Constitutive Internalization Research Articles

Dynamics and Interaction of Interleukin-4 Receptor Subunits in Living Cells

It has long been established that dimerization of Interleukin-4 receptor (IL-4R) subunits is a pivotal step for JAK/STAT signal transduction. However, ligand-induced complex formation at the surface of living cells has been challenging to observe. Here we report an experimental assay employing trisNTA dyes for orthogonal, external labeling of eGFP-tagged receptor constructs that allows the quantification of receptor heterodimerization by dual-color fluorescence cross-correlation spectroscopy. Fluorescence cross-correlation spectroscopy analysis at the plasma membrane shows that IL-4R subunit dimerization is indeed a strictly ligand-induced process. Under conditions of saturating cytokine occupancy, we determined intramembrane dissociation constants (Kd,2D) of 180 and 480 receptors per μm2 for the type-2 complexes IL-4:IL-4Rα/IL-13Rα1 and IL-13:IL-13Rα1/IL-4Rα, respectively. For the lower affinity type-1 complex IL-4:IL-4Rα/IL-2Rγ, we estimated a Kd,2D of ∼1000 receptors per μm2. The receptor densities required for effective dimerization thus exceed the typical, average expression levels by several orders of magnitude. In addition, we find that all three receptor subunits accumulate rapidly within a subpopulation of early sorting and recycling endosomes stably anchored just beneath the plasma membrane (cortical endosomes, CEs). The receptors, as well as labeled IL-4 and trisNTA ligands are specifically trafficked into CEs by a constitutive internalization mechanism. This may compensate for the inherent weak affinities that govern ligand-induced receptor dimerization at the plasma membrane. Consistently, activated receptors are also concentrated at the CEs. Our observations thus suggest that receptor trafficking may play an important role for the regulation of IL-4R-mediated JAK/STAT signaling.

Abstract 4979: A6B1 integrin internalization is increased by loss of A3B1 expression and promotes migration of human prostate cancer cells

Abstract Laminin binding integrins, A3B1 and A6B1 drive in part, cancer cell migration and metastasis. Cell migration requires dynamic internalization and recycling of surface integrins. However, whether laminin binding integrin internalization is coordinately or independently controlled is not known. In this study, we quantitated the internalization of integrin A3 and A6 and whether crosstalk influences their respective internalization and subsequent cancer cell migration. We quantitated the internalization of A6 and A3 integrins, using fluorophore conjugated antibody based internalization assays and flow cytometry. In the prostate cancer cell line DU145, we observed constitutive internalization of integrin A6 with a surface half-life of approximately 30 minutes as compared to >70 minutes for integrin A3, 45 minutes for unrelated integrin Av or 10 minutes for the unrelated transferrin receptor. Silencing A3 integrin expression resulted in a 10 percent increase in the total amount of internalized A6 integrin, with a decreased surface half-life of 20 minutes. The internalized A6 integrin showed intracellular punctate staining colocalized with the early endosome marker EEA1 and Rab4 containing vesicles. Increased integrin A6 internalization led to 40% increase in migration which was dependent on A6B1 integrin. Silencing integrin A6 expression, however, did not affect the internalization of A3 integrin indicating a unidirectional regulation of integrin internalization. The changes in the integrin internalization could be clearly ascribed to alpha subunits, as the obtained half-life of A6 and A3 integrins were independent of antibody used or integrin engagement by ligand mimetic peptides. Taken together, these data indicate that A6B1 and A3B1 integrins have different internalization kinetics and the presence of integrin A3 decreased integrin A6 internalization and ensuing cell migration. These data are consistent with previous observations in normal systems suggesting that A3B1 provides a provisional matrix during early stages of wound healing, requiring subsequent stable adhesion complexes and integrin A6 function. In cancer, the loss of A3 integrin and the increased internalization of A6 integrin may account for the “wounds that won't heal” phenotype and provide a strategy for biological intervention to block tumor progression. (Supported in part by NIH Grants CA23074 and CA159406 and the TACMASS core service of the Arizona Cancer Center) Citation Format: Lipsa Das, Todd A. Anderson, Jaime M.c. Gard, Isis C. Sroka, Stephanie R. Strautman, Raymond B. Nagle, Beatrice S. Knudsen, Anne Cress. A6B1 integrin internalization is increased by loss of A3B1 expression and promotes migration of human prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2014-4979

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the "long loop" recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation.

