The PI3K-NF-kappaB signal transduction pathway is involved in mediating the anti-inflammatory effect of IB-MECA in adjuvant-induced arthritis

The anti-inflammatory effect of adenosine was previously found to be mediated via activation of the A3 adenosine receptor (A3AR). The aim of the present study was to decipher the molecular mechanism involved with the inhibitory effect of IB-MECA, an A3AR agonist, on adjuvant-induced arthritis. The adjuvant-induced arthritis rats responded to IB-MECA treatment with a decrease in the clinical score and the pathological score of the disease. The response to IB-MECA was neutralized by the antagonist MRS 1220, confirming that the efficacy of the synthetic agonist was A3AR mediated. The A3AR protein expression level was highly expressed in the synovia, in the peripheral blood mononuclear cells and in the drain lymph node (DLN) tissues of adjuvant-induced arthritis rats in comparison with na?ve animals. Downregulation of A3AR expression was noted upon treatment with IB-MECA. Analysis of synovia and DLN protein extracts revealed a decreased expression level of PI3K, PKB/Akt, IKK, NF-kappaB and tumor necrosis factor alpha, known to affect survival and apoptosis of inflammatory cells, whereas the caspase-3 level was upregulated.Taken together, high A3AR expression is found in the synovia, in the immune cells in the DLN and in peripheral blood mononuclear cells. IB-MECA, an orally bioavailable molecule, activates the A3AR, inducing receptor downregulation and the initiation of a molecular mechanism that involves de-regulation of the PI3K-NF-kappaB signaling pathway. As a result, a potent anti-inflammatory effect manifested in the improvement of the disease clinical score and pathological score occurs. The finding that the A3AR expression level in the peripheral blood mononuclear cells and in the DLN reflects the receptor status in the remote inflammatory site suggests use of the A3AR as a follow-up biomarker.     

Trans-Golgi Network (TGN) as a regulatory node for β1-adrenergic receptor (β1AR) down-modulation and recycling

Receptor down-modulation is the key mechanism by which G protein-coupled receptors (GPCRs) prevent excessive receptor signaling in response to agonist stimulation. Recently, the trans-Golgi network (TGN) has been implicated as a key checkpoint for receptor endocytosis and degradation. Here, we investigated the involvement of the TGN in down-modulation of β1-adrenergic receptor in response to persistent isoprotenerol stimulation. Immunofluorescent staining showed that ~50% of endocytosed β1AR colocalized with TGN-46 at 5 h. Disruption of the TGN by brefeldin A (BFA) led to the robust accumulation of endocytosed β1AR in Rab11(+) recycling endosomes, inhibited β1AR entry into LAMP1(+) lysosomes, and as a result enhanced β1AR recycling to the plasma membrane. The lysosomotropic agent, chloroquine, arrested the majority of endocytosed β1AR in the TGN by 4 h. Immunoblot analysis showed that either disruption of the TGN or blockage of the lysosome prevented β1AR degradation. Co-expression of GFP-arrestin-3 in β1AR cells increased the endocytosis of β1AR and facilitated its entry to the TGN but inhibited recycling to the plasma membrane. Arrestin-3-induced inhibition of β1AR recycling was reversed by BFA treatment, whereas chloroquine induced the accumulation of arrestin-3 with β1AR in the TGN. These results demonstrate for the first time that the TGN acts as a checkpoint for both the recycling and down-regulation of β1AR and that arrestin-3 not only mediates β1AR endocytosis but also its recycling through the TGN.     

Ligand-dependent intracellular trafficking of the G protein-coupled P2Y6 receptor

Endosomal trafficking is intricately linked to G protein-coupled receptors (GPCR) fate and signaling. Extracellular uridine diphosphate (UDP) acts as a signaling molecule by selectively activating the GPCR P2Y₆. Despite the recent interest for this receptor in pathologies, such as gastrointestinal and neurological diseases, there is sparse information on the endosomal trafficking of P2Y₆ receptors in response to its endogenous agonist UDP and synthetic selective agonist 5-iodo-UDP (MRS2693). Confocal microscopy and cell surface ELISA revealed delayed internalization kinetics in response to MRS2693 vs. UDP stimulation in AD293 and HCT116 cells expressing human P2Y₆. Interestingly, UDP induced clathrin-dependent P2Y₆ internalization, whereas receptor stimulation by MRS2693 endocytosis appeared to be associated with a caveolin-dependent mechanism. Internalized P2Y₆ was associated with Rab4, 5, and 7 positive vesicles independent of the agonist. We have measured a higher frequency of receptor expression co-occurrence with Rab11-vesicles, the trans-Golgi network, and lysosomes in response to MRS2693. Interestingly, a higher agonist concentration reversed the delayed P2Y₆ internalization and recycling kinetics in the presence of MRS2693 stimulation without changing its caveolin-dependent internalization. This work showed a ligand-dependent effect affecting the P2Y₆ receptor internalization and endosomal trafficking. These findings could guide the development of bias ligands that could influence P2Y₆ signaling.     

