Internalization In Response Research Articles

Ketamine-induced reduction in mGluR5 availability is associated with an antidepressant response: an [11C]ABP688 and PET imaging study in depression.

The mechanisms of action of the rapid antidepressant effects of ketamine, an NMDA glutamate receptor antagonist, have not been fully elucidated. This study examined effects of ketamine on ligand binding to a metabotropic glutamatergic receptor (mGluR5) in individuals with major depressive disorder (MDD) and healthy controls. Thirteen healthy and thirteen MDD nonsmokers participated in two [11C]ABP688 positron emission tomography (PET) scans on the same day – before and during intravenous ketamine administration – and a third scan 1 day later. At baseline, significantly lower [11C]ABP688 binding was detected in the MDD as compared to the control group. We observed a significant ketamine-induced reduction in mGluR5 availability, (i.e. [11C]ABP688 binding), in both MDD and control subjects (average of 14±9% and 19±22%, respectively; p<0.01 for both), which persisted 24 hours later. There were no differences in ketamine-induced changes between MDD and control groups at either time point (p=0.8). A significant reduction in depressive symptoms was observed following ketamine administration in the MDD group (p<0.001), which was associated with the change in binding (p<0.04) immediately post ketamine. We hypothesize that glutamate released after ketamine administration moderates mGluR5 availability; this change appears to be related to antidepressant efficacy. The sustained decrease in binding may reflect prolonged mGluR5 internalization in response to the glutamate surge.

Src mediates endocytosis of TWIK-related acid-sensitive K+ 1 channels in PC12 cells in response to nerve growth factor.

TWIK-related acid-sensitive K(+) (TASK) channels produce background K(+) currents. We elucidated that TASK1 channels in rat adrenal medullary cells and PC12 cells are internalized in a clathrin-dependent manner in response to nerve growth factor (NGF). Here, the molecular mechanism for this internalization in PC12 cells was explored. The combination of enzyme inhibitors with tropomyosin receptor kinase A mutants revealed that the internalization was mediated by both phospholipase C and phosphatidylinositol 3-kinase pathways that converge on protein kinase C with the consequent activation of Src, a nonreceptor tyrosine kinase. The NGF-induced endocytosis of TASK1 channels did not occur in the presence of the Src inhibitor or with the expression of a kinase-dead Src mutant. Additionally, NGF induced a transient colocalization of Src with the TASK1 channel, but not the TASK1 mutant, in which tyrosine at 370 was replaced with phenylalanine. This TASK1 mutant showed no increase in tyrosine phosphorylation and markedly diminished internalization in response to NGF. We concluded that NGF induces endocytosis of TASK1 channels via tyrosine phosphorylation in its carboxyl terminus.

Novel role of reactive oxygen species-activated Trp melastatin channel-2 in mediating angiogenesis and postischemic neovascularization.

Transient receptor potential melastatin-2 (TRPM2) channel is a nonselective cation channel that mediates influx of Ca(2+) and Na(+) with relative permeability of PCa:PNa ≈0.6 in response to cellular oxidative stress. As angiogenesis and ischemic neovascularization are both significantly dependent on oxidant signaling, here we investigated the possible role of vascular endothelial growth factor (VEGF)-induced reactive oxygen species production in activating TRPM2-dependent Ca(2+) signaling and in the mechanism of angiogenesis and ischemic neovascularization. We observed that VEGF stimulation rapidly induced the association of TRPM2 and cellular Src kinase with vascular endothelial-cadherin forming a signalplex at vascular endothelial-cadherin junctions in endothelial cells. Using endothelial cells isolated from TRPM2(-/-) mice or after small interfering RNA depletion of TRPM2, we demonstrated that TRPM2-activated Ca(2+) signaling was required for cellular Src kinase-induced phosphorylation of vascular endothelial-cadherin at Y658 and Y731, the crucial sites involved in vascular endothelial-cadherin internalization in response to VEGF. VEGF-induced reactive oxygen species generation activated TRPM2-induced Ca(2+) entry, whereas the reactive oxygen species-insensitive TRPM2 mutant (C1008→A) showed impaired Ca(2+) entry. Endothelial cells depleted of TRPM2 also displayed significantly perturbed migratory phenotype and impaired activation of cellular Src in response to VEGF. TRPM2(-/-) mice reconstituted with wild-type myeloid cells demonstrated aberrant angiogenesis and neovascularization in the hindlimb ischemia model as compared with wild-type mice. VEGF-induced angiogenesis and postischemic neovascularization in mice required reactive oxygen species generation in endothelial cells and resultant TRPM2 activation. Thus, our findings provide novel insight into the role of TRPM2 in mechanism of angiogenesis and ischemic neovascularization.

