TNFalpha-induced cytoprotection requires the production of free radicals within mitochondria in C2C12 myotubes

We previously reported that tumour necrosis factor alpha (TNFalpha) can mimic classic ischemic preconditioning (IPC) in both cells and heart. However, the signalling pathways involved remain incompletely understood. One potential protective pathway could be TNFalpha-induced reactive oxygen species (ROS). We hypothesized that TNFalpha cytoprotection occurs through the generation of ROS which originate within the mitochondria. C(2)C(12) myotubes were preconditioned with either a short period of hypoxia (IPC) or a low concentration of TNFalpha (0.5 ng/ml) prior to a simulated ischemic insult. ROS generation was evaluated on cells stained with dichlorofluorescin diacetate (DCFH-DA) by flow cytometry. The source of TNFalpha-induced ROS was examined with Mitotracker Red CM-H(2)XRos. The bioenergetics of the mitochondria were evaluated by investigation of the respiratory parameters and the inner mitochondrial membrane potential. Pretreatment with TNFalpha improved cell viability compared with the simulated ischemic control (TNFalpha: 75 +/- 1% versus 34 +/- 1% for the control: p<0.001). The ROS scavenger, N-2-mercaptopropionyl-glycine (MPG), reduced the viability of TNFalpha-stimulated cells to 15 +/- 1% (p<0.001 versus TNFalpha). Similar results were obtained with IPC. TNFalpha stimulation increased ROS production mainly in the mitochondria, and this increase was abolished in the presence of MPG. Addition of TNFalpha to the cells increased State 2 respiration and modestly depolarised the membrane potential prior to the ischemic insult. In conclusion, TNFalpha-induced ROS generation can occur within the mitochondria, resulting in temporal mitochondrial perturbations which may initiate the cytoprotective effect of TNFalpha.     

Mechanisms and functions of AT(1) angiotensin receptor internalization

The type 1 (AT(1)) angiotensin receptor, which mediates the known physiological and pharmacological actions of angiotensin II, activates numerous intracellular signaling pathways and undergoes rapid internalization upon agonist binding. Morphological and biochemical studies have shown that agonist-induced endocytosis of the AT(1) receptor occurs via clathrin-coated pits, and is dependent on two regions in the cytoplasmic tail of the receptor. However, it is independent of G protein activation and signaling, and does not require the conserved NPXXY motif in the seventh transmembrane helix. The dependence of internalization of the AT(1) receptor on a cytoplasmic serine-threonine-rich region that is phosphorylated during agonist stimulation suggests that endocytosis is regulated by phosphorylation of the AT(1) receptor tail. beta-Arrestins have been implicated in the desensitization and endocytosis of several G protein-coupled receptors, but the exact nature of the adaptor protein required for association of the AT(1) receptor with clathrin-coated pits, and the role of dynamin in the internalization process, are still controversial. There is increasing evidence for a role of internalization in sustained signal generation from the AT(1) receptor. Several aspects of the mechanisms and specific function of AT(1) receptor internalization, including its precise mode and route of endocytosis, and the potential roles of cytoplasmic and nuclear receptors, remain to be elucidated.     

Ubiquitination of RIP is required for tumor necrosis factor alpha-induced NF-kappaB activation

Stimulation of cells with tumor necrosis factor (TNFalpha) triggers a recruitment of various signaling molecules, such as RIP, to the TNFalpha receptor 1 complex, leading to activation of NF-kappaB. Previous studies indicate that RIP plays an essential role for TNFalpha-induced NF-kappaB activation, but the molecular mechanism by which RIP mediates TNFalpha signals to activate NF-kappaB is not fully defined. Earlier studies suggest that RIP undergoes a ligand-dependent ubiquitination. However, it remains to be determined whether the ubiquitination of RIP is required for TNFalpha-induced NF-kappaB activation. In this study, we have identified Lys377 of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-kappaB activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNFalpha receptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNFalpha-induced NF-kappaB activation.     

