| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
RECEPTORS AND SIGNAL TRANSDUCTION
Division of Research, Ochsner Clinic Foundation, Ochsner Health System, New Orleans, Louisiana
Submitted 24 August 2006 ; accepted in final form 13 November 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
nuclear angiotensin II type 1 receptor; intracrine; intracellular
A number of transmembrane receptors, including several receptor tyrosine kinases (e.g., receptors for EGF, insulin, FGF, NGF, IL-1, erbB-4, and Her-2/neu) (25), have been reported to localize to the nucleus, either as holoproteins or protein cleavage fragments. More recently, a limited number of G protein-coupled receptors (GPCRs), including the endothelin-B and GABAB receptor, have also been found associated with cell nuclei (6, 14, 32).
Our previously published studies, in which we report nuclear accumulation of the AT1R when it is expressed with IC ANG II (8, 9), were not designed to differentiate between transport of the AT1R holoprotein versus transport of cleaved IC fragments into the nucleus. We have, therefore, expanded our approaches to investigate that question. In the present study we designed and employed an expression plasmid (pECFP/AT1R/EYFP) that encodes a fusion protein in which the AT1R is labeled at the extracellular amino terminus with ECFP and at the IC carboxy terminus with EYFP. In principle, cleavage within the AT1R leads to dissociation of the two fluorescent moieties and possible spatial separation within the cell or at the plasma membrane. We investigated this construct and related control plasmids using deconvolution microscopy and Western blot analyses to show that the AT1R undergoes cleavage within the cytoplasmic domain and transport of the cytoplasmic fragment into the nucleus. We show that the AT1R cytoplasmic domain is essential for the processing event; cleavage does not occur when the encoded protein is deleted with respect to amino acid residues 306–359. We further demonstrate that the amino-terminal extracellular domain also undergoes cleavage and can be recovered from the tissue culture media.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
pECFP-C1 and pEYFP-N1. These are otherwise referred to as pECFP and pEYFP in this study and are control vehicles into which desired fusion protein-encoding DNA sequences can be cloned (Clontech, Palo Alto, CA).
pAT1R/EYFP. pAT1R/EYFP and the encoded protein have been previously described (8, 9).
pCMV/AT1R.
This clone is also referred to as pAT1R/EYFP-N1/
EYFP. It is the same clone as above except that it is deleted with respect to the EYFP fragment. Thus the AT1R is expressed as a nonfusion protein under control of the cytomegalovirus promoter (pCMV). To produce this expression plasmid, we digested pAT1R/EYFP with AgeI/NotI, both of which digest only once in pEYFP-N1 (at sites directly flanking the EYFP gene) and not at all in AT1R. The large fragment (
5,050 bp) was purified free of the EYFP fragment (731 bp), end filled, and blunt ligated to produce pCMV/AT1R.
pEYFP-N1/EYFP.
pEYFP-N1 was digested with AgeI/NotI, both of which digest only once in pEYFP-N1 (at sites directly flanking the EYFP gene). The large fragment (4 kb) was purified free of the EYFP fragment (731 bp), end filled, and blunt ligated to produce pEYFP-N1/EYFP. This control plasmid encodes neither a fluorescent protein nor a member of the renin-angiotensin system.
pECFP/AT1R/EYFP. pECFP-C1 was digested with NheI and BglII to liberate a 755-bp ECFP-encoding fragment. The ECFP fragment was inserted into NheI/BglII-digested pEYFP-N1. This vector is designated pECFP/EYFP (note that the fluorescent moieties are not in frame). Upstream primer AT1-DF-U, 5'-GATCGAAAGCTTCTGCCACCATGGCCCTTAACTCTTCTGCT-3' (ATG start site underlined) and downstream primer AT1-DF-D, 5'-CGAGACCGAGGATCCTGCTCCACCCTCAAAACAAGACGC-3' were used to amplify AT1R from pAT1R/EYFP. The PCR product was digested with Hind III/BamHI and ligated to HindIII/BamHI-digested pECFP/EYFP, placing the AT1R in proper reading frame with both upstream ECFP and downstream EYFP sequences.
pIGF1SS/ECFP/AT1R/EYFP.
