Expression of dominant negative rab5 in HeLa cells regulates endocytic trafficking distal from the plasma membrane

Ligand-mediated endocytosis is an important regulatory mechanism of epidermal growth factor (EGF) receptor (EGFR) signal transduction. Coordinated EGFR internalization and degradation function to regulate the spatial and temporal components of EGFR-effector interactions. In an effort to better understand the molecular mechanisms that control these events, we examined the role of rab5 in the endocytic trafficking of the EGFR. Rab5 is a 25-kDa guanine nucleotide binding protein that has previously been shown to be involved in the early stages of endocytic trafficking. Using adenovirally expressed dominant negative and constitutively active rab5 [rab5(S34N) and rab5(Q79L)] in cells with endogenous EGFRs, we have found that the guanine nucleotide binding state of rab5 has no bearing on the rate of EGFR endocytosis. However, expression of dominant negative rab5 affects downstream endocytic trafficking by slowing the ligand-induced disappearance of total cellular EGFR. Using confocal microscopy to examine EGF/EGFR and rab5 localization indicates that the activity of rab5 governs whether internalized EGF/EGFR and rab5 co-localize. Transferrin, which internalizes via a constitutively internalized cell surface receptor, co-sediments with rab5(WT), but not rab5(S34N) on sucrose gradients. Taken together, these data are consistent with rab5 functioning to regulate intracellular endocytic trafficking distal from the plasma membrane.     

Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction

The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments.     

Deoxyelephantopin impedes mammary adenocarcinoma cell motility by inhibiting calpain-mediated adhesion dynamics and inducing reactive oxygen species and aggresome formation

We previously showed that deoxyelephantopin (DET), a plant sesquiterpene lactone, exhibits more profound suppression than paclitaxel (PTX) of lung metastasis of mammary adenocarcinoma TS/A cells in mice. Proteomics studies suggest that DET affects actin cytoskeletal protein networks and downregulates calpain-mediated proteolysis of several actin-associated proteins, whereas PTX mainly interferes with microtubule proteins. Here, DET was observed to significantly deregulate adhesion formation in TS/A cells, probably through inhibition of m-calpain activity. Epithelial growth factor (EGF)-mediated activation of Rho GTPase Rac1 and formation of lamellipodia in TS/A cells were remarkably suppressed by DET treatment. Further, DET impaired vesicular trafficking of EGF and induced protein carbonylation and formation of centrosomal aggregates in TS/A cells. DET-induced reactive oxygen species were observed to be the upstream stimulus for the formation of centrosomal ubiquitinated protein aggregates that might subsequently restrict cancer cell motility. PTX, however, caused dramatic morphological changes, interfered with microtubule networking, and moderately inhibited calpain-mediated cytoskeletal and focal adhesion protein cleavage in TS/A cells. This study provides novel mechanistic insights into the pharmacological action of DET against metastatic mammary cell migration and suggests that modulation of oxidative stress might be a potential strategy for treatment of metastatic breast cancer.     

MAP1A light chain-2 interacts with GTP-RhoB to control epidermal growth factor (EGF)-dependent EGF receptor signaling

Rho GTPases have been implicated in the control of several cellular functions, including regulation of the actin cytoskeleton, cell proliferation, and oncogenesis. Unlike RhoA and RhoC, RhoB localizes in part to endosomes and controls endocytic trafficking. Using a yeast two-hybrid screen and a glutathione S-transferase pulldown assay, we identified LC2, the light chain of the microtubule-associated protein MAP1A, as a novel binding partner for RhoB. GTP binding and the 18-amino acid C-terminal hypervariable domain of RhoB are critical for its binding to MAP1A/LC2. Coimmunoprecipitation and immunofluorescence experiments showed that this interaction occurs in U87 cells. Down-regulation of MAP1A/LC2 expression decreased epidermal growth factor (EGF) receptor expression and modified the signaling response to EGF treatment. We concluded that MAP1A/LC2 is critical for RhoB function in EGF-induced EGF receptor regulation. Because MAP1A/LC2 is thought to function as an adaptor between microtubules and other molecules, we postulate that the RhoB and MAP1A/LC2 interactions facilitate endocytic vesicle trafficking and regulate the trafficking of signaling molecules.     