Arrestin isoforms dictate differential kinetics of A2B adenosine receptor trafficking

Adenosine mediates the activation of adenylyl cyclase via its interaction with specific A(2A) and A(2B) adenosine receptors. Previously, we demonstrated that arrestins are involved in rapid agonist-promoted desensitization of the A(2B) adenosine receptor (A(2B)AR) in HEK293 cells. In the present study, we investigate the role of arrestins in A(2B)AR trafficking. Initial studies demonstrated that HEK293 cells stably expressing arrestin antisense constructs, which reduce endogenous arrestin levels, effectively reduced A(2B)AR internalization. A(2B)AR recycling after agonist-induced endocytosis was also significantly impaired in cells with reduced arrestin levels. Interestingly, while overexpression of arrestin-2 or arrestin-3 rescued A(2B)AR internalization and recycling, arrestin-3 promoted a significantly faster rate of recycling as compared to arrestin-2. The specificity of arrestin interaction with A(2B)ARs was further investigated using arrestins fused to the green fluorescent protein (arr-2-GFP and arr-3-GFP). Both arrestins underwent rapid translocation (<1 min) from the cytosol to the plasma membrane following A(2B)AR activation. However, longer incubations with agonist (>10 min) revealed that arr-2-GFP but not arr-3-GFP colocalized with the A(2B)AR in rab-5 and transferrin receptor containing early endosomes. At later times, the A(2B)AR but not arr-2-GFP was observed in an apparent endocytic recycling compartment. Thus, while arrestin-2 and arrestin-3 mediate agonist-induced A(2B)AR internalization with relative equal potency, arrestin isoform binding dictates the differential kinetics of A(2B)AR recycling and resensitization.     

Inhibition of receptor internalization attenuates the TNFalpha-induced ROS generation in non-phagocytic cells

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)alpha, although the events through which TNFalpha induces ROS generation are not well characterized. Here, we report that TNFalpha-induced ROS production was blocked by pretreatment with internalization inhibitor monodansyl cadaverine (MDC). Similarly, a transient expression of a GTP-binding and hydrolysis-defective dynamin mutant (dynamin(K44A)) that had been shown to be defective in internalization significantly attenuated the TNFalpha-induced intracellular ROS production. Importantly, the inhibition of receptor internalization suppressed TNFalpha signaling to mitogen-activated protein kinases (MAPKs) stimulation. Together, our results suggest that receptor internalization is somehow necessary for the TNFalpha-induced ROS generation and subsequent intracellular downstream signaling in non-phagocytes.     

An adenosine analog (IB-MECA) inhibits anchorage-dependent cell growth of various human breast cancer cell lines

A3 adenosine receptor agonists have been reported to influence cell death and survival. Here we report the effects of an A3 adenosine receptor agonist, IB-MECA, on the cell growth of human breast cancer cell lines, MCF-7 (estrogen receptor positive) and MDA-MB468 (estrogen receptor negative). Therefore, this study was aimed to investigate the expression and possible action of A3 receptor in the human breast cancer cell lines. IB-MECA, at 1-100 microM, resulted in a significant cell growth inhibition (P < 0.05) which reached the maximum at 48 h, in the cell lines. In both cell lines, agonist-induced effects were antagonized by pretreatment with a selective A3 adenosine receptor antagonist, MRS1220. Using RT-PCR method, further confirmation was provided by the presence of mRNA of A3 receptor in the cells. In addition, IB-MECA was able to inhibit forskolin-stimulated cAMP levels, which indicate the functional form of A3 receptor on the cell surface of these breast cancer cell lines. These results suggest that the inhibitory effect of IB-MECA on the growth of human breast cancer cell lines is mediated through activation of A3 adenosine receptor.     

