Regulation of Transport of the Angiotensin AT2 Receptor by a Novel Membrane-Associated Golgi Protein
http://www.100md.com
《动脉硬化血栓血管生物学》
From the Institute of Pharmacology (C.J.W.) and the First Department of Medicine (M.L.K.), University Hospital Schleswig-Holstein, Campus Kiel; the Institute of Anatomy (T.P.), University of Kiel; the Center for Cardiovascular Research/Institute of Pharmacology and Toxicology (H.F.-K., H.K., M.M., J.H.S., T.U.), Charité–University Medicine Berlin; and the Institute for Arteriosclerosis Research at the University of Muenster (M.S.), Germany.
Correspondence to Thomas Unger, MD, Center for Cardiovascular Research/Institute of Pharmacology and Toxicology, Charité–University Medicine Berlin, Hessische Str. 3-4, 10115 Berlin, Germany. E-mail thomas.unger@charite.de
Abstract
Objective— Synthesis and maturation of G protein–coupled receptors are complex events that require an intricate combination of processes including protein folding, posttranslational modifications, and transport through distinct cellular compartments. Little is known concerning the regulation of G protein–coupled receptor transport from the endoplasmic reticulum to the cell surface.
Methods and Results— Here we show that the cytoplasmatic carboxy-terminal of the angiotensin AT2 receptor (AT2R) acts independently as an endoplasmic reticulum–export signal. Using a yeast two-hybrid system, we identified a Golgi membrane–associated protein termed ATBP50 (for AT2R binding protein of 50 kDa) that binds to this motif. We also cloned ATBP60 and ATBP135 encoded by the same gene as ATBP50 that mapped to chromosomes 8p21.3. Downregulation of ATBP50 using siRNA leads to retention of AT2R in inner compartments, reduced cell surface expression, and decreased antiproliferative effects of the receptor.
Conclusion— Our results indicate that ATBP50 regulates the transport of the AT2R to cell membrane by binding to a specific motif within its cytoplasmic carboxy-terminal and thereby enabling the antiproliferative effects of the receptor.
We identified a Golgi membrane–associated protein termed ATBP50 that binds to the cytoplasmatic terminus of the angiotensin AT2 receptor. We also cloned ATBP60 and ATBP135 encoded by the same gene. Downregulation of ATBP50 leads to retention of AT2R in inner compartments, reduced cell surface expression, and decreased antiproliferative effects of AT2R.
Key Words: AT2 receptor ? ATBP50 ? receptor transport ? Golgi proteins ? ERK1/2
Introduction
The heptahelical G protein–coupled receptors (GPCRs) represent one of the largest protein families in eukaryotic cells.1 A large number of structure–function studies have defined receptor sequences that are essential for ligand binding, G protein coupling, and desensitization. In contrast, little is known concerning the requirements for the transport of these proteins to the plasma membrane. Intracellular accessory proteins can be critical for GPCR biogenesis, including aspects of receptor trafficking. Recent discoveries have identified multiple membrane-associated proteins that dictate the delivery of the receptor to the cell surface.2
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The octapeptide hormone angiotensin II (Ang II) exerts a wide variety of physiological actions such as vasoconstriction and aldosterone secretion, but it is also involved in pathological mechanisms of atherosclerosis as well as vascular and cardiac growth. Ang II mainly interacts with two receptor subtypes, designated AT1 and AT2 receptor (AT1R, AT2R), both belonging to the superfamily of GPCRs. The majority of its well-described effects are mediated by the AT1R.3 Increasing evidence indicates, however, that the AT2R subtype can modulate the effects of AT1R, including those on blood pressure,4–8 cardiac and vascular cell growth,9–11 and tissue regeneration after injury.12–15
Thus, it has been shown that stimulation or overexpression of AT2R in certain cell lines inhibits cell proliferation induced by growth factors16,17 and promotes neuronal differentiation18–21 as well as apoptosis.22–25 The growth inhibitory effects of the AT2R have been shown to be associated with the activation and/or induction of a series of phosphatases including the protein tyrosine phosphatase SHP-1, mitogen-activated protein kinase phosphatase-1 (MKP-1), and serine/threonine phosphatase 2A (PP2A), which results in the inactivation of AT1R- and/or growth factor–activated extracellular signal regulated kinase (ERK).16 In PC12W cells, long-term activation of the AT2R increases the synthesis of ceramide, which may lead to the AT2R-mediated apoptosis.26,27
However, besides the growth inhibitory function of AT2R, there is still some controversy, because it has also been reported that AT2R stimulation promotes fibrosis and growth. AT2R activation in AT2R-transfected smooth muscle cells stimulates collagen synthesis.28 Furthermore, ventricular hypertrophy induced by pressure overload was not observed in AT2R knockout mice.29
A peculiarity of the AT2R is its uncommon response to Ang II stimulation, as there is no desensitization or downregulation in response to ligand binding.30,31 In contrast, the cell surface expression of the AT2R undergoes upregulation after stimulation with Ang II, whereas the level of AT2R transcripts does not increase.32–34
Numerous studies have identified different AT2R regions responsible for the interaction with other proteins involved in AT2R activation or regulation. Among these regions, the fifth and sixth transmembrane domains were shown to directly interact with the agonist Ang II,35–38 and the third intracellular loop38–40 and the cytoplasmic tail were shown to be critical for coupling with Gi and SHP-1,41 respectively. Moreover, the cytoplasmic tail was also shown to contain a binding domain for the transcription factor promyelocytic zinc finger protein (PLZF) which affects the AT2R signaling.42 In an effort to better characterize the signaling mechanisms of the AT2R, we screened for new AT2R interacting proteins.
Methods
For details regarding cell culture, plasmid transfection of cell lines, and mouse tissues, construction of mutant and epitope-tagged ATBP and AT2R expression vectors, design of a fluorescent Golgi-marker (GM) protein, coimmunoprecipitation experiments, immunoblotting, confocal immunofluorescence microscopy, surface expression ELISA, siRNA design and transfection, proliferation assay, Northern blot analysis, RT-PCR analysis, relative quantitation of gene expression by real-time PCR, and in situ hybridization, please see the online Methods, available at http://atvb.ahajournals.org.
Yeast Two-Hybrid Screening
The mice AT2R carboxyl-terminal tail (residues 313 to 363) GAL4 binding domain fusion protein cloned in the yeast expression vector pGBKT7 was cotransformed into the yeast strain AH109 with a GAL4 activation domain fusion library of mouse 11 day embryo cDNA in pACT2 (Clontech, Heidelberg, Germany). Yeast two-hybrid screening was performed according to the Clontech Matchmaker Two-Hybrid System 3 protocol. Clones expressing both the bait and the prey were selected on growth medium lacking Leu and Trp, and the interacting proteins were identified for their adenine auxotroph complementation. The cDNA inserts from positive clones were then sequenced using the dideoxynucleotide method.
cDNA Cloning of ATBPs
Total RNA was isolated from mice embryo, and the mRNA was then affinity purified by oligo(dT)-cellulose chromatography. The cDNA synthesis was primed with oligo(dT). A kit for rapid amplification of cDNA ends (RACE) was used to identify the 5' ends of the ATBPs (Life Technologies). The following nested oligonucleotides were used for the polymerase chain reaction (PCR): AP1 primer (Life Technologies), GSP1 (5'-CAGAAGAATCCCAGGAGCCT), and GSP2 (5'-AGGCTCCTGGGATTCTTCTG). Total mice embryo RNA was used for cDNA synthesis. The PCR products were subcloned into the pcDNA3.1 vector (Invitrogen) and sequenced.
Statistical Analysis
With respect to the epidermal growth factor (EGF) stimulation experiments, a two-tailed Student t test was performed.