Metastasis Suppressor Tetraspanin CD82/KAI1 Regulates Ubiquitylation of Epidermal Growth Factor Receptor

Ligand-induced ubiquitylation of EGF receptor (EGFR) is an important regulatory mechanism that controls endocytic trafficking of the receptor and its signaling potential. Here we report that tetraspanin CD82/KAI1 specifically suppresses ubiquitylation of EGFR after stimulation with heparin-binding EGF or amphiregulin and alters the rate of recruitment of the activated receptor to EEA1-positive endosomes. The suppressive effect of CD82 is dependent on the heparin-binding domain of the ligand. Deletion of the C-terminal cytoplasmic domain of CD82 (CD82ΔC mutant) inhibits endocytic trafficking of the tetraspanin and compromises its activity toward heparin-binding EGF-activated EGFR. Reduced ubiquitylation of EGFR is accompanied by PKC-dependent increase in serine phosphorylation of c-Cbl in cells expressing elevated levels of CD82. Furthermore, phosphorylation of threonine 654 (PKC phosphorylation site) in the juxtamembrane domain of the receptor is considerably increased in CD82-expressing cells. These results describe previously unsuspected links between tetraspanin proteins and ubiquitylation of their molecular partners (e.g., EGFR). Our data identify CD82 as a new regulator of c-Cbl, which discriminatively controls the activity of this E3 ubiquitin ligase toward heparin-binding ligand-EGFR pairs. Taken together, these observations provide an important new insight into the modulatory role of CD82 in endocytic trafficking of EGF receptor.     

Protein-tyrosine phosphatase 1B modulates early endosome fusion and trafficking of Met and epidermal growth factor receptors

The endoplasmic reticulum-localized non-receptor protein-tyrosine phosphatase 1B (PTP1B) is associated with oncogenic, metabolic, and cytokine-related signaling and functionally targets multiple receptor tyrosine kinases (RTKs) for dephosphorylation. Loss of PTP1B activity leads to enhanced ligand-dependent biological activity of the Met RTK among others. Here, we demonstrate that knockdown of PTP1B or expression of a PTP1B trapping aspartic acid-to-alanine substitution (D/A) mutant delayed ligand-induced degradation of the Met and EGF RTKs. Loss of PTP1B function abrogated trafficking of Met and EGF receptor to Rab5- and phosphatidylinositol 3-phosphate (Pl3P)-positive early endosomes and subsequent trafficking through the degradative pathway. Under these conditions, internalization of the Met and EGF receptors was unaltered, suggesting a block at the level of early endosome formation. We show that the N-ethylmaleimide-sensitive factor (NSF), an essential component of the vesicle fusion machinery, was hyperphosphorylated in PTP1B knockdown or PTP1B D/A-expressing cells and was a target for PTP1B. NSF knockdown phenocopied PTP1B knockdown, demonstrating a mechanism through which PTP1B regulates endocytic trafficking. Finally, we show that PTP1B dephosphorylated NSF and that this interaction was required for physiological RTK trafficking and appropriate attenuation of downstream signaling.     

Mathematical model for the effects of epidermal growth factor receptor trafficking dynamics on fibroblast proliferation responses.

