Role of c-Cbl carboxyl terminus in serotonin 5-HT2A receptor recycling and resensitization.

The 5-hydroxytryptamine 2A receptor (5-HT(2A)R) undergoes constitutive and agonist-dependent internalization. Despite many advances in our understanding of G protein-coupled receptor trafficking, the exact mechanism of endocytic sorting of G protein-coupled receptors remains obscure. Recently, we have reported a novel finding documenting a global role for the ubiquitin ligase c-Cbl in regulating vesicular sorting of epidermal growth factor receptor (Baldys, A., Göoz, M., Morinelli, T. A., Lee, M. H., Raymond, J. R., Jr., Luttrell, L. M., and Raymond, J. R., Sr. (2009) Biochemistry 48, 1462-1473). Thus, we tested the hypothesis that c-Cbl might play a role in 5-HT(2A)R recycling. In this study, we demonstrated an association of 5-HT(2A)R with c-Cbl. Furthermore, down-regulation of c-Cbl by RNA interference blocked efficient recycling of 5-HT(2A)R to the plasma membrane. Immunofluorescence microscopy revealed that 5-HT(2A) receptors were trapped in early endosome antigen 1- and Rab11-positive sorting endosomes in cells overexpressing c-Cbl mutants lacking carboxyl termini. This inhibitory effect was associated with a relative decrease in association of c-Cbl truncation proteins with the 5-HT(2A)R, compared with that observed for the full-length c-Cbl fusion protein. Consistent with the delayed recycling, 5-HT(2A)R resensitization was greatly attenuated in the presence of c-Cbl mutants lacking carboxyl termini, as detected by changes in the cytosolic calcium. Taken together, these studies have led to the discovery that the C-terminal region of c-Cbl plays a crucial role in the temporal and spatial control of 5-HT(2A)R recycling.

Novel mechanisms in the regulation of G protein-coupled receptor trafficking to the plasma membrane.

ß(2)-adrenergic receptors (ß(2)-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated ß(2)-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced ß(2)-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. ß(2)-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, ß(2)-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between ß(2)-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.

Epidermal growth factor activates Na(+/)H(+) exchanger in podocytes through a mechanism that involves Janus kinase and calmodulin.

Sodium-proton exchanger type 1 (NHE-1) is ubiquitously expressed, is activated by numerous growth factors, and plays significant roles in regulating intracellular pH and cellular volume, proliferation and cytoskeleton. Despite its importance, little is known about its regulation in renal glomerular podocytes. In the current work, we studied the regulation of NHE-1 activity by the epidermal growth factor receptor (EGFR) in cultured podocytes. RT-PCR demonstrated mRNAs for NHE-1 and NHE-2 in differentiated podocytes, as well as for EGFR subunits EGFR/ErbB1, Erb3, and ErbB4. EGF induced concentration-dependent increases in proton efflux in renal podocytes as assessed using a Cytosensor microphysiometer, were diminished in the presence of 5-(N-methyl-N-isobutyl) amiloride or in a sodium-free solution. Furthermore, pharmacological inhibitors of Janus kinase (Jak2) and calmodulin (CaM) attenuated EGF-induced NHE-1 activity. Co-immunoprecipitation studies determined that EGF induced formation of complexes between Jak2 and CaM, as well as between CaM and NHE-1. In addition, EGF increased levels of tyrosine phosphorylation of Jak2 and CaM. The EGFR kinase inhibitor, AG1478, blocked activation of NHE-1, but did not block EGF-induced phosphorylation of Jak2 or CaM. These results suggest that EGF induces NHE-1 activity in podocytes through two pathways: (1) EGF-->EGFR-->Jak2 activation (independent of EGFR tyrosine kinase activity)-->tyrosine phosphorylation of CaM-->CaM binding to NHE-1-->conformational change of NHE-1-->activation of NHE-1; and (2) EGF-->EGFR-->EGFR kinase activation-->association of CaM with NHE-1 (independent of Jak2)-->conformational change of NHE-1-->activation of NHE-1.

Bradykinin decreases podocyte permeability through ADAM17-dependent epidermal growth factor receptor activation and zonula occludens-1 rearrangement.

