Venkateswarlu Kanamarlapudi

ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of α9 integrin promotes axon regeneration, so we have investigated α9β1 trafficking and transport in axons and at the growth cone. We have previously found that α9 and β1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied α9 and β1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of β1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.

The calcium-sensing receptor changes cell shape via a β-arrestin-1–ARNO–ARF6–ELMO protein network

G-protein-coupled receptors (GPCRs) transduce the binding of extracellular stimuli into intracellular signalling cascades that can lead to morphological changes. Here, we demonstrate that stimulation of the calcium-sensing receptor (CaSR), a GPCR that promotes chemotaxis by detecting increases in extracellular calcium, triggers plasma membrane (PM) ruffling via a pathway that involves β-arrestin 1, Arf nucleotide binding site opener (ARNO), ADP-ribosylating factor 6 (ARF6) and engulfment and cell motility protein (ELMO). Expression of dominant negative β-arrestin 1 or its knockdown with siRNA impaired the CaSR-induced PM ruffling response. Expression of a catalytically inactive ARNO also reduced CaSR-induced PM ruffling. Furthermore, β-arrestin 1 co-immunoprecipitated with the CaSR and ARNO under resting conditions. Agonist treatment did not markedly alter β-arrestin 1 binding to the CaSR or to ARNO but it did elicit the translocation and colocalisation of the CaSR, β-arrestin 1 and ARNO to membrane protrusions. Furthermore, ARF6 and ELMO, two proteins known to couple ARNO to the cytoskeleton, were required for CaSR-dependent morphological changes and translocated to the PM ruffles. These data suggest that cells ruffle upon CaSR stimulation via a mechanism that involves translocation of β-arrestin 1 pre-assembled with the CaSR or ARNO, and that ELMO plays an essential role in this CaSR-signalling-induced cytoskeletal reorganisation.

ARF6-Dependent Regulation of P2Y Receptor Traffic and Function in Human Platelets

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y1 and P2Y12 purinoceptors. Recently, we demonstrated that P2Y1 and P2Y12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y1 and P2Y12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y1 or P2Y12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling

Background: The mechanisms by which ARF6 regulate LHCGR internalization and signaling are unknown. Results: Heterotrimeric G-protein, PI 3-kinase, cytohesin ARF GEFs and ARF GAPs function upstream whereas dynamin and clathrin act downstream of ARF6 in the regulation of HLHCGR internalization and signaling. Conclusion: Dissected the molecular mechanisms underlying ARF6 involvement in LHCGR internalization and signaling. Significance: This study provides insight into the mechanisms responsible for HLHCGR regulation by ARF6. The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a Gs-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.

Agonist-induced internalisation of the glucagon-like peptide-1 receptor is mediated by the Gαq pathway

The glucagon-like peptide-1 receptor (GLP-1R) is a G-protein-coupled receptor (GPCR) and an important target in the treatment of type 2 diabetes mellitus (T2DM). Upon stimulation with agonist, the GLP-1R signals through both Gαs and Gαq coupled pathways to stimulate insulin secretion. The agonist-induced GLP-1R internalisation has recently been shown to be important for insulin secretion. However, the molecular mechanisms underlying GLP-1R internalisation remain unknown. The aim of this study was to determine the role of GLP-1R downstream signalling pathways in its internalisation. Agonist-induced human GLP-1R (hGLP-1R) internalisation and activity were examined using a number of techniques including immunoblotting, ELISA, immunofluorescence and luciferase assays to determine cAMP production, intracellular Ca(2+) accumulation and ERK phosphorylation. Agonist-induced hGLP-1R internalisation is dependent on caveolin-1 and dynamin. Inhibition of the Gαq pathway but not the Gαs pathway affected hGLP-1R internalisation. Consistent with this, hGLP-1R mutant T149M and small-molecule agonists (compound 2 and compound B), which activate only the Gαs pathway, failed to induce internalisation of the receptor. Chemical inhibitors of the Gαq pathway, PKC and ERK phosphorylation significantly reduced agonist-induced hGLP-1R internalisation. These inhibitors also suppressed agonist-induced ERK1/2 phosphorylation demonstrating that the phosphorylated ERK acts downstream of the Gαq pathway in the hGLP-1R internalisation. In summary, agonist-induced hGLP-1R internalisation is mediated by the Gαq pathway. The internalised hGLP-1R stimulates insulin secretion from pancreatic β-cells, indicating the importance of GLP-1 internalisation for insulin secretion.

