Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.     

Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25

Polarized epithelial cells maintain the polarized distribution of basolateral and apical membrane proteins through a process of receptor-mediated endocytosis, sorting, and then recycling to the appropriate membrane domain. We have previously shown that the small GTP-binding proteins, Rab11a and Rab25, are associated with the apical recycling system of Madin-Darby canine kidney cells. Here we have utilized inducible expression of wild-type, dominant negative, and constitutively active mutants to directly compare the functions of Rab25 and Rab11a in postendocytic vesicular transport. We found that a Rab11a mutant deficient in GTP binding, Rab11aS25N, potently inhibited both transcytosis and apical recycling yet failed to inhibit transferrin recycling. Similarly, expression of either wild type Rab25 or the active mutant Rab25S21V inhibited both apical recycling and transcytosis of IgA by greater than 50% but had no effect on basolateral recycling of transferrin. Interestingly, the GTPase-deficient mutant Rab11aS20V inhibited basolateral to apical transcytosis of IgA, but had no effect on either apical or basolateral recycling. These results indicate that neither Rab11a nor Rab25 function in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells cells, consistent with recent morphological observations by others. Thus, transferrin receptors must be recycled to the plasma membrane prior to sorting of apically directed cargoes into Rab11a/Rab25-positive apical recycling endosomes.     

Lipid raft-dependent glucagon-like peptide-2 receptor trafficking occurs independently of agonist-induced desensitization

The intestinotrophic and cytoprotective actions of glucagon-like peptide-2 (GLP-2) are mediated by the GLP-2 receptor (GLP-2R), a member of the class II glucagon-secretin G protein-coupled receptor superfamily. Although native GLP-2 exhibits a short circulating half-life, long-acting degradation-resistant GLP-2 analogues are being evaluated for therapeutic use in human subjects. Accordingly, we examined the mechanisms regulating signaling, internalization, and trafficking of the GLP-2R to identify determinants of receptor activation and desensitization. Heterologous cells expressing the transfected rat or human GLP-2R exhibited a rapid, dose-dependent, and prolonged desensitization of the GLP-2-stimulated cAMP response and a sustained GLP-2-induced decrease in levels of cell surface receptor. Surprisingly, inhibitors of clathrin-dependent endocytosis failed to significantly decrease GLP-2R internalization, whereas cholesterol sequestration inhibited ligand-induced receptor internalization and potentiated homologous desensitization. The hGLP-2R localized to both Triton X-100-soluble and -insoluble (lipid raft) cellular fractions and colocalized transiently with the lipid raft marker caveolin-1. Although GLP-2R endocytosis was dependent on lipid raft integrity, the receptor transiently associated with green fluorescent protein tagged-early endosome antigen 1-positive vesicles and inhibitors of endosomal acidification attenuated the reappearance of the GLP-2R on the cell surface. Our data demonstrate that GLP-2R desensitization and raft-dependent trafficking represent distinct and independent cellular mechanisms and provide new evidence implicating the importance of a clathrin- and dynamin-independent, lipid raft-dependent pathway for homologous G protein-coupled receptor internalization.     

Epidermal growth factor and membrane trafficking. EGF receptor activation of endocytosis requires Rab5a

Activated epidermal growth factor receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between signaling and endocytosis is not well understood. Here we show that EGF stimulation of NR6 cells induces a specific, rapid and transient activation of Rab5a. EGF also enhanced translocation of the Rab5 effector, early endosomal autoantigen 1 (EEA1), from cytosol to membrane. The activation of endocytosis, fluid phase and receptor mediated, by EGF was enhanced by Rab5a expression, but not by Rab5b, Rab5c, or Rab5a truncated at the NH(2) and/or COOH terminus. Dominant negative Rab5a (Rab5:N34) blocked EGF-stimulated receptor-mediated and fluid-phase endocytosis. EGF activation of Rab5a function was dependent on tyrosine residues in the COOH-terminal domain of the EGF receptor (EGFR). Removal of the entire COOH terminus by truncation (c'973 and c'991) abrogated ligand-induced Rab5a activation of endocytosis. A "kinase-dead" EGFR failed to stimulate Rab5a function. However, another EGF receptor mutant (c'1000), with the kinase domain intact and a single autophosphorylation site effectively signaled Rab5 activation. These results indicate that EGFR and Rab5a are linked via a cascade that results in the activation of Rab5a and that appears essential for internalization. The results point to an interdependent relationship between receptor activation, signal generation and endocytosis.     