Results
Identification of ATBP as Binding Protein of the AT2 Receptor
To identify AT2-interacting proteins, a yeast two-hybrid screening was set up with the AT2R carboxyl-terminal tail (residues 313 to 363) as bait protein and a cDNA library generated from mouse 11 day embryo as prey protein. Of 2x106 colonies screened, 86 fulfilled all five selection criteria (phenotypes: His +, 3 ATR, galactosidase –, Ura +, and 5-fluoro-orotic-acid +), and two of these colonies encoded for full-length cDNA termed ATBP50 for AT2 receptor binding protein of 50 kDa. To confirm the result of the two-hybrid screening, coimmunoprecipitation studies were performed with both proteins coexpressed as tagged fusion proteins in COS-7 cells. 3XFlag-tag was fused to the N terminus of AT2R, and V5-tag was fused to the C terminus of ATBP50. Cell lysates were subsequently used for coimmunoprecipitation studies with monoclonal antibodies directed against the respective tags. V5-tagged ATBP50 protein was coimmunoprecipitated with anti-3XFlag antibody when cells were cotransfected with AT2-Flag (Figure 1A, lane 2), but not in the control experiment (Figure 1A, lane 1). The other two isoforms, ATBP60 and ATBP135 (see below), were not able to interact with the AT2R (data not shown).
Figure 1. A, Specific binding of ATBP50 and ATBP50C to the AT2 receptor. Coimmunoprecipitation of ATBP50 or ATBP50C with the AT2R. COS-7 cells were transiently cotransfected with Flag-tagged AT2R and V5-tagged ATBP50 or V5-tagged ATBP50C. Proteins were chemically cross-linked, and receptor complexes were immunoprecipitated. Whole-cell extracts were subjected to SDS-PAGE, blotted, and probed for the presence of the V5 epitope and Flag epitope, respectively. Ang II stimulation was performed 48 hours after transfection for 2 hours. B, Northern blot analysis of total RNA from mice embryo. The mRNA was electrophoresed in agarose gel and blotted on nitrocellulose. The blot was hybridized with ATBP50 cDNA as a probe. C, RT-PCR analysis of AT2R, ATBP50, ATBP60, and ATBP 135. mRNA from various mouse tissues was reverse transcribed and subjected to PCR using the indicated primers with HPRT serving as control.
Furthermore, we were able to demonstrate that a C-terminal deletion mutant of ATBP (ATBP50C), which is discussed below, is still able to interact with the AT2R (Figure 1A, lane 3).
ATBP and AT2 Receptor are Coexpressed in Mouse Tissue
To examine the expression of ATBP50 in embryonic tissue, we performed Northern blots using RNA from mouse embryos with cDNA of ATBP50 as a probe. We found three mRNA isoforms detected by the probe in a comparable intensity (Figure 1B). They had an estimated size of 2.5, 3.6, and 5.9 kb, respectively. To analyze which adult tissues express ATBP, we performed isoform-specific semiquantitative RT-PCRs with RNA from various mouse tissues. A ubiquitous expression of ATBP50 was seen in all tissues tested (Figure 1C). Contrary to ATBP50, ATBP60 and ATBP135 were not detectable in brain and spleen. The strongest expression of ATBP50 was found in the uterus and adrenal gland (Figure 1C, lanes 2 and 3), tissues in which the AT2R is also predominantly expressed (Figure 1C, lanes 2 and 3). In contrast, the AT2R- and ATBP50 expressions in heart, brain, spleen, kidney, gut, and dermis were relatively weak.
More detailed investigations regarding coexpression of ATBP and AT2R were performed by in situ hybridization to evaluate the distribution patterns within different tissues using a probe common for all three isoforms. For example, in the adrenals (Figure IIA, available online at http://atvb.ahajournals.org) the expression patterns of ATBP and AT2 were strikingly similar, compatible with coexpression of both genes. This was also true for kidney, lung, heart, and brain (data not shown). We further examined tissues of AT2R-deficient mice by in situ hybridization. Although the adrenal glands of wild-type mice coexpressed the AT2 receptor and ATBP, the ATBP expression in this tissue was drastically reduced in AT2 receptor–deficient mice (Figure IIA). To substantiate this finding, we performed real-time PCR analysis on uterine tissue of wild-type– and AT2R-deficient mice, respectively. Uterus was chosen because of the strong coexpression of both proteins established in previous experiments. The ATBP mRNA expression in AT2 receptor knockout mice was reduced to <10% compared with wild-type mice (Figure IIB).
Genomic Organization of the ATBP Gene
The presence of three differently sized transcripts suggested that the ATBP gene encodes for two more transcripts than expected. A genomic BLAST search with the cDNA sequence of ATBP50 (GenBank accession number AY626781) in the mouse genome databank at NCBI showed that the gene mapped to the minus strand of the mice chromosome 8p21.3 with 10 exons spanning >47.7 kb. A search with the WGS supercontig Mm8_WIFeb01_187 sequence upstream of the ATBP50 stop codon in GENSCAN Web Server at MIT43 showed the whole gene of ATBP, spanning a region of 90.5 kb genomic DNA with 15 exons and 14 introns, codes for two additional transcripts, ATBP60 (GenBank accession number AY626782) and ATBP135 (GenBank accession number AY626781), respectively. The 5' terminus and the 3' terminus of every intron exhibit the GT-consensus and AG-consensus sequence, respectively.
All three mRNAs include exon 7 to 15 of the ATBP gene, encoding 2047 bp at the 3' end of the mRNAs. ATBP50 mRNA additionally contains exon 6 (358 bp) with the translation initiation start. Exons 4 (133 bp) and 5 (41 bp) are expressed in both ATBP60 and ATBP135 mRNA, whereas exon 3 (238 bp) is only expressed in ATBP60 mRNA and bears its translation initiation codon. Exons 1 (2311 bp) and 2 (119 bp) are exclusively expressed in ATBP135 mRNA with the translation initiation start in exon 1 (Figure 2A and 2B). Analysis of the sequence surrounding the potential translation initiation starts revealed a favorable Kozak sequence44 for ATBP50 (5'-GAAGAGATGC) and ATBP135 (5'-TTCAGGATGA) but not for ATBP60 (5'-TTTTAAATGA). A poly(A) tail and a polyadenylation signal (AATAAA) 21 nucleotides upstream from the poly(A) tail were detected in all three cDNA sequences.
Figure 2. Organization of the ATBP gene and protein. A, Genomic organization of ATBP gene. The gene spans 15 exons, spanning a region of 90 kb within chromosome 8p21.3 to 22. Numbers on top represent exons. Open boxes indicate exons transcribed in all ATBP isoforms; fasciated boxes, ATBP50-specific exons; checkered boxes, ATBP60- and ATBP135-specific exons; dotted boxes, ATBP60-specific exons; black boxes, ATBP135-specific exons. B, Predicted secondary structure of the ATBP proteins and amino acid sequence of ATBP50, ATBP60, and ATBP135 in one-letter code. The isoform specific regions are pictured in boxes with the same pattern used in Figure 2A. Within the common sequence the region of coiled-coil structures are underlined and the leucine-zipper are in bold letters.
Sequence and Structural Analysis of the ATBP Proteins
The predicted protein sequences encoded by the ATBP gene are shown in Figure 2B. Mice ATBP50, ATBP60, and ATBP135 are proteins with 440, 520 and 1209 residues and a calculated mass of 50, 60, and 135 kDa, respectively.