The Effect of Shear Stress on the Size, Structure, and Function of Human von Willebrand Factor

Clinical outcomes from ventricular assist devices (VADs) have improved significantly during recent decades, but bleeding episodes remain a common complication of long-term VAD usage. Greater understanding of the effect of the shear stress in the VAD on platelet aggregation, which is influenced by the functional activity of high molecular weight (HMW) von Willebrand factor (vWF), could provide insight into these bleeding complications. However, because VAD shear rates are difficult to assess, there is a need for a model that enables controlled shear rates to first establish the relationship between shear rates and vWF damage. Secondly, if such a dependency exists, then it is relevant to establish a rapid and quantitative assay that can be used routinely for the safety assessment of new VADs in development. Therefore, the purpose of this study was to exert vWF to controlled levels of shear using a rheometer, and flow cytometry was used to investigate the shear-dependent effect on the functional activity of vWF. Human platelet-poor plasma (PPP) was subjected to different shear rate levels ranging from 0 to 8000/s for a period of 6 h using a rheometer. A simple and rapid flow cytometric assay was used to determine platelet aggregation in the presence of ristocetin cofactor as a readout for vWF activity. Platelet aggregates were visualized by confocal microscopy. Multimers of vWF were detected using gel electrophoresis and immunoblotting. The longer PPP was exposed to high shear, the greater the loss of HMW vWF multimers, and the lower the functional activity of vWF for platelet aggregation. Confocal microscopy revealed for the first time that platelet aggregates were smaller and more dispersed in postsheared PPP compared with nonsheared PPP. The loss of HMW vWF in postsheared PPP was demonstrated by immunoblotting. Smaller vWF platelet aggregates formed in response to shear stress might be a cause of bleeding in patients implanted with VADs. The methodological approaches used herein could be useful in the design of safer VADs and other blood handling devices. In particular, we have demonstrated a correlation between the loss of HMW vWF, analyzed by immunoblotting, with platelet aggregation, assessed by flow cytometry. This suggests that flow cytometry could replace conventional immunoblotting as a simple and rapid routine test for HMW vWF loss during in vitro testing of devices.

Cytohesin 2/ARF6 regulates preadipocyte migration through the activation of ERK1/2

Preadipocyte migration is vital for the development of adipose tissue, which plays a crucial role in lipid metabolism. ADP-ribosylation factor 6 (ARF6) small GTPase, which regulates membrane trafficking, is activated by GTP-exchange factors (GEFs) such as cytohesin 2. Cytohesin 2 and ARF6 have previously been implicated in the regulation of 3T3-L1 preadipocyte migration. We investigated here the molecular mechanism underlying the cytohesin 2 and ARF6 mediated regulation of preadipocyte migration. Preadipocyte migration and the activation of ARF6 and ERK1/2 were studied by using a number of approaches, including pharmacological inhibitors, siRNA and the inhibitory peptides. The siRNA mediated down regulation of ARF6 and cytohesin 2 expression confirmed the requirement of both for migration of preadipocytes. Phosphatidylinositol 3-kinase (PI3K) and PI 4,5-bisphosphate (PIP2) have also found to be essential for the cytohesin 2/ARF6 induced preadipocyte migration. Pharmacological inhibition of the activation of ARF6, ERK1/2 or dynamin led to significant reduction in migration of 3T3-L1 preadipocytes. Furthermore, our study revealed the activation of ARF6 and ERK1/2 during migration of preadipocytes. In the migrating preadipocytes, ARF6 activation was inhibited with SecinH3 (cytohesin inhibitor) and LY294002 (PI3K inhibitor) whereas the ERK1/2 phosphorylation was inhibited with SecinH3, LY294002, PBP10 (a PIP2 sequester peptide) and PD98059 (MAPKK inhibitor). However, dynosore (dynamin inhibitor) had inhibited neither ARF6 activation nor ERK1/2 phosphorylation during preadipocyte migration. These results together suggest that cytohesin 2 activates ARF6 in a PI3K dependent manner and then the active ARF6 causes phosphorylation of ERK1/2 during preadipocyte migration.

Signal transducer and activator of transcription-3 licenses Toll-like receptor 4-dependent interleukin (IL)-6 and IL-8 production via IL-6 receptor-positive feedback in endometrial cells.