Effects of synthetic A3 adenosine receptor agonists on cell proliferation and viability are receptor independent at micromolar concentrations

The question as to whether A3 adenosine receptor (A3AR) agonists, N ⁶-(3-iodobenzyl)-adenosine-5′-N- methyluronamide (IB-MECA) and 2-chloro-N ⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA), could exert cytotoxic effects at high concentrations with or without the involvement of A3AR has been a controversial issue for a long time. The initial findings suggesting that A3AR plays a crucial role in the induction of cell death upon treatment with micromolar concentrations of IB-MECA or Cl-IB-MECA were revised, however, the direct and unequivocal evidence is still missing. Therefore, the sensitivity of Chinese hamster ovary (CHO) cells transfected with human recombinant A3AR (A3-CHO) and their counter partner wild-type CHO cells, which do not express any of adenosine receptors, to micromolar concentrations of IB-MECA and Cl-IB-MECA was studied. We observed that IB-MECA and Cl-IB-MECA exhibited a strong inhibitory effect on cell proliferation due to the blockage of cell cycle progression at G1/S and G2/M transitions in both A3-CHO and CHO cells. Further analysis revealed that IB-MECA and Cl-IB-MECA attenuated the Erk1/2 signalling irrespectively to A3AR expression. In addition, Cl-IB-MECA induced massive cell death mainly with hallmarks of a necrosis in both cell lines. In contrast, IB-MECA affected cell viability only slightly independently of A3AR expression. IB-MECA induced cell death that exhibited apoptotic hallmarks. In general, the sensitivity of A3-CHO cells to micromolar concentrations of IB-MECA and Cl-IB-MECA was somewhat, but not significantly, higher than that observed in the CHO cells. These results strongly suggest that IB-MECA and Cl-IB-MECA exert cytotoxic effects at micromolar concentrations independently of A3AR expression.     

Accelerated dephosphorylation of the beta2-adrenergic receptor by mutation of the C-terminal lysines: effects on ubiquitination, intracellular trafficking, and degradation

Agonist-mediated ubiquitination regulates some G protein-coupled receptors by targeting them to lysosomes for degradation. Phosphorylation also regulates receptor endocytosis and trafficking to lysosomes. To explore the roles of the two post-translational modifications, we mutated the three C-terminal lysines to arginines in the human beta 2-adrenergic receptor (beta 2AR) (K348/372/375R). The level of agonist-mediated ubiquitination of the mutant (3K/R) was greatly reduced compared to that of wild-type (WT) beta 2AR in whole cells and in cell-free assays. Downregulation of 3K/R also was attenuated compared to that of the WT, whereas internalization and recycling were more similar. During endocytosis, WT and 3K/R appeared in different vesicles and WT, but not 3K/R, was transported to lysosomes. Both were rapidly phosphorylated in agonist-stimulated cells, but upon agonist removal, the rate of dephosphorylation of 3K/R initially was approximately 5 times faster than that of WT. The increased rate also was observed in a cell-free, soluble assay and, thus, was not due to differences in receptor trafficking. Okadaic acid, a potent phosphatase inhibitor, reduced the level of dephosphorylation and increased the levels of lysosomal targeting and degradation of 3K/R. The reduced level of ubiquitination and rapid dephosphorylation of 3K/R appear to prevent it from being sorted to lysosomes in contrast to the phosphorylated and ubiquitinated WT beta 2AR. Our findings indicate that both phosphorylation and ubiquitination are involved in the intracellular sorting of beta 2AR between pathways of recycling to the plasma membrane and degradation in lysosomes, and that the rate of dephosphorylation may be another mechanism of regulating the sorting.     

Lipid rafts constrain basal α1A-adrenergic receptor signaling by maintaining receptor in an inactive conformation

We have reported that the α_1A-adrenergic receptor (α_1AAR) in rat-1 fibroblasts is a lipid raft protein. Here we examined whether disrupting lipid rafts by methyl-β-cyclodextrin (MCD) sequestration of cholesterol affects α_1AAR signaling. Unexpectedly, MCD increased α_1AAR-dependent basal inositol phosphate formation and p38 mitogen-activated protein kinase activation in a cholesterol-dependent manner. It also initiated internalization of surface α_1AAR, which was partially blocked by receptor inhibition. Binding assays revealed MCD-mediated increases in receptor agonist affinity as well as reciprocal decreases in inverse agonist affinity, a behavior that is usually interpreted as a shift toward the active receptor conformation. In untreated cells a fraction of the receptor was found to be present in preassociated receptor/G protein complexes, which rapidly dissociate upon receptor stimulation. Consistent with MCD-induced signaling, raft disruption resulted in an increase in receptor/G protein complexes. These results strongly suggest that lipid rafts constrain basal α_1AAR activity; however, preassembled receptor/G protein complexes could still provide a mechanism for accelerating α_1AAR signaling following stimulation.     