GDC-0152-induced autophagy promotes apoptosis in HL-60 cells

Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), have pivotal roles in renal hemodynamics, neuroendocrine signaling, blood pressure regulation, and cardiovascular homeostasis. Binding of ANP and BNP to the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) induces rapid internalization and trafficking of the receptor via endolysosomal compartments, with concurrent generation of cGMP. However, the mechanisms of the endocytotic processes of NPRA are not well understood. The present study, using ¹²⁵I-ANP binding assay and confocal microscopy, examined the function of dynamin in the internalization of NPRA in stably transfected human embryonic kidney-293 (HEK-293) cells. Treatment of recombinant HEK-293 cells with ANP time-dependently accelerated the internalization of receptor from the cell surface to the cell interior. However, the internalization of ligand–receptor complexes of NPRA was drastically decreased by the specific inhibitors of clathrin- and dynamin-dependent receptor internalization, almost 85% by monodansylcadaverine, 80% by chlorpromazine, and 90% by mutant dynamin, which are specific blockers of endocytic vesicle formation. Visualizing the internalization of NPRA and enhanced GFP-tagged NPRA in HEK-293 cells by confocal microscopy demonstrated the formation of endocytic vesicles after 5 min of ANP treatment; this effect was blocked by the inhibitors of clathrin and by mutant dynamin construct. Our results suggest that NPRA undergoes internalization via clathrin-mediated endocytosis as part of its normal itinerary, including trafficking, signaling, and metabolic degradation.     

NAK is recruited to the TNFR1 complex in a TNFalpha-dependent manner and mediates the production of RANTES: identification of endogenous TNFR-interacting proteins by a proteomic approach

Tumor necrosis factor alpha (TNFalpha) is a proinflammatory cytokine with pleiotropic immunological and biological activities. TNFalpha signaling is triggered by the engagement of soluble TNFalpha to two types of cell surface receptors, TNFR1 and TNFR2. This recruits cytosolic proteins to the intracellular domains of the receptors and initiates signaling to downstream effectors. In this study, we used a proteomic approach to identify these cytosolic proteins from affinity-purified, endogenous TNFalpha.TNFR complexes in human myelomonocytic U937 cells. Seven proteins were identified, including TRADD, TRAP2, and TRAF2, which are three proteins known to be recruited to TNFalpha receptors. NAK, RasGAP3, TRCP1, and TRCP2 were also identified. We further showed that NAK is recruited to TNFR1 in a temporally regulated and TNFalpha-dependent manner and that it mediates the TNFalpha-induced production of the chemokine RANTES (regulated on activation normal T cell expressed and secreted). These data demonstrate that NAK is a component of the TNFalpha.TNFR1 signaling complex and confirm the physiological role of NAK in the TNFalpha-mediated response.     

Tumor necrosis factor alpha (TNFalpha) induces the unfolded protein response (UPR) in a reactive oxygen species (ROS)-dependent fashion, and the UPR counteracts ROS accumulation by TNFalpha

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER overload, resulting in ER stress. To cope with ER stress, mammalian cells trigger a specific response known as the unfolded protein response (UPR). Although recent studies have indicated cross-talk between ER stress and oxidative stress, the mechanistic link is not fully understood. By using murine fibrosarcoma L929 cells, in which tumor necrosis factor (TNF) alpha induces accumulation of reactive oxygen species (ROS) and cell death, we show that TNFalpha induces the UPR in a ROS-dependent fashion. In contrast to TNFalpha, oxidative stresses by H2O2 or arsenite only induce eukaroytic initiation factor 2alpha phosphorylation, but not activation of PERK- or IRE1-dependent pathways, indicating the specificity of downstream signaling induced by various oxidative stresses. Conversely, the UPR induced by tunicamycin substantially suppresses TNFalpha-induced ROS accumulation and cell death by inhibiting reduction of cellular glutathione levels. Collectively, some, but not all, oxidative stresses induce the UPR, and pre-emptive UPR counteracts TNFalpha-induced ROS accumulation.     

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.     

Tumor necrosis factor alpha-induced pancreatic beta-cell insulin resistance is mediated by nitric oxide and prevented by 15-deoxy-Delta12,14-prostaglandin J2 and aminoguanidine. A role for peroxisome proliferator-activated receptor gamma activation and inos expression.