Recent data show that increases in bradykinin (BK) concentration contribute to the beneficial effects of angiotensin-converting enzyme inhibitor (ACEI) treatment in chronic kidney disease. However, the possible role of BK in attenuated proteinuria, often seen in ACEI-treated patients, is not well studied. Here, we report that BK decreases mouse podocyte permeability through rearrangement of the tight junction protein zonula occludens-1 (ZO-1) and identify some of the major signaling events leading to permeability change. We show that BK2 receptor (BK2R) stimulation transactivates the epidermal growth factor receptor (EGFR). EGFR transactivation is mediated by a disintegrin and metalloenzyme (ADAM) family members, which are required for both extracellular signal-regulated kinase (ERK) and EGFR activation by BK. Using a gene-silencing approach we observed that both BK-induced ERK activation and BK-induced permeability decrease in podocytes is attenuated by ADAM17 down-regulation, and we identified epiregulin (ER) as the EGFR ligand participating in ADAM-dependent BK2R-EGFR cross-talk. EGFR inhibition attenuated both ZO-1 rearrangement and BK-induced permeability decreases in podocyte. We propose that ZO-1 redistribution is an important element of BK-induced permeability change and the signaling events involved in ZO-1 rearrangement include transactivation of the EGFR via ADAM17 activation and ER shedding. Our data indicate that ADAM17 and the EGFR may be potential novel therapeutic targets in diabetic nephropathy and other chronic kidney diseases.

Critical role of ESCRT machinery in EGFR recycling.

The molecular mechanisms of EGFR vesicular trafficking to lysosomes have recently received considerable attention. It is now clear that endosomal sorting complexes required for transport (ESCRTs) are critical for EGFR degradation. Although an increasing number of membrane receptors also undergo recycling via specific pathways, little information is available regarding regulated recycling of EGFR. In this study, we investigated the roles of ESCRTs in EGFR recycling after stimulation with amphiregulin (AR). We used ESCRT small interfering RNA (siRNA) duplexes to demonstrate that AR-induced EGFR intracellular processing involves active sorting to the recycling pathway through specific members of the ESCRT family.

Essential role of c-Cbl in amphiregulin-induced recycling and signaling of the endogenous epidermal growth factor receptor.

The intracellular processing of the epidermal growth factor receptor (EGFR) induced by epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) has been studied meticulously, with the former resulting in EGFR degradation and the latter in EGFR recycling to the plasma membrane. However, little is known about how other EGF family growth factors affect the trafficking of the EGFR. Additionally, although both EGF and TGF-alpha have been shown to effectively induce initial c-Cbl (ubiquitin ligase)-mediated ubiquitination of the EGFR, limited information is available regarding the role of c-Cblin the trafficking and signaling of recycling EGFR. Thus, in this study, we investigated the roles of c-Cblin endogenous EGFR trafficking and signaling after stimulation with amphiregulin (AR). We demonstrated that a physiological concentration of AR induced recycling of the endogenous EGFR to the plasma membrane, which correlated closely with transient association of the EGFR with c-Cbl and transient EGFR ubiquitination. Most importantly, we used c-Cbl small interfering RNA (siRNA) duplexes and ac-Cbl dominant negative mutant to show that c-Cbl is critical for the efficient transition of the EGFR from early endosomes to a recycling pathway and that c-Cbl regulates the duration of extracellular signal regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) phosphorylation. These data support novel functions of c-Cbl in mediating recycling of EGF receptors to the plasma membrane, as well as in mediating the duration of activation (transient vs sustained) of ERK1/2 MAPK phosphorylation.

ADAM-17 regulates endothelial cell morphology, proliferation, and in vitro angiogenesis.

Modulation of angiogenesis is a promising approach for treating a wide variety of human diseases including ischemic heart disease and cancer. In this study, we show that ADAM-17 is an important regulator of several key steps during angiogenesis. Knocking down ADAM-17 expression using lentivirus-delivered siRNA in HUVECs inhibited cell proliferation and the ability of cells to form close contact in two-dimensional cultures. Similarly, ADAM-17 depletion inhibited the ability of HUVECs to form capillary-like networks on top of three-dimensional Matrigel as well as in co-culture with fibroblasts within a three-dimensional scaffold. In mechanistic studies, both baseline and VEGF-induced MMP-2 activation and Matrigel invasion were inhibited by ADAM-17 depletion. Based on our findings we propose that ADAM-17 is part of a novel pro-angiogenic pathway leading to MMP-2 activation and vessel formation.

Erratum: Roche, J. et al. Global Decrease of Histone H3K27 Acetylation in ZEB1-Induced Epithelial to Mesenchymal Transition in Lung Cancer Cells. Cancers, 2013, 5, 334-356.

n/a.

Isoproterenol acts as a biased agonist of the alpha-1A-adrenoceptor that selectively activates the MAPK/ERK pathway.

The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e., not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and ß2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as ß-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gαq.

Global Decrease of Histone H3K27 Acetylation in ZEB1-Induced Epithelial to Mesenchymal Transition in Lung Cancer Cells.