A PHI- and PSI-BLAST45 search with the three protein sequences in the NCBI databank showed no significant homologies to other proteins except proteins with a long coiled coil region like Golgi matrix proteins such as GM130/golgin-95,46 GCP170,47 golgin-160,48 and p115.49 An identity was found with the human mitochondrial tumor suppressor gene 1 (MTSG1) protein50 and the human ATIP.51 In the protein family database Pfam,52 we found a homology of the ATBP proteins to the CDD:pfam05483 named HOOK protein. It has been demonstrated that endogenous HOOK3 binds to Golgi.53
The ATBP proteins could be divided into two parts: an N-terminal "head" region of isoform-specific length, assumed to have a random coiled structure, and a C-terminal part of 400 amino acids identical in all three proteins. Using the program COILS (version 2.1) at EMBnet,54 the C-terminal part of the ATBP proteins were predicted to have an -helical coiled-coil motif, starting at position 214 (relative to the common part of all isoforms) and interrupted at position 260 to 261 by the strong helix breakers asparagine and proline.55 The C-terminus ends in a noncoiled-coil serine/proline-rich tail of 38 amino acids (Figure 2B). Using the program 2ZIP at DKFZ, Heidelberg, the coiled-coil region was predicted to contain two potential leucine zippers, a specialized form of coiled-coil dimerization motifs56 (Figure 2B). A hydropathy plot57 illustrated that the ATBP proteins were highly hydrophilic without any potential transmembrane domain.
ATBP Proteins Are Localized at the Golgi Matrix Through Their C-Terminal Domains
To examine the subcellular distribution of the ATBP proteins, we compared the localization of ATBP proteins with the localization of a Golgi marker (GM). For this purpose, we transiently cotransfected COS-7 cells with a DsRed2-coupled GM and one of the Flag-tagged ATBP proteins (wtATBP50 or one of the three ATBP50 deletion mutants described below, respectively). The Flag-tagged ATBP proteins were detected with fluorescein isothiocyanate (FITC)–coupled anti-Flag antibody and imaged by confocal microscopy. WtATBP50 (Figure 3A) as well as the GM (Figure 3B) were primarily located in the perinuclear region with a small amount of diffuse background staining throughout the cell. This membrane distribution is similar to what has been reported for other Golgi-Network resident proteins.58 To examine the role of ATBP domains in Golgi targeting, three EGFP-coupled deletion mutants of the ATBP50 protein were created. DsRed2-coupled GM and the mutants were cotransfected in COS-7 cells, and localization was examined by confocal microscopy. ATBP50C, a mutant lacking the C-terminal domain of the last 40 aa, lost the organized distribution and was diffusely dispersed in the nucleus and cytoplasm (Figure 3C and 3D). The same effect was seen using the mutant ATBP50NC lacking both the N and the C terminus, suggesting that the -helical coiled-coil domain alone is not needed for directing colocalization to the Golgi apparatus (data not shown). In contrast, ATBP50N, a mutant lacking the N-terminal domain, was localized at the Golgi apparatus comparable to wild type indicating that the N-terminal domain is not required for Golgi localization. As expected from the identical C terminus of the three ATBP proteins, the same feature was seen with the two other isoforms ATBP60 and ATBP135 (data not shown). These results indicate that the ATBP proteins interact with the Golgi through their noncoiled C terminus, a domain identical in all three isoforms.
Figure 3. Functional analysis of ATBP. A through D, The C-terminal truncation of ATBP leads to decreased expression at the Golgi apparatus. Coexpression of Flag-tagged wtATBP50 with DsRed-tagged GM (A and B) and of Flag-tagged ATBPC with DsRed-tagged GM (C and D) in COS-7 cells. Immunocytology was performed using a green fluorescent antibody against the Flag-tag, and immunofluorescence was viewed using a green (A and C) or a red filter (B and D). E, ATBP Proteins Homo- and Heterodimerize in vivo. COS-7 cells were transiently transfected with expression vectors encoding Flag-tagged ATBP50 in combination with V5-tagged versions of ATBP50, ATBP60, and ATBP135. Total cell extracts were immunoprecipitated (IP) with an anti-Flag antibody and electrophoresed, and the resulting blots were probed with anti-V5 antibodies to detect the various immunoprecipitated epitope-tagged ATBP proteins.
ATBP Proteins Appear as Homo- and Heterodimers In Vitro
To analyze putative dimerizations we performed coimmunoprecipitation studies with coexpressed ATBP proteins. ATBP50 was Flag-tagged and coexpressed in COS-7 cells with V5-tagged ATBP50, ATBP60, and ATBP135, respectively. Cell lysates were subsequently used for coimmunoprecipitation studies with monoclonal antibodies directed against the Flag-tag. The eluate was resolved using SDS-PAGE followed by detection with a monoclonal antibody against V5. V5-tagged ATBP50, ATBP60, and ATBP135 was coimmunoprecipitated with ATBP50 (Figure 3E). Our results allow for the possibility that all three ATBP proteins are able to form hetero- as well as homodimers with each other.
ATBP50 Is Required for AT2 Receptor Cell Surface Expression
To determine the physiological significance of the ATBP/AT2R protein interaction, we used siRNA technology to reduce endogenously expressed ATBP50 in N1E-115 cells that endogenously express the AT2R and AT1R.59 Transiently transfected EGFP-tagged AT2Rs were detected predominantly at the plasma membrane of N1E-115 cells (Figure 4A). However, when the EGFP-tagged AT2R was cotransfected with siRNA specific for ATBP50, AT2 receptor staining was increased at the endoplasmic reticulum and significantly reduced at the cell surface (Figure 4B). The cell surface distribution of the AT2R was not altered when coexpressed with Flag-tagged ATBP, indicating that overexpression of ATBP50 as such did not affect the normal plasma membrane distribution of AT2R (data not shown). We further measured the cell surface expression of the AT2R using a surface expression ELISA. A significant decrease in cell surface expression of the AT2R was observed when a C-terminal truncated AT2R (AT2RC) was used (Table I, available online at http://atvb.ahajournals.org). A strikingly similar effect was observed when ATBP50 was downregulated by siRNA (Table I). These results suggest that ATBP/AT2 receptor interaction is required for the proper targeting of AT2 receptor to the plasma membrane.
Figure 4. Interaction between ATBP50 and AT2R is required for AT2R cell surface expression. N1E-115 cells transfected with EGFP-tagged AT2R (A) and cotransfected with ATBP50 specific siRNA (B) are shown. Protein expression was detected by confocal laser microscopy.
Effect of ATBP50 on AT2 Receptor Function in N1E-115 Cells
To further investigate functional interactions between ATBP and the AT2R, we examined the ability of the AT2R to decrease the proliferative effect of EGF stimulation in absence of ATBP50. For this purpose we used the mouse neuroblastoma cell line N1E-115 for which the antiproliferative effect of the AT2R has already been shown.60 Before Ang II stimulation, the AT1R was selectively blocked by losartan (10 μmol/L). We used the activity of the ERK1/2 to measure the activation state of the growth-related mitogen-activated protein kinase cascade. The activation of ERK1/2 in these cells was visualized by Western blotting using antibodies detecting the phosphorylated (activated) form of the enzyme. As expected, EGF (50 ng/mL) induced ERK1/2 activity (Figure IA, lane 2; available online at http://atvb.ahajournals.org) compared with controls (Figure IA, lane 1). Stimulation of the AT2R by Ang II (50 nM) produced a robust and potent inhibition of ERK1/2 phosphorylation in EGF-stimulated (50 ng/mL) N1E-115 cells (Figure IA, lane 3). This effect of the AT2R was prevented by preincubation with the AT2R selective antagonist PD123319 (10 μmol/L) (Figure IA, lane 4). A knockdown of ATBP50 with siRNA reduced the AT2R ability to inhibit the EGF-induced ERK1/2 stimulation (Figure IA, lane 6), whereas control siRNA had no inhibitory effect (Figure IA, lane 5).
We then examined whether the results of reduced ERK1/2 inhibition of AT2R in the absence of ATBP50 would be reproducible in a proliferation assay with N1E-115 cells in the same experimental setup. After two days of EGF (50 ng/mL) treatment the cell proliferation rate nearly doubled compared with untreated cells (Figure IB, columns 1 and 2). This proliferation induction could be blocked with Ang II treatment (50 nM), whereas preincubation with PD123319 (10 μmol/L) was able to prevent this inhibitory effect (Figure IB, columns 3 and 4). Pretreatment of N1E-115 cells with ATBP50 specific siRNA 2 days before Ang II stimulation caused a significant reduction of the inhibitory effect of AT2R. Taken together, these data indicate that the inhibitory effect of the AT2R on EGF-induced cell proliferation requires ATBP50.