Recent studies have identified a beta-cell insulin receptor that functions in the regulation of protein translation and mitogenic signaling similar to that described for insulin-sensitive cells. These findings have raised the novel possibility that beta-cells may exhibit insulin resistance similar to skeletal muscle, liver, and fat. To test this hypothesis, the effects of tumor necrosis factor-alpha (TNFalpha), a cytokine proposed to mediate insulin resistance by interfering with insulin signaling at the level of the insulin receptor and its substrates, was evaluated. TNFalpha inhibited p70(s6k) activation by glucose-stimulated beta-cells of the islets of Langerhans in a dose- and time-dependent manner, with maximal inhibition observed at approximately 20-50 ng/ml, detected after 24 and 48 h of exposure. Exogenous insulin failed to prevent TNFalpha-induced inhibition of p70(s6k), suggesting a defect in the insulin signaling pathway. To further define mechanisms responsible for this inhibition and also to exclude cytokine-induced nitric oxide (NO) as a mediator, the ability of exogenous or endogenous insulin +/- inhibitors of nitric-oxide synthase (NOS) activity, aminoguanidine or N-monomethyl-L-arginine, was evaluated. Unexpectedly, TNFalpha and also interleukin 1 (IL-1)-induced inhibition of p70(s6k) was completely prevented by inhibitors that block NO production. Western blot analysis verified inducible NOS (iNOS) expression after TNFalpha exposure. Furthermore, the ability of IL-1 receptor antagonist protein, IRAP, to block TNFalpha-induced inhibition of p70(s6k) indicated that activation of intra-islet macrophages and the release of IL-1 that induces iNOS expression in beta-cells was responsible for the inhibitory effects of TNFalpha. This mechanism was confirmed by the ability of the peroxisome proliferator-activated receptor-gamma agonist 15-deoxy-Delta12, 14-prostaglandin J2 to attenuate TNFalpha-induced insulin resistance by down-regulating iNOS expression and/or blocking IL-1 release from activated macrophages. Overall, TNFalpha-mediated insulin resistance in beta-cells is characterized by a global inhibition of metabolism mediated by NO differing from that proposed for this proinflammatory cytokine in insulin-sensitive cells.     

Tumor necrosis factor receptor-1 can function through a G alpha q/11-beta-arrestin-1 signaling complex

Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine secreted from macrophages and adipocytes. It is well known that chronic TNFalpha exposure can lead to insulin resistance both in vitro and in vivo and that elevated blood levels of TNFalpha are observed in obese and/or diabetic individuals. TNFalpha has many acute biologic effects, mediated by a complex intracellular signaling pathway. In these studies we have identified new G-protein signaling components to this pathway in 3T3-L1 adipocytes. We found that beta-arrestin-1 is associated with TRAF2 (TNF receptor-associated factor 2), an adaptor protein of TNF receptors, and that TNFalpha acutely stimulates tyrosine phosphorylation of G alpha(q/11) with an increase in G alpha(q/11) activity. Small interfering RNA-mediated knockdown of beta-arrestin-1 inhibits TNFalpha-induced tyrosine phosphorylation of G alpha(q/11) by interruption of Src kinase activation. TNFalpha stimulates lipolysis in 3T3-L1 adipocytes, and beta-arrestin-1 knockdown blocks the effects of TNFalpha to stimulate ERK activation and glycerol release. TNFalpha also led to activation of JNK with increased expression of the proinflammatory gene, monocyte chemoattractant protein-1 and matrix metalloproteinase 3, and beta-arrestin-1 knockdown inhibited both of these effects. Taken together these results reveal novel elements of TNFalpha action; 1) the trimeric G-protein component G alpha(q/11) and the adapter protein beta-arrestin-1 can function as signaling molecules in the TNFalpha action cascade; 2) beta-arrestin-1 can couple TNFalpha stimulation to ERK activation and lipolysis; 3) beta-arrestin-1 and G alpha(q/11) can mediate TNFalpha-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression.     

NF-kappaB activation prevents apoptotic oxidative stress via an increase of both thioredoxin and MnSOD levels in TNFalpha-treated Ewing sarcoma cells

Repression of activation of c-Jun N-terminal kinase (JNK) participates in the anti-apoptotic effect of nuclear factor-kappaB (NF-kappaB) in TNFalpha-treated Ewing sarcoma cells. As oxidative stress is one of the most prominent activators of JNK, we investigated the relationship between TNFalpha-induced NF-kappaB activation and the control of oxidative stress. Inhibition of NF-kappaB activation resulted in an increase in TNFalpha-induced ROS production, lipid peroxidation and protein oxidation. Those ROS and lipid peroxides were both involved in TNFalpha-induced apoptosis, whereas only ROS elevation triggered sustained JNK activation. TNFalpha increased the level of two antioxidant enzymes, thioredoxin and manganese superoxide dismutase by an NF-kappaB-dependent mechanism. Inhibition of expression or activity of these enzymes sensitized cells to TNFalpha-induced apoptosis, indicating their functional role in protection from cell death. Thus, agents that inhibit activities of these enzymes may prove helpful in the treatment of Ewing tumors.     