Interleukin 6 (IL-6), acting via the IL-6 receptor (IL6R) and signal transducer and activator of transcription-3 (STAT3), limits neutrophil recruitment once bacterial infections are resolved. Bovine endometritis is an exemplar mucosal disease, characterized by sustained neutrophil infiltration and elevated IL-6 and IL-8, a neutrophil chemoattractant, following postpartum Gram-negative bacterial infection. The present study examined the impact of the IL6R/STAT3 signaling pathway on IL-8 production by primary endometrial cells in response to short- or long-term exposure to lipopolysaccharide (LPS) from Gram-negative bacteria. Tyrosine phosphorylation of STAT3 is required for DNA binding and expression of specific targets genes. Immunoblotting indicated constitutive tyrosine phosphorylation of STAT3 in endometrial cells was impeded by acute exposure to LPS. After 24 h exposure to LPS, STAT3 returned to a tyrosine phosphorylated state, indicating cross-talk between the Toll-like receptor 4 (TLR4) and the IL6R/STAT3 signaling pathways. This was confirmed by short interfering RNA targeting the IL6R, which abrogated the accumulation of IL-6 and IL-8, induced by LPS. Furthermore, there was a differential endometrial cell response, as the accumulation of IL-6 and IL-8 was dependent on STAT3, suppressor of cytokine signaling 3, and Src kinase signaling in stromal cells, but not epithelial cells. In conclusion, positive feedback through the IL6R amplifies LPS-induced IL-6 and IL-8 production in the endometrium. These findings provide a mechanistic insight into how elevated IL-6 concentrations in the postpartum endometrium during bacterial infection leads to marked and sustained neutrophil infiltration.

Exchange Factor EFA6R Requires C-terminal Targeting to the Plasma Membrane to Promote Cytoskeletal Rearrangement through the Activation of ADP-ribosylation Factor 6 (ARF6)

Background: The subcellular localization and cellular functions of EFA6R are unknown. Results: EFA6R requires dual targeting through the PH and CC domains to localize to the plasma membrane and function as a GEF. Conclusion: Plasma membrane-localized EFA6R stimulates actin reorganization through ARF6 activation. Significance: This study provides insight into the localization and cellular functions of EFA6R. ADP-ribosylation factor 6 (ARF6) small GTPase regulates membrane trafficking and cytoskeleton rearrangements at the plasma membrane (PM) by cycling between the GTP-bound active and GDP-bound inactive conformations. Guanine nucleotide exchange factors (GEFs) activate ARF6. The exchange factor for ARF6 (EFA6) R has been identified as a biomarker for ovarian cancer. EFA6R shares the catalytic Sec7, pleckstrin homology (PH), and coiled coil (CC) domains of the other EFA6 family GEFs. Here we report the functional characterization of EFA6R. Endogenous EFA6R was present in the plasma membrane fraction. The exogenously expressed FLAG- and GFP-tagged EFA6R were targeted to the PM. In vitro, GFP-EFA6R associated weakly but preferentially with phosphatidylinositol 4,5-bisphosphate (PIP2) through the PH domain. EFA6R required both its PH and CC domains localized at the C terminus to target the PM. Consistent with this, EFA6R lacking the CC domain (EFA6RΔCC) was released from the PM into the cytosol upon PIP2 depletion, whereas EFA6R release from the PM required both PIP2 depletion and actin destabilization. These results suggest that the dual targeting via the PH and CC domains is important for the PM localization of EFA6R. EFA6R specifically catalyzed the GTP loading of ARF6 in mammalian cells. Moreover, EFA6R regulated ARF6 localization and thereby actin stress fiber loss. The GEF activity of EFA6R was dependent on the presence of the Sec7 domain. The PH and CC domains were also required for the in vivo GEF activity of EFA6R but could be functionally replaced by the CAAX motif of K-Ras, suggesting a role for these domains in the membrane targeting of EFA6R.

ADP-ribosylation factor 6 regulates endothelin-1-induced lipolysis in adipocytes

Endothelin-1 (ET-1) induces lipolysis in adipocytes, where ET-1 chronic exposure results in insulin resistance (IR) through suppression of glucose transporter (GLUT)4 translocation to the plasma membrane and consequently glucose uptake. ARF6 small GTPase, which plays a vital role in cell surface receptors trafficking, has previously been shown to regulate GLUT4 recycling and thereby insulin signalling. ARF6 also plays a role in ET-1 promoted endothelial cell migration. However, ARF6 involvement in ET-1-induced lipolysis in adipocytes is unknown. Therefore, we investigated the role of ARF6 in ET-1-induced lipolysis in 3T3-L1 adipocytes. This was achieved by studying the effect of inhibitors for the activation of ARF6 and other signalling proteins on ET-1 induced lipolysis and ARF6 activation in the adipocytes. Our results indicate that ET-1 induces, through endothelin type A receptor (ETAR), lipolysis, the ARF6 activation and extracellular-signal regulated kinase (ERK) phosphorylation in adipocytes, further ET-1 stimulated lipolysis is inhibited by the inhibitors of ARF6 activation, ERK phosphorylation and dynamin, which is essential for endocytosis. Our studies also revealed that ARF6 acts upstream of ERK in ET-1-indcued lipolysis. In summary, we determined that ET-1 activation of ETAR signalled through ARF6, which is crucial for lipolysis.