The human IGF-1 receptor signal peptide from plasmid pIGF-I-R.8 (ATCC 59294) was amplified using upstream primer HsIGF1R-U1, 5'-GATCGAGCTAGCGCCACCATGAAGTCTGGCTCCGGAGGA-3' and downstream primer HsIGF1R-D1, 5'-GATCGAGCTAGCTGTCCACTCGTCGGCCAGAGCGA-3'. The 100-bp PCR amplification product was digested with NheI and cloned into NheI-digested pECFP/AT1R/EYFP. The clone was sequenced to confirm orientation of the signal peptide upstream of ECFP.
pECFP/AT1RCT/EYFP.
The rat AT1a receptor-encoding DNA, deleted with respect to the cytoplasmic domain (Gly306–Glu359) was PCR-amplified from AT1R/EYFP using upstream primer AT1-DF-U (see pECFP/AT1R/EYFP) and downstream primer AT1-DF-D2, 5'-CGAGACCGAGGATCCTGCAGAAAGCCGTAGAACAGAGG-3'. The product (950 bp) was digested with HindIII/BamHI and ligated to HindIII/BamHI-digested pECFP/EYFP to generate pECFP/AT1RCT/EYFP.
Cell Lines
The clonal cell line A10 (ATCC, CRL-1476) was derived from the thoracic aorta of DB1X embryonic rat and possesses many of the properties characteristic of smooth muscle cells. The cells produce spontaneous action potentials at the stationary phase of the growth cycle and exhibit an increase in activity of the enzymes myokinase and creatine phosphokinase. In addition, these cells have been shown to express both the AT1 receptor and a number of contractile proteins characteristic of VSMCs (2, 15, 21, 36, 47).
CCF-STTG1 cells (ATCC, CRL-1718) are derived from a human grade IV astrocytoma and express the glial marker GFAP (4). These are alternatively referred to CCF cells in this study.
COS-7 is an African green monkey kidney fibroblast-like cell line suitable for transfection by vectors requiring expression of SV40 T antigen. The line was derived from the CV-1 cell line (ATCC, CCL-70) by transformation with an origin-defective mutant of SV40 that codes for wild-type T antigen. We typically use this line as an initial test line for transfections (9) because it is readily transfected using commercial cationic lipids and expresses proteins from recombinant constructs at high levels. This line is alternatively referred to as COS cells in this study.
Cell Growth Measurements
A10 VSMCs, passages 15–20, were plated in DMEM high glucose at 105 cells/well on 12-well plates and permitted to attach overnight before transfection. Cells were transfected, six wells per clone or combination of clones, with various plasmids (0.5 µg DNA and 1.25 µg of lipofectamine, 2,000/well) for 4 h. Media were replaced at 24 h. At 42 h, bromodeoxyuridine (BrdU, 10 µmol/l) labeling in 1% FBS-containing medium was initiated, and labeling proceeded for 6 h (based on our pilot studies, the optimum labeling time for these cells). Where appropriate, candesartan (10–9 mol/l) and/or ANG II (10–9 mol/l) were added during the BrdU-labeling period. The labeling reagent was removed, and anti-BrdU staining was conducted using the Oncogene Research Products BrdU kit (Calbiochem-Novabiochem, San Diego, CA). Approximately 100 cells/field x 10 fields were counted (1,000 for each treatment) by a technician blinded to the study identifiers.
Transfections/Western Blot Analysis
Line A10, passages 15–20, was seeded at 2 x 105 cells/35-mm dish in DMEM and 10% FBS. Twenty-four hours after being plated, cells were transfected overnight using 0.5 µg of DNA and 1.25 µl of Lipofectamine 2000 (1 µg/µl; Invitrogen, Carlsbad, CA) in Optimem low-serum medium. COS cells were similarly transfected. CCF cells were seeded at 2 x 105 cells/35-mm dish in RPMI and 10% FBS. Twenty-four hours after being plated, cells were transfected with 3 µl of Fugene-6 (Roche Diagnostics) and 0.5 µg of DNA for 4 h in serum-free medium.