Stimulation of PC12W Cells With Ang II Leads to Strong Induction of ATBP50 mRNA
PC12W rat pheochromocytoma cells, which endogenously express both AT2R and ATBP50, were stimulated with 100 nM Ang II. A continuous increase in ATBP50 mRNA levels was observed, starting 30 minutes after stimulation (Figure IIC).
To assess whether Ang II also affects the cell surface expression of the AT2R we performed a further surface expression ELISA. In the experimental setting used, Ang II did not alter the amount of transient transfected Flag-tagged AT2R at the cell membrane (Table II, available online at http://atvb.ahajournals.org). This experiment is consistent with an additional coimmunoprecipitation experiment, where Ang II incubation did not alter the amount of ATB50 coprecipitated with Flag-tagged AT2R (Figure 1a, lane 4).
In addition, the data shown in Table II indicate, that—besides Ang II—cotransfection of ATBP50 or ATBP50C also did not alter the amount of transient transfected Flag-tagged AT2R at the cell membrane in this experimental system.
Discussion
In this study, we have identified the previously unknown ATBP50 as a new AT2R interacting protein by yeast two-hybrid screening. ATBP50 was shown to directly interact with the carboxyl-terminal tail of this receptor, and the specificity of this association was demonstrated by coimmunoprecipitation experiments. Three different ATBP transcripts (2.5 kb, 3.6 kb, and 5.9 kb) were detected by Northern blot analysis in mouse embryo tissues. These ATBP isoforms also show tissue-specific expression levels using RT-PCR analysis of multiple organs. In the adult mouse, the ATBP50 transcripts were present in all tissues tested, with abundant expression in uterus and adrenal correlating with the expression pattern of the AT2R in the adult mouse. The weak expression of ATBP50 in the liver, where the AT2R is not detectable, and the strong ATBP50 expression in the lung compared with AT2R expression suggest that ATBP has further functions that are independent of the AT2R. The putative polypeptides encoded by these gene have predicted molecular masses of 50 kDa (ATBP50), 60 kDa (ATBP60), and 135 kDa (ATBP135), respectively. Even though the N termini of all ATBPs are of different size and show no homologies, the main structural feature of the ATBP proteins is their predicted ability to form coiled-coils in the region of 400 amino acids which is identical in all isoforms. Coimmunoprecipitation experiments suggested that all three ATBP proteins are able to form hetero- and homodimers with each other, thus indicating possible complex functions besides the receptor trafficking described below.
Our study provides evidence for a functional relationship between ATBP and AT2R concerning two different aspects: First, we have shown by confocal fluorescence microscopy and ATBP50 specific siRNA that ATBP50 promotes cell surface expression of AT2R. These results were confirmed by our cell surface ELISA experiments. Together these data suggest that ATBP50 functions as a regulatory protein to modulate the plasma membrane routing and thereby the functionality of the AT2R. With respect to functionality, we observed that downregulation of ATBP50 reduced the maxi-mal inhibitory effect of AT2R on EGF-stimulated ERK1/2 activation in N1E-115 cells. This could be a direct result of a reduced presence of the receptor in the plasma membrane, a finding in accordance with Miura and Karnick who showed that the level of receptor protein expression is critical for induction of antiproliferation and apoptosis.25 The presence and expression level of ATBP50 has the potential to alter AT2R signaling. We offer a putative functional explanation for the effects of ATBP on AT2R mediated ERK1/2 inhibition by demonstrating that ATBP is necessary for AT2R cytoplasma membrane transport.
Our results indicate that ATBPs can be added to a growing list of proteins that have been identified as GPCR-binding proteins managing receptor trafficking to the cell surface. For instance, the actin binding protein filamin A (ABP-280) was found to modulate the cell surface expression of D2 and D3 receptor subtypes.61–63 Furthermore, association of the somatostatin receptor subtype 1 with Shk1 kinase–binding protein are required for targeting the receptor to the cell surface.64 In addition, Leclerc et al predicted regulatory proteins modulating the routing of the AT1R to the cell surface.65
Consistent with our bioinformatic results (eg, homology to HOOK protein), using confocal microscopy we were able to show that ATBP is located at the Golgi apparatus. Although ATBP proteins are all hydrophilic, lacking obvious hydrophobic regions capable of serving as membrane anchors, the ATBP appends to the Golgi through its C-terminal tail as evidenced by our mutation experiments.
Secondly, we demonstrate a functional interaction between ATBP and AT2R in vivo, because ATBP mRNA was markedly reduced in AT2R knockout mice, as shown by in situ hybridization and real-time PCR analysis. These results are consistent with our complementary observation that Ang II can induce ATBP50 mRNA in PC12W cells, which exclusively express AT2R but not AT1R. Therefore, ATR2 exerts positive feedback on ATBP mRNA expression. In this context it is important to note that ATBP isoforms are encoded by a single gene and characterized by N-terminal isoform-specific exons, implying that these transcripts are controlled by three different promoters. The complex mechanisms of this feedback of AT2 receptor signaling on the isoform-specific ATBP promoters and/or isoform-specific mRNA half-lives will be examined in a further project.
Therefore, our data point to the existence of a two-directional interaction between ATBP and AT2R: On the one hand, ATBP is necessary for the trafficking and function of AT2R as described above; on the other hand, AT2R positively regulates ATBP mRNA expression.
In this context it is of interest to note that besides ATBP, the C terminus of the AT2R is able to bind the recently described promyelocytic zinc finger protein (PLZF).42 After Ang II stimulation PLZF colocalizes with AT2R at the plasma membrane, followed by internalisation of AT2R and PLZF and nuclear translocation of PLZF. Therefore, it is conceivable that the transcriptional upregulation of ATBP by Ang II described here might be mediated by PLZF.
The interaction between ATBP50 and the AT2R may have direct implementations for human disease. For instance, Vervoort and coworkers have shown that mutations of the AT2R are linked to mental retardation; in particular, mutations in the receptor cytoplasmic C terminus are associated with this innateness.66 It is conceivable that dysfunctional interaction between AT2R and ATBP50 may be responsible for this disease. Another potential role of ATBP gene in human disease is suggested by its chromosomal localization at 8p21.3 as abnormalities in this region have been reported in various cancer types, thus implicating ATBP as a positional candidate gene in cancer.67
This hypothesis is further supported by the well documented antiproliferative properties of the AT2R68 that are, according to our results, closely linked to the AT2R-ATBP50 interaction.
Furthermore, Seibold et al reported that the mitochondrial tumor suppressor gene 1 (MTSG1) protein, which is identical with ATBP50, is a mitochondrion resident protein.50 However, we were not able to demonstrate that any of the ATBP proteins are localized in mitochondria. Possible explanations for this discrepancy might be the different cellular systems used (pancreatic tumor cells versus COS-7 cells) and the observation that the type of heterodimerization can influence the subcellular distribution.69
In conclusion, we have identified a novel, Golgi-localized protein, ATBP50, that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT2R and is necessary for the cell surface expression of the AT2R. ATBP50 influences the receptor-mediated signaling by regulating receptor cell surface expression. Further functions of the ATBP isoforms and the nature of their associated proteins are now under investigation. Given that the AT2R is an important mediator of the renin-angiotensin system, ATBP50 may play a significant role in cardiovascular physiology and pathophysiology as well as other conditions that are genetically linked to the AT2R, such as mental retardation.
