Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the alpha(2A) adrenergic receptor (alpha(2A) AR), with ERK activation mediated by the beta(2) adrenergic receptor (beta(2) AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the alpha(2A) AR, like that mediated by both the beta(2) AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant beta-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both alpha(2A) and beta(2) AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the beta(2) AR, the alpha(2A) AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although beta(2) AR stimulation leads to redistribution of both the beta(2) AR and EGFR, activation of the alpha(2A) AR leads to redistribution of the EGFR but the alpha(2A) AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.     

Androgen receptor is a tumor suppressor and proliferator in prostate cancer

Targeting androgens/androgen receptor (AR) functions via androgen deprivation therapy (ADT) remains the standard treatment for prostate cancer. However, most tumors eventually recur despite ADT. Here we demonstrate that the prostate AR may function as both a suppressor and a proliferator to suppress or promote prostate cancer metastasis. Results from orthotopically recombining stromal WPMY1 cells with epithelial PC3 prostate cancer cells in mice demonstrated that restoring AR in epithelial PC3 cells or knockdown of AR in stromal WPMY1 cells suppressed prostate cancer metastasis. Knockdown of the AR in epithelial CWR22rv1 prostate cancer cells also resulted in increased cell invasion in vitro and in vivo. Restoring AR in PC3 cells (PC3-AR9) results in decreased invasion in bone lesion assays and in vivo mouse models. Mice lacking the prostate epithelial AR have increased apoptosis in epithelial luminal cells and increased proliferation in epithelial basal cells. The consequences of these two contrasting results led to the expansion of CK5/CK8-positive intermediate cells, and mice developed larger and more invasive metastatic tumors in lymph nodes and died earlier than wild-type littermates. Mechanistic dissection suggested that androgens/AR might directly or indirectly modulate metastasis-related genes and suppression of TGFβ1 signals results in the partial inhibition of AR-mediated metastasis. Collectively, our understanding of these opposing roles of prostatic AR may revolutionize the way we combat prostate cancer, and allow the development of new and better therapies by targeting only the proliferative role of AR.     

雄激素受体对前列腺癌PC3细胞株增殖能力的影响

目的研究雄激素受体（AR）对雄激素非依赖性前列腺癌细胞株PC3增殖能力的影响。方法将含有AR的质粒（Pbabe-AR）稳定转染到PC3细胞,建立起新的细胞系PC3-AR,并用实时逆转录聚合酶链反应（Q—PCR）、蛋白免疫印迹试验（Western blot）和荧光素酶法确认AR的表达水平和功能;用MTT法、软琼脂糖凝胶实验检测AR对体外培养PC3细胞增殖的影响。结果PC3-AR细胞能够稳定表达有功能的AR;MTT实验显示PC3-AR细胞体外生长慢于PC3-v细胞,加入DHT以后效果更加明显。相对于PC3-v,PC3-AR在软琼脂糖凝胶中形成的克隆明显减少。结论AR能够降低PC3-AR细胞在体外的增殖能力。     

雄激素受体对前列腺癌细胞侵袭转移能力的影响及其意义

目的研究雄激素受体（AR）对雄激素非依赖性前列腺癌细胞株PC3侵袭转移能力的影响及意义。方法应用肿瘤细胞侵袭实验、创伤愈合实验以及软琼脂糖凝胶实验检测AR对PC3侵袭转移能力的影响。结果外源性AR转染到PC3（PC3-AR）后,其侵袭能力明显减弱,创伤愈合能力降低,在琼脂糖凝胶中形成克隆能力降低。结论AR明显降低雄激素非依赖性PC3的侵袭转移能力。     

Targeting the stromal androgen receptor in primary prostate tumors at earlier stages