Where indicated, at 45 h posttransfection, cells were rendered quiescent in 0.5% FBS-containing media for 3 h and then treated with ANG II (Sigma, St. Louis, MO) for 30 min before harvest. EDTA (Fisher Scientific, Fair Lawn, NJ) and 1,10-ortho-phenanthroline monohydrate (OPA) were used as indicated. Total cell extract was collected (1, 8), and protein was measured by using the Bio-Rad Protein Assay (Hercules, CA). Protein was electrophoresed on a 4%-12% NuPAGE Bis-Tris gradient gel or an 18% Novex Tris-glycine gel (Invitrogen) and transferred to Hybond-P:PVDF membrane. Specific immunoreactive proteins were detected by using ECL Plus Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ). AT1R (COOH terminus) was detected by using sheep polyclonal (ab1886) to AT1R (rat protein, amino acids 297–359) and rabbit anti-sheep IgG (both from Abcam, Cambridge, MA). AT1R (NH2 terminus) was detected by using rabbit polyclonal IgG to AT1 (N-10, sc-1173) from Santa Cruz and donkey anti-rabbit IgG (Amersham). EYFP was detected by using rabbit polyclonal antibodies to GFP (FL, sc-8334, Santa Cruz Biotechnology, Santa Cruz, CA) and donkey anti-rabbit horseradish peroxidase (NA934V, Amersham Biosciences). Mouse monoclonal anti-histone and anti-
-tubulin antibodies (Santa Cruz Biotechnology) were used as controls for nuclear and cytosolic fractions, respectively. High and low molecular weight markers were High- and Low-Range Rainbow Molecular Weight Markers (Amersham Biosciences).
Subcellular Fractionation
Chinese hamster ovary (CHO)-K1 and A10 cells were plated at 2 x 106/100-mm dish, serum deprived at 24 h of posttransfection, and (where indicated) treated with ANG II for 30 min before extract collection. Extracts were fractionated by a modification of the method of Maloney et al. (26). Specifically, plates were washed with 4-ml cold PBS and (step 1) scraped in 500 µl of CSK buffer [(in mM) 10 Tris·HCl (pH 6.8), 3 MgCl2, 100 NaCl, 1 EGTA, 300 sucrose, 1 DTT, 1 PMSF, 10 NaF, and 1 NaVO3 and phosphatase inhibitor]. Samples were transferred to a Dounce homogenizer and lysed with 30 strokes of a B-type pestle. Lysis was confirmed by microscopy using trypan blue staining. Lysate was transferred to an eppendorf and centrifuged for 10 min at 1,000 g. The pellet from this step was (step 2) resuspended in 500 µl of CSK buffer and centrifuged for 5 min at 4°C (2,000 g). The resulting pellet was then resuspended in 200 µl CSK-1% TX-100 buffer, incubated on ice for 5 min, and then centrifuged at 10,000 g for 5 min at 4°C. The supernatant was diluted with Laemmli sample buffer and represents the nuclear fraction. The supernatant from step 1 was (step 3) centrifuged at 8,000 g for 10 min at 4°C. The supernatant was (step 4) removed and centrifuged in a SW50.1 rotor (35,000 rpm for 30 min at 4°C). The resulting supernatant was diluted with 4x Laemmli sample buffer to 1x for cytoplasmic fraction.
The pellet from step 4 was resuspended in CSK-1% TX-100 buffer, incubated on ice for 30 min, and then centrifuged at 100,000 g for 30 min. The supernatant was diluted with Laemmli sample buffer (represents membrane fraction). Western blot analyses were performed (see Transfections/Western Blot Analysis; antibodies are also described) using membrane, cytosolic, and nuclear fractions.