The intracellular trafficking of the G protein-coupled receptor TPbeta depends on a direct interaction with Rab11

Intracellular trafficking pathways of cell surface receptors following their internalization are the subject of intense research efforts. However, the mechanisms by which they recycle back to the cell surface are still poorly defined. We have recently demonstrated that the small Rab11 GTPase protein is a determinant factor in controlling the recycling to the cell surface of the beta-isoform of the thromboxane A2 receptor (TPbeta) following its internalization. Here, we demonstrate with co-immunoprecipitation studies in HEK293 cells that there is a Rab11-TPbeta association occurring in the absence of agonist, which is not modulated by stimulation of TPbeta. We show with purified TPbeta intracellular domains fused to GST and HIS-Rab11 proteins that Rab11 interacts directly with the first intracellular loop and the C-tail of TPbeta. Amino acids 335-344 of the TPbeta C-tail were determined to be essential for the interaction of Rab11 with this receptor domain. This identified sequence appears to be important in directing the intracellular trafficking of the receptor from the Rab5-positive intracellular compartment to the perinuclear recycling endosome. Interestingly, our data indicate that TPbeta interacts with the GDP-bound form, and not the GTP-bound form, of Rab11 which is necessary for recycling of the receptor back to the cell surface. To our knowledge, this is the first demonstration of a direct interaction between Rab11 and a transmembrane receptor.     

Agonist-dependent internalization and trafficking of the human prostacyclin receptor: a direct role for Rab5a GTPase

The human prostacyclin receptor (hIP) undergoes rapid agonist-induced internalization by largely unknown mechanism(s). Herein the involvement of Rab5 in regulating cicaprost-induced internalization of the hIP expressed in human embryonic kidney 293 cells was investigated. Over-expression of Rab5a significantly increased agonist-induced hIP internalization. Additionally, the hIP co-localized to Rab5a-containing endocytic vesicles in response to cicaprost stimulation and there was a coincident net translocation of Rab5 from the cytosol/soluble fraction of the cell. Co-immunoprecipitation studies confirmed a direct physical interaction between the hIP and Rab5a that was augmented by cicaprost. Whilst the dominant negative Rab5a(S34N) did not show decreased interaction with the hIP or fully impair internalization, it prevented hIP sorting to endocytic vesicles. Moreover, the GTPase deficient Rab5a(Q79L) significantly increased internalization and co-localized with the hIP in enlarged endocytic vesicles. While deletion of the carboxyl terminal (C)-tail domain of the hIP did not inhibit agonist-induced internalization, co-localization or co-immunoprecipitation with Rab5a per se, receptor trafficking was altered suggesting that it contains structural determinant(s) for hIP sorting post Rab5-mediated endocytosis. Taken together, data herein and in endothelial EA.hy 926 cells demonstrate a direct role for Rab5a in agonist-internalization and trafficking of the hIP and increases knowledge of the factors regulating prostacyclin signaling.     

A tyrosine-based sorting signal regulates intracellular trafficking of protease-activated receptor-1: multiple regulatory mechanisms for agonist-induced G protein-coupled receptor internalization

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly proteolytically activated, internalized, and then sorted to lysosomes and degraded. Internalization and lysosomal sorting of activated PAR1 is critical for termination of receptor signaling. We previously demonstrated that activated PAR1 is rapidly phosphorylated and internalized via a clathrin- and dynamin-dependent pathway that is independent of arrestins. Toward understanding the mechanisms responsible for activated PAR1 internalization through clathrin-coated pits we examined the function of a highly conserved tyrosine-based motif, YXXL, localized in the cytoplasmic carboxyl tail of the receptor. A mutant PAR1 in which tyrosine 383 and leucine 386 were replaced with alanines (Y383A/L386A) was significantly impaired in agonist-triggered internalization and degradation compared with wild-type receptor. In contrast, constitutive internalization, and recycling of unactivated PAR1 Y383A/L386A mutant was not affected, suggesting that tonic cycling of the mutant receptor remained intact. Strikingly, a PAR1 C387Z truncation mutant in which the YXXL motif was exposed at the C terminus constitutively internalized and degraded in an agonist-independent manner, whereas C387Z truncation mutant in which the critical tyrosine and leucine were mutated to alanine (C387Z-Y383A/L386A) failed to internalize. Inhibition of PAR1 C387Z mutant constitutive internalization with dominant-negative K44A dynamin blocked agonist-independent degradation of the mutant receptor. Together these findings strongly suggest that internalization of activated PAR1 is controlled by multiple regulatory mechanisms involving phosphorylation and a highly conserved tyrosine-based motif, YXXL. This study is the first to describe a function for a tyrosine-based motif, YXX, in GPCR internalization and reveal novel complexities in the regulation of GPCR trafficking.     