To differentiate roles of androgen receptor (AR) in prostate stromal and epithelial cells, we have generated inducible-(ind)ARKO-TRAMP and prostate epithelial-specific ARKO TRAMP (pes-ARKO-TRAMP) mouse models, in which the AR was knocked down in both prostate epithelium and stroma or was knocked out in the prostate epithelium, respectively. We found that loss of AR in both mouse models resulted in poorly differentiated primary tumors with expanded intermediate cell populations. Interestingly, knockdown of both epithelial and stromal AR in ind-ARKO-TRAMP mice at earlier stages resulted in smaller primary prostate tumors with lower proliferation rates, and knockout of AR in pes-ARKO-TRAMP mice resulted in larger primary prostate tumors with higher proliferation rates. The differential proliferation rates, yet with similarly expanded intermediate cell populations, indicated that the prostate stromal AR might play a more dominant role than the epithelial AR to promote primary tumor proliferation at an early stage of tumor. Tissue recombination of human prostate stromal cell lines (WPMY1-v or WPMY1-ARsi) with human prostate cancer epithelial cell lines (PC3-v or PC3-AR9) further demonstrated that the AR might function as a suppressor in epithelial cells and a proliferator in stromal cells in the primary prostate tumors. The dual roles of the AR in prostate epithelium and stroma may require us to reevaluate the target and timing of androgen-deprivation therapy for prostate cancer patients and may suggest a need to develop new drugs to selectively target stromal AR in the primary prostate tumors at earlier stages.     

Src promotes destruction of c-Cbl: implications for oncogenic synergy between Src and growth factor receptors

Cellular Src and epidermal growth factor receptor (EGFR) collaborate in the progression of certain human malignancies, and their cooverexpression characterizes relatively aggressive animal tumors. Our study addressed the mode of oncogenic cooperation and reports that overexpression of c-Src in model cellular systems results in the accumulation of EGFR at the cell surface. The underlying mechanism involves inhibition of the normal, c-Cbl-regulated process of ligand-induced receptor down-regulation. In response to activation of c-Src, c-Cbl proteins undergo tyrosine phosphorylation that promotes their ubiquitylation and proteasomal destruction. Consequently, ubiquitylation of EGFR by c-Cbl is restrained in Src-transformed cells, and receptor sorting to endocytosis is impaired. In conclusion, by promoting destruction of c-Cbl, c-Src enables EGFR to evade desensitization, which explains Src-EGFR collaboration in oncogenesis.     

Dihydrotestosterone upregulates the expression of epidermal growth factor receptor and ERBB2 in androgen receptor-positive bladder cancer cells

Androgen receptor (AR) signals play important roles in bladder carcinogenesis and tumor progression. Activation of the epidermal growth factor receptor (EGFR) family, including EGFR and ERBB2, leads to bladder cancer cell growth and correlates with poor patients' prognosis. However, cross talk between AR and EGFR/ERBB2 pathways in bladder cancer remains poorly understood. In AR-positive bladder cancer UMUC3 and TCC-SUP cells, dihydrotestosterone (DHT) increased the expression of EGFR and ERBB2 both in mRNA and in protein levels, and an anti-androgen hydroxyflutamide antagonized the effect of DHT. The necessity of AR was confirmed by silencing the receptor, using short hairpin RNA (shRNA), in UMUC3 cells, as well as by expressing the receptor in AR-negative 5637 cells. Of note were much higher basal levels of EGFR and ERBB2 in UMUC3-control-shRNA than in UMUC3-AR-shRNA and those of EGFR in 5637-AR than in 5637-V. DHT additionally upregulated the levels of phosphorylation of EGFR (pEGFR) and its downstream proteins AKT (pAKT) and ERK1/2 (pERK), induced by EGF treatment, in AR-positive cells. Immunohistochemistry on cystectomy specimens showed strong associations between expressions of AR and EGFR (P=0.0136), pEGFR (P=0.0041), ERBB2 (P=0.0331), or pERK (P=0.0274), but not of pAKT (P=0.5555). The Kaplan-Meier and log-rank tests further revealed that positivity of AR (P=0.0005), EGFR (P=0.2425), pEGFR (P=0.1579), ERBB2 (P=0.2997), or pERK (P=0.1270) and negativity of pAKT (P=0.0483) were associated with tumor progression. Our results indicate that AR activation upregulates the expression of EGFR and ERBB2 in bladder cancer cells. AR signals may thus contribute to the progression of bladder cancer via regulation of the EGFR/ERBB2 pathways.     