Differential requirements of arrestin-3 and clathrin for ligand-dependent and -independent internalization of human G protein-coupled receptor 40

G protein-coupled receptor 40 (GPR40) is believed to be an attractive target to enhance insulin secretion in patients with type 2 diabetes. GPR40 has been found to couple to Gq protein, leading to the activation of phospholipase C and subsequent increases in the intracellular Ca2+ level. However, the underlying mechanisms that regulate the internalization and desensitization of GPR40 remain to be elucidated. In the present study, a construct of GPR40 fused with enhanced green fluorescent protein (EGFP) at its C-terminus was constructed for direct imaging of the localization and internalization of GPR40 by confocal microscopy. In stably transfected HEK-293 cells, GPR40 receptors underwent rapid agonist-induced internalization and constitutive ligand-independent internalization. Our data demonstrated that the agonist-mediated internalization of GPR40 was significantly blocked by hypertonic sucrose treatment and by siRNA mediated depletion of the heavy chain of clathrin. In contrast, constitutive GPR40 internalization was not affected by hypertonic sucrose or by knock-down of clathrin expression, but it was affected by treatment with methyl-β-cyclodextrin (MβCD) and nystatin. Furthermore, our results using an arrestin-3-EGFP redistribution assay and siRNA-mediated knock-down of arrestin-3 and GRK2 expression revealed that arrestin-3 and GRK2 play an essential role in the regulation of agonist-mediated GPR40 internalization, but are not involved in the regulation of constitutive GPR40 internalization. Additionally, our observation showed that upon activation by agonist, the internalized GPR40 receptors were rapidly recycled back to the plasma membrane via Rab4/Rab5 positive endosomes, whereas the constitutively internalized GPR40 receptors were recycled back to the cell surface through Rab5 positive endosomes. Because FFA receptors exhibit a high level of homology, our observations could be applicable to other members of this family.

Allosteric modulation of the G protein-coupled US28 receptor of human cytomegalovirus: Are the small-weight inverse agonist of US28 ‘camouflaged’ agonists?

The highly constitutively active G protein-coupled receptor US28 of human cytomegalovirus (HCMV) is thought to camouflage agonism by mediating constitutive endocytosis. With the use of the US28Δ300 mutant, which is largely devoid of constitutive internalization, I have demonstrated that the coupling of the receptor to its downstream signaling partners is responsible for the inverse agonism to agonism efficacy switch in some small-weight ligands of US28.

Regulation of Fibroblast Growth Factor-inducible 14 (Fn14) Expression Levels via Ligand-independent Lysosomal Degradation

Fibroblast growth factor-inducible 14 (Fn14) is a highly inducible cytokine receptor that engages multiple intracellular signaling pathways, including nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). Fn14 expression is regulated by several cytokines and growth factors, and Fn14 is transiently up-regulated after injury. In contrast, in states of chronic inflammatory disease and in some solid tumors, Fn14 is persistently up-regulated. However, the post-translational regulation of Fn14 expression has not been directly investigated. Thus, we examined Fn14 proteostasis in the presence and absence of the Fn14 ligand TNF-like weak inducer of apoptosis (TWEAK). Similar to other TNF receptor superfamily members, we found that TWEAK induces Fn14 internalization and degradation. Surprisingly, we also observed rapid, TWEAK-independent, constitutive Fn14 internalization and turnover. Fn14 levels are maintained in cell culture by ongoing synthesis and trafficking of the receptor, leading to subsequent down-regulation by lysosomal degradation. Unexpectedly, the extracellular domain of Fn14 is necessary and sufficient for constitutive turnover. Based on these findings, we propose a model in which constitutive down-regulation of Fn14 facilitates dynamic regulation of Fn14 protein levels and prevents spontaneous or inappropriate receptor signaling.

