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01/2013; , ISBN: 978-0-12-382219-2
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ABSTRACT: Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by intramolecular docking of a tethered ligand that is released by the actions of proteases, mainly of the serine protease family. Here, we evaluate four commercially available anti-PAR2 antibodies, SAM11, C17, N19 and H99, demonstrating marked differences in the ability of these reagents to detect the target receptor in Western blot, immunocytochemical and flow cytometry applications. In Western blot analysis, we evaluated antibody reactivity against both ectopic and endogenous receptors. Against material from transfected cells, we show that SAM11 and N19, and to a lesser extent C17, but not H99, are able to detect ectopic PAR2. Interestingly, these Western blot analyses indicate that N19 and C17 detect conformations of ectopic PAR2 distinct to those recognised by SAM11. Significantly, our data also indicate that Western blot signal detected by SAM11 and C17, and much of the signal detected by N19, against cells endogenously expressing PAR2 is non-specific. Despite confounding non-specific signals, we were able to discern N19 reactivity against endogenous PAR2 as a broad smear that we also observed in ectopically expressing human and mouse cells and that is sensitive to loss of N-glycosylation. In immunocytochemistry analysis, each antibody is able to detect ectopic PAR2 although it appears that H99 detects only a subset of the ectopically expressed receptor. In addition, SAM11 and N19 are able to detect both ectopic and endogenous cell surface PAR2 by flow cytometry. In summary: (1) each antibody can detect ectopic PAR2 by immunocytochemical analysis with SAM11 and N19 suitable for cell surface detection of both ectopic and endogenous receptor by flow cytometry; (2) in Western blot analysis, N19, SAM11 and C17 can detect ectopically expressed PAR2, with only N19 able to detect the endogenous receptor by this technique and (3) in each of these approaches, appropriate controls are essential to ensure that non-specific reactivity is identified.
Archiv für Experimentelle Pathologie und Pharmakologie 07/2012; 385(9):861-73. · 2.65 Impact Factor
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ABSTRACT: Stimulation of the androgen receptor via bioavailable androgens, including testosterone and testosterone metabolites, is a key driver of prostate development and the early stages of prostate cancer. Androgens are hydrophobic and as such require carrier proteins, including sex hormone-binding globulin (SHBG), to enable efficient distribution from sites of biosynthesis to target tissues. The similarly hydrophobic corticosteroids also require a carrier protein whose affinity for steroid is modulated by proteolysis. However, proteolytic mechanisms regulating the SHBG/androgen complex have not been reported. Here, we show that the cancer-associated serine proteases, kallikrein-related peptidase (KLK)4 and KLK14, bind strongly to SHBG in glutathione S-transferase interaction analyses. Further, we demonstrate that active KLK4 and KLK14 cleave human SHBG at unique sites and in an androgen-dependent manner. KLK4 separated androgen-free SHBG into its two laminin G-like (LG) domains that were subsequently proteolytically stable even after prolonged digestion, whereas a catalytically equivalent amount of KLK14 reduced SHBG to small peptide fragments over the same period. Conversely, proteolysis of 5α-dihydrotestosterone (DHT)-bound SHBG was similar for both KLKs and left the steroid binding LG4 domain intact. Characterization of this proteolysis fragment by [(3)H]-labeled DHT binding assays revealed that it retained identical affinity for androgen compared with full-length SHBG (dissociation constant = 1.92 nM). Consistent with this, both full-length SHBG and SHBG-LG4 significantly increased DHT-mediated transcriptional activity of the androgen receptor compared with DHT delivered without carrier protein. Collectively, these data provide the first evidence that SHBG is a target for proteolysis and demonstrate that a stable fragment derived from proteolysis of steroid-bound SHBG retains binding function in vitro.
Endocrinology 04/2012; 153(7):3179-89. · 4.46 Impact Factor
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ABSTRACT: Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.
Journal of Biological Chemistry 02/2012; 287(13):9792-803. · 4.77 Impact Factor
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ABSTRACT: Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.
Journal of Biological Chemistry 12/2011; 286(49):42303-15. · 4.77 Impact Factor
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ABSTRACT: Discovered in the 1990s, protease activated receptors(1) (PARs) are membrane-spanning cell surface proteins that belong to the G protein coupled receptor (GPCR) family. A defining feature of these receptors is their irreversible activation by proteases; mainly serine. Proteolytic agonists remove the PAR extracellular amino terminal pro-domain to expose a new amino terminus, or tethered ligand, that binds intramolecularly to induce intracellular signal transduction via a number of molecular pathways that regulate a variety of cellular responses. By these mechanisms PARs function as cell surface sensors of extracellular and cell surface associated proteases, contributing extensively to regulation of homeostasis, as well as to dysfunctional responses required for progression of a number of diseases. This review examines common and distinguishing structural features of PARs, mechanisms of receptor activation, trafficking and signal termination, and discusses the physiological and pathological roles of these receptors and emerging approaches for modulating PAR-mediated signaling in disease.