Statin Inhibits IKs Internalization in Response to Prolonged Stress Stimulus

Statins are among the most commonly prescribed drug classes, and their use is expected to increase due to recent changes in therapy guidelines. Although statins have been suggested to shorten QT interval in heart failure patients, the effect of this drug class on cardiac electrophysiology has been incompletely studied. Here we study the effect of statins on one of the major repolarizing currents in the heart, IKs. We show that in response to a known stress stimulus (sustained alpha-AR stimulation), IKs undergoes clathrin-dependent internalization. Statin treatment inhibits this internalization. Our results suggest that statin regulates IKs internalization by inhibiting activation of Rab5, a GTPase involved in endocytic pathway regulation. We observe no significant effect of statin treatment on unstimulated IKs function and acute receptor regulation. Our results suggest that statins may prevent pathological QT prolongation in heart failure patients by inhibiting pathological remodeling and IKs internalization, while maintaining physiological trafficking and function of the channel.

S1PR1 Tyr143 phosphorylation downregulates endothelial cell surface S1PR1 expression and responsiveness.

Activation of sphingosine-1-phosphate receptor 1 (S1PR1) plays a key role in repairing endothelial barrier function. We addressed the role of phosphorylation of the three intracellular tyrosine residues of S1PR1 in endothelial cells in regulating the receptor responsiveness and endothelial barrier function regulated by sphingosine 1-phosphate (S1P)-mediated activation of S1PR1. We demonstrated that phosphorylation of only Y143 site was required for S1PR1 internalization in response to S1P. Maximal S1PR1 internalization was seen in 20 min but S1PR1 returned to the cell surface within 1 h accompanied by Y143-dephosphorylation. Cell surface S1PR1 loss paralleled defective endothelial barrier enhancement induced by S1P. Expression of phospho-defective (Y143F) or phospho-mimicking (Y143D) mutants, respectively, failed to internalize or showed unusually high receptor internalization, consistent with the requirement of Y143 in regulating cell surface S1PR1 expression. Phosphorylation of the five S1PR1 C-terminal serine residues did not affect the role of Y143 phosphorylation in signaling S1PR1 internalization. Thus, rapid reduction of endothelial cell surface expression of S1PR1 subsequent to Y143 phosphorylation is a crucial mechanism of modulating S1PR1 signaling, and hence the endothelial barrier repair function of S1P.

DYRK1A-mediated phosphorylation of GluN2A at Ser(1048) regulates the surface expression and channel activity of GluN1/GluN2A receptors.

N-methyl-D-aspartate glutamate receptors (NMDARs) play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs were modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser1048 of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser1048 hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser1048 increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons.

Abstract 333: Maturation Of ß-adrenergic Receptor (ß-AR) Signaling In Human Induced Pluripotent Stem Cell-derived Cardiomyocytes (hipsc-cms)

