Cell Tissue Res DOI 10.1007/s00441-014-2056-9
REGULAR ARTICLE
The intracellular carboxyl tail of the PAR-2 receptor controls intracellular signaling and cell death Zhihui Zhu & Rolf Stricker & Rong yu Li & Gregor Zündorf & Georg Reiser
Received: 22 June 2014 / Accepted: 6 November 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The protease-activated receptors are a group of unique G protein-coupled receptors, including PAR-1, PAR2, PAR-3 and PAR-4. PAR-2 is activated by multiple trypsinlike serine proteases, including trypsin, tryptase and coagulation proteases. The clusters of phosphorylation sites in the PAR-2 carboxyl tail are suggested to be important for the binding of adaptor proteins to initiate intracellular signaling to Ca2+ and mitogen-activated protein kinases. To explore the functional role of PAR-2 carboxyl tail in controlling intracellular Ca2+, ERK and AKT signaling, a series of truncated mutants containing different clusters of serines/threonines were generated and expressed in HEK293 cells. Firstly, we observed that lack of the complete C-terminus of PAR-2 in a mutated receptor gave a relatively low level of localization on the cell plasma membrane. Secondly, the shortened carboxyl tail containing 13 amino acids was sufficient for receptor internalization. Thirdly, the cells expressing truncation mutants showed deficits in their capacity to couple to intracellular Ca2+ and ERK and AKT signaling upon trypsin challenge. In addition, HEK293 cells carrying different PAR-2 truncation mutants displayed decreased levels of cell survival after long-lasting trypsin stimulation. In summary, the PAR-2 carboxyl tail was found to control the receptor localization, internalization, intracellular Ca2+ responses and signaling to ERK and AKT. The latter can be considered to be important for cell death control.
Electronic supplementary material The online version of this article (doi:10.1007/s00441-014-2056-9) contains supplementary material, which is available to authorized users. Z. Zhu : R. Stricker : R. yu Li : G. Zündorf : G. Reiser (*) Institut für Neurobiochemie, Otto-von-Guericke-Universität Magdeburg, Medizinische Fakultät, Leipziger Straße 44, 39120 Magdeburg, Germany e-mail: [email protected]
Keywords PAR-2 . Carboxyl tail . Truncation mutants . Ca2+ . ERK-AKT pathway Abbreviations AP Activating peptide AKT Protein kinase B AMPK Adenosine monophosphate-activated protein kinase ERK Extracellular signal-regulated kinase FCS Fetal calf serum GPCRs G protein coupled receptors HBSS Hank’s buffered salt solution PAR Protease-activated receptors Ser Serine SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Thr Threonine
Introduction The human genome encodes more than 800 G proteincoupled receptors (GPCRs) (Ostermaier et al. 2014). GPCRs are important 7-transmembrane domain proteins that sense signaling molecules, such as hormones (Mayo et al. 2000) and neurotransmitters (Roth et al. 1998) and connect intracellular signaling to physiological functions. To date, GPCRs are the main targets of drugs, accounting for 50–60 % of the current drug targets (Lundstrom 2009). Recently, many studies have provided insights into linkage between the GPCRs structure and cellular functions. Basically, the structure of GPCRs includes three main parts: N-terminus and three extracellular loops that form the extracellular region, 7 transmembrane helices and 3 intracellular loops plus the carboxyl terminal tail,
Cell Tissue Res
which form the intracellular region. Classically, the extracellular region is believed to regulate ligand access, the transmembrane region is thought to construct the structural core and the intracellular region is confirmed to interact with cytosolic signaling proteins (Venkatakrishnan et al. 2013). Protease-activated receptors (PAR) are a unique family of GPCRs because of the distinct mechanism of activation. Activation of PAR is initiated by the cleavage within the Nterminus by serine proteases, resulting in the new tethered ligands binding to the second loop of the extracellular region (Macfarlane et al. 2001; Ossovskaya and Bunnett 2004). In the PAR family, four isoforms have been identified, namely PAR-1, PAR-2, PAR-3 and PAR-4 (Hollenberg and Compton 2002). Amongst them, PAR-1, PAR-3 and PAR-4 can be activated by thrombin; however, PAR-2 is the only receptor that cannot be activated by thrombin but stimulated by trypsin (Macfarlane et al. 2001; Ossovskaya and Bunnett 2004). PAR2 is activated by multiple trypsin-like serine proteases including trypsin, tryptase and coagulation proteases (Russo et al. 2009). It is known that PAR-2 mediates the cellular effects through activation of heterotrimeric G proteins, including Gq, Gi and G12/13 but not Gs (Coughlin 2005; Russo et al. 2009). Several studies have revealed that the predominant α subunit involved in mediating PAR effects are the pertussis-toxininsensitive Gαq/G11 and G12/G13 subunits (Vaidyula and Rao 2003). The response to activation of these G proteins is the elevation of intracellular Ca2+ via the Gαq/phospholipase C/inositol trisphosphate (IP3) pathway, as has been shown for PAR-2 in cultured hippocampal neurons (Smith-Swintosky et al. 1997). Another separate signaling arm initiated by PAR-2 activation goes through the recruitment of β-arrestins (β-arrestin 1 and β-arrestin 2) as scaffolds. β-arrestins act as molecular switches