ARF6 activated by the LHCG receptor through the cytohesin family of guanine nucleotide exchange factors mediates the receptor internalization and signaling

The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a G(s)-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization.     

Role for CD74 and CXCR4 in clathrin-dependent endocytosis of the cytokine MIF

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that plays a role in innate and adaptive immunity. Depending on the cellular context and disease state, MIF signaling is mediated by its receptors CXCR2, CXCR4 and/or CD74. Although it is known that MIF is endocytosed, the exact mechanism has remained unknown. In exploring the mechanism of MIF endocytosis with biologically active Alexa(546)MIF, pathway-specific inhibitors (monodansylcadaverine, MDC; chlorpromazine, CPZ; dynasore; dominant-negative dynamin, bafilomycin, nocodazole) and receptor overexpression and blockade approaches, we identified a clathrin/dynamin-dependent endocytosis pathway as the main track for MIF internalization. MIF endocytosis was rapid and colocalization with both early and late endosomal vesicles in a microtubule- and acidification-dependent manner was observed. LDL endocytosis (which is clathrin-mediated) served as a control and was similarly inhibited by MDC or dynasore. When MIF endocytosis was compared to that of transferrin, acetylated LDL, and choleratoxin B (the latter internalized by a clathrin-independent pathway) by colocalization studies, the MIF internalization pathway clearly resembled that of LDL but also shared early trafficking with transferrin, whereas no colocalization with choleratoxin was noted. To identify the receptors involved in MIF endocytosis, we focused on CD74 and CXCR4 which form a heteromeric complex. Ectopic overexpression of CD74 in HEK293 and HeLa cells, which do not endogenously express CD74, led to a marked acceleration of MIF endocytosis while pharmacological blockade of CXCR4, which is endogenously expressed on these cells, with AMD3100 led to a 20% reduction of MIF endocytosis in HEK293-CD74 transfectants, whereas in untransfected cells, a blockade of 40% was observed. Of note, both CD74 and CXCR4 strongly colocalize with Alexa(546)MIF both on the plasma membrane and in endosomal compartments. Moreover, MIF-stimulated AKT signaling, which was previously shown to involve both CD74 and CXCR4, was reduced by endocytosis inhibitors, indicating that MIF signaling is at least in part due to endosomal signaling mechanisms. Thus, MIF uptake follows a rapid LDL-like, clathrin- and dynamin-dependent endocytosis pathway, which is dependent on the receptors CD74 and CXCR4 and leads to the initiation of endosomal signaling responses.     

Flotillins Regulate Membrane Mobility of the Dopamine Transporter but Are Not Required for Its Protein Kinase C Dependent Endocytosis

Flotillins were proposed to mediate clathrin‐independent endocytosis, and recently, flotillin‐1 was implicated in the protein kinase C (PKC)‐triggered endocytosis of the dopamine transporter (DAT). Since endocytosis of DAT was previously shown to be clathrin‐mediated, we re‐examined the role of clathrin coat proteins and flotillin in DAT endocytosis using DAT tagged with the hemagglutinin epitope (HA) in the extracellular loop and a quantitative HA antibody uptake assay. Depletion of flotillin‐1, flotillin‐2 or both flotillins together by small interfering RNAs (siRNAs) did not inhibit PKC‐dependent internalization and degradation of HA‐DAT. In contrast, siRNAs to clathrin heavy chain and μ2 subunit of clathrin adaptor complex AP‐2 as well as a dynamin inhibitor Dyngo‐4A significantly decreased PKC‐dependent endocytosis of HA‐DAT. Similarly, endocytosis and degradation of DAT that is not epitope‐tagged were highly sensitive to the clathrin siRNAs and dynamin inhibition but were not affected by flotillin knockdown. Very little co‐localization of DAT with flotillins was observed in cells ectopically expressing DAT and in cultured mouse dopaminergic neurons. Depletion of flotillins increased diffusion rates of HA‐DAT in the plasma membrane, suggesting that flotillin‐organized microdomains may regulate the lateral mobility of DAT. We propose that clathrin‐mediated endocytosis is the major pathway of PKC‐dependent internalization of DAT, and that flotillins may modulate functional association of DAT with plasma membrane rafts rather than mediate DAT endocytosis .     