hiPSC-CMs are a novel model system for cardiovascular diseases, with proven utility in cardiac channelopathies, however, how accurately hiPSC-CMs recapitulate the signaling pathways regulating contractility and remodeling of adult cardiomyocytes remains to be determined. As ß-ARs are key regulators of both contractility and remodeling, we examined ß-AR signaling at different stages of hiPSC-CM maturation. hiPSCs, derived from healthy controls, were studied at 0, 14, 30, and 60d after cardiac induction under isoproterenol (ISO) stimulation ± ß1 or ß2 antagonists. Flag-tagged ß-ARs were transfected into hiPSC-CMs for trafficking studies. Compared to the ß1:ß2 ratio of 70:30 in adult ventricular cardiomyocytes, ß2-AR expression was higher in early stage (d14-30) hiPSC-CMs; ß1-ARs increased with maturation (d14-d60), reflective of the normal developmental pattern. Downstream signaling also matured from d14-60, regulating both function (PLB and TnI) and remodeling (ERK). The majority of cAMP generation and PKA activation at d30 was through ß2-ARs; by d60-90, ß1-AR signaling contributed equally. In contrast to studies in rodent cells, ß2-ARs did not show compartmentalization of PKA signaling. Between d30-60, components of the SR (RYR, PLB and CSQ) and caveolae (Cav3) matured, suggesting development of functional compartmentalization machinery. Finally, hiPSC-CMs showed ß2-AR downregulation and internalization in response to ISO, an important component of the normal cardiomyocyte response to pathologic stimulation. Thus, hiPSC-CM ß-AR signaling matures with time after cardiac induction, recapitulating many of the features of post-natal and several of adult cardiomyocytes. hiPSC-CM models of human cardiomyopathies are a reasonable platform for studying ß-AR signaling. Furthermore, the maturation of ß-AR signaling can be used as a read-out for hiPSC-CM maturity.

Proteomic analysis of the epidermal growth factor receptor (EGFR) interactome and post-translational modifications associated with receptor endocytosis in response to EGF and stress.

Aberrant expression, activation, and stabilization of epidermal growth factor receptor (EGFR) are causally associated with several human cancers. Post-translational modifications and protein-protein interactions directly modulate the signaling and trafficking of the EGFR. Activated EGFR is internalized by endocytosis and then either recycled back to the cell surface or degraded in the lysosome. EGFR internalization and recycling also occur in response to stresses that activate p38 MAP kinase. Mass spectrometry was applied to comprehensively analyze the phosphorylation, ubiquitination, and protein-protein interactions of wild type and endocytosis-defective EGFR variants before and after internalization in response to EGF ligand and stress. Prior to internalization, EGF-stimulated EGFR accumulated ubiquitin at 7 K residues and phosphorylation at 7 Y sites and at S(1104). Following internalization, these modifications diminished and there was an accumulation of S/T phosphorylations. EGFR internalization and many but not all of the EGF-induced S/T phosphorylations were also stimulated by anisomycin-induced cell stress, which was not associated with receptor ubiquitination or elevated Y phosphorylation. EGFR protein interactions were dramatically modulated by ligand, internalization, and stress. In response to EGF, different E3 ubiquitin ligases became maximally associated with EGFR before (CBL, HUWE1, and UBR4) or after (ITCH) internalization, whereas CBLB was distinctively most highly EGFR associated following anisomycin treatment. Adaptin subunits of AP-1 and AP-2 clathrin adaptor complexes also became EGFR associated in response to EGF and anisomycin stress. Mutations preventing EGFR phosphorylation at Y(998) or in the S(1039) region abolished or greatly reduced EGFR interactions with AP-2 and AP-1, and impaired receptor trafficking. These results provide new insight into spatial, temporal, and mechanistic aspects of EGFR regulation.

New Insights on Plant Cell Elongation: A Role for Acetylcholine

We investigated the effect of auxin and acetylcholine on the expression of the tomato expansin gene LeEXPA2, a specific expansin gene expressed in elongating tomato hypocotyl segments. Since auxin interferes with clathrin-mediated endocytosis, in order to regulate cellular and developmental responses we produced protoplasts from tomato elongating hypocotyls and followed the endocytotic marker, FM4-64, internalization in response to treatments. Tomato protoplasts were observed during auxin and acetylcholine treatments after transient expression of chimerical markers of volume-control related compartments such as vacuoles. Here we describe the contribution of auxin and acetylcholine to LeEXPA2 expression regulation and we support the hypothesis that a possible subcellular target of acetylcholine signal is the vesicular transport, shedding some light on the characterization of this small molecule as local mediator in the plant physiological response.

