Physical and functional interactions of two naturally-occurring mutant variants of the P2Y12 receptor
Date
2019
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
G-protein-coupled receptors (GPCRs) constitute the largest family of receptors in humans, responding to a wide range of stimuli, and responsible for nearly every physiological process. Their pharmacological significance is unprecedented with around a third of FDA-approved drugs targeting these receptors (Sriram & Insel 2018). ☐ In platelets, GPCRs play a vital role in their activation and aggregation. Platelets are anucleate cells that contribute to both protective hemostasis and pathophysiological thrombosis. Humans express two purinergic receptors, P2Y1 and P2Y12, and two protease-activated receptors, PAR1 and PAR4, on the plasma membrane of platelets (Abbracchio et al 2006; Coughlin 2005). P2Y12 is of particular significance as it is the target of clinically effective anti-thrombotic drugs and is the focus of this study. ☐ While P2Y1 and PAR1 are required for the initiation of platelet activation, P2Y12 and PAR4 are important for stable responsiveness. PAR4 is activated by thrombin, the most potent platelet activator, and signals through Gαq which initiates intracellular events leading to the release of dense granules (Coughlin 2005). Dense granules release a variety of signaling molecules extracellularly including ADP, which is the agonist for P2Y12. P2Y12 is coupled to Gαi and therefore decreases cAMP levels in the cell (Abbracchio et al 2006). Both PAR4 and P2Y12 can independently activate Akt, but synergy between the two receptors is required for maximal Akt activation for stable thrombi in vivo (Li et al 2011). ☐ Our understanding of how GPCRs operate has changed in recent years. Historically thought to exist as monomeric entities transducing signals through a single G protein and adopting only one of two conformational states (on or off), GPCRs are now understood to form dimers (Gurevich & Gurevich 2008), couple to more than one G protein (Wenzel-Seifert & Seifert 2000), and adopt multiple agonist-dependent conformations of varying levels of activity (Swaminath et al 2005). ☐ Increasing evidence supports that dimerization plays a pivotal role in normal cell signaling and further diversifies receptor responses. While it has become clear that class C GPCRs form obligate homo- and heterodimers, the characterization of class A oligomers in both their size and function, remains elusive (Gurevich & Gurevich 2008). Our lab has shown that class A GPCRs, P2Y12 and PAR4 directly and specifically associate with each other in a functionally relevant manner (Khan et al 2014). Disrupting their heterodimerization significantly decreased phospho-Akt levels when the receptors were co-expressed heterologously in HEK293T cells. ☐ Recently, our lab became interested in naturally-occurring mutant variants of P2Y12 that have been identified in two patients found to have platelet aggregation defects. The first patient had a lifelong history of bleeding and was found to be compound heterozygous for two mutant variants of P2Y12: R256Q and R265W. These receptors have been shown to have normal surface expression and agonist binding but have a Gαi defect in platelets (Cattaneo et al 2003). Given that the mutations are not present cytosolically where we would expect direct physical association with the G protein, these mutations may affect coupling through altering receptor structure. The second patient was heterozygous for a mutation resulting in an aspartic acid to asparagine substitution at amino acid position 121 of P2Y12 (D121N) (Kostyak et al 2018). It is surprising that isolated platelets from this subject are virtually unresponsive to agonist ADP, given that the subject is heterozygous for the D121N mutation. It is expected that this individual would express a wild type P2Y12 receptor encoded by the remaining gene copy and therefore be able to functionally compensate. This mutation is located in the highly conserved DRY motif of GPCRs which is thought to be important for G protein coupling, receptor conformational states, and trafficking (Nygaard et al 2009). This mutation may structurally alter the receptor in a manner resulting in its apparent dominant negative phenotype. ☐ Interestingly, the crystal structure of P2Y12 packed as a dimer (Zhang et al 2014b), and treatment of cells with the active metabolite of a P2Y12 antagonist decreased the proportion of apparent P2Y12 oligomers resolved on SDS-PAGE (running as higher-than-expected molecular weight bands on immunoblots) (Savi et al 2006). This is suggestive that the functional unit of P2Y12 is at least a dimer, if not a higher-ordered oligomer. The aforementioned P2Y12 mutant variants may have an altered ability to homodimerize or heterodimerize with PAR4, in-turn affecting their function and providing an explanation for the observed phenotype in patients. ☐ Using fluorescence correlation spectroscopy (FCS), my lab has found the predominant species of P2Y12 to be a monomer on the plasma membrane of live HEK293T cells for the first time. Interestingly, we found that P2Y12 mutant variants R256Q and R265W have a higher tendency to homodimerize compared to the wild-type receptor. Furthermore, P2Y12R256Q, when expressed in isolation was unable to reduce forskolin-induced cAMP activity, suggesting an inability to couple Gαi (Barnawi 2017). Since P2Y12R265W was found to exhibit a more severe homodimerization defect relative to P2Y12R256Q, I decided to further investigate potential structural and functional properties of this receptor. Additionally, I aimed to characterize the functional deficits and dimerization potential of P2Y12D121N to begin to understand the potential dominant-negative function of this mutation in platelet aggregation. ☐ Here, I found that both P2Y12 mutant variants R265W and D121N exhibit constitutive activity in phosphorylation of Akt, which may be explained through cooperation with PAR4. Consistent with this hypothesis, I found that P2Y12R265W may physically associate with PAR4. The tendency of P2Y12R265W, relative to WT P2Y12, to heterodimerize with PAR4 as well as P2Y12D121N/PAR4 interactions, have yet to be investigated. Additionally, the DRY motif has been found to participate in interactions that maintain GPCRs in an inactive state. A charge-neutral mutation of aspartic acid to asparagine may induce constitutive activity. ☐ Through FCS, I confirmed previous findings from my lab that P2Y12R265W forms increased homodimers relative to wild type receptor, whereas P2Y12D121N does not homodimerize. It has yet to be determined whether P2Y12D121N may dimerize with the wild-type receptor. Finally, I show that neither P2Y12R265W or P2Y12D121N have a defect in Gαi coupling, as revealed through a bioluminescence-based cAMP assay in live cells. ☐ It was surprising to us that P2Y12R265W exhibited no defect in forskolin-induced cAMP inhibition, as we expected its increased homodimerization would preclude either activation of G protein or its association with the receptor; however, this further highlights the complex relationship between GPCR structure and signaling. Our results are suggestive that constitutive activity of these P2Y12 mutant variants promotes agonist-independent desensitization. This would severely reduce ADP-dependent platelet aggregation as observed in the compound heterozygous P2Y12-R256Q/R265W patient and the P2Y12D121N heterozygous individual.
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Keywords
P2Y12 receptor, Mutant variants