LPS or ethanol triggers clathrin‐ and rafts/caveolae‐dependent endocytosis of TLR4 in cortical astrocytes

Toll‐like receptor 4 (TLR4) activation and signalling in glial cells play critical roles in neurological disorders and in alcohol‐induced brain damage. TLR4 endocytosis upon lipopolysaccharide (LPS) stimulation regulates which signalling pathway is activated, the MyD88‐dependent or the TIR‐domain‐containing adapter‐inducing interferon‐β (TRIF)‐dependent pathway. However, it remains elusive whether ethanol‐induced TLR4 signalling is associated with receptor internalization and trafficking, and which endocytic pathway(s) are used in cortical astrocytes. Using the adenoviral over‐expression of TLR4^GFP, confocal microscopy and the imagestream technique, we show that upon ethanol or LPS stimulation, TLR4 co‐localizes with markers of the clathrin and caveolin endocytic pathways, and that this endocytosis is dependent on dynamin. Using chlorpromazin and filipin as inhibitors of the clathrin and rafts/caveolae endocytic pathways, respectively, we demostrate that TRIF‐dependent signalling relies on an intact clathrin pathway, whereas disruption of rafts/caveolae inhibits the MyD88‐ and TRIF‐dependent signalling pathways. Immunofluorescence studies also suggest that lipid rafts and clathrin cooperate for appropriate TLR4 internalization. We also show that ethanol can trigger similar endocytic pathways as LPS does, although ethanol delays clathrin internalization and alters TLR4 vesicular trafficking. Our results provide new insights into the effects of ethanol or LPS on TLR4 signalling in cortical astrocytes, events that may underlie neuroinflammation and brain damage.

     

Cell entry of bovine ephemeral fever virus requires activation of Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB pathways as well as Cox‐2‐mediated PGE2/EP receptor signalling to enhance clathrin‐mediated virus endocytosis

Although we have previously demonstrated that cell entry of bovine ephemeral fever virus (BEFV) follows a clathrin‐mediated and dynamin 2‐dependent endocytosis pathway, the cellular mechanism mediating virus entry remains unknown. Here, we report that BEFV triggers simultaneously Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB signalling pathways in the stage of virus binding to induce clathrin and dynamin 2 expressions, while vesicular stomatitis virus only activates Src‐JNK signalling to enhance its entry. Activation of these pathways by ultraviolet‐inactivated BEFV suggests a role for virus binding but not viral internalization and gene expression. By blocking these signalling pathways with specific inhibitors, BEFV‐induced expressions of clathrin and dynamin 2 were significantly diminished. By labelling BEFV with 3,3′‐dilinoleyloxacarbocyanine perchlorate to track viral entry, we found that virus entry was hindered by both Src and Akt inhibitors, suggesting that these signalling pathways are crucial for efficient virus entry. In addition, BEFV also triggers Cox‐2‐catalysed prostaglandin E₂ (PGE₂) synthesis and induces expressions of G‐protein‐coupled E‐prostanoid (EP) receptors 2 and 4, leading to amplify signal cascades of Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB, which elevates both clathrin and dynamin 2 expressions. Furthermore, pretreatment of cells with adenylate cyclase (cAMP) inhibitor SQ22536 reduced BEFV‐induced Src phosphorylation as well as clathrin and dynamin 2 expressions. Our findings reveal for the first time that BEFV activates the Cox‐2‐mediated PGE₂/EP receptor signalling pathways, further enhancing Src‐JNK‐AP1 in a cAMP‐dependent manner and PI3K‐Akt‐NF‐κB in a cAMP‐independent manner. Accordingly, BEFV stimulates PGE₂/EP receptor signalling amplifying Src‐JNK‐AP1 and PI3K‐Akt‐NF‐κB pathways in an autocrine or paracrine fashion to enhance virus entry.     

Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation.     

Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling

Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.     