Conditioned Media
VSMCs were grown to 85%-90% confluence, after which media were changed to serum-free media for 24 h before ANG II treatment. Cells were treated with ANG II (10–7 mol/l) for 1 h (with fresh additions every 15 min). Conditioned media were collected and centrifuged to remove cells and particulates. An equal volume of 15% TCA and carrier BSA to a final concentration of 2 µg/ml were added to precipitate protein (17). Samples were incubated at 4°C overnight and then centrifuged at 11,000 rpm at 4°C for 30 min. Pellets were resuspended in 50 µl of lysis buffer, diluted with 20 µl of sample buffer, and electrophoresed as described in Transfections/Western Blot Analysis for Western blot assays.
Transfections/Imaging
Transfections of plasmid DNA:polycationic lipid complexes were as described previously (see Transfections/Western Blot Analysis). Transfections were performed on glass bottom dishes (No. 1.5, poly-D-lysine coated) from MatTek (Ashland, MA).
Three-dimensional Deconvolution Microscopy
Deconvolution microscopy was performed using a Zeiss Axiovert 200 M microscope, xenon light source with automated Z-axis, and appropriate filters. Constrained iterative and nearest neighbor algorithms were performed by using Slidebook 4.1.0 software (Intelligent Imaging Innovations, Denver, CO). All images were captured with a 63X PlanApo oil lens (1.4 numerical apperature) in three dimensions at 0.5-µm steps through the vertical z-axis (10–15 z-axis planes captured per image) and deconvolved to render confocal images. Images shown here are approximately midnuclear in the z-axis.
Statistics and Correlation Coefficients
Pearson correlation coefficients were determined by using Slidebook 4.1.0 software. Images were deconvolved, segment masks were applied for yellow and cyan fluorescence, intensities and cross-channel statistical analyses were performed, and correlation coefficients were calculated.
Groups were compared using a one-way ANOVA with Tukey-Kramer or Bonferroni multiple-comparison post hoc tests.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Our results show that the fluorescent moieties of double-labeled AT1R (ECFP/AT1R/EYFP) are coincidental when expressed in transfected untreated COS cells (Fig. 1, row 1). However, ANG II treatment alters the distribution of fluorescence. When expressed in ANG II-treated COS cells, double-labeled AT1R was found to be cleaved with corresponding transport of the yellow fluorescent moiety to the nucleus (Fig. 1, row 2). The cyan fluorescent moiety does not accumulate in cell nuclei. Moreover the periphery of cells is devoid of cyan fluorescence, suggesting that the cyan fluorescent moiety may be cleaved from the plasma membrane and released into the medium, perhaps with a portion of the extracellular AT1R domain. The same fluorescent distribution is observed as early as 48 h and as late as 96 h posttransfection.
|
|
|
CT/EYFP-transfected cells, we find that the YFP domain is not visibly processed and there is no accumulation of yellow fluorescence in the nucleus (Fig. 1, row 5; and Table 2). Therefore, the AT1R carboxy terminus is necessary for processing and transport of the yellow fluorescent moiety to the nucleus; the yellow fluorescent domain is not itself cleaved in the absence of the AT1R carboxy terminus; the proteolytic activity is not nonspecific. Transient Transfections, Cell Proliferation: ANG II Stimulates pECFP/AT1R/EYFP-Transfected Cells
Since the distribution of yellow fluorescence from pECFP/AT1R/EYFP follows closely that which we have observed in previous studies with pAT1R/EYFP, we suspected that ECFP/AT1R/EYFP retains biological function, as does AT1R/EYFP. Nevertheless, we performed a series of experiments to verify that ECFP/AT1R/EYFP stimulates A10 VSMC proliferation in the presence of ANG II. Exogenous ANG II treatment significantly increases proliferation (1.44-fold) of untransfected A10 VSMCs, consistent with the existence of endogenous receptor in these cells (Fig. 2). ANG II increases proliferation of both pAT1R/EYFP- and pECFP/AT1R/EYFP-transfected cells 1.7-fold over corresponding untreated cells and in a candesartan-sensitive fashion. Receptor transfection in no case increases baseline proliferation in the absence of ANG II treatment. These studies show that ECFP/AT1R/EYFP, like AT1R/EYFP, retains native biological properties.