Crystal structure of rab11 in complex with rab11 family interacting protein 2

The small GTPase Rab11 regulates the recycling of endosomes to the plasma membrane via interactions with the Rab11 family of interacting proteins (FIPs). FIPs contain a highly conserved Rab binding domain (RBD) at their C termini whose structure is unknown. Here, we have determined the crystal structure of the RBD of FIP2 in complex with Rab11(GTP) by single wavelength anomalous diffraction methods. The overall structure is a heterotetramer with dyad symmetry, arranged as a Rab11-(FIP2)2-Rab11 complex. FIP2 forms a central alpha-helical coiled coil, with both helices contributing to the Rab11 binding patch on equivalent and opposite sides of the homodimer. Switch 1 of Rab11 is embedded between the two helices, while switch 2 remains flexible and is peripherally associated with the effector. The complex reveals the structural basis for Rab11 recognition by FIPs and suggests the molecular mechanisms underlying endocytic recycling pathways.     

Rab11BP/Rabphilin-11, a downstream target of rab11 small G protein implicated in vesicle recycling.

Rab11 small G protein has been implicated in vesicle recycling, but its upstream regulators or downstream targets have not yet been identified. We isolated here a downstream target of Rab11, named rabphilin-11, from bovine brain. Moreover, we isolated from a rat brain cDNA library its cDNA, which encoded a protein with a M(r) of 100,946 and 908 amino acids (aa). Rabphilin-11 bound GTP-Rab11 more preferentially than GDP-Rab11 at the N-terminal region and was specific for Rab11 and inactive for other Rab and Rho small G proteins. Both GTP-Rab11 and rabphilin-11 were colocalized at perinuclear regions, presumably the Golgi complex and recycling endosomes, in Madin-Darby canine kidney cells. In HeLa cells cultured on fibronectin, both the proteins were localized not only at perinuclear regions but also along microtubules, which were oriented toward membrane lamellipodia. Treatment of HeLa cells with nocodazole caused disruption of microtubules and dispersion of GTP-Rab11 and rabphilin-11. Overexpression of the C-terminal fragment of rabphilin-11 (aa 607-730), lacking the GTP-Rab11 binding domain, in HeLa cells reduced accumulation of transferrin at perinuclear regions and cell migration. Rabphilin-11 turned out to be a rat counterpart of recently reported bovine Rab11BP. These results indicate that rabphilin-11 is a downstream target of Rab11 which is involved in vesicle recycling.     

Rab11 mediates selective recycling and endocytic trafficking in Trypanosoma brucei

Trypanosoma brucei possesses a streamlined secretory system that guarantees efficient delivery to the cell surface of the critical glycosyl‐phosphatidylinositol (GPI)‐anchored virulence factors, variant surface glycoprotein (VSG) and transferrin receptor (TfR). Both are thought to be constitutively endocytosed and returned to the flagellar pocket via TbRab11+ recycling endosomes. We use conditional knockdown with established reporters to investigate the role of TbRab11 in specific endomembrane trafficking pathways in bloodstream trypanosomes. TbRab11 is essential. Ablation has a modest negative effect on general endocytosis, but does not affect turnover, steady state levels or surface localization of TfR. Nor are biosynthetic delivery to the cell surface and recycling of VSG affected. TbRab11 depletion also causes increased shedding of VSG into the media by formation of nanotubes and extracellular vesicles. In contrast to GPI‐anchored cargo, TbRab11 depletion reduces recycling of the transmembrane invariant surface protein, ISG65, leading to increased lysosomal turnover. Thus, TbRab11 plays a critical role in recycling of transmembrane, but not GPI‐anchored surface proteins. We proposed a two‐step model for VSG turnover involving release of VSG‐containing vesicles followed by GPI hydrolysis. Collectively, our results indicate a critical role of TbRab11 in the homeostatic maintenance of the secretory/endocytic system of bloodstream T. brucei.      