Pharmacology [?] Therapeutics 01/2011; 130(3):248-82. · 8.56 Impact Factor
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ABSTRACT: Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca²⁺ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor.
PLoS ONE 01/2011; 6(11):e28018. · 4.09 Impact Factor
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ABSTRACT: CUB-domain-containing protein 1 (CDCP1) is an integral membrane glycoprotein with potential as a marker and therapeutic target for a number of cancers. Here we examine mechanisms regulating cellular processing of CDCP1. By analyzing cell lines exclusively passaged non-enzymatically and through use of a panel of protease inhibitors, we demonstrate that full-length 135 kDa CDCP1 is post-translationally processed in a range of cell lines by a mechanism involving serine protease activity, generating a C-terminal 70-kDa fragment. Immunopurification and N-terminal sequencing of this cell-retained fragment and detailed mutagenesis, show that proteolytic processing of CDCP1 occurs at two sites, Arg-368 and Lys-369. We show that the serine protease matriptase is an efficient, but not essential, cellular processor of CDCP1 at Arg-368. Importantly, we also demonstrate that proteolysis induces tyrosine phosphorylation of 70-kDa CDCP1 and recruitment of Src and PKCdelta to this fragment. In addition, Western blot and mass spectroscopy analyses show that an N-terminal 65-kDa CDCP1 ectodomain is shed intact from the cell surface. These data provide new insights into mechanisms regulating CDCP1 and suggest that the biological role of this protein and, potentially, its function in cancer, may be mediated by both 70-kDa cell retained and 65-kDa shed fragments, as well as the full-length 135-kDa protein.
Journal of Biological Chemistry 08/2010; 285(34):26162-73. · 4.77 Impact Factor
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ABSTRACT: The serine proteases of the trypsin-like (S1) family play critical roles in many key biological processes including digestion, blood coagulation, and immunity. Members of this family contain N- or C-terminal domains that serve to tether the serine protease catalytic domain directly to the plasma membrane. These membrane-anchored serine proteases are proving to be key components of the cell machinery for activation of precursor molecules in the pericellular microenvironment, playing vital functions in the maintenance of homoeostasis. Substrates activated by membrane-anchored serine proteases include peptide hormones, growth and differentiation factors, receptors, enzymes, adhesion molecules and viral coat proteins. In addition, new insights into our understanding of the physiological functions of these proteases and their involvement in human pathology have come from animal models and patient studies. The present review discusses emerging evidence for the diversity of this fascinating group of membrane serine proteases as potent modifiers of the pericellular microenvironment through proteolytic processing of diverse substrates. We also discuss the functional consequences of the activities of these proteases on mammalian physiology and disease.
Biochemical Journal 06/2010; 428(3):325-46. · 4.90 Impact Factor
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ABSTRACT: Prostate cancer is the most common cancer and a leading cause of death among male patients in industrial countries. Kallikrein-related peptidase 4 (KLK4) is a trypsin-like serine protease predominantly expressed in basal and secretory cells of the prostate gland which is overexpressed in prostate tumours. KLK4 overexpression increases in vitro cell migration and proliferation rates and has the proteolytic ability to activate many tumorigenic and metastatic pathways including the protease activated receptors (PARs). A lack of KLK4-specific small-molecule inhibitors available for therapeutic development prompted us to examine the sunflower trypsin inhibitor (SFTI), a small (1.4 kDa) circular Bowman-Birk serine protease inhibitor, as a potential bioscaffold to regulate KLK4 activity.
Here we demonstrate that the amidolytic (but not proteolytic) activity of KLK4 was blocked by SFTI. We then used SFTI as a scaffold for designing inhibitors with improved specificity and the ability to block KLK4’s proteolytic activity. Initially we probed KLK4’s active site using a 125 tetra-peptide sparse matrix library, selecting the optimal substrate sequence to guide re-engineering of SFTI. The resulting inhibitor (SFTI-FCQR) showed highly selective inhibition of KLK4 against peptide substrates (Ki of 3.6 ± 0.3 nM) and the macromolecular substrate fibrinogen, blocked KLK4 initiated calcium flux from PAR2 and displayed remarkable stability in cell based assays with a half life of four days.