Pharmacological evidence of bradykinin regeneration from extended sequences that behave as peptidase–activated B2 receptor agonists

While bradykinin (BK) is known to be degraded by angiotensin converting enzyme (ACE), we have recently discovered that Met-Lys-BK-Ser-Ser is paradoxically activated by ACE. We designed and evaluated additional “prodrug” peptides extended around the BK sequence as potential ligands that could be locally activated by vascular or blood plasma peptidases. BK regeneration was estimated using the contractility of the human umbilical vein as model of vascular functions mediated by endogenous B2 receptors (B2Rs) and the endocytosis of the fusion protein B2R-green fluorescent protein (B2R-GFP) expressed in Human Embryonic Kidney 293 cells. Of three BK sequences extended by a C-terminal dipeptide, BK-His-Leu had the most desirable profile, exhibiting little direct affinity for the receptor but a significant one for ACE (as shown by competition of [3H]BK binding to B2R-GFP or of [3H]enalaprilat to recombinant ACE, respectively). The potency of the contractile effect of this analog on the vein was reduced 18-fold by the ACE inhibitor enalaprilat, pharmacologically evidencing BK regeneration in situ. BK-Arg, a potential substrate of arginine carboxypeptidases, had a low affinity for B2Rs and its potency as a contractile agent was reduced 15-fold by tissue treatment with an inhibitor of these enzymes, Plummer’s inhibitor. B2R-GFP internalization in response to 100 nM of the extended peptides recapitulated these findings, as enalaprilat selectively inhibited the effect of BK-His-Leu and Plummer’s inhibitor, that of BK-Arg. The two peptidase inhibitors did not affect BK-induced effects in either assay. The novel C-terminally extended BKs had no or very little affinity for the kinin B1 receptor (competition of [3H]Lys-des-Arg9-BK binding). The feasibility of peptidase-activated B2R agonists is illustrated by C-terminal extensions of the BK sequence.

Collapsin Response Mediator Protein 2 (CRMP2) Interacts with N-Methyl-d-aspartate (NMDA) Receptor and Na+/Ca2+ Exchanger and Regulates Their Functional Activity

Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-d-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca(2+) dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, TAT-CBD3, but not CBD3 without TAT or TAT-scramble peptide, inhibited increases in cytosolic Ca(2+) mediated by the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. TAT-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, TAT-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca(2+) influx into neurons, was strongly suppressed by TAT-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to TAT-CBD3 exposure. Moreover, CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that TAT-CBD3 protects against glutamate-induced Ca(2+) dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of TAT-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.

Congestive heart failure in rats is associated with increased collecting duct vasopressin sensitivity and vasopressin type 2 receptor reexternalization

A number of studies have shown that rats with congestive heart failure (CHF) have increased protein levels of the vasopressin (AVP)-regulated water channel aquaporin-2 (AQP2) even during conditions with unchanged circulating levels of AVP, suggesting an increase in the sensitivity of the AVP type 2 (V2) receptor in experimental CHF. The present study was aimed at investigating AVP signaling in rats with moderate CHF (left ventricular end diastolic pressure >10 mmHg; normal plasma AVP levels) induced by ligation of the left anterior descending coronary artery. Sham-operated rats were used as controls. Western blotting analyses revealed an increased abundance of AQP2 in renal cortex (+33 ± 9% of sham; P < 0.05) and in inner medulla (IM) (+54 ± 15% of sham; P < 0.05) in CHF rats compared with sham-operated controls. Dose-response studies on isolated collecting ducts (CDs) showed an increased accumulation of cAMP in response to AVP in CHF rats compared with controls. V2 receptor surface-binding studies in isolated IMCDs showed a marked and comparable AVP-induced V2 receptor internalization in response to AVP in both CHF and control rats. As expected V2 receptor surface binding remained low after AVP challenge in control rats. In contrast to this, V2 receptor surface binding returned to pre-AVP levels within 30 min in the CHF rats, indicating an obtained recycling ability of the V2 receptor in CHF. Together the results indicate the presence of an increased AVP sensitivity in the CDs from CHF rats, associated with an acquired recycling ability of the V2 receptor.

Regulation of Metabotropic Glutamate Receptor 7 (mGluR7) Internalization and Surface Expression by Ser/Thr Protein Phosphatase 1

The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.

