptor-mediated signaling release nucleotides basally and/or after mechanical stimulation. Moreover, cell surface expression of enzymes that metabolize or interconvert nucleotides confounds the interpretation of results obtained with exogenously presented nucleotides. Although radioligand binding assays provide useful means to study P2Y receptor binding properties in ways that circumvent some of these problems, lack of high-affinity, stable, and selective antagonists for these receptors has largely prevented the development of such direct assays.
A recent goal of our laboratory has been purification of the P2Y receptor subtypes to near homogeneity. These purified receptors allow unequivocal determination of ligand binding selectivities and also provide a well controlled system to study both G protein selectivity and influences of other proteins on the signaling activities of P2Y receptors. Thus, recombinant P2Y receptors are solubilized in detergent, purified, and reconstituted into model phospholipid vesicles with heterotrimeric G proteins. Our data to date indicate maintenance of the native structure and signaling properties of the two ADP-activated receptors of platelets, the P2Y1 and P2Y12 receptors, when studied in purified form. This approach was initially applied by Elliot Ross and coworkers to address questions of the molecular mechanism(s) of β-adrenergic receptor/G protein interaction . Detergent-solubilized β-adrenergic receptors maintained ligand affinities equivalent to those of the natively expressed receptor, and hormone-promoted activation of G proteins was observed when these signaling partners were combined by reconstitution in model phospholipid vesicles. This approach has been greatly simplified with the advent of molecular techniques designed to epitope-tag proteins and through the use of high-level expression systems. Application of this technology to purinergic receptors provides a reliable and precise means to study the ligand and G protein selectivity of P2Y receptors in an environment independent of nucleotide interconversion, release, or metabolism.
Our lab has attempted to purify several P2Y receptors, including the human P2Y1, human P2Y2, human P2Y2 fused to its cognate G protein Gαq (P2Y2-Gq), human P2Y12, and an avian P2Y receptor . The avian P2Y receptor is unique among all species members of the P2Y family in that it couples equally well to both Gαq and Gαi, essentially encompassing the properties of the two mammalian subfamilies, the P2Y1-like Gq-coupled and the P2Y12-like Gi-coupled families [12, 13]. Among other physiological actions, the P2Y2 receptor is critical in lung airway physiology and is a current target for the development of cystic fibrosis therapeutics [14, 15]. The P2Y1 receptor and the P2Y12 receptor are mediators of the physiological action of ADP in platelets, and activation of both receptors is required to initiate and sustain platelet aggregation [16, 17, 18]. A clinically important drug clopidogrel targets the P2Y12 receptor, and many more therapeutic agents currently are in development targeting both the P2Y1 and P2Y12 receptors .
Receptor expression and solubilization
Functional reconstitution of purified receptors
Retention of native signaling activities has been confirmed with several of the purified P2Y receptors after reconstitution with heterotrimeric G proteins in model phospholipid vesicles. The initial step in forming protein-containing phospholipid vesicles is the preparation of detergent/phospholipid mixed micelles. The type of lipids used and their relative amounts is critical for proper membrane fusion, protein incorporation, and orientation of integralmembrane proteins (for a detailed review, see Racker ). Phosphatidylethanolamine, phosphatidylserine, and cholesteryl hemisuccinate, which all have been used previously in successful reconstitution experiments with GPCRs, are dried and then resuspended in deoxycholatecontaining buffer. This preparation is then combined with 15 pmol of purified receptor, 50 pmol of Ga, and 150 pmol of Gβ1γ2 and immediately loaded onto a G-50 Sephadex column. The void volume, which contains the vesicles, is eluted and collected. Although G-50 Sephadex resin does not separate the formed vesicles from free proteins of the molecular sizes used in these studies, contamination of vesicles with free protein is inconsequential. Alternatively, we have prepared vesicles with either an ACA-34 column or Sephacryl S-300 resin, both of which readily separate vesicles from free proteins.
Activities of purified P2Y receptors
To assess steady-state GTP hydrolysis, GTP labeled at the γ-phosphate with 32P is incubated with vesicles reconstituted with P2Y receptors and heterotrimeric G proteins. Liberated [32P]Pi is then quantified as described previously to determine basal activity and activities promoted by P2Y receptor activation and/or stimulation of GTPase activity by RGS proteins or effectors such as PLC-β, which are GAPs [20, 21]. As shown in previous publications from our laboratory, both the purified P2Y1 and the P2Y12 receptors maintain activities expected based on previous studies of the native receptors [20, 21]. Briefly, 2MeSADP was the most potent agonist tested in reconstitutions with purified P2Y1 receptor and Gαqβ1γ2, while ATP was a partial agonist. Both RGS4 and PLC-β were effective GAPs in the P2Y1 receptor/Gq reconstitution experiments . P2Y12 reconstitution experiments also revealed 2MeSADP as the most potent agonist tested while the cognate P2Y12 agonist, ADP, was three orders of magnitude less potent. ATP exhibited no agonist activity but inhibited the action of 2MeSADP at the P2Y12 receptor. Following reconstitution with Gβ1γ2 and Gα-subunits of the Gi and Gq families, we demonstrated selective coupling of the P2Y12 receptor to Gαi2 .
Utility of purified receptor preparations
The successful purification and functional reconstitution of P2Y receptors allows studies of P2Y signaling and its regulation in a system free of nucleotide release, interconversion, or metabolism, which is critical in studies of nucleotide-based agonists and antagonists. Indeed, we have functionally reconstituted an entire P2Y receptor-G protein-effector system by illustrating that an effector of the P2Y1 receptor, PLC-β1, acts as a GAP on Gαq in phospholipid vesicles composed of the P2Y1 receptor and Gαqβ1γ2 .
RGS proteins in a reconstituted GPCR/G protein platelet model
RGS proteins are classically defined as GTPase activating proteins. However, RGS proteins likely play more complex roles in GPCR signaling. In addition to their conserved RGS box domain, members of a number of RGS subfamilies contain multiple domains that allow protein-protein interactions, potentially conferring to these proteins activities in addition to their capacity to promote GTP hydrolysis by Gα-subunits. For example, the aforementioned GoLoco domain, as well as PDZ and PTB domains exist in RGS12 and RGS14, a GGL domain exists in RGS6, 7, 9, and 11 that confers to these RGS proteins Ligand binding and signaling activities of purified P2Y receptors 47 capacity to exist as functional heterodimers with Gβ5, and a DH/PH domain exists in the RGS protein p115RhoGEF that confers activity as a guanine nucleotide exchange factor for Rho (for an RGS review, see Neubig and Siderovski ).
Despite high level expression of various P2Y receptors in Sf9 insect cells, only a subset of these receptors proved to be solubilized by detergents. Of the three receptors that were successfully solubilized, only two were active upon reconstitution with their cognate heterotrimeric G proteins. The reason for the insolubility/inactivity of some receptors is unknown. Analyses of the two active receptors, the P2Y1 and the P2Y12 receptors, have provided novel and unequivocal insight into the signaling properties of these receptors. Future studies of RGS proteins, GDIs and Gβγ dimers in our reconstitution systems should lead to further delineation of the intricacies of P2Y signaling.
This work was supported by grants GM38213, HL34322, and HL54889 from the National Institutes of Health and the American Heart Association.
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