Rab11A and Rab11B distinctly regulate protease‐activated receptor‐1 recycling and constitutive degradation (802.10)

Protease‐activated receptor‐1 (PAR1) is a G protein‐coupled receptor (GPCR) expressed throughout the vasculature. Thrombin irreversibly activates PAR1, therefore receptor signaling and expression is tightly regulated. Upon activation, PAR1 is rapidly internalized and sorted to lysosomes for degradation, a process critical for signal termination. Whereas naïve PAR1 is constitutively internalized, generating an intracellular pool of PAR1 that recycles and repopulates the cell surface with uncleaved PAR1 after thrombin stimulation, a process critical for rapid recovery of thrombin signaling. The mechanisms that mediate recycling of naive PAR1 are not known. Using a siRNA library screen of 140 membrane trafficking proteins, we identified Rab11B as a key regulator of PAR1 trafficking. We confirmed that siRNA depletion of Rab11B, and not Rab11A reduced PAR1 surface expression, but did not affect expression of the thromboxane GPCRs, TPα and TPβ. Rab11B knockdown had no affect on PAR1 constitutive internalization, but blocked PAR1 recycling resulting in loss of surface expression and enhanced receptor degradation that was blocked by lysosomal protease inhibitors. In contrast to Rab11B, knockdown of Rab11A by siRNA disrupted PAR1 sorting from endosomes to lysosomes leading to accumulation, without affecting surface expression, suggesting that Rab11A and Rab11B distinctly regulate PAR1 trafficking. The mechanisms by which Rab11A and Rab11B distinctly regulate intracellular trafficking of PAR1 are not known and currently being investigated. Given that elevated PAR1 expression contributes to malignancy of certain cancers, understanding the mechanisms that control PAR1 surface expression could aid in the development of therapeutic approaches.Grant Funding Source: Supported by NIH R01 GM090689, Western States Affiliate AHA Postdoctoral Fellowship and the NIGMS In

EGF inhibits constitutive internalization and palmitoylation-dependent degradation of membrane-spanning procancer CDCP1 promoting its availability on the cell surface

Many cancers are dependent on inappropriate activation of epidermal growth factor receptor (EGFR), and drugs targeting this receptor can improve patient survival, although benefits are generally short-lived. We reveal a novel mechanism linking EGFR and the membrane-spanning, cancer-promoting protein CDCP1 (CUB domain-containing protein 1). Under basal conditions, cell surface CDCP1 constitutively internalizes and undergoes palmitoylation-dependent degradation by a mechanism in which it is palmitoylated in at least one of its four cytoplasmic cysteines. This mechanism is functional in vivo as CDCP1 is elevated and palmitoylated in high-grade serous ovarian tumors. Interestingly, activation of the EGFR system with EGF inhibits proteasome-mediated, palmitoylation-dependent degradation of CDCP1, promoting recycling of CDCP1 to the cell surface where it is available to mediate its procancer effects. We also show that mechanisms inducing relocalization of CDCP1 to the cell surface, including disruption of its palmitoylation and EGF treatment, promote cell migration. Our data provide the first evidence that the EGFR system can function to increase the lifespan of a protein and also promote its recycling to the cell surface. This information may be useful for understanding mechanisms of resistance to EGFR therapies and assist in the design of treatments for EGFR-dependent cancers.

Serum IgE clearance is facilitated by human FcεRI internalization

The high-affinity IgE receptor FcεRI is constitutively expressed in mast cells and basophils and is required for transmitting stimulatory signals upon engagement of IgE-bound allergens. FcεRI is also constitutively expressed in dendritic cells (DCs) and monocytes in humans; however, the specific functions of the FcεRI expressed by these cells are not completely understood. Here, we found that FcεRI expressed by human blood DC antigen 1-positive (BDCA1+) DCs and monocytes, but not basophils, traffics to endolysosomal compartments under steady-state conditions. Furthermore, IgE bound to FcεRI on BDCA1+ DCs was rapidly endocytosed, transported to the lysosomes, and degraded in vitro. IgE injected into mice expressing human FcεRIα (FCER1A-Tg mice) was endocytosed by conventional DCs and monocytes, and endocytosis was associated with rapid clearance of circulating IgE from these mice. Importantly, this rapid IgE clearance was dependent on monocytes or DCs but not basophils. These findings strongly suggest that constitutive internalization of human FcεRI by DCs and monocytes distinctively contributes to serum IgE clearance.