Agonist activated adrenocorticotropin receptor internalizes via a clathrin-mediated G protein receptor kinase dependent mechanism

The physiological effects of the pituitary hormone, adrenocorticotropic hormone (ACTH) on the adrenal are mediated by the melanocortin 2 receptor (MC2R), a G protein coupled receptor (GPCR) that signals via adenylate cyclase to elevate intracellular cyclic AMP (cAMP) levels. The function and expression of the receptor is likely to be a major determinant of the response to ACTH. Following repeated stimulation, the cAMP signal is diminished or desensitized. Prolonged desensitization may involve internalization of the receptor. Internalization may occur by at least two mechanisms--receptor mediated endocytosis via clathrin-coated pits and by caveolae mediated internalization. The mode of internalization for the endogenous MC2R in Y1 cells was determined using radiolabelled ACTH. Treatment of Y1 cells with hypertonic sucrose or with concanavalin A, which inhibit clathrin-mediated endocytosis, blocked internalization. Filipin and nystatin, which inhibit caveolae formation, did not influence internalization. A dominant negative GRK2 inhibited internalization whilst the protein kinase A (PKA) consensus site mutant MC2R (S208A) internalized normally. However, dominant negative V53D beta-arrestin-1 did not inhibit ACTH internalization in Y1 cells. In conclusion, it appears that the MC2R in Y1 cells internalizes by a G protein coupled receptor kinase (GRK) dependent clathrin-coated pit mechanism.     

EGFR and HER2 receptor kinase signaling mediate epithelial cell invasion by Candida albicans during oropharyngeal infection

The fungus Candida albicans is the major cause of oropharyngeal candidiasis (OPC). A key feature of this disease is fungal invasion of oral epithelial cells, a process that can occur by active penetration and fungal-induced endocytosis. Two invasins, Als3 and Ssa1, induce epithelial cell endocytosis of C. albicans, in part by binding to E-cadherin. However, inhibition of E-cadherin function only partially reduces C. albicans endocytosis, suggesting that there are additional epithelial cell receptors for this organism. Here, we show that the EGF receptor (EGFR) and HER2 function cooperatively to induce the endocytosis of C. albicans hyphae. EGFR and HER2 interact with C. albicans in an Als3- and Ssa1-dependent manner, and this interaction induces receptor autophosphorylation. Signaling through both EGFR and HER2 is required for maximal epithelial cell endocytosis of C. albicans in vitro. Importantly, oral infection with C. albicans stimulates the phosphorylation of EGFR and HER2 in the oral mucosa of mice, and treatment with a dual EGFR and HER2 kinase inhibitor significantly decreases this phosphorylation and reduces the severity of OPC. These results show the importance of EGFR and HER2 signaling in the pathogenesis of OPC and indicate the feasibility of treating candidal infections by targeting the host cell receptors with which the fungus interacts.     

Insulin-induced surface redistribution regulates internalization of the insulin receptor and requires its autophosphorylation.

The role of insulin-induced receptor autophosphorylation in its internalization was analyzed by comparing 125I-labeled insulin (125I-insulin) internalization in Chinese hamster ovary (CHO) cell lines transfected with normal (CHO.T) or mutated insulin receptors. In four cell lines with a defect of insulin-induced autophosphorylation, 125I-insulin internalization was impaired. By contrast, in CHO.T cells and in two other CHO cell lines with amino acid deletions or insertions that do not perturb autophosphorylation, 125I-insulin internalization was not affected. A morphological analysis showed that the inhibition is linked to the ligand-specific surface redistribution in which the insulin-receptor complexes leave microvilli and concentrate on nonvillous segments of the membrane where endocytosis occurs.     