While this manuscript was in preparation, Nouet et al have described a protein named ATIP, which is identical to ATBP50, as an AT2 receptor interacting protein.51
Acknowledgments
Parts of this work have been supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 415: Pathophysiology of signal transduction pathways). We thank Michael Bader (Max Delbrück Center for Molecular Medicine (MDC), Berlin Buch, Germany) for the AT2R knockout mice.
Received August 10, 2004; accepted November 1, 2004.
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Correspondence to Thomas Unger, MD, Center for Cardiovascular Research/Institute of Pharmacology and Toxicology, Charité–University Medicine Berlin, Hessische Str. 3-4, 10115 Berlin, Germany. E-mail thomas.unger@charite.de
Abstract
Objective— Synthesis and maturation of G protein–coupled receptors are complex events that require an intricate combination of processes including protein folding, posttranslational modifications, and transport through distinct cellular compartments. Little is known concerning the regulation of G protein–coupled receptor transport from the endoplasmic reticulum to the cell surface.
Methods and Results— Here we show that the cytoplasmatic carboxy-terminal of the angiotensin AT2 receptor (AT2R) acts independently as an endoplasmic reticulum–export signal. Using a yeast two-hybrid system, we identified a Golgi membrane–associated protein termed ATBP50 (for AT2R binding protein of 50 kDa) that binds to this motif. We also cloned ATBP60 and ATBP135 encoded by the same gene as ATBP50 that mapped to chromosomes 8p21.3. Downregulation of ATBP50 using siRNA leads to retention of AT2R in inner compartments, reduced cell surface expression, and decreased antiproliferative effects of the receptor.
Conclusion— Our results indicate that ATBP50 regulates the transport of the AT2R to cell membrane by binding to a specific motif within its cytoplasmic carboxy-terminal and thereby enabling the antiproliferative effects of the receptor.
We identified a Golgi membrane–associated protein termed ATBP50 that binds to the cytoplasmatic terminus of the angiotensin AT2 receptor. We also cloned ATBP60 and ATBP135 encoded by the same gene. Downregulation of ATBP50 leads to retention of AT2R in inner compartments, reduced cell surface expression, and decreased antiproliferative effects of AT2R.
Key Words: AT2 receptor ? ATBP50 ? receptor transport ? Golgi proteins ? ERK1/2
Introduction
The heptahelical G protein–coupled receptors (GPCRs) represent one of the largest protein families in eukaryotic cells.1 A large number of structure–function studies have defined receptor sequences that are essential for ligand binding, G protein coupling, and desensitization. In contrast, little is known concerning the requirements for the transport of these proteins to the plasma membrane. Intracellular accessory proteins can be critical for GPCR biogenesis, including aspects of receptor trafficking. Recent discoveries have identified multiple membrane-associated proteins that dictate the delivery of the receptor to the cell surface.2
See page 15
The octapeptide hormone angiotensin II (Ang II) exerts a wide variety of physiological actions such as vasoconstriction and aldosterone secretion, but it is also involved in pathological mechanisms of atherosclerosis as well as vascular and cardiac growth. Ang II mainly interacts with two receptor subtypes, designated AT1 and AT2 receptor (AT1R, AT2R), both belonging to the superfamily of GPCRs. The majority of its well-described effects are mediated by the AT1R.3 Increasing evidence indicates, however, that the AT2R subtype can modulate the effects of AT1R, including those on blood pressure,4–8 cardiac and vascular cell growth,9–11 and tissue regeneration after injury.12–15
Thus, it has been shown that stimulation or overexpression of AT2R in certain cell lines inhibits cell proliferation induced by growth factors16,17 and promotes neuronal differentiation18–21 as well as apoptosis.22–25 The growth inhibitory effects of the AT2R have been shown to be associated with the activation and/or induction of a series of phosphatases including the protein tyrosine phosphatase SHP-1, mitogen-activated protein kinase phosphatase-1 (MKP-1), and serine/threonine phosphatase 2A (PP2A), which results in the inactivation of AT1R- and/or growth factor–activated extracellular signal regulated kinase (ERK).16 In PC12W cells, long-term activation of the AT2R increases the synthesis of ceramide, which may lead to the AT2R-mediated apoptosis.26,27
However, besides the growth inhibitory function of AT2R, there is still some controversy, because it has also been reported that AT2R stimulation promotes fibrosis and growth. AT2R activation in AT2R-transfected smooth muscle cells stimulates collagen synthesis.28 Furthermore, ventricular hypertrophy induced by pressure overload was not observed in AT2R knockout mice.29
A peculiarity of the AT2R is its uncommon response to Ang II stimulation, as there is no desensitization or downregulation in response to ligand binding.30,31 In contrast, the cell surface expression of the AT2R undergoes upregulation after stimulation with Ang II, whereas the level of AT2R transcripts does not increase.32–34
Numerous studies have identified different AT2R regions responsible for the interaction with other proteins involved in AT2R activation or regulation. Among these regions, the fifth and sixth transmembrane domains were shown to directly interact with the agonist Ang II,35–38 and the third intracellular loop38–40 and the cytoplasmic tail were shown to be critical for coupling with Gi and SHP-1,41 respectively. Moreover, the cytoplasmic tail was also shown to contain a binding domain for the transcription factor promyelocytic zinc finger protein (PLZF) which affects the AT2R signaling.42 In an effort to better characterize the signaling mechanisms of the AT2R, we screened for new AT2R interacting proteins.
Methods
For details regarding cell culture, plasmid transfection of cell lines, and mouse tissues, construction of mutant and epitope-tagged ATBP and AT2R expression vectors, design of a fluorescent Golgi-marker (GM) protein, coimmunoprecipitation experiments, immunoblotting, confocal immunofluorescence microscopy, surface expression ELISA, siRNA design and transfection, proliferation assay, Northern blot analysis, RT-PCR analysis, relative quantitation of gene expression by real-time PCR, and in situ hybridization, please see the online Methods, available at http://atvb.ahajournals.org.
Yeast Two-Hybrid Screening
The mice AT2R carboxyl-terminal tail (residues 313 to 363) GAL4 binding domain fusion protein cloned in the yeast expression vector pGBKT7 was cotransformed into the yeast strain AH109 with a GAL4 activation domain fusion library of mouse 11 day embryo cDNA in pACT2 (Clontech, Heidelberg, Germany). Yeast two-hybrid screening was performed according to the Clontech Matchmaker Two-Hybrid System 3 protocol. Clones expressing both the bait and the prey were selected on growth medium lacking Leu and Trp, and the interacting proteins were identified for their adenine auxotroph complementation. The cDNA inserts from positive clones were then sequenced using the dideoxynucleotide method.
cDNA Cloning of ATBPs
Total RNA was isolated from mice embryo, and the mRNA was then affinity purified by oligo(dT)-cellulose chromatography. The cDNA synthesis was primed with oligo(dT). A kit for rapid amplification of cDNA ends (RACE) was used to identify the 5' ends of the ATBPs (Life Technologies). The following nested oligonucleotides were used for the polymerase chain reaction (PCR): AP1 primer (Life Technologies), GSP1 (5'-CAGAAGAATCCCAGGAGCCT), and GSP2 (5'-AGGCTCCTGGGATTCTTCTG). Total mice embryo RNA was used for cDNA synthesis. The PCR products were subcloned into the pcDNA3.1 vector (Invitrogen) and sequenced.
Statistical Analysis
With respect to the epidermal growth factor (EGF) stimulation experiments, a two-tailed Student t test was performed.