We apply a mathematical model for receptor-mediated cell uptake and processing of epidermal growth factor (EGF) to analyze and predict proliferation responses to fibroblastic cells transfected with various forms of the EGF receptor (EGFR) to EGF. The underlying conceptual hypothesis is that the mitogenic signal generated by EGF/EGFR binding on the cell surface, via stimulation of receptor tyrosine kinase activity, is attenuated when the receptors are downregulated and growth factor is depleted by endocytic internalization and subsequent intracellular degradation. Hence, the cell proliferation rate ought to depend on receptor/ligand binding and trafficking parameters as well as on intrinsic receptor signal transduction properties. The goal of our modeling efforts is to formulate this hypothesis in quantitative terms. The mathematical model consists of kinetic equations for binding, internalization, degradation, and recycling of EGF and EGFR, along with an expression relating DNA synthesis rate to EGF/EGFR complex levels. Parameter values have been previously determined from independent binding and trafficking kinetic experiments on B82 fibroblasts transfected with wild-type and mutant EGFR. We show that this model can successfully interpret literature data for EGF-dependent growth of NR6 fibroblasts transfected with wild-type EGFR. Moreover, it successfully predicts the literature observation that NR6 cells transfected with a delta 973 truncation mutant EGFR, which is kinase-active but internalization-deficient, require an order of magnitude lower EGF concentration than cells with wild-type EGFR for half-maximal proliferation rate. This result demonstrates that it may be feasible to genetically engineer mammalian cell lines with reduced growth factor requirements by a rational, nonempirical approach. We explore by further model computations the possibility of exploiting other varieties of EGFR mutants to alter growth properties of fibroblastic cells, based on relationships between changes in the primary structure of the EGF receptor and the rates of specific receptor/ligand binding and trafficking processes. Our studies show that the ability to predict cell proliferation as a function of serum growth factors such as EGF could lead to the designed development of cells with optimized growth responses. This approach may also aid in elucidation of mechanisms underlying loss of normal cell proliferation control in malignant transformation, by demonstrating that receptor trafficking dynamics may in some cases play as important a role as intrinsic signal transduction in determining the overall resulting mitogenic response.     

Nifedipine-sensitive vascular reactivity of femoral arteries in WKY: the effects of pertussis toxin pretreatment and endothelium removal

Maintenance of norepinephrine (NE)-induced contraction is dependent on Ca(2+) influx through L-type voltage-dependent Ca(2+) channels (VDCC), which is opposed by nitric oxide. Adrenergic receptors are coupled with different G proteins, including inhibitory G proteins (Gi) that can be inactivated by pertussis toxin (PTX). Our study was aimed to investigate the effects of endothelium removal, PTX pretreatment and acute VDCC blockade by nifedipine on the contractions of femoral arteries stimulated by norepinephrine. We used 12-week-old male WKY, half of the rats being injected with PTX (10 microg/kg i.v., 48 h before the experiment), which considerably reduced their blood pressure (BP). Contractions of isolated arteries were measured using Mulvany-Halpern myograph. NE dose-response curves determined in femoral arteries from PTX-treated WKY rats were shifted to the right compared to those from control WKY. On the contrary, removal of endothelium augmented NE dose-response curves shifting them to the left. Acute VDCC blockade by nifedipine (10(-7) M) abolished all differences in NE dose-response curves which were dependent on the presence of either intact endothelium or functional Gi proteins because all NE dose-response curves were identical to the curve seen in vessels with intact endothelium from PTX-treated animals. We can conclude that BP reduction after PTX injection is accompanied by the attenuation of NE-induced contraction of femoral arteries irrespective of endothelium presence. Moreover, our data indicate that both vasodilator action of endothelium and Gi-dependent vasoconstrictor effect of norepinephrine operate via the control of Ca(2+) influx through VDCC.     

Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells

Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.     

Role of pertussis toxin-sensitive G proteins in stimulus-dependent human eosinophil degranulation.

Stimulation of human normodense eosinophils with immobilized secretory IgA (sIgA) or IgG, or with the soluble stimulus, FMLP, triggers the exocytotic release of the granule protein, eosinophil-derived neurotoxin (EDN). In this report, we demonstrate that these stimuli also provoke an increase in phospholipase C-mediated phosphoinositide breakdown in eosinophils. Pretreatment of eosinophils with pertussis toxin (PTX) for 2 h irreversibly abolished the increases in phospholipase C activity and EDN release induced by immobilized sIgA or FMLP. In contrast, PTX treatment only transiently inhibited eosinophil activation induced by immobilized IgG. Maximal inhibition of IgG-stimulated phosphoinositide hydrolysis and EDN release occurred after 2 h of PTX pretreatment with PTX, followed by a gradual recovery of cellular responsiveness to immobilized IgG as the duration of PTX pretreatment was extended to 16 h. Activated PTX catalyzed the in vitro ADP-ribosylation of 41- and 44-kDa proteins in eosinophil membranes. A 2-h pretreatment of intact cells with PTX markedly reduced the pools of unmodified 41- and 44-kDa substrates available for subsequent ADP-ribosylation in vitro, suggesting that both proteins were substrates for PTX in intact eosinophils. Continuous exposure of eosinophils to PTX for times ranging from 2 to 15 h resulted in the gradual reappearance of unmodified 44-kDa protein, whereas the levels of unmodified 41-kDa protein were persistently reduced in PTX-treated cells. The time course of the decline and reappearance of unmodified 44-kDa substrate in PTX-treated eosinophils closely paralleled the changes in the responsiveness of these cells to immobilized IgG. These results suggest that the receptors for sIgA, FMLP, or IgG transduce activating signals for eosinophil degranulation through differential coupling to at least two PTX-sensitive G proteins.     