|
To determine whether the AT1 receptor processing evident in deconvolved fluorescent images could be corroborated by electrophoretic protein analyses, extracts from transfected CCF cells were evaluated. Western blots were probed with anti-GFP antibody (polyclonal, generated to full-length GFP), which reacts with EYFP and ECFP proteins (Fig. 3). Extracts from pEYFP-N1-transfected cells, as predicted, produce a band of 29–30 kDa. pAT1R/EYFP-transfected cells produce a predominant broad band between 66 and 97 kDa (predicted molecular weight based on linear regression of protein markers is 85 kDa). This is in good agreement with the minimum theoretical molecular weight of 71 kDa. Of course, the AT1R typically has a molecular weight between 41 and 58 kDa (with as high as 75 kDa documented), depending on posttranslational modifications (16, 32). pECFP/AT1R/EYFP-transfected CCF cells show a predominant cluster of immunoreactive proteins of >97 kDa (consistent with the theoretical minimum size of 101 kDa). Cells transfected with either pAT1R/EYFP or pECFP/AT1R/EYFP likewise demonstrate a smaller immunoreactive fragment of 36 kDa. This fragment size is consistent with the size of the IC EYFP domain together with the IC cytoplasmic carboxy terminus of the AT1R, suggesting that the receptor is cleaved either 1) within the IC receptor domain and close to the membrane or 2) within the seventh transmembrane-spanning region.
|
|
Processing of ECFP/AT1R/EYFP: Effects of Metalloprotease Inhibitors
The canonical receptor tyrosine kinase model (e.g., ErbB-4) involves cleavage of the extracellular receptor domain by a metalloprotease, such as TNF--converting enzyme (TACE) followed by cleavage of the IC domain [receptor intramembrane proteolysis (RIP)] by a secretase enzyme (6). To determine whether metalloproteases might be involved in cleavage of the AT1R, we transfected A10 cells with pAT1R/EYFP or pECFP/AT1R/EYFP, treated with the metal-chelating metalloprotease inhibitors EDTA or OPA, and evaluated extracts for accumulation of the cleavage fragment CT-CF. Both EDTA and OPA inhibit accumulation of the CT-CF (Fig. 5). Furthermore, both EDTA and OPA inhibit accumulation of nuclear yellow fluorescence by image analyses (Table 1).
|
To determine whether native, unmodified AT1R is cleaved, we sought to identify a low molecular weight fragment on high percentage polyacrylamide gels following transfection of CHO-K1 cells, which lack detectable endogenous AT1R (10, 21, 24, 39, 40), with a plasmid that encodes the native AT1R protein. Cells were transfected with pEYFP-N1/EYFP (control) or pCMV/AT1R. Where indicated, cells were treated with ANG II (10–6 mol/l for 30 min) before collecting extracts. Extracts were electrophoresed on an 18% gel with appropriate markers, and immunoblots were performed using anti-AT1R antibody (Fig. 6). We identified the presence of a 6- to 7-kDa fragment in pCMV/AT1R-transfected cells, which was not present in control extracts. This was consistent with the size of the cytoplasmic domain of the unmodified, native AT1R. Moreover, the intensity of this fragment was amplified (3.2-fold, P < 0.05, n = 3 experiments; Fig. 6A, lanes 7–9) with ANG II treatment. In cells treated with EDTA (Fig. 6B, lanes 11–12) or OPA (lanes 14–15) for 12 h before harvest, no detectable cleavage fragment accumulates..
|
Extracts were collected from CHO-K1 and A10 cells and fractionated into cytoplasmic, membrane, and nuclear components, and Western blot analyses were performed (Fig. 7). The carboxy-terminal tail was identified using anti-AT1R (carboxy terminal) antibodies. The 7-kDa cleavage fragment is detected in nuclear and cytoplasmic fractions predominantly and only in A10 cells.