Sorting of the FGF receptor 1 in a human glioma cell line

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase promoting tumor growth in a variety of cancers, including glioblastoma. Binding of FGFs triggers the intracellular Ras/Raf/ERK signaling pathway leading to cell proliferation. Down-regulation of FGFR1 and, consequently, inactivation of its signaling pathways represent novel treatment strategies for glioblastoma. In this study, we investigated the internalization and endocytic trafficking of FGFR1 in the human glioma cell line U373. Stimulation with FGF-2 induced cell rounding accompanied by increased BrdU and pERK labeling. The overexpression of FGFR1 (without FGF treatment) resulted in enhanced phosphorylated FGFR1 suggesting receptor autoactivation. Labeled ligand (FGF-2-Cy5.5) was endocytosed in a clathrin- and caveolin-dependent manner. About 25 % of vesicles carrying fluorescently tagged FGFR1 represented early endosomes, 15 % transferrin-positive recycling endosomes and 40 % Lamp1-positive late endosomal/lysosomal vesicles. Stimulation with FGF-2 increased the colocalization rate in each of these vesicle populations. The treatment with the lysosomal inhibitor leupeptin resulted in FGFR1 accumulation in lysosomes, but did not enhance receptor recycling as observed in neurons. Analysis of vesicle distributions revealed an accumulation of recycling endosomes in the perinuclear region. In conclusion, the shuttling of receptor tyrosine kinases can be directly visualized by overexpression of fluorescently tagged receptors which respond to ligand stimulation and follow the recycling and degradation pathways similarly to their endogenous counterparts.     

Cell-based, high-content screen for receptor internalization, recycling and intracellular trafficking

A variety of physiologically important receptors are internalized and then recycled back to the plasma membrane by the endocytic recycling compartment. These include the transferrin receptor and many G-protein coupled receptors (GPCRs). The internalization of GPCRs is a result of agonist stimulation. A cell-based fluorescent imaging assay is described that detects and quantifies the presence of fluorescently labeled receptors and macromolecules in the recycling compartment. This High Content Screening application is conducted on the ArrayScan II System that includes fluorescent reagents, imaging instrumentation and the informatics tools necessary to screen for compounds that affect receptor internalization, recycling and GPCR activation. We demonstrate the Receptor Internalization and Trafficking application by quantifying (i) the internalization and recycling of the transferrin receptor using a fluorescently labeled ligand and (ii) the internalization of a physiologically functional model GPCR, a GFP-parathyroid hormone receptor chimera. These assays give high signal-to-noise ratios, broad dynamic ranges between stimulated and unstimulated conditions and low variability across different screening runs. Thus, the Receptor Internalization and Trafficking application, in conjunction with the ArrayScan II System, forms the basis of a robust, information-rich and automated screen for GPCR activation.     

A3 adenosine receptor activation decreases mortality and renal and hepatic injury in murine septic peritonitis

The role of A3 adenosine receptors (ARs) in sepsis and inflammation is controversial. In this study, we determined the effects of A3AR modulation on mortality and hepatic and renal dysfunction in a murine model of sepsis. To induce sepsis, congenic A3AR knockout mice (A3AR KO) and wild-type control (A3AR WT) mice were subjected to cecal ligation and double puncture (CLP). A3AR KO mice had significantly worse 7-day survival compared with A3AR WT mice. A3AR KO mice also demonstrated significantly higher elevations in plasma creatinine, alanine aminotransferase, aspartate aminotransferase, keratinocyte-derived chemokine, and TNF-alpha 24 h after induction of sepsis compared with A3AR WT mice. Renal cortices from septic A3AR KO mice exhibited increased mRNA encoding proinflammatory cytokines and enhanced nuclear translocation of NF-kB compared with samples from A3AR WT mice. A3AR WT mice treated with N6-(3-iodobenzyl)ADO-5'N-methyluronamide (IB-MECA; a selective A3AR agonist) or 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191; a selective A3AR antagonist) had improved or worsened 7-day survival after induction of sepsis, respectively. Moreover, A3AR WT mice treated with IB-MECA or MRS-1191 showed acutely improved or worsened, respectively, renal and hepatic function following CLP. IB-MECA significantly reduced mortality in mice lacking the A1AR or A2aAR but not the A3AR, demonstrating specificity of IB-MECA in activating A3ARs and mediating protection against sepsis-induced mortality. We conclude that endogenous or exogenous A3AR activation confers significant protection from murine septic peritonitis primarily by attenuating the hyperacute inflammatory response in sepsis.     