Results
Identification of ATBP as Binding Protein of the AT2 Receptor
To identify AT2-interacting proteins, a yeast two-hybrid screening was set up with the AT2R carboxyl-terminal tail (residues 313 to 363) as bait protein and a cDNA library generated from mouse 11 day embryo as prey protein. Of 2x106 colonies screened, 86 fulfilled all five selection criteria (phenotypes: His +, 3 ATR, galactosidase –, Ura +, and 5-fluoro-orotic-acid +), and two of these colonies encoded for full-length cDNA termed ATBP50 for AT2 receptor binding protein of 50 kDa. To confirm the result of the two-hybrid screening, coimmunoprecipitation studies were performed with both proteins coexpressed as tagged fusion proteins in COS-7 cells. 3XFlag-tag was fused to the N terminus of AT2R, and V5-tag was fused to the C terminus of ATBP50. Cell lysates were subsequently used for coimmunoprecipitation studies with monoclonal antibodies directed against the respective tags. V5-tagged ATBP50 protein was coimmunoprecipitated with anti-3XFlag antibody when cells were cotransfected with AT2-Flag (Figure 1A, lane 2), but not in the control experiment (Figure 1A, lane 1). The other two isoforms, ATBP60 and ATBP135 (see below), were not able to interact with the AT2R (data not shown).
Figure 1. A, Specific binding of ATBP50 and ATBP50C to the AT2 receptor. Coimmunoprecipitation of ATBP50 or ATBP50C with the AT2R. COS-7 cells were transiently cotransfected with Flag-tagged AT2R and V5-tagged ATBP50 or V5-tagged ATBP50C. Proteins were chemically cross-linked, and receptor complexes were immunoprecipitated. Whole-cell extracts were subjected to SDS-PAGE, blotted, and probed for the presence of the V5 epitope and Flag epitope, respectively. Ang II stimulation was performed 48 hours after transfection for 2 hours. B, Northern blot analysis of total RNA from mice embryo. The mRNA was electrophoresed in agarose gel and blotted on nitrocellulose. The blot was hybridized with ATBP50 cDNA as a probe. C, RT-PCR analysis of AT2R, ATBP50, ATBP60, and ATBP 135. mRNA from various mouse tissues was reverse transcribed and subjected to PCR using the indicated primers with HPRT serving as control.
Furthermore, we were able to demonstrate that a C-terminal deletion mutant of ATBP (ATBP50C), which is discussed below, is still able to interact with the AT2R (Figure 1A, lane 3).
ATBP and AT2 Receptor are Coexpressed in Mouse Tissue
To examine the expression of ATBP50 in embryonic tissue, we performed Northern blots using RNA from mouse embryos with cDNA of ATBP50 as a probe. We found three mRNA isoforms detected by the probe in a comparable intensity (Figure 1B). They had an estimated size of 2.5, 3.6, and 5.9 kb, respectively. To analyze which adult tissues express ATBP, we performed isoform-specific semiquantitative RT-PCRs with RNA from various mouse tissues. A ubiquitous expression of ATBP50 was seen in all tissues tested (Figure 1C). Contrary to ATBP50, ATBP60 and ATBP135 were not detectable in brain and spleen. The strongest expression of ATBP50 was found in the uterus and adrenal gland (Figure 1C, lanes 2 and 3), tissues in which the AT2R is also predominantly expressed (Figure 1C, lanes 2 and 3). In contrast, the AT2R- and ATBP50 expressions in heart, brain, spleen, kidney, gut, and dermis were relatively weak.
More detailed investigations regarding coexpression of ATBP and AT2R were performed by in situ hybridization to evaluate the distribution patterns within different tissues using a probe common for all three isoforms. For example, in the adrenals (Figure IIA, available online at http://atvb.ahajournals.org) the expression patterns of ATBP and AT2 were strikingly similar, compatible with coexpression of both genes. This was also true for kidney, lung, heart, and brain (data not shown). We further examined tissues of AT2R-deficient mice by in situ hybridization. Although the adrenal glands of wild-type mice coexpressed the AT2 receptor and ATBP, the ATBP expression in this tissue was drastically reduced in AT2 receptor–deficient mice (Figure IIA). To substantiate this finding, we performed real-time PCR analysis on uterine tissue of wild-type– and AT2R-deficient mice, respectively. Uterus was chosen because of the strong coexpression of both proteins established in previous experiments. The ATBP mRNA expression in AT2 receptor knockout mice was reduced to <10% compared with wild-type mice (Figure IIB).
Genomic Organization of the ATBP Gene
The presence of three differently sized transcripts suggested that the ATBP gene encodes for two more transcripts than expected. A genomic BLAST search with the cDNA sequence of ATBP50 (GenBank accession number AY626781) in the mouse genome databank at NCBI showed that the gene mapped to the minus strand of the mice chromosome 8p21.3 with 10 exons spanning >47.7 kb. A search with the WGS supercontig Mm8_WIFeb01_187 sequence upstream of the ATBP50 stop codon in GENSCAN Web Server at MIT43 showed the whole gene of ATBP, spanning a region of 90.5 kb genomic DNA with 15 exons and 14 introns, codes for two additional transcripts, ATBP60 (GenBank accession number AY626782) and ATBP135 (GenBank accession number AY626781), respectively. The 5' terminus and the 3' terminus of every intron exhibit the GT-consensus and AG-consensus sequence, respectively.
All three mRNAs include exon 7 to 15 of the ATBP gene, encoding 2047 bp at the 3' end of the mRNAs. ATBP50 mRNA additionally contains exon 6 (358 bp) with the translation initiation start. Exons 4 (133 bp) and 5 (41 bp) are expressed in both ATBP60 and ATBP135 mRNA, whereas exon 3 (238 bp) is only expressed in ATBP60 mRNA and bears its translation initiation codon. Exons 1 (2311 bp) and 2 (119 bp) are exclusively expressed in ATBP135 mRNA with the translation initiation start in exon 1 (Figure 2A and 2B). Analysis of the sequence surrounding the potential translation initiation starts revealed a favorable Kozak sequence44 for ATBP50 (5'-GAAGAGATGC) and ATBP135 (5'-TTCAGGATGA) but not for ATBP60 (5'-TTTTAAATGA). A poly(A) tail and a polyadenylation signal (AATAAA) 21 nucleotides upstream from the poly(A) tail were detected in all three cDNA sequences.
Figure 2. Organization of the ATBP gene and protein. A, Genomic organization of ATBP gene. The gene spans 15 exons, spanning a region of 90 kb within chromosome 8p21.3 to 22. Numbers on top represent exons. Open boxes indicate exons transcribed in all ATBP isoforms; fasciated boxes, ATBP50-specific exons; checkered boxes, ATBP60- and ATBP135-specific exons; dotted boxes, ATBP60-specific exons; black boxes, ATBP135-specific exons. B, Predicted secondary structure of the ATBP proteins and amino acid sequence of ATBP50, ATBP60, and ATBP135 in one-letter code. The isoform specific regions are pictured in boxes with the same pattern used in Figure 2A. Within the common sequence the region of coiled-coil structures are underlined and the leucine-zipper are in bold letters.
Sequence and Structural Analysis of the ATBP Proteins
The predicted protein sequences encoded by the ATBP gene are shown in Figure 2B. Mice ATBP50, ATBP60, and ATBP135 are proteins with 440, 520 and 1209 residues and a calculated mass of 50, 60, and 135 kDa, respectively.
A PHI- and PSI-BLAST45 search with the three protein sequences in the NCBI databank showed no significant homologies to other proteins except proteins with a long coiled coil region like Golgi matrix proteins such as GM130/golgin-95,46 GCP170,47 golgin-160,48 and p115.49 An identity was found with the human mitochondrial tumor suppressor gene 1 (MTSG1) protein50 and the human ATIP.51 In the protein family database Pfam,52 we found a homology of the ATBP proteins to the CDD:pfam05483 named HOOK protein. It has been demonstrated that endogenous HOOK3 binds to Golgi.53
The ATBP proteins could be divided into two parts: an N-terminal "head" region of isoform-specific length, assumed to have a random coiled structure, and a C-terminal part of 400 amino acids identical in all three proteins. Using the program COILS (version 2.1) at EMBnet,54 the C-terminal part of the ATBP proteins were predicted to have an -helical coiled-coil motif, starting at position 214 (relative to the common part of all isoforms) and interrupted at position 260 to 261 by the strong helix breakers asparagine and proline.55 The C-terminus ends in a noncoiled-coil serine/proline-rich tail of 38 amino acids (Figure 2B). Using the program 2ZIP at DKFZ, Heidelberg, the coiled-coil region was predicted to contain two potential leucine zippers, a specialized form of coiled-coil dimerization motifs56 (Figure 2B). A hydropathy plot57 illustrated that the ATBP proteins were highly hydrophilic without any potential transmembrane domain.