that are capable of modifying the signals generated by receptors (DeWire et al. 2007). On the one hand, downstream targets of the Gαq/Ca2+ signaling arm are directly inhibited by β-arrestins; on the other hand, signal transduction pathways conducted through G protein and β-arrestins are synergistic. For example, PAR-2 activates adenosine monophosphate-activated protein kinase (AMPK), a key regulator of cellular energy balance, through Ca2+-dependent kinase β, while it inhibits AMPK through interaction with β-arrestins (Wang et al. 2010). The C terminus of GPCRs is suggested to be important for binding multiple signaling protein adaptors (Ferguson et al. 1996; Krupnick and Benovic 1998; Lefkowitz 1998). The binding of β-arrestins to GPCRs confers desensitization, internalization and intracellular signaling (Ostermaier et al. 2014). For example, the activation of extracellular signal-regulated kinase (ERK) via PAR-2 relies on the β-arrestin-dependent internalization of the receptor and subsequently formed stable complexes
containing β-arrestins and receptors to conduct the signaling cascades (DeWire et al. 2007). It has been reported that phosphorylation of the C terminus played a role in desensitization of activated PAR-2 signaling (Ricks and Trejo 2009). PAR-2 activation caused a rapid and robust increase in phosphorylation of PAR-2 wild-type, rather than mutants in which all serines and threonines in the cytoplasmic tail were converted to alanines (Ricks and Trejo 2009), indicating that the important sites of PAR-2 phosphorylation are located in the cytoplasmic tail. Previous studies showed that pharmacological inhibitors of protein kinase C enhanced PAR-2-mediated calcium responses in transformed rat kidney epithelial cells and Berkeley rat intestinal epithelial (hBRIE 380) cells, implying a role for phosphorylation in PAR-2 regulation (Böhm et al. 1996). However, one important study of the PAR-2 carboxyl tail points out that, besides the serine and threonine phosphorylation sites, the other amino acids present in the PAR-2 carboxyl tail also contribute to receptor internalization and intracellular Ca2+ signaling (Seatter et al. 2004). Given that the carboxyl tail of PAR-2 orchestrates intracellular signaling networks to regulate cellular functions, we hypothesized: (1) loss of the different regions of PAR-2 carboxyl tail will influence the receptor localization and internalization; and (2) the different fragments of amino acids residues containing different clusters of serines/threonines of the PAR2 carboxyl tail will differently affect intracellular signaling and cell survival. Our results provide novel insights into the connections between the fragments of amino acid residues containing different phosphorylation sites in the PAR-2 carboxyl tail and control of intracellular signaling and cell death.
Materials and methods Plasmid constructs The full-length rat PAR-2 cDNA was amplified and cloned into the pEGFPN1 receptor (Clontech). The verified plasmid of PAR-2 with GFP fluorescence tag linked to C-terminus was used as the template to generate the PAR-2 C-tail truncation mutants. In brief, two complementary oligonucleotides containing the desired gene fragment coding the indicated mutants were synthesized. All the mutants were generated using site-directed mutagenesis kit (Stratagene, Amsterdam, Netherlands). The specific sequences for the primers used for all the mutants were: S348Z forward, 5’-TTTG TCTA CTAC TTTG TTAA GCTT CGAA TTCT GCAG-3’ and reverse, 5’-CTGC AGAA TTCG AAGC TTAA CAAA GTAG TAGA CAAA; C361Z, forward, 5’-GCCA GAAA CGCG CTCC TCAA GCTT CGAA TTCT GCAG-3’ and reverse, 5’-CTGC AGAA TTCG AAGC TTGCA GGAG CGCG TTTC TGGC-3’; K368Z forward, 5’-CGAA GCGT
Cell Tissue Res
CCGC ACCG TGAA GCTT CGAA TTCT GCAG-3’ and reverse, 5’-CTGC AGAA TTCG AAGCTTCA CGGT GCGG ACGC TTCG-3’; S379Z forward, 5’- TCGC TCAC TCCA ACAA GAAG TCTTC GAAT TCTG CAG-3’ and reverse, 5’-CTGC AGAA TTCG AAGC TTCG TGTT GGAG GTGA GCGA-3’; S386Z forward, 5’-TCCA GGAA ATC CAGC TCT AAGC TTCG TTCG AATT CTG CAG-3’ and reverse, 5’-CTGCAG AATT CGAA GCTTAGAG CTGG ATTT CCTG GA-3’. All constructs were verified by DNA sequencing (SEQLAB, Göttingen, Germany). The program used to obtain the mutated gene was started with the initial denaturation at 98 °C for 30 s, followed by denaturation at 95 °C for 30 s. Then, the annealing temperatures were at 55–57 °C for 40–60 s and the flexible time for extension was set based on the length of gene fragments (2 min/kb).
Na2HPO4 × 2H2O, 0.34 mM, MgSO4 × 7H2O, 0.41 mM. NaCl, 87.4 mM; HEPES, 10 mM, CaCl 2 , 1.25 mM; NaHCO3, 4.2 mM; glucose, 5.6 mM; pH=7.3). For live-cell imaging, the cells were incubated on stage in a chamber with 5 % CO2 at 37 °C in HBSS buffer in a Zeiss inverted LSM 510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany). During the whole process, the same cells were monitored and photographed before or after application of trypsin. First, the cells of interest were selected and imaged to document the original state of the receptor without any challenges. Thereafter, trypsin was carefully added into the HBSS buffer to the cells. The final concentration of trypsin was 100 nM. Fluorescence images were captured sequentially at 5, 10, 20 and 30 min at excitation of 488 nm to detect receptor internalization upon stimulation by trypsin with a LSM510 laser scanning confocal microscope.