The epithelial to mesenchymal transition (EMT) enables epithelial cells with a migratory mesenchymal phenotype. It is activated in cancer cells and is involved in invasion, metastasis and stem-like properties. ZEB1, an E-box binding transcription factor, is a major suppressor of epithelial genes in lung cancer. In the present study, we show that in H358 non-small cell lung cancer cells, ZEB1 downregulates EpCAM (coding for an epithelial cell adhesion molecule), ESRP1 (epithelial splicing regulatory protein), ST14 (a membrane associated serine protease involved in HGF processing) and RAB25 (a small G-protein) by direct binding to these genes. Following ZEB1 induction, acetylation of histone H4 and histone H3 on lysine 9 (H3K9) and 27 (H3K27) was decreased on ZEB1 binding sites on these genes as demonstrated by chromatin immunoprecipitation. Of note, decreased H3K27 acetylation could be also detected by western blot and immunocytochemistry in ZEB1 induced cells. In lung cancers, H3K27 acetylation level was higher in the tumor compartment than in the corresponding stroma where ZEB1 was more often expressed. Since HDAC and DNA methylation inhibitors increased expression of ZEB1 target genes, targeting these epigenetic modifications would be expected to reduce metastasis.

Sustained activation of protein kinase C induces delayed phosphorylation and regulates the fate of epidermal growth factor receptor.

It is well established that acute activation of members of the protein kinase C (PKC) family induced by activation of cellular receptors can transduce extracellular stimuli to intracellular signaling. However, the functions of sustained activation of PKC are not well studied. We have previously shown that sustained activation of classical PKC isoforms over 15-60 min induced the formation of the pericentrion, a subset of recycling endosomes that are sequestered perinuclearly in a PKC- and phospholipase D (PLD)-dependent manner. In this study, we investigated the role of this process in the phosphorylation of EGFR on threonine 654 (Thr-654) and in the regulation of intracellular trafficking and fate of epidermal growth factor receptor (EGFR). Sustained stimulation of the angiotensin II receptor induced translocation of the EGFR to the pericentrion, which in turn prevents full access of EGF to the EGFR. These effects required PKC and PLD activities, and direct stimulation of PKC with phorbol esters was sufficient to reproduce these effects. Furthermore, activation of PKC induced delayed phosphorylation of EGFR on Thr-654 that coincided with the formation of the pericentrion and which was dependent on PLD and endocytosis of EGFR. Thus, Thr-654 phosphorylation required the formation of the pericentrion. On the other hand, using a T654A mutant of EGFR, we find that the phosphorylation on Thr-654 was not required for translocation of EGFR to the pericentrion but was required for protection of EGFR from degradation in response to EGF. Taken together, these results demonstrate a novel role for the pericentrion in the regulation of EGFR phosphorylation, which in turn is important for the fates of EGFR.

TRPP2 and TRPV4 form an EGF-activated calcium permeable channel at the apical membrane of renal collecting duct cells.

OBJECTIVE: Regulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear. METHODS AND RESULTS: We report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\TRPV4 channel mediated increase in intracellular calcium. CONCLUSION: We conclude that in the absence of cilia, an EGF activated TRPP2\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.

Sustained receptor stimulation leads to sequestration of recycling endosomes in a classical protein kinase C- and phospholipase D-dependent manner.

Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. It is also well established that ligand stimulation of many plasma membrane receptors leads to their internalization. However, stimulus-induced regulation of endosomal trafficking has not received much attention. In previous studies, we showed that sustained stimulation of protein kinase C (PKC) with phorbol esters led to sequestration of recycling endosomes in a juxtanuclear region. In this study, we investigated whether G-protein-coupled receptors that activate PKC exerted effects on endosomal trafficking. Stimulation of cells with serotonin (5-hydroxytryptamine (5-HT)) led to sequestration of the 5-HT receptor (5-HT2AR) into a Rab11-positive juxtanuclear compartment. This sequestration coincided with translocation of PKC as shown by confocal microscopy. Mechanistically the observed sequestration of 5-HT2AR was shown to require continuous PKC activity because it was inhibited by pretreatment with classical PKC inhibitor Gö6976 and could be reversed by posttreatment with this inhibitor. In addition, classical PKC autophosphorylation was necessary for receptor sequestration. Moreover inhibition of phospholipase D (PLD) activity and inhibition of PLD1 and PLD2 using dominant negative constructs also prevented this process. Functionally this sequestration did not affect receptor desensitization or resensitization as measured by intracellular calcium increase. However, the PKC- and PLD-dependent sequestration of receptors resulted in co-sequestration of other plasma membrane proteins and receptors as shown for epidermal growth factor receptor and protease activated receptor-1. This led to heterologous desensitization of those receptors and diverted their cellular fate by protecting them from agonist-induced degradation. Taken together, these results demonstrate a novel role for sustained receptor stimulation in regulation of intracellular trafficking, and this process requires sustained stimulation of PKC and PLD.