|
As mentioned earlier, no product conforming to the predicted size of ECFP/AT1R (less the carboxy-terminal cytoplasmic fragment) is detected in cell extracts prepared from pECFP/AT1R/EYFP-transfected cells, prompting us to question whether the extracellular domain of the AT1R might also be cleaved from the membrane. To test this hypothesis, CHO-K1 cells, which lack detectable AT1R (10, 21, 24, 39, 40), were transfected with pAT1R/EYFP or pECFP/AT1R/EYFP, and conditioned media were collected (15). Western blot analysis of protein concentrated from the conditioned media was conducted (Fig. 8). A band of 33 kDa is detected using both anti-GFP (cross-reacts with ECFP) and anti-AT1R (amino terminal specific) antibodies in proteins collected from pECFP/AT1R/EYFP-transfected cells. This size is consistent with that expected for CFP (30 kDa) plus amino terminus of AT1R (amino acids 1–28, 3 kDa), suggesting cleavage at or near the extracellular domain/TM1 juxtaposition. As expected, this band is not observed in proteins collected from pAT1R/EYFP-transfected cells. Without the CFP component, the 3-kDa amino-terminal cleavage fragment is not visible within the limitations of standard Western blot procedures.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
In addition to the many examples of single-pass transmembrane receptors, which as holoproteins or processed fragments translocate to the nucleus (5, 7, 11, 13, 22, 23, 25, 31, 34, 38, 40, 41, 44–46), a few multipass seven-transmembrane GPCRs, including the growth hormone-releasing hormone, endothelin-B, and GABAB receptors, have recently been found either to be processed or to be transported to the nucleus or both (6, 14, 16, 30).
Our previously published studies indicate that the expression of the single-fluorescent moiety fusion protein, AT1R/EYFP, stimulates proliferation of A10 VSMCs. The present study indicates that the expression of the related double-fluorescent protein, ECFP/AT1R/EYFP, similarly stimulates cell proliferation in ANG II-treated CCF and A10 cells. ECFP/AT1R/EYFP also undergoes cleavage with transport of the YFP domain to the nucleus and accumulation of a 36-kDa fragment, CT-CF. Interestingly, CT-CF accumulates regardless of ANG II treatment, but the quantity is amplified following ANG II treatment. However, the fragment only significantly translocates to the nucleus in ANG II-treated cells. Therefore, ANG II seems to have a role in both accumulation and transport of the CT-CF.
A number of different regulatory domains and functions map to the carboxy terminus of the AT1R. Specific residues in the carboxy-terminal tail play roles in G protein coupling and receptor uptake, whereas phosphorylation of serine and threonine residues by PKA and PKC may result in uncoupling from G proteins and receptor desensitization. Using a carboxy-terminal deletion mutant (309–359), which is very similar to the mutant that we generated and used in this study, Inagami and associates (33) demonstrated that the mutant AT1 receptor shares a similar ANG II-binding affinity and maximum binding value as wild-type but markedly reduced G protein interaction. This suggests that the carboxy-terminal cytoplasmic domain is involved in G protein coupling but not in cell-surface presentation or ANG II binding. Our imaging studies suggest that ECFP/AT1RCT/EYFP is properly transported to the plasma membrane upon ANG II treatment but that the fluors remain coincidental, and there is no accumulation of nuclear fluorescence indicating no cleavage and no nuclear transport. Our immunoblot studies corroborate this conclusion. Importantly, therefore, the cytoplasmic carboxy terminal domain is obligatory for processing.