Developmental variation in Rab11-dependent trafficking in Trypanosoma brucei

In Trypanosoma brucei, endocytosis is developmentally regulated and is substantially more active in the mammalian infective stage, where it likely plays a role in immune evasion. The small GTPase TbRAB11 is highly expressed in the mammalian stage and mediates recycling of glycosylphosphatidylinositol-anchored proteins, including the variant surface glycoprotein (VSG) and the transferrin receptor, plus trafficking of internalized anti-VSG antibody and transferrin. No function has been assigned to TbRAB11 in the procyclic (insect) stage trypanosome. The importance of TbRAB11 to both bloodstream and procyclic form viability was assessed by RNA interference (RNAi). Suppression of TbRAB11 in the bloodstream form was rapidly lethal and led to cells with round morphology and an enlarged flagellar pocket. TbRAB11 RNAi was also lethal in procyclic forms, which also became rounded, but progression to cell death was significantly slower and the flagellar pocket remained normal. In bloodstream forms, silencing of TbRAB11 had no effect on exocytosis of newly synthesized VSG, fluid-phase endocytosis, or transferrin uptake, but export of internalized transferrin was inhibited. Lectin endocytosis assays revealed a block to postendosomal transport mediated by suppressing TbRAB11. By contrast, in procyclic forms, depletion of TbRAB11 blocks both fluid-phase endocytosis and internalization of surface proteins. In normal bloodstream forms, most VSG is recycled, but in procyclics, internalized surface proteins accumulated in the lysosome. These data demonstrate that TbRAB11 controls recycling and is essential in both life stages of T. brucei but that its primary role is subject to developmental variation.     

Distinct Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound Rab4 and Rab5.

Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiled-coil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rab-binding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTP-bound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.     

Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases

G protein-coupled receptors (GPCRs) are integral membrane proteins that, in response to activation by extracellular stimuli, regulate intracellular second messenger levels via their coupling to heterotrimeric G proteins. GPCR activation also initiates a series of molecular events that leads to G protein-coupled receptor kinase-mediated receptor phosphorylation and the binding of beta-arrestin proteins to the intracellular face of the receptor. beta-Arrestin binding not only contributes to the G protein-uncoupling of GPCRs, but also mediates the targeting of many GPCRs for endocytosis in clathrin-coated pits. Several GPCRs internalize as a stable complex with beta-arrestin and the stability of this complex appears to regulate, at least in part, whether the receptors are dephosphorylated in early endosomes and recycled back to the cell surface as fully functional receptors, retained in early endosomes or targeted for degradation in lysosomes. More recently, it has become appreciated that the movement of GPCRs through functionally distinct intracellular membrane compartments is regulated by a variety of Rab GTPases and that the activity of these Rab GTPases may influence GPCR function. Moreover, it appears that GPCRs are not simply passive cargo molecules, but that GPCR activation may directly influence Rab GTPase activity and as such, GPCRs may directly control their own targeting between intracellular compartments. This review provides a synopsis of the current knowledge regarding the role of beta-arrestins and Rab GTPases in regulating the intracellular trafficking and function of GPCRs.     

MUC1 intra-cellular trafficking is clathrin, dynamin, and rab5 dependent

MUC1, a transmembrane glycoprotein, is abnormally over-expressed in most human adenocarcinomas. MUC1 association with cytoplasmic cell signal regulators and nuclear accumulation are important for its tumor related activities. Little is known about how MUC1 translocates from the cell membrane to the cytoplasm. In this study, live cell imaging was used to study MUC1 intracellular trafficking. The interaction between EGFR and MUC1 was mapped by FRET analysis and EGF stimulated MUC1 endocytosis was observed directly through live cell imaging. MUC1-CT endocytosis was clathrin and dynamin dependent. Rab5 over-expression resulted in decreased cell membrane localization of MUC1, with accumulation of MUC1 endocytic vesicles in the peri-nuclear region. Conversely, over-expression of a Rab5 dominant negative mutant (S34N) resulted in redistribution of MUC1 from the peri-nuclear region to the cytoplasm. Collectively, these results indicated that MUC1 intra-cellular trafficking occurs through a regulated process that was stimulated by direct EGFR and MUC1 interaction, mediated by clathrin coated pits that were dynamin dependent and regulated by Rab5.     

Conserved structure and adjacent location of the thrombin receptor and protease-activated receptor 2 genes define a protease-activated receptor gene cluster.