ATBP Proteins Are Localized at the Golgi Matrix Through Their C-Terminal Domains
To examine the subcellular distribution of the ATBP proteins, we compared the localization of ATBP proteins with the localization of a Golgi marker (GM). For this purpose, we transiently cotransfected COS-7 cells with a DsRed2-coupled GM and one of the Flag-tagged ATBP proteins (wtATBP50 or one of the three ATBP50 deletion mutants described below, respectively). The Flag-tagged ATBP proteins were detected with fluorescein isothiocyanate (FITC)–coupled anti-Flag antibody and imaged by confocal microscopy. WtATBP50 (Figure 3A) as well as the GM (Figure 3B) were primarily located in the perinuclear region with a small amount of diffuse background staining throughout the cell. This membrane distribution is similar to what has been reported for other Golgi-Network resident proteins.58 To examine the role of ATBP domains in Golgi targeting, three EGFP-coupled deletion mutants of the ATBP50 protein were created. DsRed2-coupled GM and the mutants were cotransfected in COS-7 cells, and localization was examined by confocal microscopy. ATBP50C, a mutant lacking the C-terminal domain of the last 40 aa, lost the organized distribution and was diffusely dispersed in the nucleus and cytoplasm (Figure 3C and 3D). The same effect was seen using the mutant ATBP50NC lacking both the N and the C terminus, suggesting that the -helical coiled-coil domain alone is not needed for directing colocalization to the Golgi apparatus (data not shown). In contrast, ATBP50N, a mutant lacking the N-terminal domain, was localized at the Golgi apparatus comparable to wild type indicating that the N-terminal domain is not required for Golgi localization. As expected from the identical C terminus of the three ATBP proteins, the same feature was seen with the two other isoforms ATBP60 and ATBP135 (data not shown). These results indicate that the ATBP proteins interact with the Golgi through their noncoiled C terminus, a domain identical in all three isoforms.
Figure 3. Functional analysis of ATBP. A through D, The C-terminal truncation of ATBP leads to decreased expression at the Golgi apparatus. Coexpression of Flag-tagged wtATBP50 with DsRed-tagged GM (A and B) and of Flag-tagged ATBPC with DsRed-tagged GM (C and D) in COS-7 cells. Immunocytology was performed using a green fluorescent antibody against the Flag-tag, and immunofluorescence was viewed using a green (A and C) or a red filter (B and D). E, ATBP Proteins Homo- and Heterodimerize in vivo. COS-7 cells were transiently transfected with expression vectors encoding Flag-tagged ATBP50 in combination with V5-tagged versions of ATBP50, ATBP60, and ATBP135. Total cell extracts were immunoprecipitated (IP) with an anti-Flag antibody and electrophoresed, and the resulting blots were probed with anti-V5 antibodies to detect the various immunoprecipitated epitope-tagged ATBP proteins.
ATBP Proteins Appear as Homo- and Heterodimers In Vitro
To analyze putative dimerizations we performed coimmunoprecipitation studies with coexpressed ATBP proteins. ATBP50 was Flag-tagged and coexpressed in COS-7 cells with V5-tagged ATBP50, ATBP60, and ATBP135, respectively. Cell lysates were subsequently used for coimmunoprecipitation studies with monoclonal antibodies directed against the Flag-tag. The eluate was resolved using SDS-PAGE followed by detection with a monoclonal antibody against V5. V5-tagged ATBP50, ATBP60, and ATBP135 was coimmunoprecipitated with ATBP50 (Figure 3E). Our results allow for the possibility that all three ATBP proteins are able to form hetero- as well as homodimers with each other.
ATBP50 Is Required for AT2 Receptor Cell Surface Expression
To determine the physiological significance of the ATBP/AT2R protein interaction, we used siRNA technology to reduce endogenously expressed ATBP50 in N1E-115 cells that endogenously express the AT2R and AT1R.59 Transiently transfected EGFP-tagged AT2Rs were detected predominantly at the plasma membrane of N1E-115 cells (Figure 4A). However, when the EGFP-tagged AT2R was cotransfected with siRNA specific for ATBP50, AT2 receptor staining was increased at the endoplasmic reticulum and significantly reduced at the cell surface (Figure 4B). The cell surface distribution of the AT2R was not altered when coexpressed with Flag-tagged ATBP, indicating that overexpression of ATBP50 as such did not affect the normal plasma membrane distribution of AT2R (data not shown). We further measured the cell surface expression of the AT2R using a surface expression ELISA. A significant decrease in cell surface expression of the AT2R was observed when a C-terminal truncated AT2R (AT2RC) was used (Table I, available online at http://atvb.ahajournals.org). A strikingly similar effect was observed when ATBP50 was downregulated by siRNA (Table I). These results suggest that ATBP/AT2 receptor interaction is required for the proper targeting of AT2 receptor to the plasma membrane.
Figure 4. Interaction between ATBP50 and AT2R is required for AT2R cell surface expression. N1E-115 cells transfected with EGFP-tagged AT2R (A) and cotransfected with ATBP50 specific siRNA (B) are shown. Protein expression was detected by confocal laser microscopy.
Effect of ATBP50 on AT2 Receptor Function in N1E-115 Cells
To further investigate functional interactions between ATBP and the AT2R, we examined the ability of the AT2R to decrease the proliferative effect of EGF stimulation in absence of ATBP50. For this purpose we used the mouse neuroblastoma cell line N1E-115 for which the antiproliferative effect of the AT2R has already been shown.60 Before Ang II stimulation, the AT1R was selectively blocked by losartan (10 μmol/L). We used the activity of the ERK1/2 to measure the activation state of the growth-related mitogen-activated protein kinase cascade. The activation of ERK1/2 in these cells was visualized by Western blotting using antibodies detecting the phosphorylated (activated) form of the enzyme. As expected, EGF (50 ng/mL) induced ERK1/2 activity (Figure IA, lane 2; available online at http://atvb.ahajournals.org) compared with controls (Figure IA, lane 1). Stimulation of the AT2R by Ang II (50 nM) produced a robust and potent inhibition of ERK1/2 phosphorylation in EGF-stimulated (50 ng/mL) N1E-115 cells (Figure IA, lane 3). This effect of the AT2R was prevented by preincubation with the AT2R selective antagonist PD123319 (10 μmol/L) (Figure IA, lane 4). A knockdown of ATBP50 with siRNA reduced the AT2R ability to inhibit the EGF-induced ERK1/2 stimulation (Figure IA, lane 6), whereas control siRNA had no inhibitory effect (Figure IA, lane 5).
We then examined whether the results of reduced ERK1/2 inhibition of AT2R in the absence of ATBP50 would be reproducible in a proliferation assay with N1E-115 cells in the same experimental setup. After two days of EGF (50 ng/mL) treatment the cell proliferation rate nearly doubled compared with untreated cells (Figure IB, columns 1 and 2). This proliferation induction could be blocked with Ang II treatment (50 nM), whereas preincubation with PD123319 (10 μmol/L) was able to prevent this inhibitory effect (Figure IB, columns 3 and 4). Pretreatment of N1E-115 cells with ATBP50 specific siRNA 2 days before Ang II stimulation caused a significant reduction of the inhibitory effect of AT2R. Taken together, these data indicate that the inhibitory effect of the AT2R on EGF-induced cell proliferation requires ATBP50.