Disabled-2 (Dab2) inhibits Wnt/β-catenin signalling by binding LRP6 and promoting its internalization through clathrin

Canonical Wnt signalling requires caveolin-dependent internalization of low-density lipoprotein receptor-related protein 6 (LRP6). Here we report that the tumour suppressor and endocytic adaptor disabled-2 (Dab2), previously described as an inhibitor of Wnt/β-catenin signalling, selectively recruits LRP6 to the clathrin-dependent endocytic route, thereby sequestering it from caveolin-mediated endocytosis. Wnt stimulation induces the casein kinase 2 (CK2)-dependent phosphorylation of LRP6 at S1579, promoting its binding to Dab2 and internalization with clathrin. LRP6 receptor mutant (S1579A), deficient in CK2-mediated phosphorylation and Dab2 binding, fails to associate with clathrin, and thus escapes the inhibitory effects of Dab2 on Wnt/β-catenin signalling. Our data suggest that the S1579 site of LRP6 is a negative regulatory point during LRP6-mediated dorsoventral patterning in zebrafish and in allograft mouse tumour models. We conclude that the tumour suppressor functions of Dab2 involve modulation of canonical Wnt signalling by regulating the endocytic fate of the LRP6 receptor.

Immunolocalization of Metabotropic Glutamate Receptor 5 in Isolated Rabbit Cardiomyocytes and Cultured H9C2 Cells: Receptor Clustering and Internalization with Simulated Ischemia–Induced Contracture

Background L-glutamate protects hearts against myocardial stunning and infarction while glutamate receptors antagonists abolish the protection. We report immunolocalization (IL) of glutamate Receptor 5 (mGluR5) in isolated rabbit normoxic (rode-shaped) or ischemic contracture (round-shape) cardiomyocytes and in H9C2 cells. Methods Collagenase-dispersed rabbit heart cells, attached and fixed on glass slides, underwent blocking of the non-specific binding, labeling with mGluR5 antibody, biotinylated secondary antibody and ultra-Vidin Texas Red as a fluorescent probe, and examined under fluorescent microscopy. H9C2 cells were cultured on cover slips and treated as above and examined under fluorescent and confocal microscopy. Results Positive IL of mGluR5were evenly distributed on cell membranes of normoxic rabbit cardiomyocytes but clustered and internalized in cells underwent contracture. IL of mGluR5 in cultured H9C2 cells was positive on the surface membranes and on the mitochondria around the myofibrils. Conclusions mGluR5 are localized on membranes of rode-shaped cardiomyocytes while undergo clustering and internalization during contracture. mGluR5 found in H9C2 on cells membranes and mitochondria. Further studies are warranted to elucidate the cellular and molecular signaling mechanisms of mGluR5 activation (or blockade) and internalization in response to oxidative stress.

Neurotensin Triggers Dopamine D2 Receptor Desensitization through a Protein Kinase C and β-Arrestin1-dependent Mechanism

The peptide neurotensin (NT) is known to exert a potent excitatory effect on the dopaminergic system by inhibiting D2 dopamine (DA) receptor (D2R) function. This regulation is dependent on activation of PKC, a well known effector of the type 1 NT receptor (NTR1). Because PKC phosphorylation of the D2R has recently been shown to induce its internalization, we hypothesized that NT acts to reduce D2R function through heterologous desensitization of the D2R. In the present study, we first used HEK-293 cells to demonstrate that NT induces PKC-dependent D2R internalization. Furthermore, internalization displayed faster kinetics in cells expressing the D2R short isoform, known to act as an autoreceptor in DA neurons, than in cells expressing the long isoform, known to act as a postsynaptic D2R. In patch clamp experiments on cultured DA neurons, overexpression of a mutant D2S lacking three key PKC phosphorylation sites abrogated the ability of NT to reduce D2R-mediated cell firing inhibition. Short interfering RNA-mediated inhibition of β-arrestin1 and dynamin2, proteins important for receptor desensitization, reduced agonist-induced desensitization of D2R function, but only the inhibition of β-arrestin1 reduced the effect of NT on D2R function. Taken together, our data suggest that NT acutely regulates D2 autoreceptor function and DA neuron excitability through PKC-mediated phosphorylation of the D2R, leading to heterologous receptor desensitization.