Cytotoxicity of TNFalpha is regulated by integrin-mediated matrix signaling

Cytokines of the tumor necrosis factor (TNF) family regulate inflammation and immunity, and a subset of this family can also induce cell death in a context-dependent manner. Although TNFalpha is cytotoxic to certain tumor cell lines, it induces apoptosis in normal cells only when NFkappaB signaling is blocked. Here we show that the matricellular protein CCN1/CYR61 can unmask the cytotoxic potential of TNFalpha without perturbation of NFkappaB signaling or de novo protein synthesis, leading to rapid apoptosis in the otherwise resistant primary human fibroblasts. CCN1 acts through binding to integrins alpha(v)beta(5), alpha(6)beta(1), and syndecan-4, triggering the generation of reactive oxygen species (ROS) through a Rac1-dependent mechanism via 5-lipoxygenase and the mitochondria, leading to the biphasic activation of JNK necessary for apoptosis. Mice with the genomic Ccn1 locus replaced with an apoptosis-defective Ccn1 allele are substantially resistant to TNFalpha-induced apoptosis in vivo. These results indicate that CCN1 may act as a physiologic regulator of TNFalpha cytotoxicity, providing the contextual cues from the extracellular matrix for TNFalpha-mediated cell death.     

Tumor necrosis factor alpha-mediated insulin resistance, but not dedifferentiation, is abrogated by MEK1/2 inhibitors in 3T3-L1 adipocytes

Tumor necrosis factor-alpha (TNFalpha) has been implicated as a contributing mediator of insulin resistance observed in pathophysiological conditions such as obesity, cancer-induced cachexia, and bacterial infections. Previous studies have demonstrated that TNFalpha confers insulin resistance by promoting phosphorylation of serine residues on insulin receptor substrate 1 (IRS-1), thereby diminishing subsequent insulin-induced tyrosine phosphorylation of IRS-1. However, little is known about which signaling molecules are involved in this process in adipocytes and about the temporal sequence of events that ultimately leads to TNFalpha-stimulated IRS-1 serine phosphorylation. In this study, we demonstrate that specific inhibitors of the MAP kinase kinase (MEK)1/2-p42/44 mitogen-activated protein (MAP) kinase pathway restore insulin signaling to normal levels despite the presence of TNFalpha. Additional experiments show that MEK1/2 activity is required for TNFalpha-induced IRS-1 serine phosphorylation, thereby suggesting a mechanism by which these inhibitors restore insulin signaling. We observe that TNFalpha requires 2.5-4 h to markedly reduce insulin-triggered tyrosine phosphorylation of IRS-1 in 3T3-L1 adipocytes. Although TNFalpha activates p42/44 MAP kinase, maximal stimulation is observed within 10-30 min. To our surprise, p42/44 activity returns to basal levels well before IRS-1 serine phosphorylation and insulin resistance are observed. These activation kinetics suggest a mechanism of p42/44 action more complicated than a direct phosphorylation of IRS-1 triggered by the early spike of TNFalpha-induced p42/44 activity. Chronic TNFalpha treatment (> 72 h) causes adipocyte dedifferentiation, as evidenced by the loss of triglycerides and down-regulation of adipocyte-specific markers. We observe that this longer term TNFalpha-mediated dedifferentiation effect utilizes alternative, p42/44 MAP kinase-independent intracellular pathways. This study suggests that TNFalpha-mediated insulin resistance, but not adipocyte dedifferentiation, is mediated by the MEK1/2-p42/44 MAP kinase pathway.     

Inhibition of tumor necrosis factor alpha-mediated NFkappaB activation and leukocyte adhesion,with enhanced endothelial apoptosis,by G protein-linked receptor (TP) ligands

Tumor necrosis factor (TNF) alpha is a critical mediator of inflammation; however, TNFalpha is rarely released alone and the "cross-talk" between different classes of inflammatory mediators is largely unexplored. Thromboxane A(2) (TXA(2)) is released during I/R injury and exerts its effects via a G protein-linked receptor (TP). In this study, we found that TXA(2) mimetics stimulate leukocyte adhesion molecule (LAM) expression on endothelium via TPbeta. The potential interaction between TXA(2) and TNFalpha in altering endothelial survival and LAM expression was examined. IBOP, a TXA(2) mimetic, attenuated TNFalpha-induced LAM expression in vitro, in a concentration-dependent manner, by preventing TNFalpha-enhanced gene expression, and also reduced TNFalpha-induced leukocyte adhesion to endothelium both in vitro and in vivo. IBOP abrogated TNFalpha-induced NFkappaB activation in endothelial cells, as determined by reduced IkappaB phosphorylation and NFkappaB nuclear translocation, by inhibiting the assembly of signaling intermediates with the intracellular domain of TNF receptors 1 and 2 in response to TNFalpha. This inhibition resulted from the Galpha(q)-mediated enhancement of STAT1 activation and was reversed by anti-STAT1 antisense oligonucleotides. TNFalpha-mediated TNFR1-FADD association and caspase 8 activation were not inhibited by IBOP co-stimulation, however, resulting in a 2.6-fold increase in endothelial cell apoptosis. By stimulating the vessel wall and inducing endothelial cell apoptosis, TXA(2), in combination with TNFalpha, may hamper the angiogenic response during inflammation or ischemia, thus reducing revascularization and tissue viability.     