SFTI’s constrained geometry and circular backbone prevent the use of linear peptide libraries for screening beyond the P1-P4 residues. Therefore we set out to further optimize SFTI-FCQR in silico. Screening of a SFTI-FCQR-based library using the Charmm27 force field allowed design of a second generation KLK4 inhibitor (SFTI-FCQR-N14) with greatly enhanced potency (IC50 = 63.5 ± 2.4 pM), specificity and undiminished half-life. Pharmacokinetic analysis in mice has shown 68.9% bioavailability by intravenous perfusion (3 mg/kg) and a plasma clearance time of 4 hours.
This study is the first demonstration that efficient substrate sequences for a target protease can be directly grafted onto the SFTI scaffold to enhance inhibition. In addition, SFTI-FCQR-N14 provides a lead molecule for development of a KLK4-targeted prostate cancer therapeutic and is currently undergoing in vivo assessment using a prostate cancer mouse model.
ASMR Queensland Postgraduate Student Conference, Brisbane, Queensland; 06/2010
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ABSTRACT: The classical action of androgen receptor (AR) is to regulate gene transcriptional processes via AR nuclear translocation, response element binding and recruitment of, or crosstalk with, transcription factors. AR also utilises non-classical, non-genomic mechanisms of signal transduction. These precede gene transcription or protein synthesis, and involve steroid-induced modulation of cytoplasmic or cell membrane-bound regulatory proteins. Despite many decades of investigation, the role of AR in gene regulation of cells and tissues remains only partially characterised. AR exerts most of its effects in sex hormone-dependent tissues of the body, but the receptor is also expressed in many tissues not previously thought to be androgen sensitive. Thus it is likely that a complex, more over-arching, role for AR exists. Each AR domain co-ordinates a multitude of individual and vital roles via a diverse array of interacting partner molecules that are necessary for cellular and tissue development and maintenance. Aberrant AR activity, promoted by mutations or binding partner misregulation, can present as many clinical manifestations including androgen insensitivity syndrome and prostate cancer. In the case of malignant prostate cancer, treatment generally revolves around androgen deprivation therapies designed to interfere with AR action and the androgen signalling axis. Androgen therapies for prostate cancer often fail, highlighting a real need for increased research into AR function.
The international journal of biochemistry & cell biology 11/2009; 42(6):813-27. · 4.89 Impact Factor
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ABSTRACT: The androgen receptor (AR) is involved in the development and maintenance of the normal prostate and the development and progression of prostate cancer (PCa). Caveolin-1 (cav-1) is an AR co-regulator. The expression of this integral membrane protein is upregulated in PCa and correlates positively with its development. This review focuses on the likely interactive roles of AR and cav-1, with particular reference to progression to androgen-insensitivity in PCa. The classical role of AR is modulation of gene transcription by binding specific DNA sequences called androgen response elements in the promoter regions of target genes. To carry out this role, AR interacts with many co-regulator proteins which either enhance or repress its activation. Altered expression or misregulated activation of a co-regulator protein may significantly alter AR activity and the basal transcription rate of androgen responsive genes. Cav-1 has roles in cell signalling and trafficking, roles that are important in PCa survival, metastasis, and the development of multidrug resistant phenotypes. Although cav-1 appears to increase AR genomic activity and increase tumor cell survival, there is also mounting evidence that cav-1 can manipulate rapid, non-genomic AR signalling at the plasma membrane. By increasing our understanding of cav-1 as an AR co-regulator, we may be able to reinstate appropriate transcriptional responses to androgen signalling and minimise misregulated AR activity, thus permitting more effective targeted therapies for PCa.
International Union of Biochemistry and Molecular Biology Life 10/2009; 61(10):961-70. · 3.51 Impact Factor
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ABSTRACT: The function of CUB domain-containing protein 1 (CDCP1), a recently described transmembrane protein expressed on the surface of hematopoietic stem cells and normal and malignant cells of different tissue origin, is not well defined. The contribution of CDCP1 to tumor metastasis was analyzed by using HeLa carcinoma cells overexpressing CDCP1 (HeLa-CDCP1) and a high-disseminating variant of prostate carcinoma PC-3 naturally expressing high levels of CDCP1 (PC3-hi/diss). CDCP1 expression rendered HeLa cells more aggressive in experimental metastasis in immunodeficient mice. Metastatic colonization by HeLa-CDCP1 was effectively inhibited with subtractive immunization-generated, CDCP1-specific monoclonal antibody (mAb) 41-2, suggesting that CDCP1 facilitates relatively late stages of the metastatic cascade. In the chick embryo model, time- and dose-dependent inhibition of HeLa-CDCP1 colonization by mAb 41-2 was analyzed quantitatively to determine when and where CDCP1 functions during metastasis. Quantitative PCR and immunohistochemical analyses indicated that CDCP1 facilitated tumor cell survival soon after vascular arrest. Live cell imaging showed that the function-blocking mechanism of mAb 41-2 involved enhancement of tumor cell apoptosis, confirmed by attenuation of mAb 41-2-mediated effects with the caspase inhibitor z-VAD-fmk. Under proapoptotic conditions in vitro, CDCP1 expression conferred HeLa-CDCP1 cells with resistance to doxorubicin-induced apoptosis, whereas ligation of CDCP1 with mAb 41-2 caused additional enhancement of the apoptotic response. The functional role of naturally expressed CDCP1 was shown by mAb 41-2-mediated inhibition of both experimental and spontaneous metastasis of PC3-hi/diss. These findings confirm that CDCP1 functions as an antiapoptotic molecule and indicate that during metastasis CDCP1 facilitates tumor cell survival likely during or soon after extravasation.