Ligand-induced internalization of the orexin OX(1) and cannabinoid CB(1) receptors assessed via N-terminal SNAP and CLIP-tagging.

Many G protein-coupled receptors internalize following agonist binding. The studies were designed to identify novel means to effectively quantify this process using the orexin OX(1) receptor and the cannabinoid CB(1) receptor as exemplars. The human OX(1) and CB(1) receptors were modified to incorporate both epitope tags and variants (SNAP and CLIP) of the enzyme O(6)-alkylguanine-DNA-alkyltransferase within their extracellular, N-terminal domain. Cells able to regulate expression of differing amounts of these constructs upon addition of an antibiotic were developed and analysed. Cell surface forms of each receptor construct were detected by both antibody recognition of the epitope tags and covalent binding of fluorophores to the O(6)-alkylguanine-DNA-alkyltransferase variants. Receptor internalization in response to agonists but not antagonists could be monitored by each approach but sensitivity was up to six- to 10-fold greater than other approaches when employing a novel, time-resolved fluorescence probe for the SNAP tag. Sensitivity was not enhanced, however, for the CLIP tag, possibly due to higher levels of nonspecific binding. These studies demonstrate that highly sensitive and quantitative assays that monitor cell surface CB(1) and OX(1) receptors and their internalization by agonists can be developed based on introduction of variants of O(6)-alkylguanine-DNA-alkyltransferase into the N-terminal domain of the receptor. This should be equally suitable for other G protein-coupled receptors.

Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling

The expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin II localized to swelling-induced membrane blebs with actin just before retraction, and MLCK inhibition led to persistent blebbing and attenuated cell volume recovery. At the base of the cell, MLCK also localized to dynamic actin-coated rings and patches upon swelling, which were associated with uptake of the membrane marker FM4-64X, consistent with sites of membrane internalization. Hypotonic exposure evoked increased biochemical association of the cell volume regulator Src with MLCK and with the endocytosis regulators cortactin and dynamin, which colocalized within these structures. Inhibition of either Src or MLCK led to altered patch and ring lifetimes, consistent with the concept that Src and MLCK form a swelling-induced protein complex that regulates volume recovery through membrane turnover and compensatory endocytosis under osmotic stress.

EBP50 inhibits the anti-mitogenic action of the parathyroid hormone type 1 receptor in vascular smooth muscle cells

Parathyroid hormone-related protein (PTHrP) and the parathyroid hormone type 1 receptor (PTH1R) are important regulators of vascular remodeling. PTHrP expression is associated to increased proliferation of vascular smooth muscle cells (VSMC). In contrast, signaling via the PTH1R inhibits cell growth. The mechanisms regulating the dual effect of PTHrP and PTH1R on VSMC proliferation are only partially understood. In this study we examined the role of the adaptor protein ezrin–radixin–moesin-binding phosphoprotein (EBP50) on PTH1R expression, trafficking, signaling and control of A10 cell proliferation. In normal rat vascular tissues, EBP50 was restricted to the endothelium with little expression in VSMC. EBP50 expression significantly increased in VSMC following angioplasty in parallel with PTHrP. Interestingly, PTHrP was able to induce EBP50 expression. In the clonal rat aortic smooth muscle cell line A10, EBP50 increased the recruitment of PTH1R to the cell membrane and delayed its internalization in response to PTHrP(1–36). This effect required an intact C-terminal motif in the PTH1R. In naïve A10 cells, PTHrP(1–36) stimulated cAMP production but not intracellular calcium release. In contrast, PTHrP(1–36) induced both cAMP and calcium signaling in A10 cells over-expressing EBP50. Finally, EBP50 attenuated the induction of p27 kip1 and the anti-proliferative effect of PTHrP(1–36). In summary, this study demonstrates the dynamic expression of EBP50 in vessels following injury and the effects of EBP50 on PTH1R function in VSMC. These findings highlight one of the mechanisms leading to increased VSMC proliferation and have important implication in the understanding of the molecular events leading to restenosis.

Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells

Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.