A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells

We have previously shown that overexpression of LIM kinase1 (LIMK1) resulted in a marked retardation of the internalization of the receptor-mediated endocytic tracer, Texas red-labeled epidermal growth factor (EGF) in low-invasive human breast cancer cell MCF-7. We thereby postulate that LIMK1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor (EGFR) in tumor cells by reorganizing and influencing actin-filament dynamics. In the present study, we further assessed the effect of wild-type LIMK1, a kinase-deficient dominant negative mutant of LIMK1 (DN-LIMK1) and an active, unphosphorylatable cofilin mutant (S3A cofilin) on internalization of EGF-EGFR in MDA-MB-231, a highly invasive human breast cancer cell line. We demonstrate here that a marked delay in the receptor-mediated internalization of Texas red-labeled EGF was observed in the wild-type LIMK1 transfectants, and that most of the internalized EGF staining were accumulated within transferrin receptor-positive early endosomes even after 30 min internalization. In contrast, the expression of dominant-negative LIMK1 mutant rescued the efficient endocytosis of Texas red-EGF, and large amounts of Texas red-EGF staining already reached LIMPII-positive late endosomes/lysosomal vacuoles after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGFR trafficking, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15–30 min after internalization. Taken together, our novel findings presented in this paper implicate that LIMK1 signaling indeed plays a pivotal role in the regulation of EGFR trafficking through the endocytic pathway in invasive tumor cells.     

Engagement of the Small GTPase Rab31 Protein and Its Effector,Early Endosome Antigen 1, Is Important for Trafficking of the Ligand-bound Epidermal Growth Factor Receptor from the Early to the Late Endosome

Rab31 is a member of the Rab5 subfamily of Rab GTPases. Although localized largely to the trans-Golgi network, it shares common guanine nucleotide exchange factors and effectors with other Rab5 subfamily members that have been implicated in endocytic membrane traffic. We investigated whether Rab31 also has a role in the trafficking of the ligand-bound EGF receptor (EGFR) internalized through receptor-mediated endocytosis. We found that loss of Rab31 inhibits, but overexpression enhances, EGFR trafficking to the late endosomes and that the effect of Rab31 silencing could be specifically rescued by overexpression of a silencing-resistant form of Rab31. Rab31 was found to interact with the EGFR by coimmunoprecipitation and affinity pulldown analyses, and the primarily trans-Golgi network-localized Rab31 has increased colocalization with the EGFR in A431 cells 30 min after pulsing with EGF. A glycerol gradient sedimentation assay suggested that Rab31 is sequestered into a high molecular weight complex after stimulation with EGF, as was early endosome antigen 1 (EEA1), a factor responsible for endosomal tethering and fusion events. We found that loss of EEA1 reduced the interaction between Rab31 and the EGFR and abrogated the effect of Rab31 overexpression on the trafficking of the EGFR. Likewise, loss of GAPex5, a Rab31 guanine nucleotide exchange factor that has a role in ubiquitination and degradation of the EGFR, reduced the interaction of Rab31 with the EGFR and its effect on EGFR trafficking. Taken together, our results suggest that Rab31 is an important regulator of endocytic trafficking of the EGFR and functions in an EGFR trafficking complex that includes EEA1 and GAPex5.     

Endosomal accumulation of the activated epidermal growth factor receptor (EGFR) induces apoptosis

Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.     