Molecular Cancer Research 09/2009; 7(8):1197-211. · 4.29 Impact Factor
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ABSTRACT: An estimated 25%-40% of infertile men have idiopathic infertility associated with deficient sperm numbers and quality. Here, we identify the membrane-anchored serine protease PRSS21, also known as testisin, to be a novel proteolytic factor that directs epididymal sperm cell maturation and sperm-fertilizing ability. PRSS21-deficient spermatozoa show decreased motility, angulated and curled tails, fragile necks, and dramatically increased susceptibility to decapitation. These defects reflect aberrant maturation during passage through the epididymis, because histological and electron microscopic structural analyses showed an increased tendency for curled and detached tails as spermatozoa transit from the corpus to the cauda epididymis. Cauda epididymal spermatozoa deficient in PRSS21 fail to mount a swelling response when exposed to hypotonic conditions, suggesting an impaired ability to respond to osmotic challenges facing maturing spermatozoa in the female reproductive tract. These data suggest that aberrant regulation of PRSS21 may underlie certain secondary male infertility syndromes, such as "easily decapitated" spermatozoa in humans.
Biology of Reproduction 08/2009; 81(5):921-32. · 4.01 Impact Factor
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Joakim E Swedberg,
Laura V Nigon,
Janet C Reid,
Simon J de Veer,
Carina M Walpole,
Carson R Stephens,
Terry P Walsh,
Thomas K Takayama, John D Hooper,
Judith A Clements,
Ashley M Buckle,
Jonathan M Harris
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ABSTRACT: Human kallikrein-related peptidase 4 (KLK4/prostase), a trypsin-like serine protease, is a potential target for prostate cancer treatment because of its proteolytic ability to activate many tumorigenic and metastatic pathways including the protease activated receptors (PARs). Currently there are no KLK4-specific small-molecule inhibitors available for therapeutic development. Here we re-engineer the naturally occurring sunflower trypsin inhibitor to selectively block the proteolytic activity of KLK4 and prevent stimulation of PAR activity in a cell-based system. The re-engineered inhibitor was designed using a combination of molecular modeling and sparse matrix substrate screening.
Chemistry & biology 07/2009; 16(6):633-43. · 6.52 Impact Factor
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ABSTRACT: In the last few years dysregulated expression of the cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) has been associated with several cancers and this cell surface molecule has been recognized both as a tumor marker and as a potential target to disrupt progression of cancer. Here we summarize what is known about CDCP1 including its structural features, expression in normal and cancerous tissues, and the in vitro experiments and studies in animal models that have provided the key insights into its potential role in tumor formation and metastasis in humans. We conclude by highlighting opportunities and challenges in targeting CDCP1 in cancer.
International Union of Biochemistry and Molecular Biology Life 07/2009; 61(7):723-30. · 3.51 Impact Factor
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ABSTRACT: Maintaining the body's levels of iron within precise boundaries is essential for normal physiological function. Alterations of these levels below or above the healthy limit lead to a systemic deficiency or overload in iron. The type-two transmembrane serine protease (TTSP), matriptase-2 (also known as TMPRSS6), is attracting significant amounts of interest due to its recently described role in iron homeostasis. The finding of this regulatory role for matriptase-2 was originally derived from the observation that mice deficient in this protease present with anemia due to elevated levels of hepcidin and impaired intestinal iron absorption. Further in vitro analysis has demonstrated that matriptase-2 functions to suppress bone morphogenetic protein stimulation of hepcidin transcription through cell surface proteolytic processing of the bone morphogenetic protein co-receptor hemojuvelin. Consistently, the anemic phenotype of matriptase-2 knockout mice is mirrored in humans with matripase-2 mutations. Currently, 14 patients with iron-refractory iron deficiency anemia (IRIDA) have been reported to harbor various genetic mutations that abrogate matriptase-2 proteolytic activity. In this review, after overviewing the membrane anchored serine proteases, in particular the TTSP family, we summarize the identification and characterization of matriptase-2 and describe its functional relevance in iron metabolism.