Intracellular Trafficking of the Human Cytomegalovirus-Encoded 7-trans-Membrane Protein Homologs pUS27 and pUL78 during Viral Infection: A Comparative Analysis

Human cytomegalovirus (HCMV) encodes four G protein-coupled receptor (GPCR) homologs, termed pUS27, pUS28, pUL33, and pUL78. In contrast to the extensively characterized vGPCRs pUS28 and pUL33, knowledge concerning pUS27 and pUL78 is limited. Previous studies already demonstrated constitutive internalization of pUS27 and pUL78, as well as an association with the endosomal machinery, however, these results were mainly obtained using transiently transfected cells. To explore the subcellular localization of both receptors during viral infection, we constructed recombinant HCMVs expressing tagged vGPCRs. Colocalization analyses revealed a predominant association of pUS27 or pUL78 with the trans-Golgi network or the endoplasmic reticulum, respectively. Intriguingly, our data emphasize that protein sorting is highly regulated by viral functions as we detected dramatic changes in the colocalization of pUS27 and pUL78 with endosomal markers during progression of HCMV replication. Furthermore, we observed cell type-dependent differences in trafficking of both vGPCRs between fibroblasts and epithelial cells. Most importantly, infection experiments with a recombinant HCMV carrying tagged versions of pUS27 and pUL78 simultaneously, revealed that these two proteins do not colocalize during viral infection. This contrasts to results of transient expression experiments. In conclusion, our results highlight the importance to investigate vGPCR trafficking in a viral context.

Intracellular localization of the M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization is mediated by a C-terminal tryptophan-based motif.

The M1 muscarinic acetylcholine receptor (M1-mAChR, encoded by CHRM1) is a G-protein-coupled membrane receptor that is activated by extracellular cholinergic stimuli. Recent investigations have revealed the intracellular localization of M1-mAChR. In this study, we observed constitutive internalization of M1-mAChR in mouse neuroblastoma N1E-115 cells without agonist stimulation. Constitutive internalization depended on dynamin, clathrin and the adaptor protein-2 (AP-2) complex. A WxxI motif in the M1-mAChR C-terminus is essential for its constitutive internalization, given that replacement of W(442) or I(445) with alanine residues abolished constitutive internalization. This WxxI motif resembles YxxΦ, which is the canonical binding motif for the μ2 subunit of the AP-2 complex. The M1-mAChR C-terminal WxxI motif interacted with AP-2 μ2. W442A and I445A mutants of the M1-mAChR C-terminal sequence lost AP-2-μ2-binding activity, whereas the W442Y mutant bound more effectively than wild type. Consistent with these results, W442A and I445A M1-mAChR mutants selectively localized to the cell surface. By contrast, the W442Y receptor mutant was found only at intracellular sites. Our data indicate that the cellular distribution of M1-mAChR is governed by the C-terminal tryptophan-based motif, which mediates constitutive internalization.

The stem cell-expressed receptor Lgr5 possesses canonical and functionally active molecular determinants critical to β-arrestin-2 recruitment.

Lgr5 is a membrane protein related to G protein-coupled receptors (GPCR)s whose expression identifies stem cells in multiple tissues and is strongly correlated with cancer. Despite the recent identification of endogenous ligands for Lgr5, its mode of signaling remains enigmatic. The ability to couple to G proteins and βarrestins are classical molecular behaviors of GPCRs that have yet to be observed for Lgr5. Therefore, the goal of this study was to determine if Lgr5 can engage a classical GPCR behavior and elucidate the molecular determinants of this process. Structural analysis of Lgr5 revealed several motifs consistent with its ability to recruit βarr2. Among them, a “SSS” serine cluster located at amino acid position 873-875 within the C-terminal tail (C-tail), is in a region consistent with other GPCRs that bind βarr2 with high-affinity. To test its functionality, a ligand-independent βarr2 translocation assay was implemented. We show that Lgr5 recruits βarr2 and that the “SSS” amino acids (873-875) are absolutely critical to this process. We also demonstrate that for full efficacy, this cluster requires other Lgr5 C-tail serines that were previously shown to be important for constitutive and βarr2 independent internalization of Lgr5. These data are proof of principle that a classical GPCR behavior can be manifested by Lgr5. The existence of alternative ligands or missing effectors of Lgr5 that scaffold this classical GPCR behavior and the downstream signaling pathways engaged should be considered. Characterizing Lgr5 signaling will be invaluable for assessing its role in tissue maintenance, repair, and disease.