Apoptosis induced by crocidolite asbestos in human lung epithelial cells involves inactivation of Akt and MAPK pathways.

Exposure of human lung epithelial (A549) cells to asbestos fibers causes apoptosis, which is largely attributed to release of iron and generation of reactive oxygen species (ROS) within the cells. To mimic the highly oxidative environment generated by asbestos exposure in the absence of the actual fibers, we used two chemicals; buthione sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and ferric ammonium citrate (FAC), a source of iron. Here, we report that exposure of A549 cells to crocidolite asbestos led to a significant time-dependent inactivation of signaling proteins, i.e. Akt and all mitogen-activated protein kinases (MAPKs) (p38, ERK1/2 and SAPK/JNK), and subsequently to apoptosis. Unlike crocidolite treatment, the use of BSO and FAC, independently or combined, did not change the phosphorylation status of proteins, nor did it induce apoptosis. Taken together, our results presented herein point to the possibility that crocidolite-induced apoptosis of human lung epithelial cells is not a mere consequence of generation of oxidants but also requires inactivation of major cell growth and differentiation pathways.

Identification of a putative nuclear localization sequence within ANG II AT(1A) receptor associated with nuclear activation.

Angiotensin II (ANG II) type 1 (AT(1)) receptors, similar to other G protein-coupled receptors, undergo desensitization and internalization, and potentially nuclear localization, subsequent to agonist interaction. Evidence suggests that the carboxy-terminal tail may be involved in receptor nuclear localization. In the present study, we examined the carboxy-terminal tail of the receptor for specific regions responsible for the nuclear translocation phenomenon and resultant nuclear activation. Human embryonic kidney cells stably expressing either a wild-type AT(1A) receptor-green fluorescent protein (AT(1A)R/GFP) construct or a site-directed mutation of a putative nuclear localization sequence (NLS) [K307Q]AT(1A)R/GFP (KQ/AT(1A)R/GFP), were examined for differences in receptor nuclear trafficking and nuclear activation. Receptor expression, intracellular signaling, and ANG II-induced internalization of the wild-type/GFP construct and of the KQ/AT(1A)R/GFP mutant was similar. Laser scanning confocal microscopy showed that in cells expressing the AT(1A)R/GFP, trafficking of the receptor to the nuclear area and colocalization with lamin B occurred within 30 min of ANG II (100 nM) stimulation, whereas the KQ/AT(1A)R/GFP mutant failed to demonstrate nuclear localization. Immunoblotting of nuclear lysates with an anti-GFP antibody confirmed these observations. Nuclear localization of the wild-type receptor correlated with increase transcription for both EGR-1 and PTGS-2 genes while the nuclear-deficient KQ/AT(1A)R/GFP mutant demonstrated increases for only the EGR-1 gene. These results suggest that a NLS (KKFKKY; aa307-312) is located within the cytoplasmic tail of the AT(1A) receptor and that nuclear localization of the receptor corresponds with specific activation of transcription for the COX-2 gene PTGS-2.

Role of iron in inactivation of epidermal growth factor receptor after asbestos treatment of human lung and pleural target cells.

Although the mechanism by which asbestos causes cancer remains unknown, iron associated with asbestos is thought to play a role in the pathogenic effects of fibers. Here, we examined the effects of asbestos on the epidermal growth factor receptor (EGFR) in human lung epithelial (A549) cells, human pleural mesothelial (MET5A) cells, and normal human small airway epithelial (SAEC) cells. Treatment of A549, MET5A, and SAEC cells with asbestos caused a significant reduction of EGFR tyrosine phosphorylation. This was both time- (15 min to 24 h) and concentration-dependent (1.5, 3, and 6 mug/cm(2)) in A549 cells. Also, treatment with 6 mug/cm(2) crocidolite for 24 h diminished the phosphorylation levels of human EGFR 2 (HER2). Exposure of A549 cells to 6 mug/cm(2) crocidolite for 3-24 h resulted in no detectable Y1045 phosphorylation and no apparent degradation of the EGFR. Inhibition of fiber endocytosis resulted in a considerable inhibition of EGFR dephosphorylation. Removal of iron from asbestos by desferrioxamine B or phytic acid inhibited asbestos-induced decreases in EGFR phosphorylation. The effects of crocidolite, amosite, and chrysotile on the EGFR phosphorylation state appeared to be directly related to the amount of iron mobilized from these fibers. These results strongly suggest that iron plays an important role in asbestos-induced inactivation of EGFR.