Stimulation of PC12W Cells With Ang II Leads to Strong Induction of ATBP50 mRNA
PC12W rat pheochromocytoma cells, which endogenously express both AT2R and ATBP50, were stimulated with 100 nM Ang II. A continuous increase in ATBP50 mRNA levels was observed, starting 30 minutes after stimulation (Figure IIC).
To assess whether Ang II also affects the cell surface expression of the AT2R we performed a further surface expression ELISA. In the experimental setting used, Ang II did not alter the amount of transient transfected Flag-tagged AT2R at the cell membrane (Table II, available online at http://atvb.ahajournals.org). This experiment is consistent with an additional coimmunoprecipitation experiment, where Ang II incubation did not alter the amount of ATB50 coprecipitated with Flag-tagged AT2R (Figure 1a, lane 4).
In addition, the data shown in Table II indicate, that—besides Ang II—cotransfection of ATBP50 or ATBP50C also did not alter the amount of transient transfected Flag-tagged AT2R at the cell membrane in this experimental system.
Discussion
In this study, we have identified the previously unknown ATBP50 as a new AT2R interacting protein by yeast two-hybrid screening. ATBP50 was shown to directly interact with the carboxyl-terminal tail of this receptor, and the specificity of this association was demonstrated by coimmunoprecipitation experiments. Three different ATBP transcripts (2.5 kb, 3.6 kb, and 5.9 kb) were detected by Northern blot analysis in mouse embryo tissues. These ATBP isoforms also show tissue-specific expression levels using RT-PCR analysis of multiple organs. In the adult mouse, the ATBP50 transcripts were present in all tissues tested, with abundant expression in uterus and adrenal correlating with the expression pattern of the AT2R in the adult mouse. The weak expression of ATBP50 in the liver, where the AT2R is not detectable, and the strong ATBP50 expression in the lung compared with AT2R expression suggest that ATBP has further functions that are independent of the AT2R. The putative polypeptides encoded by these gene have predicted molecular masses of 50 kDa (ATBP50), 60 kDa (ATBP60), and 135 kDa (ATBP135), respectively. Even though the N termini of all ATBPs are of different size and show no homologies, the main structural feature of the ATBP proteins is their predicted ability to form coiled-coils in the region of 400 amino acids which is identical in all isoforms. Coimmunoprecipitation experiments suggested that all three ATBP proteins are able to form hetero- and homodimers with each other, thus indicating possible complex functions besides the receptor trafficking described below.
Our study provides evidence for a functional relationship between ATBP and AT2R concerning two different aspects: First, we have shown by confocal fluorescence microscopy and ATBP50 specific siRNA that ATBP50 promotes cell surface expression of AT2R. These results were confirmed by our cell surface ELISA experiments. Together these data suggest that ATBP50 functions as a regulatory protein to modulate the plasma membrane routing and thereby the functionality of the AT2R. With respect to functionality, we observed that downregulation of ATBP50 reduced the maxi-mal inhibitory effect of AT2R on EGF-stimulated ERK1/2 activation in N1E-115 cells. This could be a direct result of a reduced presence of the receptor in the plasma membrane, a finding in accordance with Miura and Karnick who showed that the level of receptor protein expression is critical for induction of antiproliferation and apoptosis.25 The presence and expression level of ATBP50 has the potential to alter AT2R signaling. We offer a putative functional explanation for the effects of ATBP on AT2R mediated ERK1/2 inhibition by demonstrating that ATBP is necessary for AT2R cytoplasma membrane transport.
Our results indicate that ATBPs can be added to a growing list of proteins that have been identified as GPCR-binding proteins managing receptor trafficking to the cell surface. For instance, the actin binding protein filamin A (ABP-280) was found to modulate the cell surface expression of D2 and D3 receptor subtypes.61–63 Furthermore, association of the somatostatin receptor subtype 1 with Shk1 kinase–binding protein are required for targeting the receptor to the cell surface.64 In addition, Leclerc et al predicted regulatory proteins modulating the routing of the AT1R to the cell surface.65
Consistent with our bioinformatic results (eg, homology to HOOK protein), using confocal microscopy we were able to show that ATBP is located at the Golgi apparatus. Although ATBP proteins are all hydrophilic, lacking obvious hydrophobic regions capable of serving as membrane anchors, the ATBP appends to the Golgi through its C-terminal tail as evidenced by our mutation experiments.
Secondly, we demonstrate a functional interaction between ATBP and AT2R in vivo, because ATBP mRNA was markedly reduced in AT2R knockout mice, as shown by in situ hybridization and real-time PCR analysis. These results are consistent with our complementary observation that Ang II can induce ATBP50 mRNA in PC12W cells, which exclusively express AT2R but not AT1R. Therefore, ATR2 exerts positive feedback on ATBP mRNA expression. In this context it is important to note that ATBP isoforms are encoded by a single gene and characterized by N-terminal isoform-specific exons, implying that these transcripts are controlled by three different promoters. The complex mechanisms of this feedback of AT2 receptor signaling on the isoform-specific ATBP promoters and/or isoform-specific mRNA half-lives will be examined in a further project.
Therefore, our data point to the existence of a two-directional interaction between ATBP and AT2R: On the one hand, ATBP is necessary for the trafficking and function of AT2R as described above; on the other hand, AT2R positively regulates ATBP mRNA expression.
In this context it is of interest to note that besides ATBP, the C terminus of the AT2R is able to bind the recently described promyelocytic zinc finger protein (PLZF).42 After Ang II stimulation PLZF colocalizes with AT2R at the plasma membrane, followed by internalisation of AT2R and PLZF and nuclear translocation of PLZF. Therefore, it is conceivable that the transcriptional upregulation of ATBP by Ang II described here might be mediated by PLZF.
The interaction between ATBP50 and the AT2R may have direct implementations for human disease. For instance, Vervoort and coworkers have shown that mutations of the AT2R are linked to mental retardation; in particular, mutations in the receptor cytoplasmic C terminus are associated with this innateness.66 It is conceivable that dysfunctional interaction between AT2R and ATBP50 may be responsible for this disease. Another potential role of ATBP gene in human disease is suggested by its chromosomal localization at 8p21.3 as abnormalities in this region have been reported in various cancer types, thus implicating ATBP as a positional candidate gene in cancer.67
This hypothesis is further supported by the well documented antiproliferative properties of the AT2R68 that are, according to our results, closely linked to the AT2R-ATBP50 interaction.
Furthermore, Seibold et al reported that the mitochondrial tumor suppressor gene 1 (MTSG1) protein, which is identical with ATBP50, is a mitochondrion resident protein.50 However, we were not able to demonstrate that any of the ATBP proteins are localized in mitochondria. Possible explanations for this discrepancy might be the different cellular systems used (pancreatic tumor cells versus COS-7 cells) and the observation that the type of heterodimerization can influence the subcellular distribution.69
In conclusion, we have identified a novel, Golgi-localized protein, ATBP50, that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT2R and is necessary for the cell surface expression of the AT2R. ATBP50 influences the receptor-mediated signaling by regulating receptor cell surface expression. Further functions of the ATBP isoforms and the nature of their associated proteins are now under investigation. Given that the AT2R is an important mediator of the renin-angiotensin system, ATBP50 may play a significant role in cardiovascular physiology and pathophysiology as well as other conditions that are genetically linked to the AT2R, such as mental retardation.
While this manuscript was in preparation, Nouet et al have described a protein named ATIP, which is identical to ATBP50, as an AT2 receptor interacting protein.51
Acknowledgments
Parts of this work have been supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 415: Pathophysiology of signal transduction pathways). We thank Michael Bader (Max Delbrück Center for Molecular Medicine (MDC), Berlin Buch, Germany) for the AT2R knockout mice.
Received August 10, 2004; accepted November 1, 2004.
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