Dopamine Transporter Endocytic Trafficking in Striatal Dopaminergic Neurons: Differential Dependence on Dynamin and the Actin Cytoskeleton

Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss. Longstanding evidence supports PKC-stimulated DAT trafficking in heterologous expression studies. However, PKC-stimulated DAT internalization is not readily observed in cultured dopaminergic neurons. Moreover, conflicting reports implicate both classic and nonclassic endocytic mechanisms mediating DAT trafficking. Prior DAT trafficking studies relied primarily upon chronic gene disruption and dominant-negative protein expression, or were performed in cell lines and cultured neurons, yielding results difficult to translate to adult dopaminergic neurons. Here, we use newly described dynamin inhibitors to test whether constitutive and PKC-stimulated DAT internalization are dynamin-dependent in adult dopaminergic neurons. Ex vivo biotinylation studies in mouse striatal slices demonstrate that acute PKC activation drives native DAT surface loss, and that surface DAT surprisingly partitions between endocytic-willing and endocytic-resistant populations. Acute dynamin inhibition reveals that constitutive DAT internalization is dynamin-independent, whereas PKC-stimulated DAT internalization is dynamin-dependent. Moreover, total internal reflection fluorescence microscopy experiments demonstrate that constitutive DAT internalization occurs equivalently from lipid raft and nonraft microdomains, whereas PKC-stimulated DAT internalization arises exclusively from lipid rafts. Finally, DAT endocytic recycling relies on a dynamin-dependent mechanism that acts in concert with the actin cytoskeleton. These studies are the first comprehensive investigation of native DAT trafficking in ex vivo adult neurons, and reveal that DAT surface dynamics are governed by complex multimodal mechanisms.

The Chemokine Receptor CCR1 Is Constitutively Active, Which Leads to G Protein-independent, β-Arrestin-mediated Internalization

Activation of G protein-coupled receptors by their associated ligands has been extensively studied, and increasing structural information about the molecular mechanisms underlying ligand-dependent receptor activation is beginning to emerge with the recent expansion in GPCR crystal structures. However, some GPCRs are also able to adopt active conformations in the absence of agonist binding that result in the initiation of signal transduction and receptor down-modulation. In this report, we show that the CC-type chemokine receptor 1 (CCR1) exhibits significant constitutive activity leading to a variety of cellular responses. CCR1 expression is sufficient to induce inhibition of cAMP formation, increased F-actin content, and basal migration of human and murine leukocytes. The constitutive activity leads to basal phosphorylation of the receptor, recruitment of β-arrestin-2, and subsequent receptor internalization. CCR1 concurrently engages Gαi and β-arrestin-2 in a multiprotein complex, which may be accommodated by homo-oligomerization or receptor clustering. The data suggest the presence of two functional states for CCR1; whereas receptor coupled to Gαi functions as a canonical GPCR, albeit with high constitutive activity, the CCR1·β-arrestin-2 complex is required for G protein-independent constitutive receptor internalization. The pertussis toxin-insensitive uptake of chemokine by the receptor suggests that the CCR1·β-arrestin-2 complex may be related to a potential scavenging function of the receptor, which may be important for maintenance of chemokine gradients and receptor responsiveness in complex fields of chemokines during inflammation.

Up-regulation of GABAB Receptor Signaling by Constitutive Assembly with the K+ Channel Tetramerization Domain-containing Protein 12 (KCTD12)

GABA(B) receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA(B) receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA(B1a), GABA(B1b), and GABA(B2) form fully functional heteromeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA(B) receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA(B) receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA(B) receptor-mediated K(+) current response. In summary, our experiments support that the up-regulation of functional GABA(B) receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.

Differential regulation of macropinocytosis in macrophages by cytokines: Implications for foam cell formation and atherosclerosis

A key event during the formation of lipid-rich foam cells during the progression of atherosclerosis is the uptake of modified low-density lipoproteins (LDL) by macrophages in response to atherogenic mediators in the arterial intima. In addition to scavenger receptor-dependent uptake of LDL, macropinocytosis is known to facilitate the uptake of LDL through the constitutive and passive internalization of large quantities of extracellular solute. In this study we confirm the ability of macropinocytosis to facilitate the uptake of modified LDL by human macrophages and show its modulation by TGF-β, IFN-γ, IL-17A and IL-33. Furthermore we show that the TGF-β-mediated inhibition of macropinocytosis is a Smad-2/-3-independent process.