Haematologica 05/2009; 94(6):840-9. · 6.42 Impact Factor
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ABSTRACT: The Tweety proteins comprise a family of chloride ion channels with three members identified in humans (TTYH1-3) and orthologues in fly and murine species. In humans, increased TTYH2 expression is associated with cancer progression, whereas fly Tweety is associated with developmental processes. Structurally, Tweety proteins are characterized by five membrane-spanning domains and N-glycan modifications important for trafficking to the plasma membrane, where these proteins are oriented with the amino terminus located extracellularly and the carboxyl terminus cytoplasmically. In addition to N-glycosylation, ubiquitination mediated by the HECT type E3 ubiquitin ligase Nedd4-2 is a post-translation modification important in regulating membrane proteins. In the present study, we performed a comprehensive analysis of the ability of each of TTYH1-3 to interact with Nedd4-2 and to be ubiquitinated and regulated by this ligase. Our data indicate that Nedd4-2 binds to two family members, TTYH2 and TTYH3, which contain consensus PY ((L/P)PXY) binding sites for HECT type E3 ubiquitin ligases, but not to TTYH1, which lacks this motif. Consistently, Nedd4-2 ubiquitinates both TTYH2 and TTYH3. Importantly, we have shown that endogenous TTYH2 and Nedd4-2 are binding partners and demonstrated that the TTYH2 PY motif is essential for these interactions. We have also shown that Nedd4-2-mediated ubiquitination of TTYH2 is a critical regulator of cell surface and total cellular levels of this protein. These data, indicating that Nedd4-2 differentially interacts with and regulates TTYH1-3, will be important for understanding mechanisms controlling Tweety proteins in physiology and disease.
Journal of Biological Chemistry 07/2008; 283(35):24000-10. · 4.77 Impact Factor
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ABSTRACT: The Tweety proteins are a family of recently identified putative Cl(-) channels predicted to be modified by N-glycosylation and, controversially, to contain five or six membrane-spanning domains, leading to the contentious proposal that members of this family do not share the same topology at the plasma membrane. In humans, three family members have been identified, designated TTYH1 (Tweety homologue 1), TTYH2 and TTYH3. To gain greater insight into the arrangement of membrane-spanning domains and cellular processing of Tweety proteins, in the present study we have examined the sequence homology, hydrophobicity and N-glycan content of members of this family and performed N-glycosylation site-mutagenesis studies on TTYH2 and TTYH3. Based on these observations we propose a structure for Tweety family proteins which incorporates five membrane-spanning domains with a topology at the cell surface in which the N-terminus is located extracellularly and the C-terminus cytoplasmically. Our results also suggest that N-glycosylation is important, but not essential, in the processing of members of the Tweety family with results indicating that, although incomplete N-glycosylation mediates reduced expression and increased ubiquitination of TTYH2, N-glycosylation is not the determining factor for TTYH2 trafficking to the plasma membrane. This information will be important for the characterization of Tweety family proteins in normal physiology and disease.
Biochemical Journal 06/2008; 412(1):45-55. · 4.90 Impact Factor
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ABSTRACT: Kallikrein-related peptidase 4 (KLK4) is one of the 15 members of the human KLK family and a trypsin-like, prostate cancer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasma membrane primarily by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. Here we show, using Ca(2+) flux assays, that KLK4 signals via both PAR-1 and PAR-2 but not via PAR-4. Dose-response analysis over the enzyme concentration range 0.1-1000 nM indicated that KLK4-induced Ca(2+) mobilization via PAR-1 is more potent than via PAR-2, whereas KLK4 displayed greater efficacy via the latter PAR. We confirmed the specificity of KLK4 signaling via PAR-2 using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressing and small interfering RNA-mediated receptor knockdown cell lines. Consistently, confocal microscopy analyses indicated that KLK4 initiates loss of PAR-2 from the cell surface and receptor internalization. Immunohistochemical analysis indicated the co-expression of agonist and PAR-2 in primary prostate cancer and bone metastases, suggesting that KLK4 signaling via this receptor will have pathological relevance. These data provide insight into KLK4-mediated cell signaling and suggest that signals induced by this enzyme via PARs may be important in prostate cancer.
Journal of Biological Chemistry 06/2008; 283(18):12293-304. · 4.77 Impact Factor