Inhibition of dynamin prevents CCL2-mediated endocytosis of CCR2 and activation of ERK1/2

The magnitude and duration of G protein-coupled receptor (GPCR) signals are regulated through desensitization mechanisms. In leukocytes, ligand binding to chemokine receptors leads to Ca²⁺ mobilization and ERK activation through pertussis toxin-sensitive G proteins, as well as to phosphorylation of the GPCR. After interaction with the endocytic machinery (clathrin, adaptin), the adaptor β-arrestin recognizes the phosphorylated GPCR tail and quenches signaling to receptors. The molecular mechanisms that lead to receptor endocytosis are not universal amongst the GPCR, however, and the precise spatial and temporal events in the internalization of the CCR2 chemokine receptor remain unknown. Here we show that after ligand binding, CCR2 internalizes rapidly and reaches early endosomes, and later, lysosomes. Knockdown of clathrin by RNA interference impairs CCR2 internalization, as does treatment with the dynamin inhibitor, dynasore. Our results show that CCR2 internalization uses a combination of clathrin-dependent and -independent pathways, as observed for other chemokine receptors. Moreover, the use of dynasore allowed us to confirm the existence of a dynamin-sensitive element that regulates ERK1/2 activation. Our results indicate additional complexity in the link between receptor internalization and cell signaling.     

TTP specifically regulates the internalization of the transferrin receptor

Different plasma membrane receptors are internalized through saturable/noncompetitive pathways, suggesting cargo-specific regulation. Here, we report that TTP (SH3BP4), a SH3-containing protein, specifically regulates the internalization of the transferrin receptor (TfR). TTP interacts with endocytic proteins, including clathrin, dynamin, and the TfR, and localizes selectively to TfR-containing coated-pits (CCP) and -vesicles (CCV). Overexpression of TTP specifically inhibits TfR internalization, and causes the formation of morphologically aberrant CCP, which are probably fission impaired. This effect is mediated by the SH3 of TTP, which can bind to dynamin, and it is rescued by overexpression of dynamin. Functional ablation of TTP causes a reduction in TfR internalization, and reduced cargo loading and size of TfR-CCV. Tyrosine phosphorylation of either TTP or dynamin prevents their interaction, pointing to a possible mechanism of exclusion of TTP from some CCP. Thus, TTP might represent one of the long sought for molecules that allow cargo-specific control of clathrin endocytosis.     

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.     

Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.     

Characterization of RANTES- and aminooxypentane-RANTES-triggered desensitization signals reveals differences in recruitment of the G protein-coupled receptor complex.

The trafficking of lymphocyte populations is a complex process controlled by a vast array of molecules. In this process, cells must be able to sense small changes in chemoattractant gradients. Migration through a chemotactic gradient probably employs an on-off mechanism in which chemokine receptor desensitization, internalization, and recycling may be important steps. This multistep process requires the coordinated action of many factors, including G protein-coupled receptor kinases, arrestins, clathrin, and GTP-hydrolyzing proteins such as dynamin. In this report, we show that RANTES and its derivative, aminooxypentane (AOP)-RANTES, a potent RANTES antagonist as well as an inhibitor of HIV-1 infection, both promote CCR5 desensitization involving G protein-coupled receptor kinases-2 and beta-arrestin equally well. An important difference between the two molecules is that (AOP)-RANTES is more efficient than RANTES in promoting Ser/Thr phosphorylation of the receptor and association of G protein-coupled receptor kinases-2, beta-arrestin, and clathrin to the CCR5. After stimulation with either ligand, we observe rapid, transient association of dynamin to CCR5, implicating this protein in receptor sensitization, but this association is faster and longer-lasting following (AOP)-RANTES stimulation. In summary, we show that chemokine receptor internalization takes place through the formation of clathrin vesicles and involves dynamin activity. We provide compelling evidence that the differences between RANTES and (AOP)-RANTES in G alpha i activation condition subsequent signaling events, including internalization and receptor recycling.     

Plasticity of B cell receptor internalization upon conditional depletion of clathrin

B cell antigen receptor (BCR) association with lipid rafts, the actin cytoskeleton, and clathrin-coated pits influences B cell signaling and antigen presentation. Although all three cellular structures have been separately implicated in BCR internalization, the relationship between them has not been clearly defined. In this study, internalization pathways were characterized by specifically blocking each potential mechanism of internalization. BCR uptake was reduced by approximately 70% in B cells conditionally deficient in clathrin heavy chain expression. Actin or raft antagonists were both able to block the residual, clathrin-independent BCR internalization. These agents also affected clathrin-dependent internalization, indicating that clathrin-coated pits, in concert with mechanisms dependent on rafts and actin, mediate the majority of BCR internalization. Clustering G(M1) gangliosides enhanced clathrin-independent BCR internalization, and this required actin. Thus, although rafts or actin independently did not mediate BCR internalization, they apparently cooperate to promote some internalization even in the absence of clathrin. Simultaneous inhibition of all BCR uptake pathways resulted in sustained tyrosine phosphorylation and activation of the extracellular signal-regulated kinase (ERK), strongly suggesting that downstream BCR signaling can occur without receptor translocation to endosomes and that internalization leads to signal attenuation.     