Pharmacological characterization of DPTN and other selective A3 adenosine receptor antagonists

The A₃ adenosine receptor (AR) is emerging as an attractive drug target. Antagonists are proposed for the potential treatment of glaucoma and asthma. However, currently available A₃AR antagonists are potent in human and some large animals, but weak or inactive in mouse and rat. In this study, we re-synthesized a previously reported A₃AR antagonist, DPTN, and evaluated its affinity and selectivity at human, mouse, and rat ARs. We showed that DPTN, indeed, is a potent A₃AR antagonist for all three species tested, albeit a little less selective for mouse and rat A₃AR in comparison to the human A₃AR. DPTN’s K_i values at respective A₁, A_2A, A_2B, and A₃ receptors were (nM) 162, 121, 230, and 1.65 (human); 411, 830, 189, and 9.61 (mouse); and 333, 1147, 163, and 8.53 (rat). Its antagonist activity at both human and mouse A₃ARs was confirmed in a cyclic AMP functional assay. Considering controversial use of currently commercially available A₃AR antagonists in rats and mice, we also re-examined other commonly used and selective A₃AR antagonists under the same experimental conditions. The K_i values of MRS1523 were shown to be 43.9, 349, and 216 nM at human, mouse, and rat A₃ARs, respectively. MRS1191 and MRS1334 showed incomplete inhibition of [¹²⁵I]I-AB-MECA binding to mouse and rat A₃ARs, while potent human A₃AR antagonists, MRS1220, MRE3008F20, PSB10, PSB-11, and VUF5574 were largely inactive. Thus, we demonstrated that DPTN and MRS1523 are among the only validated A₃AR antagonists that can be possibly used (at an appropriate concentration) in mouse or rat to confirm an A₃AR-related mechanism or function.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09823-5.     

Binding of the beta2 adrenergic receptor to N-ethylmaleimide-sensitive factor regulates receptor recycling

Following agonist stimulation, most G protein-coupled receptors become desensitized and are internalized, either to be degraded or recycled back to the cell surface. What determines the fate of a specific receptor type after it is internalized is poorly understood. Here we show that the rapidly recycling beta2 adrenergic receptor (beta2AR) binds via a determinant including the last three amino acids in its carboxyl-terminal tail to the membrane fusion regulatory protein, N-ethylmaleimide-sensitive factor (NSF). This is documented by in vitro overlay assays and by cellular coimmunoprecipitations. Receptors bearing mutations in any of the last three residues fail to interact with NSF. After stimulation with the agonist isoproterenol, a green fluorescent protein fusion of NSF colocalizes with the wild type beta2AR but not with a tail-mutated beta2AR. The beta2AR-NSF interaction is required for efficient internalization of the receptors and for their recycling to the cell surface. Mutations in the beta2AR tail that ablate NSF binding reduce the efficiency of receptor internalization upon agonist stimulation. Upon subsequent treatment of cells with the antagonist propranolol, wild type receptors return to the cell surface, while tail-mutated receptors remain sequestered. Thus, the direct binding of the beta2AR to NSF demonstrates how, after internalization, the fate of a receptor is reliant on a specific interaction with a component of the cellular membrane-trafficking machinery.     

Structural analysis of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the γ-phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. Over the past several years, structural studies employing X-ray crystallography have advanced our understanding of the molecular mechanisms by which RTKs recognize their ligands and are activated by dimerization and tyrosine autophosphorylation. This review will highlight the key results that have emerged from these structural studies.     

EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate

EGF-R [EGF (epidermal growth factor) receptor] ligands can promote or inhibit cell growth. The biological outcome of receptor activation is dictated, at least in part, by ligand-specified patterns of endocytic trafficking. EGF-R trafficking downstream of the ligands EGF and TGF-alpha (transforming growth factor-alpha) has been investigated extensively. However, less is known about EGF-R fates induced by the ligands BTC (betacellulin) and AR (amphiregulin). We undertook comparative analyses to identify ligand-specific molecular events that regulate EGF-R trafficking and degradation. EGF (17 nM) and BTC (8.5 nM) induced significant EGF-R degradation, with or without ectopic expression of the ubiquitin ligase Cbl. Human recombinant AR (17 nM) failed to affect receptor degradation in either case. Notably, levels of ligand-induced EGF-R ubiquitination did not correlate strictly with receptor degradation. Dose-response experiments revealed that AR at a saturating concentration was a partial agonist at the EGF-R, with approx. 40% efficacy (relative to EGF) at inducing receptor tyrosine phosphorylation, ubiquitination and association with Cbl. EGF-R down-regulation and degradation also were compromised upon cell stimulation with AR (136 nM). These outcomes correlated with decreased degradation of the Cbl substrate and internalization inhibitor hSprouty2. Downstream of the hSprouty2 checkpoint in AR-stimulated cells, Cbl-free EGF-R was incorporated into endosomes from which Cbl-EGF-R complexes were excluded. Our results suggest that the AR-specific EGF-R fate results from decreased hSprouty2 degradation and reduced Cbl recruitment to underphosphorylated EGF-R, two effects that impair EGF-R trafficking to lysosomes.     

Met receptor tyrosine kinase degradation is altered in response to the leucine-rich repeat of the Listeria invasion protein internalin B

Entry of the bacterial pathogen Listeria monocytogenes into host epithelial cells is critical for infection and virulence. One major pathway for Listeria entry involves binding of the bacterial protein Internalin B to the host receptor tyrosine kinase Met (hepatocyte growth factor receptor). Activation of Met and downstream signaling cascades is critical for Listeria entry. Internalin B is composed of several structural domains including an N-terminal leucine-rich repeat that is sufficient for binding Met and stimulating downstream signal transduction. Internalin B is monomeric, whereas the leucine-rich repeat is dimeric when expressed as an isolated fragment. The different quaternary states of Internalin B and the leucine-rich repeat suggest that these two Met ligands might cause distinct biological effects. Here we demonstrate that Internalin B and the leucine-rich repeat fragment exhibit agonist properties that differentially influence Met down-regulation in lysosomes. Specifically, Met stability is increased in response to the leucine-rich repeat fragment compared with Internalin B. Interestingly, Internalin B and the leucine-rich repeat stimulate equivalent rates of clathrin-mediated Met internalization. However, the leucine-rich repeat is defective in promoting lysosomal down-regulation of Met and instead enhances receptor recycling to the cell surface. In addition, the leucine-rich repeat causes prolonged Met activation (phosphorylation) and increased cell motility compared with Internalin B. Taken together, our findings indicate that individual domains of Internalin B differentially regulate Met trafficking. The ability of the leucine-rich repeat fragment to promote Met recycling could account for the increased cell motility induced by this ligand.     

Ubiquitination of fibroblast growth factor receptor 1 is required for its intracellular sorting but not for its endocytosis

Endocytosis and targeting of growth factor receptors for lysosomal degradation have been associated with ubiquitination of the intracellular part of the receptors. To elucidate the role of receptor ubiquitination in internalization and sorting of fibroblast growth factor receptor (FGFR), we constructed several mutants of FGFR1 in which lysines, potential ubiquitination sites, were substituted for arginines. Substitution of all lysine residues in the intracellular part of FGFR1 resulted in inactivation of the tyrosine kinase domain of the receptor. However, several multilysine FGFR1 mutants, where up to 26 of 29 lysines in the intracellular part of the receptor were mutated, retained tyrosine kinase activity. The active multilysine mutants were poorly ubiquitinated, but internalized normally, indicating that ubiquitination of the receptor is not required for endocytosis. In contrast, degradation of the multilysine mutants was dramatically reduced as the mutants were inefficiently transported to lysosomes but rather sorted to recycling endosomes. The altered sorting resulted in sustained signaling. The duration of FGFR1 signaling seems to be tightly regulated by receptor ubiquitination and subsequent sorting to the lysosomes for degradation.     

Macropinocytosis of the PDGF β-receptor promotes fibroblast transformation by H-RasG12V

Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. The extent to which the endocytic trafficking routes can contribute to such RTK hyperactivation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation were synergistic in stimulating phosphatidylinositol (PI) 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of PI 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in nontransformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor promoted macropinocytosis of both receptors and increased their activation in nontransformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis.     

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.