Cell culture and transient/stable transfection
Quantification of the level of PAR-2 mutant on the plasma membrane and PAR-2 internalization
HEK 293 cells were grown in Dulbecco’s modified Eagle’s medium/Ham’s F-12 1:1 medium (Biochrom, Berlin, Germany) with 10 % heat-inactivated fetal calf serum, 100 units per ml penicillin and 100 μg/mL streptomycin at 37 °C and 5 % CO2. One day before transfection, HEK 293 cells were plated on a 6-well plate. For the transient transfection, HEK 293 cells with 70 % of confluence were transfected with 4 μg of PAR-2 truncation mutants plasmids with 4 μL of MATra-A reagent according to the manual (IBA, Göttingen, Germany) for 36 h. After that, the transfected HEK293 cells were deprived of fetal calf serum (FCS) for 6 h. To activate the truncation mutants of PAR-2, cells were exposed to 100 nM of trypsin (Sigma, Steinheim, Germany) or 50 μM of the specific agonist rat PAR-2 activating peptide (AP) SLIGAL (Polypeptide, Strasbourg, France) in the absence of FCS for 30 min and then the cells were harvested for western blot. To generate the stable cell lines of PAR-2 truncations, the plasmids carrying PAR-2 gene fragments were transfected into HEK 293 cells with DOTAP liposomal transfection reagent according to the manufacturer’s protocol (Roche Diagnostics, Germany). Then, 24 h post transfection, G418 was added to select the positively transfected cell lines. The stably transfected HEK 293 cells carrying the different PAR-2 truncations were used in receptor internalization assay. Internalization assay For imaging of trypsin-induced receptor internalization, the experiment was carried out with the method similar to one used before in our laboratory (Tulapurkar et al. 2005). Briefly, HEK 293 cells stably transfected with PAR-2 mutants with GFP tag were grown till 60–70 % confluence. After that, cells were gently washed twice with pre-heated Hank’s buffered salt solution (HBSS) buffer (KCl, 50 mM, KH2PO4, 0.44 mM,
GFP fluorescence intensity of confocal images was analyzed using the Zeiss LSM 510meta software histo macro, as described before (Ecke et al. 2008). First, the whole cell was selected as region of interest and the GFP fluorescence intensity was obtained as the sum of receptor expression on the plasma membrane and in the cytosol; the value was recorded as V1. Then the cytosol was selected as region of interest and the value of GFP fluorescence intensity was calculated as V2. The difference V1 − V2 was taken as the level of mutated receptors expressed on the plasma membrane. For quantification of receptor internalization, regions of interest were set in cytosol of single cells and the average fluorescence intensities in the regions of interest were determined over time. The intensity values for 30 min were compared to the starting value at 0 min. Western blot The transiently transfected cells were lysed on ice by lysis buffer (50 mM Tris, 150 mM NaCl, 0.25 % Na-deoxycholate, 1 mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail tablets (pH 7.4) (Roche Diagnostics). After centrifugation for 20 min at 15,000 rpm, the protein of the lysates was quantified by the Bradford method. Next, 30 μg of protein were separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by electro-transfer to nitrocellulose membranes. The membranes were blocked and incubated overnight with the primary antibody (PhosphoERK, 1:1000; Phospho-AKT, 1:1000; AKT 1:2000; ERK 1:1000; Cell Signaling, Frankfurt, Germany; or GAPDH, 1:2000; Millipore, Temecula, CA, USA) diluted in TBST buffer (20 mM Tris/HCl, pH 7.6, 137 mM NaCl, 0.1 % Tween 20) at 4 °C, followed by 1 h incubation with the
Cell Tissue Res
secondary antibodies conjugated to horseradish peroxidase at 25 °C. After washing, the immune complexes were detected by the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). Immunoreactive bands were visualized on the Chemi Doc™ XRS+imaging system (BioRad, München, Germany). The optical density of the bands was quantified by Quantity One quantification software (BioRad).
medium was applied to each well containing the transfected cells without disturbance. After 1 h incubation at 37 °C in a humidified atmosphere (37 °C, 5 % CO2), the absorbance at 450 nm was measured using a micro-plate reader (Molecular Devices). The wells containing only the medium plus WST-1 solution were set as blank.
Calcium measurement
All experiments were carried out at least three times and results are expressed as means±SEM. Comparisons between tests were done by Student’s t test between two groups or by one-way ANOVA followed by Dunnett’s test or by Newman– Keuls test among different groups.
To monitor the intracellular Ca2+ mobilization by the different PAR-2 truncations, the stable transfected HEK293 cells carrying different mutants of PAR-2 were challenged under a series of different concentrations of trypsin. For calcium measurements, cells were grown on 22-mm coverslides, as described by Ecke et al. (2008). The intracellular Ca2+ concentrations were measured with an imaging system (Agilent Technologies/TILL Photonics, Gräfelfing, Germany) attached to a Zeiss Axioscope microscope (Carl Zeiss). For the experiments, cells were loaded with the Ca2+ sensitive Fura-2 AM (2 μM, 0.02 % Pluronic, 30 min at 25 °C), which was from Life Technologies (Carlsbad, CA, USA). The dye remains intracellular after cleavage by non-specific esterase activity. Fluorescence signals were acquired at 510 nm emission during alternating excitation at 340 nm (Fura-2 bound to free Ca2+) and 380 nm (Ca2+-free Fura-2 molecule) every 3 s. The ratio of the emission intensities of 340 nm and 380 nm excitations is attributed to the intracellular calcium concentration. Cells were exposed to HBSS buffer for 89 s. At the time point of 90 s, trypsin was applied to the cells. Then, the Ca2+ responses were monitored for 210 s to obtain the respective maximum value during this time. Only cells with an obviously membrane-localized GFP signal were taken for data analysis. For each measurement, at least 15 cells were selected and analyzed. The data were obtained and analyzed from three independent experiments. WST-1 assay The reagent WST-1 is a colorimetric assay for the nonradioactive quantification of cellular proliferation, viability and cytotoxicity. The WST-1 assay was applied to evaluate cell survival. In detail, HEK 293 cells were stably transfected with PAR-2 mutants and we selected the 100 % transfected monoclones by G418 geneticin treatments. The stable transfected cells were seeded on 96-well plates for 24 h. The next day, cells were deprived for FCS overnight before treatment with 50 μM of PAR-2 agonist SLIGAL (Polypeptide). Cells were exposed to an agonist for 48 h in FCS-free medium. Afterwards, WST-1 assays were performed to check the cell survival among the different mutants. In brief, 10 μl of WST-1 (Roche, Dassel, Germany) solutions with 90 μl of serum-free
Statistical analysis
Results Construction of PAR-2 truncations mutants To examine the functional role of PAR-2 carboxyl tail in receptor internalization and to find out which cluster of serines/threonines is important for signaling and cell survival, the various truncation mutants were generated by PCR mutagenesis. Briefly, the template DNA used to amplify the PAR-2 truncation mutants is the rat wild-type PAR-2 receptor inserted into the pEGFP-N1 vector. The GFP tag was linked to the Cterminus of PAR-2. PAR-2 carboxyl tail truncation mutants were generated by deletion of certain gene fragments coding for the indicated residues. From the PAR-2 carboxyl tail s e q u e n c e V 3 4 8 S K D F R D Q A R N A L L C R S V RT VKRMQISLTSNFKFSRKSSYSSSSTSVKTSY397 the truncations were generated. The resulting mutants obtained were designated 348-Del, 361-Del, 368-Del, 379-Del and 386-Del. The number at the respective mutant indicates the C-terminal amino acid residue of the truncated receptor. For example, in 348-Del, the C-terminal amino acid residue is V348, while in 361-Del it is L361. The cellular localization and internalization of PAR-2 carboxyl tail mutants In unstimulated cells, the major portion of the truncated receptors was located clearly on the plasma membrane (Fig. 1b, c, g, h), except for the 348-Del truncation, in which all the amino acids of the C-terminus were deleted (Fig. 1a). To check if differences existed in the rate of expression of the mutant receptors on the plasma membrane, we quantified the level of mutated receptors on the plasma membrane. However, we excluded from the calculation the 348-Del mutant, since 348-Del is distributed mainly in the cytosol with a very low level of localization on the cell plasma membrane. The
Cell Tissue Res Fig. 1 Receptor internalization of PAR-2 carboxyl tail truncation mutants. HEK 293 cells were stably transfected with PAR-2 mutants with GFP tag (green fluorescence), designated as 348Del, 361-Del, 368-Del, 379-Del, 386-Del and PAR-2 (wild-type receptors). The transfected cells were cultured until 70 % confluence and then the cells were used for receptor internalization assay. Briefly, the transfected cells were monitored in the absence or presence of 100 nM trypsin in HBSS for up to 30 min at 37 °C to visualize receptor trafficking. Images were processed with Zeiss confococal microscopy software. Pictures (a–c) and (g–i) were taken at 0 min. From the same cultures the corresponding pictures (d–f) and (j–l) were taken at 30 min showing the same field. The white arrows point out the real-time trafficking of the mutated internalized receptors into the cytosol. The experiments were repeated at least three times and similar results were obtained (Scale bar 20 μm)
quantification data in Fig. 2a confirmed that there were no differences concerning the level of the mutant receptors expressed on the plasma membrane. Importantly, the level for these mutants was comparable to that of the wild-type receptor. The PAR-2 carboxyl tail truncation mutants 361Del, 368-Del, 379-Del and 386-Del, which lack different phosphorylation sites of serines or threonines at the carboxyl tail region, were localized mainly on the plasma membrane (Fig. 1b, c, g, h), like the wild-type receptors (Fig. 1i). To investigate which part of the PAR-2 carboxyl tail with different series of serines and threonines as possible phosphorylation sites is essential for the receptor internalization, HEK cells transfected with the truncation mutants of PAR-2 were treated with 100 nM of trypsin
for up to 30 min. Under the challenge with trypsin for 5 min, PAR-2 wild-type receptor showed small levels of internalization, as indicated by white arrows in the pictures (supplementary Fig. 1). However, other mutant receptors apparently need more than 5 min to internalize (supplementary Fig. 1). Interestingly, 30 min treatment with trypsin failed to cause any obvious changes in receptor localization in the 348-Del mutant (Fig. 1d). For the other mutant receptors, the internalization pattern observed (Fig. 1e, f, j, k) was similar to that of PAR-2 wild-type receptor (Fig. 1l). The internalized receptors (green fluorescence) were well localized beneath the plasma membrane and/or finally formed clusters of intracellular vesicles, as marked by white arrows
Cell Tissue Res
Ca2+ responses induced by different concentrations of trypsin in PAR-2 mutants
Fig. 2 Quantification of expression of mutated PAR-2 receptors on the plasma membrane and of receptor internalization. a The level of the mutated receptors expressed on the plasma membrane. b Receptor internalization. The quantification was performed by evaluating the GFP fluorescence intensities, as described in “Materials and methods”. In (b), Student’s t test was used for analysis of significance of differences between the GFP fluorescence intensities at times of 0 and 30 min. Data shown represent the mean±SEM (*p
REGULAR ARTICLE
The intracellular carboxyl tail of the PAR-2 receptor controls intracellular signaling and cell death Zhihui Zhu & Rolf Stricker & Rong yu Li & Gregor Zündorf & Georg Reiser
Received: 22 June 2014 / Accepted: 6 November 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The protease-activated receptors are a group of unique G protein-coupled receptors, including PAR-1, PAR2, PAR-3 and PAR-4. PAR-2 is activated by multiple trypsinlike serine proteases, including trypsin, tryptase and coagulation proteases. The clusters of phosphorylation sites in the PAR-2 carboxyl tail are suggested to be important for the binding of adaptor proteins to initiate intracellular signaling to Ca2+ and mitogen-activated protein kinases. To explore the functional role of PAR-2 carboxyl tail in controlling intracellular Ca2+, ERK and AKT signaling, a series of truncated mutants containing different clusters of serines/threonines were generated and expressed in HEK293 cells. Firstly, we observed that lack of the complete C-terminus of PAR-2 in a mutated receptor gave a relatively low level of localization on the cell plasma membrane. Secondly, the shortened carboxyl tail containing 13 amino acids was sufficient for receptor internalization. Thirdly, the cells expressing truncation mutants showed deficits in their capacity to couple to intracellular Ca2+ and ERK and AKT signaling upon trypsin challenge. In addition, HEK293 cells carrying different PAR-2 truncation mutants displayed decreased levels of cell survival after long-lasting trypsin stimulation. In summary, the PAR-2 carboxyl tail was found to control the receptor localization, internalization, intracellular Ca2+ responses and signaling to ERK and AKT. The latter can be considered to be important for cell death control.
Electronic supplementary material The online version of this article (doi:10.1007/s00441-014-2056-9) contains supplementary material, which is available to authorized users. Z. Zhu : R. Stricker : R. yu Li : G. Zündorf : G. Reiser (*) Institut für Neurobiochemie, Otto-von-Guericke-Universität Magdeburg, Medizinische Fakultät, Leipziger Straße 44, 39120 Magdeburg, Germany e-mail: [email protected]
Keywords PAR-2 . Carboxyl tail . Truncation mutants . Ca2+ . ERK-AKT pathway Abbreviations AP Activating peptide AKT Protein kinase B AMPK Adenosine monophosphate-activated protein kinase ERK Extracellular signal-regulated kinase FCS Fetal calf serum GPCRs G protein coupled receptors HBSS Hank’s buffered salt solution PAR Protease-activated receptors Ser Serine SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Thr Threonine
Introduction The human genome encodes more than 800 G proteincoupled receptors (GPCRs) (Ostermaier et al. 2014). GPCRs are important 7-transmembrane domain proteins that sense signaling molecules, such as hormones (Mayo et al. 2000) and neurotransmitters (Roth et al. 1998) and connect intracellular signaling to physiological functions. To date, GPCRs are the main targets of drugs, accounting for 50–60 % of the current drug targets (Lundstrom 2009). Recently, many studies have provided insights into linkage between the GPCRs structure and cellular functions. Basically, the structure of GPCRs includes three main parts: N-terminus and three extracellular loops that form the extracellular region, 7 transmembrane helices and 3 intracellular loops plus the carboxyl terminal tail,
Cell Tissue Res
which form the intracellular region. Classically, the extracellular region is believed to regulate ligand access, the transmembrane region is thought to construct the structural core and the intracellular region is confirmed to interact with cytosolic signaling proteins (Venkatakrishnan et al. 2013). Protease-activated receptors (PAR) are a unique family of GPCRs because of the distinct mechanism of activation. Activation of PAR is initiated by the cleavage within the Nterminus by serine proteases, resulting in the new tethered ligands binding to the second loop of the extracellular region (Macfarlane et al. 2001; Ossovskaya and Bunnett 2004). In the PAR family, four isoforms have been identified, namely PAR-1, PAR-2, PAR-3 and PAR-4 (Hollenberg and Compton 2002). Amongst them, PAR-1, PAR-3 and PAR-4 can be activated by thrombin; however, PAR-2 is the only receptor that cannot be activated by thrombin but stimulated by trypsin (Macfarlane et al. 2001; Ossovskaya and Bunnett 2004). PAR2 is activated by multiple trypsin-like serine proteases including trypsin, tryptase and coagulation proteases (Russo et al. 2009). It is known that PAR-2 mediates the cellular effects through activation of heterotrimeric G proteins, including Gq, Gi and G12/13 but not Gs (Coughlin 2005; Russo et al. 2009). Several studies have revealed that the predominant α subunit involved in mediating PAR effects are the pertussis-toxininsensitive Gαq/G11 and G12/G13 subunits (Vaidyula and Rao 2003). The response to activation of these G proteins is the elevation of intracellular Ca2+ via the Gαq/phospholipase C/inositol trisphosphate (IP3) pathway, as has been shown for PAR-2 in cultured hippocampal neurons (Smith-Swintosky et al. 1997). Another separate signaling arm initiated by PAR-2 activation goes through the recruitment of β-arrestins (β-arrestin 1 and β-arrestin 2) as scaffolds. β-arrestins act as molecular switches that are capable of modifying the signals generated by receptors (DeWire et al. 2007). On the one hand, downstream targets of the Gαq/Ca2+ signaling arm are directly inhibited by β-arrestins; on the other hand, signal transduction pathways conducted through G protein and β-arrestins are synergistic. For example, PAR-2 activates adenosine monophosphate-activated protein kinase (AMPK), a key regulator of cellular energy balance, through Ca2+-dependent kinase β, while it inhibits AMPK through interaction with β-arrestins (Wang et al. 2010). The C terminus of GPCRs is suggested to be important for binding multiple signaling protein adaptors (Ferguson et al. 1996; Krupnick and Benovic 1998; Lefkowitz 1998). The binding of β-arrestins to GPCRs confers desensitization, internalization and intracellular signaling (Ostermaier et al. 2014). For example, the activation of extracellular signal-regulated kinase (ERK) via PAR-2 relies on the β-arrestin-dependent internalization of the receptor and subsequently formed stable complexes
containing β-arrestins and receptors to conduct the signaling cascades (DeWire et al. 2007). It has been reported that phosphorylation of the C terminus played a role in desensitization of activated PAR-2 signaling (Ricks and Trejo 2009). PAR-2 activation caused a rapid and robust increase in phosphorylation of PAR-2 wild-type, rather than mutants in which all serines and threonines in the cytoplasmic tail were converted to alanines (Ricks and Trejo 2009), indicating that the important sites of PAR-2 phosphorylation are located in the cytoplasmic tail. Previous studies showed that pharmacological inhibitors of protein kinase C enhanced PAR-2-mediated calcium responses in transformed rat kidney epithelial cells and Berkeley rat intestinal epithelial (hBRIE 380) cells, implying a role for phosphorylation in PAR-2 regulation (Böhm et al. 1996). However, one important study of the PAR-2 carboxyl tail points out that, besides the serine and threonine phosphorylation sites, the other amino acids present in the PAR-2 carboxyl tail also contribute to receptor internalization and intracellular Ca2+ signaling (Seatter et al. 2004). Given that the carboxyl tail of PAR-2 orchestrates intracellular signaling networks to regulate cellular functions, we hypothesized: (1) loss of the different regions of PAR-2 carboxyl tail will influence the receptor localization and internalization; and (2) the different fragments of amino acids residues containing different clusters of serines/threonines of the PAR2 carboxyl tail will differently affect intracellular signaling and cell survival. Our results provide novel insights into the connections between the fragments of amino acid residues containing different phosphorylation sites in the PAR-2 carboxyl tail and control of intracellular signaling and cell death.
Materials and methods Plasmid constructs The full-length rat PAR-2 cDNA was amplified and cloned into the pEGFPN1 receptor (Clontech). The verified plasmid of PAR-2 with GFP fluorescence tag linked to C-terminus was used as the template to generate the PAR-2 C-tail truncation mutants. In brief, two complementary oligonucleotides containing the desired gene fragment coding the indicated mutants were synthesized. All the mutants were generated using site-directed mutagenesis kit (Stratagene, Amsterdam, Netherlands). The specific sequences for the primers used for all the mutants were: S348Z forward, 5’-TTTG TCTA CTAC TTTG TTAA GCTT CGAA TTCT GCAG-3’ and reverse, 5’-CTGC AGAA TTCG AAGC TTAA CAAA GTAG TAGA CAAA; C361Z, forward, 5’-GCCA GAAA CGCG CTCC TCAA GCTT CGAA TTCT GCAG-3’ and reverse, 5’-CTGC AGAA TTCG AAGC TTGCA GGAG CGCG TTTC TGGC-3’; K368Z forward, 5’-CGAA GCGT
Cell Tissue Res
CCGC ACCG TGAA GCTT CGAA TTCT GCAG-3’ and reverse, 5’-CTGC AGAA TTCG AAGCTTCA CGGT GCGG ACGC TTCG-3’; S379Z forward, 5’- TCGC TCAC TCCA ACAA GAAG TCTTC GAAT TCTG CAG-3’ and reverse, 5’-CTGC AGAA TTCG AAGC TTCG TGTT GGAG GTGA GCGA-3’; S386Z forward, 5’-TCCA GGAA ATC CAGC TCT AAGC TTCG TTCG AATT CTG CAG-3’ and reverse, 5’-CTGCAG AATT CGAA GCTTAGAG CTGG ATTT CCTG GA-3’. All constructs were verified by DNA sequencing (SEQLAB, Göttingen, Germany). The program used to obtain the mutated gene was started with the initial denaturation at 98 °C for 30 s, followed by denaturation at 95 °C for 30 s. Then, the annealing temperatures were at 55–57 °C for 40–60 s and the flexible time for extension was set based on the length of gene fragments (2 min/kb).
Na2HPO4 × 2H2O, 0.34 mM, MgSO4 × 7H2O, 0.41 mM. NaCl, 87.4 mM; HEPES, 10 mM, CaCl 2 , 1.25 mM; NaHCO3, 4.2 mM; glucose, 5.6 mM; pH=7.3). For live-cell imaging, the cells were incubated on stage in a chamber with 5 % CO2 at 37 °C in HBSS buffer in a Zeiss inverted LSM 510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany). During the whole process, the same cells were monitored and photographed before or after application of trypsin. First, the cells of interest were selected and imaged to document the original state of the receptor without any challenges. Thereafter, trypsin was carefully added into the HBSS buffer to the cells. The final concentration of trypsin was 100 nM. Fluorescence images were captured sequentially at 5, 10, 20 and 30 min at excitation of 488 nm to detect receptor internalization upon stimulation by trypsin with a LSM510 laser scanning confocal microscope.
Cell culture and transient/stable transfection
Quantification of the level of PAR-2 mutant on the plasma membrane and PAR-2 internalization
HEK 293 cells were grown in Dulbecco’s modified Eagle’s medium/Ham’s F-12 1:1 medium (Biochrom, Berlin, Germany) with 10 % heat-inactivated fetal calf serum, 100 units per ml penicillin and 100 μg/mL streptomycin at 37 °C and 5 % CO2. One day before transfection, HEK 293 cells were plated on a 6-well plate. For the transient transfection, HEK 293 cells with 70 % of confluence were transfected with 4 μg of PAR-2 truncation mutants plasmids with 4 μL of MATra-A reagent according to the manual (IBA, Göttingen, Germany) for 36 h. After that, the transfected HEK293 cells were deprived of fetal calf serum (FCS) for 6 h. To activate the truncation mutants of PAR-2, cells were exposed to 100 nM of trypsin (Sigma, Steinheim, Germany) or 50 μM of the specific agonist rat PAR-2 activating peptide (AP) SLIGAL (Polypeptide, Strasbourg, France) in the absence of FCS for 30 min and then the cells were harvested for western blot. To generate the stable cell lines of PAR-2 truncations, the plasmids carrying PAR-2 gene fragments were transfected into HEK 293 cells with DOTAP liposomal transfection reagent according to the manufacturer’s protocol (Roche Diagnostics, Germany). Then, 24 h post transfection, G418 was added to select the positively transfected cell lines. The stably transfected HEK 293 cells carrying the different PAR-2 truncations were used in receptor internalization assay. Internalization assay For imaging of trypsin-induced receptor internalization, the experiment was carried out with the method similar to one used before in our laboratory (Tulapurkar et al. 2005). Briefly, HEK 293 cells stably transfected with PAR-2 mutants with GFP tag were grown till 60–70 % confluence. After that, cells were gently washed twice with pre-heated Hank’s buffered salt solution (HBSS) buffer (KCl, 50 mM, KH2PO4, 0.44 mM,
GFP fluorescence intensity of confocal images was analyzed using the Zeiss LSM 510meta software histo macro, as described before (Ecke et al. 2008). First, the whole cell was selected as region of interest and the GFP fluorescence intensity was obtained as the sum of receptor expression on the plasma membrane and in the cytosol; the value was recorded as V1. Then the cytosol was selected as region of interest and the value of GFP fluorescence intensity was calculated as V2. The difference V1 − V2 was taken as the level of mutated receptors expressed on the plasma membrane. For quantification of receptor internalization, regions of interest were set in cytosol of single cells and the average fluorescence intensities in the regions of interest were determined over time. The intensity values for 30 min were compared to the starting value at 0 min. Western blot The transiently transfected cells were lysed on ice by lysis buffer (50 mM Tris, 150 mM NaCl, 0.25 % Na-deoxycholate, 1 mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail tablets (pH 7.4) (Roche Diagnostics). After centrifugation for 20 min at 15,000 rpm, the protein of the lysates was quantified by the Bradford method. Next, 30 μg of protein were separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by electro-transfer to nitrocellulose membranes. The membranes were blocked and incubated overnight with the primary antibody (PhosphoERK, 1:1000; Phospho-AKT, 1:1000; AKT 1:2000; ERK 1:1000; Cell Signaling, Frankfurt, Germany; or GAPDH, 1:2000; Millipore, Temecula, CA, USA) diluted in TBST buffer (20 mM Tris/HCl, pH 7.6, 137 mM NaCl, 0.1 % Tween 20) at 4 °C, followed by 1 h incubation with the
Cell Tissue Res
secondary antibodies conjugated to horseradish peroxidase at 25 °C. After washing, the immune complexes were detected by the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). Immunoreactive bands were visualized on the Chemi Doc™ XRS+imaging system (BioRad, München, Germany). The optical density of the bands was quantified by Quantity One quantification software (BioRad).
medium was applied to each well containing the transfected cells without disturbance. After 1 h incubation at 37 °C in a humidified atmosphere (37 °C, 5 % CO2), the absorbance at 450 nm was measured using a micro-plate reader (Molecular Devices). The wells containing only the medium plus WST-1 solution were set as blank.
Calcium measurement
All experiments were carried out at least three times and results are expressed as means±SEM. Comparisons between tests were done by Student’s t test between two groups or by one-way ANOVA followed by Dunnett’s test or by Newman– Keuls test among different groups.