Inhibition of clathrin/dynamin-dependent internalization interferes with LPS-mediated TRAM-TRIF-dependent signaling pathway

Recognition of lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) activates two district proinflammatory signaling pathway and initiates LPS internalization. To investigate roles of LPS internalization, a traditionally regarded metabolic pathway for LPS, in regulation of these two pathways, three internalization inhibitors, monodansylcadaverine (MDC, a clathrin inhibitor), dynasore (DS, a dynamin inhibitor) and chloroquine (CQ, an endosome acidifying maturation inhibitor) were applied to induce internalization dysfunction in macrophages. Results showed MDC and DS affected LPS internalization but did not interfere with their colocalization. Additionally, they decreased cytokines and chemokines release and inhibited signaling molecules activation mediated by TRAM-TRIF-dependent pathway as determined by protein array. In contrast, CQ did not inhibit LPS internalization but affected the colocalization. It also suppressed macrophage activation mediated by both MyD88-dependent and TRAM-TRIF-dependent pathways. The above data indicated that LPS internalization was clathrin/dynamin dependent and it was essential for activation of TRAM-TRIF-dependent signaling pathway.     

Induction of phosphatidylinositol 3-kinase-mediated endocytosis by salt stress leads to intracellular production of reactive oxygen species and salt tolerance

Salt imposes immediate problems for plant cells, such as osmotic stress, impaired ion homeostasis and sodium toxicity, followed by a secondary oxidative stress caused by generation of reactive oxygen species (ROS). Here, we analyzed the production of ROS during salt stress. We show that salt stress triggered plasma membrane internalization, resulting in the production of ROS within endosomes. The intracellular ROS were produced by NADPH oxidase in response to the ionic but not the osmotic stress. Both endocytosis and ROS production were suppressed in phosphatidylinositol (PtdIns) 3-kinase (PI3K) mutants, PI3K being a key regulator of vesicle trafficking in animals and plants, and by wortmannin, which is a specific inhibitor of PI3K and PI4K. Endocytosis and the production of ROS were rescued by supplementation of seedlings with exogenous PtdIns 3-phosphate (PtdIns3P), less with PtdIns4P, but not with PtdIns(4,5)P(2). Surprisingly, despite reduced oxidative stress, the mutants and the wortmannin-treated plants exhibited a phenotype overly sensitive to salt, as also resulted from treatment with diphenyleneiodonium, a suicide inhibitor of NADPH oxidase, suggesting a positive role for ROS in salt tolerance. In summary, our results show that salt stress responses, such as increased plasma membrane endocytosis and the intracellular production of ROS, are coordinated by phospholipid-regulated signaling pathways, and suggest that ROS act in the signal transduction of the salt tolerance response.     

Distinct regulation of internalization and mitogen-activated protein kinase activation by two isoforms of the dopamine D2 receptor

Two isoforms of the dopamine D2 receptor, D2L (long) and D2S (short), differ by the insertion of a 29-amino acid specific to D2L within the putative third intracellular loop of the receptor. Here, we examined D2 receptor-mediated MAPK activation in association with receptor internalization. Overexpression of beta-arrestin 1 and 2 increased the D2S-mediated activation of MAPK, whereas it did not affect the activation of MAPK by D2L. Expression of a dominant negative beta-arrestin 2 (319-418) mutant and of a dominant negative dynamin I (K44A) mutant inhibited the activation of MAPK by D2S, but not the activation of MAPK by D2L. Treatment with inhibitors of internalization, i.e. concanavalin A and monodansylcadaverin, blocked D2S-mediated MAPK activation but not D2L-mediated activation. By confocal microscopy, we observed beta-arrestin 1 and 2, translocated to the plasma membrane and colocalized with D2L and D2S receptors upon stimulation with dopamine, and this was followed by the translocation of receptors into endocytic vesicles. Moreover, the expression of the beta-arrestin 2 (319-418) mutant blocked the internalization of both D2L and D2S. In addition, although K44A dynamin mutant expression did not alter D2L internalization, it completely blocked the internalization of D2S. The stimulation of D2L induces activation of MAPK via transactivation of the platelet-derived growth factor receptor, whereas D2S does not. Taken together, these data suggest that D2L activates MAPK signaling by mobilizing the growth factor receptor, platelet-derived growth factor receptor, whereas D2S appears to activate MAPK signaling by mobilizing clathrin-mediated endocytosis in a beta-arrestin/dynamin-dependent manner.