A clathrin, caveolae, and dynamin-independent endocytic pathway requiring free membrane cholesterol drives HIV-1 internalization and infection in polarized trophoblastic cells

In human trophoblastic cells, a correlation between early endosomal trafficking of HIV-1 and virus infection was previously documented. However, if HIV-1 is massively internalized in these cells, the endocytic pathway(s) responsible for viral uptake is still undefined. Here we address this vital question. Amongst all the putative endocytic pathways present in polarized trophoblastic cells, we demonstrate that HIV-1 infection of these cells is independent of clathrin-mediated endocytosis and macropinocytosis. Importantly, treatment with the cholesterol-sequestering drug filipin severely impairs virus internalization, whereas the cholesterol-depleting compound methyl-beta-cyclodextrin has no impact on this pathway. Moreover, viral internalization is unaffected by overexpression of a mutant dynamin 2 or treatment with a kinase or tyrosine phosphatase inhibitor. Thus, HIV-1 infection in polarized trophoblastic cells occurs primarily via a clathrin, caveolae, and dynamin-independent pathway requiring free cholesterol. Notably, even though HIV-1 did not initially co-localize with transferrin, some virions migrate at later time points to transferrin-enriched endosomes, suggesting an unusual transit from the non-classical pathway to early endosomes. Finally, virus internalization in these cells does not involve the participation of microtubules but relies partly on actin filaments. Collectively these findings provide unprecedented information on the route of HIV-1 internalization in polarized human trophoblasts.     

Inhibition of receptor internalization by monodansylcadaverine selectively blocks p55 tumor necrosis factor receptor death domain signaling

The 55-kDa receptor for tumor necrosis factor (TR55) triggers multiple signaling cascades initiated by adapter proteins like TRADD and FAN. By use of the primary amine monodansylcadaverine (MDC), we addressed the functional role of tumor necrosis factor (TNF) receptor internalization for intracellular signal distribution. We show that MDC does not prevent the interaction of the p55 TNF receptor (TR55) with FAN and TRADD. Furthermore, the activation of plasmamembrane-associated neutral sphingomyelinase activation as well as the stimulation of proline-directed protein kinases were not affected in MDC-treated cells. In contrast, activation of signaling enzymes that are linked to the "death domain" of TR55, like acid sphingomyelinase and c-Jun-N-terminal protein kinase as well as TNF signaling of apoptosis in U937 and L929 cells, are blocked in the presence of MDC. The results of our study suggest a role of TR55 internalization for the activation of select TR55 death domain signaling pathways including those leading to apoptosis.     

Bovine Ephemeral Fever Virus Uses a Clathrin-Mediated and Dynamin 2-Dependent Endocytosis Pathway That Requires Rab5 and Rab7 as Well as Microtubules

The specific cell pathways involved in bovine ephemeral fever virus (BEFV) cell entry have not been determined. In this work, colocalization of the M protein of BEFV with clathrin or dynamin 2 was observed under a fluorescence microscope. To better understand BEFV entry, we carried out internalization studies with a fluorescently labeled BEFV by using a lipophilic dye, 3,30-dilinoleyloxacarbocyanine perchlorate (DiO), further suggesting that BEFV uses a clathrin-mediated endocytosis pathway. Our results suggest that clathrin-mediated and dynamin 2-dependent endocytosis is an important avenue of BEFV entry. Suppression of Rab5 or Rab7a through the use of a Rab5 dominant negative mutant and Rab7a short hairpin RNA (shRNA) demonstrated that BEFV requires both early and late endosomes for endocytosis and subsequent infection in MDBK and Vero cells. Treatment of BEFV-infected cells with nocodazole significantly decreased the M protein synthesis and viral yield, indicating that microtubules play an important role in BEFV productive infection, likely by mediating trafficking of BEFV-containing endosomes. Furthermore, BEFV infection was strongly blocked by different inhibitors of endosomal acidification, suggesting that virus enters host cells by clathrin-mediated and dynamin 2-dependent endocytosis in a pH-dependent manner.     