Regulatory Role of the GTP-Binding Protein, Go, in the Mechanism of Exocytosis in Adrenal Chromaffin Cells

To elucidate the possible involvement of GTP-binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and antibodies against the G proteins (Gi and G(o)) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine-evoked catecholamine release. High K(+)-, histamine-, or gamma-aminobutyric acid-evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10(-4) M acetylcholine was decreased significantly in PTX-treated cells. In digitonin-permeabilized cells, PTX pretreatment resulted in a decrease of the half-maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP-gamma-S (a nonhydrolyzable GTP analogue) inhibited Ca(2+)-dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the alpha subunit of the PTX-sensitive G protein, G(o). Treatment with similar antibodies against the alpha subunit of Gi had no effect. These findings suggest that G(o) directly controls the Ca(2+)-triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.     

Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living cells.

In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries.     

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

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

rab7 activity affects epidermal growth factor:epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family. Ligand (epidermal growth factor or EGF) binding to the EGFR results in the coordinated activation and integration of biochemical signaling events to mediate cell growth, migration, and differentiation. One mechanism the cell utilizes to orchestrate these events is ligand-mediated endocytosis through the canonical clathrin-mediated endocytic pathway. Identification of proteins that regulate the intracellular movement of the EGF.EGFR complex is an important first step in dissecting how specificity of EGFR signaling is conferred. We examined the role of the small molecular weight guanine nucleotide-binding protein (G-protein) rab7 as a regulator of the distal stages of the endocytic pathway. Through the transient expression of activating and inactivating mutants of rab7 in HeLa cells, we have determined that rab7 activity directly correlates with the rate of radiolabeled EGF and EGFR degradation. Furthermore, when inhibitory mutants of rab7 are expressed, the internalized EGF.EGFR complex accumulates in high-density endosomes that are characteristic of the late endocytic pathway. Thus, we conclude that rab7 regulates the endocytic trafficking of the EGF.EGFR complex by regulating its lysosomal degradation.     

Epidermal growth factor receptors destined for the nucleus are internalized via a clathrin-dependent pathway

The epidermal growth factor (EGF) transduces its actions via the EGF receptor (EGFR), which can traffic from the plasma membrane to either the cytoplasm or the nucleus. However, the mechanism by which EGFR reaches the nucleus is unclear. To investigate these questions, liver cells were analyzed by immunoblot of cell fractions, confocal immunofluorescence and real time confocal imaging. Cell fractionation studies showed that EGFR was detectable in the nucleus after EGF stimulation with a peak in nuclear receptor after 10 min. Movement of EGFR to the nucleus was confirmed by confocal immunofluorescence and labeled EGF moved with the receptor to the nucleus. Small interference RNA (siRNA) was used to knockdown clathrin in order to assess the first endocytic steps of EGFR nuclear translocation in liver cells. A mutant dynamin (dynamin K44A) was also used to determine the pathways for this traffic. Movement of labeled EGF or EGFR to the nucleus depended upon dynamin and clathrin. This identifies the pathway that mediates the first steps for EGFR nuclear translocation in liver cells.     

Inactivation of G(i) proteins by pertussis toxin diminishes the effectiveness of adrenergic stimuli in conduit arteries from spontaneously hypertensive rats

Treatment with pertussis toxin (PTX) which eliminates the activity of G(i) proteins effectively reduces blood pressure (BP) and vascular resistance in spontaneously hypertensive rats (SHR). In this study we have compared the functional characteristics of isolated arteries from SHR with and without PTX-treatment (10 microg/kg i.v., 48 h before the experiment). Rings of thoracic aorta, superior mesenteric artery and main pulmonary artery were studied under isometric conditions to measure the reactivity of these vessels to receptor agonists and to transmural electrical stimuli. We have found that the treatment of SHR with PTX had no effect on endothelium-dependent relaxation of thoracic aorta induced by acetylcholine. In PTX-treated SHR, the maximum contraction of mesenteric artery to exogenous noradrenaline was reduced and the dose-response curve to cumulative concentration of noradrenaline was shifted to the right. Similarly, a reduction in the magnitude of neurogenic contractions elicited by electrical stimulation of perivascular nerves was observed in the mesenteric artery from PTX-treated SHR. PTX treatment of SHR also abolished the potentiating effect of angiotensin II on neurogenic contractions of the main pulmonary artery. These results indicate that PTX treatment markedly diminishes the effectiveness of adrenergic stimuli in vasculature of SHR. This could importantly affect BP regulation in genetic hypertension.