Upstream Stimulatory Factors 1 and 2 Mediate the Transcription of Angiotensin II Binding and Inhibitory Protein

The angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP/Agtrap) promotes constitutive internalization of the AT1R so as to specifically inhibit the pathological activation of its downstream signaling yet preserve the base-line physiological signaling activity of the AT1R. Thus, tissue-specific regulation of Agtrap expression is relevant to the pathophysiology of cardiovascular and renal disease. However, the regulatory mechanism of Agtrap gene expression has not yet been fully elucidated. In this study, we show that the proximal promoter region from -150 to +72 of the mouse Agtrap promoter, which contains the X-box, E-box, and GC-box consensus motifs, is able to elicit substantial transcription of the Agtrap gene. Among these binding motifs, we showed that the E-box specifically binds upstream stimulatory factor (Usf) 1 and Usf2, which are known E-box-binding transcription factors. It is indicated that the E-box-Usf1/Usf2 binding regulates Agtrap expression because of the following: 1) mutation of the E-box to prevent Usf1/Usf2 binding reduces Agtrap promoter activity; 2) knockdown of Usf1 or Usf2 affects both endogenous Agtrap mRNA and Agtrap protein expression, and 3) the decrease in Agtrap mRNA expression in the afflicted kidney by unilateral ureteral obstruction is accompanied by changes in Usf1 and Usf2 mRNA. Furthermore, the results of siRNA transfection in mouse distal convoluted tubule cells and those of unilateral ureteral obstruction in the afflicted mouse kidney suggest that Usf1 decreases but Usf2 increases the Agtrap gene expression by binding to the E-box. The results also demonstrate a functional E-box-USF1/USF2 interaction in the human AGTRAP promoter, thereby suggesting that a strategy of modulating the E-box-USF1/USF2 binding has novel therapeutic potential.

Differences in the signaling pathways of α(1A)- and α(1B)-adrenoceptors are related to different endosomal targeting.

AimsTo compare the constitutive and agonist-dependent endosomal trafficking of α1A- and α1B-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype.MethodsUsing CypHer5 technology and VSV-G epitope tagged α1A- and α1B-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot).Results and ConclusionsConstitutive as well as agonist-induced trafficking of α1A and α1B ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α1A-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to β-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α1B-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-β-cyclodextrin

Pertuzumab counteracts the inhibitory effect of ErbB2 on degradation of ErbB3

Overexpression of ErbB2 and ErbB3 is found in several human cancers, and ErbB2-ErbB3 heterodimers are known as the most potent signaling units among ErbB dimers. While ErbB2 probably undergoes weak endocytosis, ErbB3 is readily internalized even in the absence of added ligand and without requirement for kinase activity. Overexpression of ErbB2 has been demonstrated to inhibit epidermal growth factor-induced internalization and degradation of epidermal growth factor receptor. This happens due to epidermal growth factor receptor-ErbB2 dimerization and can be counteracted by the anti-ErbB2 antibody pertuzumab, which binds the dimerization arm of ErbB2. Pertuzumab does also inhibit ErbB2-ErbB3 dimerization, but to what extent this has effect on constitutive and/or ligand-induced downregulation of ErbB3 is not known. In this study, we demonstrate that expression of ErbB2 as such did not block constitutive internalization of ErbB3, but that heregulin-induced degradation of ErbB3 was significantly slowed in cells expressing high levels of ErbB2. Incubation with pertuzumab did, however, counteract this effect. This indicates that the formation of ErbB2-ErbB3 heterodimers inhibits downregulation of ErbB3 and supports the notion that pertuzumab inhibits ErbB2 dimerization. The inhibitory effect of pertuzumab on ligand-induced ErbB2-ErbB3 heterodimerization was confirmed by the observation that pertuzumab inhibited heregulin-induced phosphorylation of ErbB3 in cells expressing ErbB2 and efficiently reduced heregulin-induced downstream signaling in cells expressing low levels of ErbB2. Altogether the results indicate that pertuzumab can be a valuable therapeutic agent not only in cancers overexpressing ErbB2 but also in cancers co-expressing ErbB2 and ErbB3.

Palmitoylation of Protease-activated Receptor-1 Regulates Adaptor Protein Complex-2 and -3 Interaction with Tyrosine-based Motifs and Endocytic Sorting

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface.