p24A, a Type I Transmembrane Protein, Controls ARF1-dependent Resensitization of Protease-activated Receptor-2 by Influence on Receptor Trafficking
2007; Elsevier BV; Volume: 282; Issue: 41 Linguagem: Inglês
10.1074/jbc.m703205200
ISSN1083-351X
AutoresWeibo Luo, Yingfei Wang, Georg Reiser,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoProtease-activated receptor-2 (PAR-2), the second member of the G protein-coupled PAR family, is irreversibly activated by trypsin or tryptase and then targeted to lysosomes for degradation. Intracellular presynthesized receptors stored at the Golgi apparatus repopulate the cell surface after trypsin stimulation, thereby leading to rapid resensitization to trypsin signaling. However, the molecular mechanisms of the exocytic trafficking of PAR-2 from the Golgi apparatus to the plasma membrane remain largely unclear. Here we show that p24A, a type I transmembrane protein, which is a crucial constituent of the Golgi apparatus, associates with PAR-2 at the Golgi apparatus. The protein interaction occurs between the N-terminal region of p24A (residues 1-105; p24A-GL (GOLD domain with a small linker)) and the second extracellular loop of PAR-2. After receptor activation, PAR-2 dissociates from p24A. Importantly, we found that ADP-ribosylation factor 1 regulated the dissociation process and initiated PAR-2 trafficking to the plasma membrane. Conversely, overexpression of the fragment p24A-GL, but not other mutants containing the functional coiled-coil domain of p24A, arrested PAR-2 at the Golgi apparatus and inhibited receptor trafficking to the plasma membrane, which consequently prevented resensitization of PAR-2. These findings identify a new function of p24A as a regulator of signal-dependent trafficking that regulates the life cycle of PAR-2, Thus, we reveal a new molecular mechanism underlying resensitization of PAR-2. Protease-activated receptor-2 (PAR-2), the second member of the G protein-coupled PAR family, is irreversibly activated by trypsin or tryptase and then targeted to lysosomes for degradation. Intracellular presynthesized receptors stored at the Golgi apparatus repopulate the cell surface after trypsin stimulation, thereby leading to rapid resensitization to trypsin signaling. However, the molecular mechanisms of the exocytic trafficking of PAR-2 from the Golgi apparatus to the plasma membrane remain largely unclear. Here we show that p24A, a type I transmembrane protein, which is a crucial constituent of the Golgi apparatus, associates with PAR-2 at the Golgi apparatus. The protein interaction occurs between the N-terminal region of p24A (residues 1-105; p24A-GL (GOLD domain with a small linker)) and the second extracellular loop of PAR-2. After receptor activation, PAR-2 dissociates from p24A. Importantly, we found that ADP-ribosylation factor 1 regulated the dissociation process and initiated PAR-2 trafficking to the plasma membrane. Conversely, overexpression of the fragment p24A-GL, but not other mutants containing the functional coiled-coil domain of p24A, arrested PAR-2 at the Golgi apparatus and inhibited receptor trafficking to the plasma membrane, which consequently prevented resensitization of PAR-2. These findings identify a new function of p24A as a regulator of signal-dependent trafficking that regulates the life cycle of PAR-2, Thus, we reveal a new molecular mechanism underlying resensitization of PAR-2. G protein-coupled receptors (GPCRs) 3The abbreviations used are: GPCR, G protein-coupled receptor; AP, activating peptide; ARF1, ADP-ribosylation factor 1; COP, coat protein; C-tail, carboxyl tail; ER, endoplasmic reticulum; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin epitope; HEK, human embryonic kidney cells; PAR-2, protease-activated receptor-2; RT, reverse transcription; siRNA, small interfering RNA; GOLD, Golgi dynamics. constitute a large family of seven-transmembrane domain spanning receptors that regulate diverse biological responses within cells via heterotrimeric G proteins and downstream effectors (1Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell Biol. 2002; 3: 639-650Crossref PubMed Scopus (2125) Google Scholar). Protease-activated receptor-2 (PAR-2), the second member of the G protein-coupled PAR family, is involved in inflammation, pain, cell proliferation, and anti-apoptosis (2Cottrell G.S. Amadesi S. Schmidlin F. Bunnett N. Biochem. Soc. Trans. 2003; 31: 1191-1197Crossref PubMed Google Scholar, 3Steinhoff M. Buddenkotte J. Shpacovitch V. Rattenholl A. Moormann C. Vergnolle N. Luger T.A. Hollenberg M.D. Endocr. Rev. 2005; 26: 1-43Crossref PubMed Scopus (440) Google Scholar, 4Wang Y. Luo W. Reiser G. Biochem. J. 2007; 401: 65-78Crossref PubMed Scopus (47) Google Scholar). A unique activation mechanism is responsible for PAR-2 activation by irreversible proteolytic cleavage by serine proteases, such as trypsin and tryptase (2Cottrell G.S. Amadesi S. Schmidlin F. Bunnett N. Biochem. Soc. Trans. 2003; 31: 1191-1197Crossref PubMed Google Scholar). Thus, a new N terminus is unmasked acting as a tethered ligand, which can interact with the second extracellular loop of the receptor and thereby initiate multiple signal transductions. A synthetic receptor-activating peptide (AP) with the sequence identical to that of the tethered ligand domain can also fully activate the receptor, bypassing receptor proteolysis (2Cottrell G.S. Amadesi S. Schmidlin F. Bunnett N. Biochem. Soc. Trans. 2003; 31: 1191-1197Crossref PubMed Google Scholar). Upon activation, PAR-2 is rapidly sorted to early endosomes and then translocated to lysosomes where it is degraded (5Dery O. Thoma M.S. Wong H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 18524-18535Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Therefore, PAR-2 resensitization requires the repopulation of cell-surface receptors from the presynthesized and newly synthesized PAR-2 that are stored at the Golgi apparatus (6Böhm S.K. Khitin L.M. Grady E.F. Aponte G. Payan D.G. Bunnett N.W. J. Biol. Chem. 1996; 271: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). It has been shown that the GTPase Rab11a is partially involved in resensitization of PAR-2 (7Roosterman D. Schmidlin F. Bunnett N.W. Am. J. Physiol. 2003; 284: C1319-C1329Crossref PubMed Scopus (68) Google Scholar). However, the precise molecular mechanisms underlying exocytic PAR-2 transport from the Golgi apparatus to the plasma membrane are still poorly characterized. The mammalian p24 family, which includes type I transmembrane proteins of the early secretory pathway, is divided into p25 (α), p24A (β), p23 (γ), and p26 (δ) subfamilies (8Dominguez M. Dejgaard K. Fullekrug J. Dahan S. Fazel A. Paccaud J.P. Thomas D.Y. Bergeron J.J. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (292) Google Scholar). p24A, a major member of the p24 family, has been shown to localize in membranes of the intermediate compartment, cis-Golgi network, and endoplasmic reticulum (ER) (8Dominguez M. Dejgaard K. Fullekrug J. Dahan S. Fazel A. Paccaud J.P. Thomas D.Y. Bergeron J.J. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (292) Google Scholar, 9Emery G. Rojo M. Gruenberg J. J. Cell Sci. 2000; 113: 2507-2516Crossref PubMed Google Scholar, 10Blum R. Feick P. Puype M. Vandekerckhove J. Klengel R. Nastainczyk W. Schulz I. J. Biol. Chem. 1996; 271: 17183-17189Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 11Blum R. Pfeiffer F. Feick P. Nastainczyk W. Kohler B. Schafer K.H. Schulz I. J. Cell Sci. 1999; 112: 537-548Crossref PubMed Google Scholar). The protein p24A has a large N terminus at the lumen and a highly conserved, short cytoplasmic C-tail at the cytosol. The cytoplasmic tail of p24A, containing conserved hydrophobic (FF) and basic (RR) motifs, is able to bind to coat protein (COP) I and COPII subunits (8Dominguez M. Dejgaard K. Fullekrug J. Dahan S. Fazel A. Paccaud J.P. Thomas D.Y. Bergeron J.J. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (292) Google Scholar, 12Contreras I. Ortiz-Zapater E. Aniento F. Plant J. 2004; 38: 685-698Crossref PubMed Scopus (64) Google Scholar, 13Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar). This suggests that p24A is an important constituent of coatomers (14Kaiser C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3783-3785Crossref PubMed Scopus (46) Google Scholar). The importance of p24A on transport vesicles is also supported by the evidence that p24A, via its cytoplasmic tail, is able to interact with ADP-ribosylation factor 1 (ARF1) (12Contreras I. Ortiz-Zapater E. Aniento F. Plant J. 2004; 38: 685-698Crossref PubMed Scopus (64) Google Scholar, 15Gommel D.U. Memon A.R. Heiss A. Lottspeich F. Pfannstiel J. Lechner J. Reinhard C. Helms J.B. Nickel W. Wieland F.T. EMBO J. 2001; 20: 6751-6760Crossref PubMed Scopus (83) Google Scholar, 16Majoul I. Straub M. Hell S.W. Duden R. Soling H.D. Dev. Cell. 2001; 1: 139-153Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), resulting in initiation of transport vesicle assembly and cargo packaging (17D'Souza-Schorey C. Chavrier P. Nat. Rev. Mol. Cell Biol. 2006; 7: 347-358Crossref PubMed Scopus (1071) Google Scholar). The coiled-coil domain located at the N terminus of p24A has been shown to mediate the formation of a hetero-oligomeric complex between p24 proteins, which is a prerequisite for localization and cycling of p24 proteins in the early secretory pathway (9Emery G. Rojo M. Gruenberg J. J. Cell Sci. 2000; 113: 2507-2516Crossref PubMed Google Scholar, 18Ciufo L.F. Boyd A. J. Biol. Chem. 2000; 275: 8382-8388Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 19Gommel D. Orci L. Emig E.M. Hannah M.J. Ravazzola M. Nickel W. Helms J.B. Wieland F.T. Sohn K. FEBS Lett. 1999; 447: 179-185Crossref PubMed Scopus (66) Google Scholar). These previous studies suggest that p24A, acting as cargo receptor as well as coat protein receptor, is involved in biogenesis of transport vesicles and subsequent protein trafficking between the ER and the Golgi apparatus and within the Golgi apparatus (13Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar, 20Schimmoller F. Singer-Kruger B. Schroder S. Kruger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (284) Google Scholar, 21Stamnes M.A. Craighead M.W. Hoe M.H. Lampen N. Geromanos S. Tempst P. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8011-8015Crossref PubMed Scopus (196) Google Scholar, 22Carney G.E. Bowen N.J. Biol. Cell. 2004; 96: 271-278Crossref PubMed Google Scholar). However, no specific cargo proteins for p24A have been identified so far (22Carney G.E. Bowen N.J. Biol. Cell. 2004; 96: 271-278Crossref PubMed Google Scholar). Conversely, data from Caenorhabditis elegans and Saccharomyces cerevisiae implicate that p24A functions as quality control in the early secretory pathway but might not directly mediate cargo protein sorting (23Wen C. Greenwald I. J. Cell Biol. 1999; 145: 1165-1175Crossref PubMed Scopus (78) Google Scholar, 24Springer S. Chen E. Duden R. Marzioch M. Rowley A. Hamamoto S. Merchant S. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4034-4039Crossref PubMed Scopus (96) Google Scholar). Therefore, the precise functions of p24A are still a puzzle. Here we show for the first time that a GPCR, PAR-2, can be identified as a specific cargo protein of p24A. Moreover, we determined the interaction domains on both partner proteins. Importantly, studies on the physiological function of the interaction revealed that p24A regulates exocytic trafficking of PAR-2 from the Golgi apparatus to the plasma membrane. p24A serves in the biosynthetic pathway as a signal-dependent retention and release component to control subsequent receptor resensitization, which is initiated by activation of the small GTPase ARF1. Our findings provide novel functional insights into the physiological role of p24A, and also elucidate a molecular mechanism underlying PAR-2 resensitization. Plasmid Constructs—The FLAG tag (DYKDDDDK) followed by trypsin cleavage site of human PAR-2 amplified by PCR was cloned into pEAK-HA (hemagglutinin epitope) vector, and then the proopiomelanocortin signal sequence was introduced to the N terminus of the FLAG-PAR-2 fusion construct. pVL1392-EL2-GST, pcDNA-p24Amyc, pEGFP-p24A, and pEGFP-p24A deletion mutants all were constructed by standard PCR cloning. All of the cDNAs were mutated at the stop codon and conjugated with the tag at the C terminus. Primer sequences are available on request. Other constructs have been described previously (25Luo W. Wang Y. Hanck T. Stricker R. Reiser G. J. Biol. Chem. 2006; 281: 7927-7936Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). pGEX-VHS-GAT and pGEX-VHS were kindly provided by J. S. Bonifacino (National Institutes of Health). The DNA sequences of plasmid constructs were confirmed to be in-frame by ABI 310 sequencer. Cell Culture and Transfection—Human embryonic kidney (HEK) cells 293 and primary rat astrocytes were cultured as described (25Luo W. Wang Y. Hanck T. Stricker R. Reiser G. J. Biol. Chem. 2006; 281: 7927-7936Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 26Wang Y. Luo W. Stricker R. Reiser G. J. Neurochem. 2006; 98: 1046-1060Crossref PubMed Scopus (54) Google Scholar) and transfected using magnet-assisted transfection (IBA GmbH, Germany) (4Wang Y. Luo W. Reiser G. Biochem. J. 2007; 401: 65-78Crossref PubMed Scopus (47) Google Scholar, 25Luo W. Wang Y. Hanck T. Stricker R. Reiser G. J. Biol. Chem. 2006; 281: 7927-7936Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To activate PAR-2, cells were rinsed with Hanks' solution and stimulated with the specific peptide agonist, human PAR-2 AP SLIGKV-NH2 (100 μm, Bachem, for human PAR-2), or with the most potent peptide agonist 2-furoyl-LIGRLO-NH2 (50 μm, Bachem, for rat PAR-2) in serum-free medium. To prevent receptor resensitization, cells were pretreated with 10 μg/ml brefeldin A (Calbiochem) for 30 min prior to agonist stimulation. Protein-Protein Interaction in Vitro and in Vivo—To detect protein interaction in vitro, we performed the glutathione S-transferase (GST) pulldown assay (25Luo W. Wang Y. Hanck T. Stricker R. Reiser G. J. Biol. Chem. 2006; 281: 7927-7936Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Cell extracts containing Myc-tagged p24A or green fluorescent protein (GFP)-tagged p24A derivatives were incubated overnight with GST fusion protein of PAR-2 derivatives immobilized on glutathione-Sepharose beads (GE Healthcare), followed by Western blot analysis with anti-Myc antibody (Invitrogen) or anti-GFP antibody (Cell Signaling Technology). Immunoprecipitation was also carried out as described (25Luo W. Wang Y. Hanck T. Stricker R. Reiser G. J. Biol. Chem. 2006; 281: 7927-7936Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) to test protein interaction in vivo. Cell extracts were incubated overnight with antibodies indicated in the presence of protein A-Sepharose (GE Healthcare) or protein G-agarose beads (Sigma), followed by Western blot analysis with anti-Myc antibody (Invitrogen), anti-HA antibody (Cell Signaling Technology), or anti-PAR-2 (C-17) antibody (Santa Cruz Biotechnology). Immunofluorescence—Immunofluorescence analysis was carried out by using an LSM510 confocal laser scanning microscope (Carl Zeiss, Germany) (25Luo W. Wang Y. Hanck T. Stricker R. Reiser G. J. Biol. Chem. 2006; 281: 7927-7936Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Detailed protocols are described in the Supplemental Material. siRNA—siRNA-based p24A knockdown, performed using magnet-assisted transfection (IBA GmbH, Germany) in HEK293-PAR-2-HA cells (25Luo W. Wang Y. Hanck T. Stricker R. Reiser G. J. Biol. Chem. 2006; 281: 7927-7936Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), was evaluated by reverse transcription (RT)-PCR and Western blot at 48 h after transfection. p24A siRNA labeled with Alexa Fluor 488 (5′-CCGGAUGUCCACCAUGACUdTdT-3′) was designed based on the mRNA sequence (GenBank™ accession number X92098) and synthesized by Qiagen. ARF1 ON-TARGETplus SMARTpool siRNA containing four individual siRNAs that target different regions of ARF1 open reading frame was obtained from Dharmacon (5′-UGACAGAGAGCGUGUGAAC-3′;5′-CGGCCGAGAUCACAGACAA-3′;5′-ACGAUCCUCUACAAGCUUA-3′;5′-GAACCAGAAGUGAACGCGA-3′). Control siRNA labeled with Alexa Fluor 488 (5′-UUCUCCGAACGUGUCACGUdTdT-3′), which could not target any genes, was obtained from Qiagen. Cellular ARF1-GTPase Assay—Recombinant fusion proteins GST-VHS-GAT and GST-VHS as well as GST were expressed in Escherichia coli BL21 cells, extracted in lysis buffer (50 mm Tris/HCl, pH 8.0, 150 mm NaCl, 10% glycerol, 0.1% Triton X-100) supplemented with 1 mg/ml lysozyme (Sigma) and protease inhibitor mixture (Roche Diagnostics; one tablet per 50 ml), and purified with glutathione-Sepharose beads (GE Healthcare). Primary rat astrocytes were treated with 50 μm 2-furoyl-LIGRLO-NH2 for the indicated times and lysed in modified RIPA buffer (26Wang Y. Luo W. Stricker R. Reiser G. J. Neurochem. 2006; 98: 1046-1060Crossref PubMed Scopus (54) Google Scholar). The resulting cell lysates were incubated overnight with equal amounts of GST fusion proteins immobilized on glutathione-Sepharose beads, followed by Western blot with anti-ARF1 antibody (Epitomics). Total levels of each ARF1 in 2% of whole cell lysates used for precipitation were also determined by Western blot. Calcium Imaging—PAR-2-induced intracellular calcium mobilization in single cells was determined using the calcium-sensitive fluorescent dye Fura-2 AM (2 μm) (27Luo W. Wang Y. Reiser G. Brain Res. 2005; 1047: 159-167Crossref PubMed Scopus (22) Google Scholar). Cells expressing GFP or GFP-tagged truncated p24As were chosen by the fluorescence microscope at 460-nm excitation wavelength. Statistical Analysis—Data were expressed as mean ± S.E. Differences within multiple groups were examined by one-way analysis of variance. p < 0.05 was considered significant. Analysis of the Interaction of PAR-2 with p24A—In a profile search for PAR-2-interacting proteins in the yeast two-hybrid system, we used human PAR-2 as bait (25Luo W. Wang Y. Hanck T. Stricker R. Reiser G. J. Biol. Chem. 2006; 281: 7927-7936Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). There we identified p24A (residues 43-181, GenBank™ accession number X92098), a type I transmembrane protein of the early secretory pathway, to interact with PAR-2. To confirm the finding from the yeast system, we then performed the co-immunoprecipitation assay in transfected HEK293 cells co-expressing PAR-2-HA and p24Amyc. PAR-2-HA was precipitated by anti-HA antibody from transfected HEK293 cell lysates, and the interaction of PAR-2-HA with p24A was analyzed by Western blot using the anti-Myc antibody. As shown in Fig. 1A, p24A was specifically immunoprecipitated by PAR-2-HA in HEK293-PAR-2-HA + p24Amyc cells (lane 6) but not in the negative control wild-type HEK293 cells (lane 4) and HEK293-p24Amyc cells (lane 5). The nitrocellulose membrane was reprobed with the anti-HA antibody to confirm the specificity of the immunoprecipitation of PAR-2-HA. These results suggest that p24A interacts with PAR-2 within cells. Next, we asked whether the interaction of PAR-2 with p24A could occur in a relevant physiological system and whether this interaction was species-specific. We performed immunoprecipitation experiments with anti-p24A antibody in primary rat astrocytes. The interaction between PAR-2 and p24A was determined by Western blot using the anti-PAR-2 antibody. As shown in Fig. 1B, the anti-p24A antibody strongly precipitated PAR-2 from rat astrocytes (lane 2), whereas the control antibody (IgG) did not (lane 1). Immunoprecipitation by anti-PAR-2 antibody from rat astrocyte lysates served as positive control (Fig. 1B, lane 3). Similar results were also observed in analogous experiments carried out with primary rat cortical neurons (Fig. 1C). These results confirm the existence of an endogenous interaction between PAR-2 and p24A in a native system. Moreover, this interaction is not restricted to the human proteins. Analysis of the Domains of PAR-2 and p24A Responsible for the Interaction—To map the domain of PAR-2 responsible for the interaction with p24A, we next performed in vitro GST pulldown assays. Wild-type and truncated PAR-2-GST fusion proteins, which are depicted in Fig. 2A, were generated in Sf9 cells and purified by glutathione-Sepharose beads. Either wild-type or truncated PAR-2-GST fusion proteins on glutathione-Sepharose beads were further incubated overnight with the lysate of HEK293 cells expressing p24Amyc, and then the precipitates were analyzed for p24Amyc by Western blot using the anti-Myc antibody. As shown in Fig. 2B, p24A interacted with both PAR-2Δ(246-397)-GST (Δ(246-397)) and PAR-2Δ(1-213)-GST (Δ(1-213)) with binding capacities comparable with that of the wild-type PAR-2-GST (WT). The lack of interaction of p24A with GST confirmed the specificity of the precipitation (Fig. 2B). Because p24A is an intracellular protein, it is primarily thought that the intracellular loops and the carboxyl tail (C-tail) of the receptor would be required for the interaction. Therefore, we tested the interaction of p24A with the intracellular loops and the C-tail of PAR-2 in the GST pulldown assay. Interestingly, all the intracellular loops and the C-tail of PAR-2 failed to interact with p24A (Fig. 2C). Instead, we made the astonishing discovery that the second extracellular loop of PAR-2 (EL2) strongly interacted with p24A (Fig. 2D). Next, we determined the domain of p24A responsible for the interaction with PAR-2 in the GST pulldown assay. The wild-type and a series of truncated p24A-GFP fusion proteins were constructed and expressed in HEK293 cells, as depicted in Fig. 2E. Cell extracts containing the various p24A derivatives were incubated overnight with the wild-type PAR-2-GST immobilized on glutathione-Sepharose beads. Subsequent Western blot analysis, using the anti-GFP antibody, demonstrated that deletion of the N-terminal luminal part of p24A (ΔN) completely abolished the interaction with PAR-2-GST in the GST pulldown assay (Fig. 2F). However, proteins with C-terminal deletion (ΔC) and deletions of both the transmembrane domain and the C-terminal domain (ΔCT) in either case efficiently interacted with PAR-2-GST (Fig. 2F). The lack of interaction of PAR-2-GST with GFP confirmed the specificity of precipitation (Fig. 2F). These data strongly suggest that the N terminus of p24A is required for the interaction with PAR-2. The Golgi dynamics (GOLD) domain located at the N terminus of p24A is supposed to bind to cargo proteins (28Anantharaman V. Aravind L. Genome Biol. 2002; 3 (research0023)Google Scholar). Thus, we further tested whether the GOLD domain is involved in the interaction with PAR-2 in the GST pulldown assay. As expected, p24AΔGOLD-GFP (ΔG) failed to interact with PAR-2 (Fig. 2G, Pull-down), suggesting that the GOLD domain is required for the interaction with PAR-2. However, the single GOLD domain (G) only weakly bound to PAR-2 (Fig. 2G). Therefore, the GOLD domain is necessary but not sufficient to interact with PAR-2. Further experiments demonstrated that p24A-GL-GFP (GL), which contains the GOLD domain and a neighboring linker, indeed strongly interacted with PAR-2 (Fig. 2G, Pull-down). Identical results were also observed when we used PAR-2EL2-GST in the GST pulldown assay, showing that p24A-GL-GFP directly bound to the second extracellular loop of PAR-2 (supplemental Fig. 1). Therefore, these findings clearly demonstrate that p24A-GL (residues 1-105) is responsible for the interaction with PAR-2. Taken together, our data show that the N-terminal region of p24A (residues 1-105) specifically interacts with the second extracellular loop (EL2) of PAR-2. p24A Interacts with PAR-2 at the Golgi Apparatus—To localize the interaction of PAR-2 with p24A in the cells, we next performed in vivo immunofluorescence staining in co-transfected HEK293 cells. Using the antibody against HA, PAR-2-HA was predominantly detected at the plasma membrane and located in intracellular stores as well (Fig. 3A). By staining with the anti-Myc antibody, p24Amyc was shown to be distributed in the cytosol but strongly located at the Golgi apparatus (Fig. 3B), as revealed by co-localization with the Golgi apparatus marker GM130 (Fig. 3C) in the merged picture (Fig. 3D). The merged image clearly shows that p24A closely co-localized with the intracellular PAR-2, but not with the cell-surface receptor (Fig. 3D, indicated by arrowheads). Moreover, GM130 staining demonstrates that GM130 additionally co-localized with the intracellular PAR-2 and p24A (Fig. 3D, indicated by arrowheads). Therefore, these data clearly indicate that the interaction of PAR-2 with p24A occurs at the Golgi apparatus. In further experiments we used p24A small interfering RNA (siRNA). p24A siRNA was shown to specifically knock down p24A mRNA and protein expression. The levels were reduced by ∼60% in HEK293-PAR-2-HA cells (Fig. 4A). Immunostaining demonstrated that p24A knockdown completely eliminated the intracellular PAR-2 but had no effect on cell-surface receptors (Fig. 4B). Interestingly, we found that GM130 staining became diffuse in the cytosol in p24A-deficient HEK293-PAR-2-HA cells (Fig. 4B, arrowheads), indicating that the Golgi apparatus was disrupted by p24A knockdown. However, p24A knockdown had no effect on the organization of the ER, as shown by staining with the ER marker BiP (Fig. 4B). These results reveal that p24A is a crucial structural component of the Golgi apparatus. The redistribution of the intracellular PAR-2 might be due to the disruption of the structure of the Golgi apparatus by p24A siRNA. ARF1 Regulates the Interaction of PAR-2 with p24A—To examine the functional significance of the interaction, we evaluated whether activation of PAR-2 affects the interaction with p24A. As shown by the co-immunoprecipitation experiment in Fig. 5A, the precipitation of p24A was significantly reduced at 30 min after stimulation with the specific peptide agonist of PAR-2, the