To monitor the intracellular Ca2+ mobilization by the different PAR-2 truncations, the stable transfected HEK293 cells carrying different mutants of PAR-2 were challenged under a series of different concentrations of trypsin. For calcium measurements, cells were grown on 22-mm coverslides, as described by Ecke et al. (2008). The intracellular Ca2+ concentrations were measured with an imaging system (Agilent Technologies/TILL Photonics, Gräfelfing, Germany) attached to a Zeiss Axioscope microscope (Carl Zeiss). For the experiments, cells were loaded with the Ca2+ sensitive Fura-2 AM (2 μM, 0.02 % Pluronic, 30 min at 25 °C), which was from Life Technologies (Carlsbad, CA, USA). The dye remains intracellular after cleavage by non-specific esterase activity. Fluorescence signals were acquired at 510 nm emission during alternating excitation at 340 nm (Fura-2 bound to free Ca2+) and 380 nm (Ca2+-free Fura-2 molecule) every 3 s. The ratio of the emission intensities of 340 nm and 380 nm excitations is attributed to the intracellular calcium concentration. Cells were exposed to HBSS buffer for 89 s. At the time point of 90 s, trypsin was applied to the cells. Then, the Ca2+ responses were monitored for 210 s to obtain the respective maximum value during this time. Only cells with an obviously membrane-localized GFP signal were taken for data analysis. For each measurement, at least 15 cells were selected and analyzed. The data were obtained and analyzed from three independent experiments. WST-1 assay The reagent WST-1 is a colorimetric assay for the nonradioactive quantification of cellular proliferation, viability and cytotoxicity. The WST-1 assay was applied to evaluate cell survival. In detail, HEK 293 cells were stably transfected with PAR-2 mutants and we selected the 100 % transfected monoclones by G418 geneticin treatments. The stable transfected cells were seeded on 96-well plates for 24 h. The next day, cells were deprived for FCS overnight before treatment with 50 μM of PAR-2 agonist SLIGAL (Polypeptide). Cells were exposed to an agonist for 48 h in FCS-free medium. Afterwards, WST-1 assays were performed to check the cell survival among the different mutants. In brief, 10 μl of WST-1 (Roche, Dassel, Germany) solutions with 90 μl of serum-free
Statistical analysis
Results Construction of PAR-2 truncations mutants To examine the functional role of PAR-2 carboxyl tail in receptor internalization and to find out which cluster of serines/threonines is important for signaling and cell survival, the various truncation mutants were generated by PCR mutagenesis. Briefly, the template DNA used to amplify the PAR-2 truncation mutants is the rat wild-type PAR-2 receptor inserted into the pEGFP-N1 vector. The GFP tag was linked to the Cterminus of PAR-2. PAR-2 carboxyl tail truncation mutants were generated by deletion of certain gene fragments coding for the indicated residues. From the PAR-2 carboxyl tail s e q u e n c e V 3 4 8 S K D F R D Q A R N A L L C R S V RT VKRMQISLTSNFKFSRKSSYSSSSTSVKTSY397 the truncations were generated. The resulting mutants obtained were designated 348-Del, 361-Del, 368-Del, 379-Del and 386-Del. The number at the respective mutant indicates the C-terminal amino acid residue of the truncated receptor. For example, in 348-Del, the C-terminal amino acid residue is V348, while in 361-Del it is L361. The cellular localization and internalization of PAR-2 carboxyl tail mutants In unstimulated cells, the major portion of the truncated receptors was located clearly on the plasma membrane (Fig. 1b, c, g, h), except for the 348-Del truncation, in which all the amino acids of the C-terminus were deleted (Fig. 1a). To check if differences existed in the rate of expression of the mutant receptors on the plasma membrane, we quantified the level of mutated receptors on the plasma membrane. However, we excluded from the calculation the 348-Del mutant, since 348-Del is distributed mainly in the cytosol with a very low level of localization on the cell plasma membrane. The
Cell Tissue Res Fig. 1 Receptor internalization of PAR-2 carboxyl tail truncation mutants. HEK 293 cells were stably transfected with PAR-2 mutants with GFP tag (green fluorescence), designated as 348Del, 361-Del, 368-Del, 379-Del, 386-Del and PAR-2 (wild-type receptors). The transfected cells were cultured until 70 % confluence and then the cells were used for receptor internalization assay. Briefly, the transfected cells were monitored in the absence or presence of 100 nM trypsin in HBSS for up to 30 min at 37 °C to visualize receptor trafficking. Images were processed with Zeiss confococal microscopy software. Pictures (a–c) and (g–i) were taken at 0 min. From the same cultures the corresponding pictures (d–f) and (j–l) were taken at 30 min showing the same field. The white arrows point out the real-time trafficking of the mutated internalized receptors into the cytosol. The experiments were repeated at least three times and similar results were obtained (Scale bar 20 μm)
quantification data in Fig. 2a confirmed that there were no differences concerning the level of the mutant receptors expressed on the plasma membrane. Importantly, the level for these mutants was comparable to that of the wild-type receptor. The PAR-2 carboxyl tail truncation mutants 361Del, 368-Del, 379-Del and 386-Del, which lack different phosphorylation sites of serines or threonines at the carboxyl tail region, were localized mainly on the plasma membrane (Fig. 1b, c, g, h), like the wild-type receptors (Fig. 1i). To investigate which part of the PAR-2 carboxyl tail with different series of serines and threonines as possible phosphorylation sites is essential for the receptor internalization, HEK cells transfected with the truncation mutants of PAR-2 were treated with 100 nM of trypsin
for up to 30 min. Under the challenge with trypsin for 5 min, PAR-2 wild-type receptor showed small levels of internalization, as indicated by white arrows in the pictures (supplementary Fig. 1). However, other mutant receptors apparently need more than 5 min to internalize (supplementary Fig. 1). Interestingly, 30 min treatment with trypsin failed to cause any obvious changes in receptor localization in the 348-Del mutant (Fig. 1d). For the other mutant receptors, the internalization pattern observed (Fig. 1e, f, j, k) was similar to that of PAR-2 wild-type receptor (Fig. 1l). The internalized receptors (green fluorescence) were well localized beneath the plasma membrane and/or finally formed clusters of intracellular vesicles, as marked by white arrows
Cell Tissue Res
Ca2+ responses induced by different concentrations of trypsin in PAR-2 mutants
Fig. 2 Quantification of expression of mutated PAR-2 receptors on the plasma membrane and of receptor internalization. a The level of the mutated receptors expressed on the plasma membrane. b Receptor internalization. The quantification was performed by evaluating the GFP fluorescence intensities, as described in “Materials and methods”. In (b), Student’s t test was used for analysis of significance of differences between the GFP fluorescence intensities at times of 0 and 30 min. Data shown represent the mean±SEM (*p