The ubiquitin-proteasome pathway regulates the availability of the GH receptor

GH promotes not only longitudinal growth in children but is active throughout life in protein, fat, and carbohydrate metabolism. The multiple actions of GH start when GH binds to the cell surface-expressed GH receptor. Effectiveness of the hormone depends both on its presence in the circulation and the availability of receptors at the cell surface of target cells. In this study, we examined the role of the ubiquitin-proteasome pathway in regulating GH receptor availability. We show that receptor turnover is rapid, and almost 3-fold prolonged in the internalization-deficient mutant GH receptor (F327A). Using a monovalent GH antagonist, B2036, we could quantify the internalization of the nonactivated receptor. By comparing internalization of the receptor with shedding of the GH-binding protein, we show that in Chinese hamster lung cell lines, internalization followed by lysosomal degradation is the major pathway for receptor degradation and that the ubiquitin-proteasome pathway controls this process. Inhibition of endocytosis resulted in a 200% increase in receptor availability at the cell surface at steady state.     

Role of protein kinase Calpha in melatonin signal transduction

Melatonin induces nuclear exclusion of the androgen receptor (AR) via activation of protein kinase C (PKC). The specific members of the PKC superfamily involved in AR nuclear exclusion were investigated in prostate cancer PC3 cells stably transfected with the wild-type androgen receptor (PC3-AR). PKCalpha was essentially cytoplasmic whereas PKCbeta and PKCepsilon were essentially membranal, suggesting their constitutive activity in the PC3-AR cells. Melatonin treatment induced membrane association of PKCalpha in a time and dose dependent manner. The PKCalpha and PKCbeta1 specific inhibitor GO6976 and the PKCbeta isoform-specific inhibitor hispidin had no effects on AR localization under basal conditions. However, GO6976 but not hispidin negated the melatonin-mediated nuclear exclusion of the AR. These data indicate that PKCalpha activation is a critical step in AR nuclear exclusion by melatonin. They also imply that PKCalpha-activation is a potentially effective way to control of the AR activity in prostate cancer cells.     

Bovine Parainfluenza Virus Type 3 (BPIV3) Enters HeLa Cells via Clathrin-Mediated Endocytosis in a Cholesterol- and Dynamin-Dependent Manner

Bovine parainfluenza virus 3 (BPIV3) is a crucial causative agent of respiratory disease in young and adult cattle. No specific therapies are available for BPIV3 infection. Understanding the internalization pathway of the virus will provide a new strategy for the development of antiviral therapy. Here, the mechanism of BPIV3 entry into HeLa cells was analyzed using RNA silencing and pharmacological inhibitors. Treatment of HeLa cells with hypertonic medium prevented BPIV3 internalization. These results indicated that BPIV3 entered HeLa cells via receptor-mediated endocytosis. Moreover, removing cell membrane cholesterol through MβCD treatment hampered viral penetration but not viral replication. In addition, BPIV3 infection was inhibited by pretreatment with dynasore or chlorpromazine (CPZ) or knockdown of dynamin II or clathrin heavy chain. However, virus entry was unaffected by nystatin, EIPA, wortmannin, or cytochalasin D treatment or caveolin-1 knockdown. These data demonstrated that the entry of BPIV3 into HeLa cells was dependent on clathrin-mediated endocytosis but not on caveolae-mediated endocytosis or the macropinocytosis pathway. Many viruses are transported to endosomes, which provide an acidic environment and release their genome upon separation from primary endocytic vesicles. However, we found that BPIV3 infection required endosomal cathepsins, but not a low pH. In summary, we show, for the first time, that BPIV3 enters HeLa cells through the clathrin-mediated endocytosis pathway, presenting novel insights into the invasion mechanism of Paramyxoviridae.     