Clathrin-dependent endocytosis is required for TrkB-dependent Akt-mediated neuronal protection and dendritic growth

Endocytosis of Trk (tropomyosin-related kinase) receptors is critical for neurotrophin signal transduction and biological functions. However, the mechanism governing endocytosis of TrkB (tropomyosin-related kinase B) and the specific contributions of TrkB endocytosis to downstream signaling are unknown. In this study, we report that blocking clathrin, dynamin, or AP2 in cultured neurons of the central nervous system inhibited brain-derived neurotrophic factor (BDNF)-induced activation of Akt but not ERK. Treating neurons with the clathrin inhibitor monodansylcadaverine or a peptide that blocks dynamin function specifically abrogated Akt pathway activation in response to BDNF but did not affect the response of other downstream effectors or the up-regulation of immediate early genes neuropeptide Y and activity-regulated cytoskeleton-associated protein. Similar effects were found in neurons expressing small interfering RNA to silence AP2 or a dominant negative form of dynamin that inhibits clathrin-mediated endocytosis. In PC12 cells, ERK but not Akt activation required TrkA endocytosis following stimulation with nerve growth factor, whereas the opposite was true when TrkA-expressing neurons were stimulated with nerve growth factor in the central nervous system. Thus, the specific effects of internalized Trk receptors probably depend on the presence of cell type-specific modulators of neurotrophin signaling and not on differences inherent to Trk receptors themselves. Endocytosis-dependent activation of Akt in neurons was found to be critical for BDNF-supported survival and dendrite outgrowth. Together, these results demonstrate the functional requirement of clathrin- and dynamin-dependent endocytosis in generating the full intracellular response of neurons to BDNF in the central nervous system.     

Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin

Clathrin-mediated endocytosis is a fundamental cellular process conserved from yeast to mammals and is an important endocytic route for the internalization of many specific cargos, including activated growth factor receptors. Here we examined changes in tyrosine phosphorylation, a representative output of growth factor receptor signaling, in cells in which endocytic clathrin-coated pits are frozen at a deeply invaginated state, that is, cells that lack dynamin (fibroblasts from dynamin 1, dynamin 2 double conditional knockout mice). The major change observed in these cells relative to wild-type cells was an increase in the phosphorylation state, and thus activation, of activated Cdc42-associated kinase (Ack), a nonreceptor tyrosine kinase. Ack is concentrated at clathrin-coated pits, and binds clathrin heavy chain via two clathrin boxes. RNA interference-based approaches and pharmacological manipulations further demonstrated that the phosphorylation of Ack requires both clathrin assembly into endocytic clathrin-coated pits and active Cdc42. These findings reveal a link between progression of clathrin-coated pits to endocytic vesicles and an activation-deactivation cycle of Ack.     

Protease-activated receptor-1 down-regulation: a mutant HeLa cell line suggests novel requirements for PAR1 phosphorylation and recruitment to clathrin-coated pits

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly activated by a proteolytic mechanism, then internalized and degraded in lysosomes. The latter is critical for temporal fidelity of thrombin signaling. Toward understanding PAR1 down-regulation, we first investigated the pathway of PAR1 internalization. Activated PAR1 was rapidly recruited to clathrin-coated pits, where it colocalized with transferrin receptor (TfnR). Dominant-negative dynamin and clathrin hub mutants both blocked PAR1 internalization. Blockade of PAR1 internalization with dynamin K44A also inhibited activation-dependent PAR1 degradation. Thus activated PAR1 internalizes via clathrin-coated pits together with receptors that recycle and is then sorted away from such receptors and delivered to lysosomes. In the course of these studies we identified a mutant HeLa cell line, designated JT1, that was defective in PAR1 internalization. PAR1 signaled robustly in JT1 cells but was not phosphorylated or recruited to clathrin-coated pits after activation. Internalization of TfnR was intact in JT1 cells and internalization of beta(2)-adrenergic receptor, a GPCR that internalizes and recycles, was present but perhaps reduced. Taken together, these studies suggest that PAR1 is internalized in a dynamin- and clathrin-dependent manner like TfnR and beta(2)-adrenergic receptor but requires a distinct gene product for recruitment into this pathway.