human PAR-2 AP, compared with that in unstimulated cells (0 min). The reduction in interaction was similarly observed at 60 min after receptor activation (Fig. 5A). The data summarized in the plot demonstrate that activation of PAR-2 significantly reduced the interaction with p24A by 64% at 30 min and by 57% at 60 min, respectively (Fig. 5A). Similar results were observed in HEK293-PAR-2-HA + p24Amyc cells treated with the PAR-2 agonist protease trypsin (data not shown). Therefore, these data indicate that p24A is dissociated from PAR-2 after receptor activation. Next, we asked by which mechanisms the cell-surface PAR-2 signals are transmitted to p24A, to induce protein dissociation from PAR-2 after receptor activation. p24A is able to interact with inactive ARF1-GDP (12Contreras I. Ortiz-Zapater E. Aniento F. Plant J. 2004; 38: 685-698Crossref PubMed Scopus (64) Google Scholar, 16Majoul I. Straub M. Hell S.W. Duden R. Soling H.D. Dev. Cell. 2001; 1: 139-153Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), and activation of ARF1 would initiate transport vesicle assembly and cargo packaging, thereby resulting in protein trafficking along the secretory pathway (17D'Souza-Schorey C. Chavrier P. Nat. Rev. Mol. Cell Biol. 2006; 7: 347-358Crossref PubMed Scopus (1071) Google Scholar). Therefore, we hypothesized that activated ARF1 might regulate protein dissociation between PAR-2 and p24A. To test this assumption, rat astrocytes were stimulated with rat PAR-2 AP (50 μm) for the indicated time periods (Fig. 5B), and then cell lysates were used to measure the activated ARF1. The latter was precipitated specifically by GST-VHS-GAT in a GST pulldown assay. The GAT domain of GGA3 (Golgilocalized, γ ear-containing ARF-binding protein 3) has been shown to specifically interact with the activated ARF1 (29Dell'Angelica E.C. Puertollano R. Mullins C. Aguilar R.C. Vargas J.D. Hartnell L.M. Bonifacino J.S. J. Cell Biol. 2000; 149: 81-94Crossref PubMed Scopus (326) Google Scholar) and can thus serve as a tool to demonstrate ARF1 activation. Activation of PAR-2 by PAR-2 AP time-dependently induced ARF1 activation in rat astrocytes (Fig. 5B). The maximal activation of ARF1 was observed at 20 min after stimulation with PAR-2 AP (summary in Fig. 5B), which was an early event prior to protein dissociation between PAR-2 and p24A. GST-VHS, the domain of GGA3 with no ARF1 binding, and GST were not able to pull down ARF1-GTP (Fig. 5B), confirming the specificity of precipitation by the GAT domain. Therefore, these data clearly demonstrate that ARF1 can be activated after stimulation with PAR-2 agonists. To further study whether activated ARF1 initiates protein dissociation between PAR-2 and p24A after receptor activation, we tested the effect of brefeldin A on the interaction of PAR-2 with p24A. Brefeldin A is an inhibitor of the guanine nucleotide exchange factors (GEFs) and can prevent ARF1 activation within cells (30Morinaga N. Adamik R. Moss J. Vaughan M. J. Biol. Chem. 1999; 274: 17417-17423Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). To this aim, HEK293-PAR-2-HA + p24Amyc cells were pretreated with brefeldin A, followed by human PAR-2 AP stimulation. Here we found that brefeldin A completely prevented protein dissociation of p24A from PAR-2 after PAR-2 activation, both at 30 and 60 min (Fig. 5C), indicating that activation of ARF1 results in protein dissociation of PAR-2 from p24A. When the cell-surface PAR-2 has been activated and internalized, the intracellular PAR-2 should be triggered to be sorted to the plasma membrane for receptor resensitization. Brefeldin A has been shown previously to inhibit PAR-2 resensitization (6
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