Nanoconjugation modulates the trafficking and mechanism of antibody induced receptor endocytosis

Treatment with monoclonal antibody (mAbs) is a viable therapeutic option in cancer. Recently, these mAbs such as cetuximab, herceptin, etc., have been used as targeting agents to selectively deliver chemotherapeutics to cancerous cells. However, mechanisms of nanoparticles-mAbs interactions with the target cells and its effect on intracellular trafficking and mechanism are currently unknown. In this paper, we demonstrate that the distinct patterning and dynamics of anti-EGFR (epidermal growth factor receptor) antibody cetuximab (C225)- induced EGFR internalization in pancreatic cancer cells with variable receptor expression is altered upon nanoconjugation. Nanoconjugation uniformly enhanced C225-induced EGFR endocytosis in PANC-1, AsPC-1, and MiaPaca-2 cells, influenced its compartmentalization and regulated the involvement of dynamin-2 in the endocytic processes. Receptor endocytosis and its intracellular trafficking were monitored by confocal microscopy and transmission electron microscopy. The role of dynamin-2 in EGFR endocytosis was determined after overexpressing either wild-type dynamin-2 or mutant dynamin-2 in pancreatic cancer cells followed by tracking the receptor-antibody complex internalization by confocal microscopy. Significantly, these findings demonstrate that the nanoconjugation cannot be construed as an innocuous reaction involved in attaching the targeting agent to the nanoparticle, instead it may distinctly alter the cellular processes at the molecular level, at least antibody induced receptor endocytosis. This information is critical for successful design of a nanoparticle-based targeted drug delivery system for future clinical translation.     

MT1 melatonin receptor internalization underlies melatonin-induced morphologic changes in Chinese hamster ovary cells and these processes are dependent on Gi proteins, MEK 1/2 and microtubule modulation

Abstract:  Melatonin induces cellular differentiation in numerous cell types. Data show that multiple mechanisms are involved in these processes that are cell-type specific and may be receptor dependent or independent. The focus of this study was to specifically assess the role of human MT1 melatonin receptors in cellular differentiation using an MT1-Chinese hamster ovary (CHO) model; one that reproducibly produces measurable morphologic changes in response to melatonin. Using multiple approaches, we show that melatonin induces MT1-CHO cells to hyperelongate through a MEK 1/2, and ERK 1/2-dependent mechanism that is dependent upon MT1 receptor internalization, Gi protein activation, and clathrin-mediated endocytosis. Using immunoprecipitation analysis, we show that MT1 receptors form complexes with Gi_α 2,3, Gq_α, β-arrestin-2, MEK 1/2, and ERK 1/2 in the presence of melatonin. We also show that MEK and ERK activity that is induced by melatonin is dependent on Gi protein activation, clathrin-mediated endocytosis and is modulated by microtubules. We conclude from these studies that melatonin-induced internalization of human MT1 melatonin receptors in CHO cells is responsible for activating both MEK 1/2 and ERK 1/2 to drive these morphologic changes. These events, as mediated by melatonin, require Gi protein activation and endocytosis mediated through clathrin, to form MT1 receptor complexes with β-arrestin-2/MEK 1/2 and ERK 1/2. The MT1-CHO model is invaluable to mapping out signaling cascades as mediated through MT1 receptors especially because it separates out MEK/ERK 1/2 activation by MT1 receptors from that of receptor tyrosine kinases.     

Visualization of the thyrotropin-releasing hormone receptor and its ligand during endocytosis and recycling.