BACKGROUND: Thrombin is a serine protease that elicits a variety of cellular responses. Molecular cloning of a thrombin receptor revealed a G protein-coupled receptor that is activated by a novel proteolytic mechanism. Recently, a second protease-activated receptor was discovered and dubbed PAR2. PAR2 is highly related to the thrombin receptor by sequence and, like the thrombin receptor, is activated by cleavage of its amino terminal exodomain. Also like the thrombin receptor, PAR2 can be activated by the hexapeptide corresponding to its tethered ligand sequence independent of receptor cleavage. Thus, functionally, the thrombin receptor and PAR2 constitute a fledgling receptor family that shares a novel proteolytic activation mechanism. To further explore the relatedness of the two known protease-activated receptors and to examine the possibility that a protease-activated gene cluster might exist, we have compared the structure and chromosomal locations of the thrombin receptor and PAR2 genes. MATERIALS AND METHODS: The genomic structures of the two protease-activated receptor genes were determined by analysis of lambda phage, P1 bacteriophage, and bacterial artificial chromosome (BAC) genomic clones. Chromosomal location was determined with fluorescent in situ hybridization (FISH) on metaphase chromosomes, and the relative distance separating the two genes was evaluated both by means of two-color FISH and analysis of YACs and BACs containing both genes. RESULTS: Analysis of genomic clones revealed that the two protease-activated receptor genes share a two-exon genomic structure in which the first exon encodes 5'-untranslated sequence and signal peptide, and the second exon encodes the mature receptor protein and 3'-untranslated sequence. The two receptor genes also share a common locus with the two human genes located at 5q13 and the two mouse genes at 13D2, a syntenic region of the mouse genome. These techniques also suggest that the physical distance separating these two genes is less than 100 kb. CONCLUSIONS: The fact that the thrombin receptor and PAR2 genes share an identical structure and are located within approximately 100 kb of each other in the genome demonstrates that these genes arose from a gene duplication event. These results define a new protease-activated receptor gene cluster in which new family members may be found.     

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor

The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR(2)), which is required for post-endocytic sorting of PAR(2) to lysosomes, where degradation arrests signaling. The mechanisms of post-endocytic sorting of ubiquitinated receptors are incompletely understood. Here, we investigated the role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), in post-endocytic sorting and signaling of PAR(2). In HEK-PAR(2) cells, PAR(2) activating peptide (PAR(2)-AP) induced PAR(2) trafficking from the cell surface to early endosomes containing endogenous HRS, and then to lysosomes. HRS overexpression or knockdown with small interfering RNA caused formation of enlarged HRS-positive endosomes, where activated PAR(2) and c-Cbl accumulated, and PAR(2) failed to traffic to lysosomes. Overexpression of HRS prevented PAR(2)-AP-induced degradation of PAR(2), as determined by Western blotting. Overexpression of HRS mutant lacking an ubiquitin-binding motif similarly caused retention of PAR(2) in enlarged endosomes. Moreover, HRS overexpression or knockdown caused retention of ubiquitin-resistant PAR(2)Delta14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR(2)Delta14K/R. HRS overexpression or knockdown similarly prevented lysosomal trafficking and recycling of calcitonin receptor-like receptor, a non-ubiquitinated receptor that traffics to lysosomes after sustained activation and recycles after transient activation. Thus, HRS plays a critically important role in the post-endocytic sorting of single receptors, PAR(2) and CLR, to both degradative and recycling pathways. This sorting role for HRS is independent of its ubiquitin-interacting motif, and it can regulate trafficking of both ubiquitinated and non-ubiquitinated PAR(2) and non-ubiquitinated CLR. The ultimate sorting decision to degradative or recycling pathways appears to occur downstream from HRS.     

Rab GTPases regulating receptor trafficking at the late endosome-lysosome membranes

Lysosomes serve key degradative functions for the turnover of membrane lipids and protein components. Its biogenesis is principally dependent on exocytic traffic from the late endosome via the trans-Golgi network, and it also receives cargo to be degraded from the endocytic pathway. Membrane trafficking to the late endosome-lysosome is tightly regulated to maintain the amplitude of signalling events and cellular homeostasis. Key coordinators of lysosomal traffic include members of the Rab small GTPase family. Amongst these, Rab7, Rab9 and the more recently studied Rab22B/31 have all been reported to regulate membrane trafficking processed at the late endosome-lysosome system. We discuss what is known about the roles of these Rab proteins and their interacting partners on the regulation of traffic of important receptor proteins such as the epidermal growth factor receptor (EGFR) and the mannose 6-phosphate receptor (M6PR), in association with the late endosome-lysosome system. Better knowledge of EGFR and M6PR traffic in this regard may aid in understanding the pathological processes, such as oncogenic transformations associated with these receptors. Copyright ? 2012 John Wiley & Sons, Ltd.