Differential Endosomal Sorting of a Novel P2Y₁₂ Purinoreceptor Mutant

Abstract

P2Y 12 receptor internalization and recycling play an essential role in ADP-induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y 12 (P341A) whose P2Y 12 receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild-type (WT) P2Y 12 recycling and investigated P2Y 12 -P341A receptor traffic. Treatment with ADP resulted in delayed Rab5-dependent internalization of P341A when compared with WT P2Y 12 . While WT P2Y 12 rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7-positive endosomes with considerable agonist-dependent accumulation in the trans-Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT-P2Y 12 . Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y 12 receptor traffic and explain the compromised receptor function in the platelets of the P2Y 12 -P341A-expressing patient. The trafficking of G protein-coupled receptors (GPCRs) involves a series of highly coordinated events. While some GPCRs, such as the protease-activated receptor (PAR) family, become degraded upon internalization (1), most are efficiently recycled back to the plasma membrane. Studies from our laboratory investigating purinergic GPCR function in human platelets have demonstrated efficient internalization, desensitization and rapid resensitization of P2Y 12 in response to ADP (2-4). Exposure of platelets to ADP results in simultaneous activation of P2Y 1 and P2Y 12 receptors, which couple to G q and G i , respectively, and act synergistically to mediate platelet activation and aggregation (5). The regulation of P2Y 12 is G protein-coupled receptor kinase (GRK) dependent with subsequent recruitment to clathrin-coated pits (CCPs) in an arrestin-dependent manner (3,6). Following internalization and receptor dephosphorylation, P2Y 12 receptors are recycled back to the plasma membrane (2). Efficient recycling of both P2Y 1 and P2Y 12 receptors is required for the maintenance of receptor responsiveness to ADP in platelets (2). As the mechanisms that underlie P2Y 12 receptor recycling remain largely unknown, we sought to define the endocytic traffic route of this receptor and identify key proteins that regulate this process.Bioinformatic analysis of the protein sequences of an array of GPCR families including the β 1 AR and β 2 AR subtypes, chemokine CXCR2, ET A endothelin receptor, P2Y 1 and P2Y 12 has revealed the presence of C-terminal type I, type ...

Raft-like membranes from the trans-Golgi network and endosomal compartments

“…70) and a mutant P2Y 12 G protein-coupled receptor 71 . With the growing interest in mapping the molecular components of isolated organelles by lipidomic and proteomic studies, this protocol is potentially useful in that it represents a strategy for preparing a highly purified membrane fraction from a dynamic cellular compartment.…”

Pathophysiological consequences of receptor mistraffic: Tales from the platelet P2Y 12 receptor

“…Activation of P2Y 12 receptors stimulates ARF6 activity which in turn regulates Nm23-H1, an nucleoside diphosphate kinase to facilitate dynamin-dependent fission of clathrin-coated vesicles. Following internalization, the P2Y 12 receptor undergoes rab5-dependent trafficking to a sorting compartment from which it is rapidly recycled (18). Rapid recycling of the P2Y 12 receptor had previously been described in human platelets although the mechanism at that time was unclear (8).…”

“…In relation to receptor traffic, the wild type P2Y 12 receptor undergoes significant agonistindependent internalization where it colocalizes with transferrin, a marker for early and recycling endosomes (31). This pool of receptor, which recycles in a rab4-dependent manner, can readily recycle back to the cell surface (18). Whether like other platelet GPCRs (54,55) this agonist-independent traffic may provide an internal pool of receptor able to mobilise to the cell surface and support receptor signalling requires further study.…”

“…These ligands can bind to a number of PDZ domaincontaining regulatory proteins with these interactions regulating various aspects of receptor function including stabilization, signaling and trafficking (62,63). P2Y 12 was not able to recycle and resensitize following agonist exposure (18,32). This was in contrast to the wild type receptor which recycles back to the cell surface in a rab4/11-dependent manner rapidly following agonist-dependent internalization facilitating receptor resensitization (18).…”

“…P2Y 12 was not able to recycle and resensitize following agonist exposure (18,32). This was in contrast to the wild type receptor which recycles back to the cell surface in a rab4/11-dependent manner rapidly following agonist-dependent internalization facilitating receptor resensitization (18). Therefore the integrity of the PDZ-ligand of the P2Y 12 …”