Endocytosis and recycling of both thyrotropin-releasing hormone (TRH) and its G-protein-coupled receptor were visualized by conventional and confocal fluorescence microscopy in pituitary cells using a rhodamine-labeled TRH analog (Rhod-TRH) and indirect immunofluorescent staining of cells stably transfected with an epitope-tagged TRH receptor (TRHR). The epitope-tagged TRHR was confined to the cell surface prior to agonist treatment. Both Rhod-TRH and TRHR were also localized on the plasma membrane after agonist binding at 0 degrees C. Ligand binding at 37 degrees C resulted in rapid endocytosis, and both Rhod-TRH and the epitope-tagged TRHR appeared in cytoplasmic vesicles within 5 min. Fluorescently labeled TRH and transferrin colocalized in the same endocytotic vesicles, and internalization of Rhod-TRH and TRHR was inhibited by hypertonic medium, suggesting that endocytosis occurred by a clathrin-dependent mechanism. Internalized TRHRs returned to the membrane within 20 min after removal of TRH, and cycloheximide did not block receptor recycling. A mutant TRHR truncated at Cys335 signaled but did not internalize Rhod-TRH, confirming the importance of the carboxyl terminus of the TRHR in receptor-mediated endocytosis. Thus, the TRH-TRHR complex is endocytosed via clathrin-coated vesicles and the receptor is recycled to the plasma membrane.     

YRRL motifs in the cytoplasmic domain of the thrombopoietin receptor regulate receptor internalization and degradation

Thrombopoietin (Tpo), acting through the c-Mpl receptor, promotes the survival and proliferation of hematopoietic stem and progenitor cells and drives megakaryocyte differentiation. The proproliferation and survival signals activated by Tpo must therefore be tightly regulated to prevent uncontrolled cell growth. In this work, we determined the mechanisms that control Tpo-stimulated c-Mpl internalization and defined the processes leading to its degradation. Stimulation of BaF-Mpl cells with Tpo leads to rapid, clathrin-dependent endocytosis of the receptor. Using small interfering RNA (siRNA), we found that inhibition of adaptor protein 2 (AP2), which mediates endocytosis of transmembrane proteins, strongly attenuates Tpo-stimulated c-Mpl internalization. AP2 interacts with YXXPhi motifs and we identified 2 such motifs in c-Mpl (Y(8)RRL and Y(78)RRL) and investigated Tpo-stimulated internalization of receptors bearing point mutations at these sites. After Tpo stimulation, internalization was greatly reduced in c-Mpl Y(78)F and c-Mpl Y(8+78)F, and these cell lines also exhibited increased proliferation and increased strength and duration of Jak2, STAT5, AKT, and ERK1/2 activation in response to Tpo. We also found that the Y(8)RRL motif regulates Tpo-stimulated lysosomal degradation of c-Mpl. Our data establishes that c-Mpl cytoplasmic YRRL motifs are responsible for both Tpo-mediated internalization via interactions with AP2 and lysosomal targeting after endocytosis.     

Receptor-specific mechanisms of desensitization of β-adrenergic receptor function

β-Adrenergic receptor (βAR)-specific, agonist-induced desensitization of adenylate cyclase can be shown in most mammalian cells examined to involve at least three reactions. An initial ‘uncoupling’ reaction leads to a 40–60% loss of catecholamine-stimulated adenylate cyclase activity at a time when no detectable loss of βAR has occurred. This process precedes by 45–90 sec the appearance of βAR in cytoplasmic vesicles. Such βAR exhibit ligand binding properties consistent with their existence on the inside of membrane vesicles; thus, they appear to be formed by a process of agonist-induced βAR internalization (endocytosis). A third process results in the loss of βAR, at least in some cases due to receptor degradation. In general, agonist-induced desensitization or down-regulation reactions do not require protein synthesis. Recovery from the desensitized states does not require protein synthesis, whereas recovery from βAR down-regulation (degraded receptors) requires new receptor synthesis. Agonist-induced βAR desensitization and down-regulation reactions appear to have much in common with the process of polypeptide hormone-induced receptor down-regulation. The availability of a large number of ligands (agonists, partial agonists and antagonists) for the βAR should allow the use of this receptor system to gain unique insights into the general processes of ligand-induced, cell surface receptor endocytosis.