Although there have not been any previous reports directly demonstrating that the AT1R undergoes biologically functional proteolytic cleavage, there exists some indirect supporting evidence. Modrall and colleagues (29) postulated that receptor downregulation might occur independently of receptor endocytosis. Using endocytosis-deficient mutants (carboxy terminus deleted), they showed that receptors were downregulated both by 125I-labeled ANG II endocytosis and by radioligand assays for AT1 receptor binding sites. They further demonstrated that the endocytosis-deficient mutant receptors (309–359 and 311–359) were fully capable of rapid downregulation comparable with that of the wild-type receptor. They suggest, therefore, that an alternative pathway, that of receptor degradation, might be responsible for loss of cell-surface receptor. Our studies, showing nuclear accumulation of the carboxy-terminal cytoplasmic fragment of the AT1R, accompanied by alterations in signal transduction and cell proliferation, suggest that the degradation of plasma membrane receptor actually represents a biologically functional-regulated proteolysis.
The ANG II type 2 (AT2) receptor, in addition to its traditional plasma membrane function, appears to have a unique and important IC role. The AT2 receptor, which shares only 32% amino acid sequence homology with the AT1 receptor, has recently been shown to bind to a transcription factor and to drive its localization to the nucleus. The rat AT2 receptor carboxy terminus binds to the transcription factor promyelocytic zinc finger protein (PLZF) on yeast two-hybrid screens of a human heart library (37). Confocal microscopy showed that ANG II induces cytosolic PLZF to colocalize with AT2R at the plasma membrane and then drives the receptor and PLZF to internalize. PLZF slowly appears in the nucleus, whereas AT2R accumulates in the perinuclear region. Nuclear PLZF binds to a number of genes that contribute to protein synthesis, and the authors suggest that these AT2 receptor-mediated changes in gene regulation could, in effect, contribute to cardiac hypertrophy. The AT2 receptor, in whole or in part, does not appear to translocate into the nucleus, unlike the AT1R, yet this is an example of a receptor that, through the cytoplasmic carboxy terminus, serves a unique IC chaperone function and, thus, contributes to alterations in gene expression.
The existence of prototypical plasma membrane receptors within cell nuclei and accompanying evidence that at least some of these are involved in transcriptional regulation of gene expression prompt us to ask why some receptors have evolved to occupy multiple cellular locations and presumably perform several functions. The most reasonable answers are that nuclear accumulation of a conventional "plasma membrane" receptor or receptor product may 1) contribute to the amplification of a downstream response to an external stimulus, 2) prolong a response to a stimulus, and/or 3) reduce the degeneracy of ligand-receptor signaling pathways and nuclear responses that are shared by multiple cell-surface receptors (i.e., increase specificity).
The discovery of traditional plasma membrane receptors in the nucleus is a conundrum that has plagued scientists working in related disciplines for several years. Although clear evidence exists for the presence of uncleaved holoreceptors, including those for FGF-1, ErbB-3, and epidermal growth factor receptor in the nucleus, the mechanism by which protein possessing hydrophobic domains, and associated with a lipid bilayer, may escape the membrane and acquire soluble properties that permit nuclear transport and nuclear function remains a mystery. Receptors, such as that for ErbB-4, Alzheimer precursor protein, and now AT1R, that undergo cleavage to release soluble cytoplasmic domains, which are then transported to the cell nucleus, pose less of a conceptual challenge.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
2. Anand-Srivastava MB, Palaparti A. Angiotensin-II-induced enhanced expression of Gi proteins is attenuated by losartan in A10 vascular smooth muscle cells: role of AT1 receptors. Can J Physiol Pharmacol 81: 150–158, 2003.[CrossRef][Web of Science][Medline]
3. Baldwin JM. The probable arrangement of the helices in G protein-coupled receptors. EMBO J 12: 1693–1703, 1993.[Web of Science][Medline]
4. Barna BP, Chou SM, Jacobs B, Ransohoff RM, Hahn JF, Bay JW. Enhanced DNA synthesis of human glial cells exposed to human leukocyte products. J Neuroimmunol 10: 151–158, 1985.[CrossRef][Web of Science][Medline]
5. Bergman A, Religa D, Karlstrom H, Laudon H, Winblad B, Lannfelt L, Lundkvist J, Naslund J. APP intracellular domain formation and unaltered signaling in the presence of familial Alzheimer's disease mutations. Exp Cell Res 287: 1–9, 2003.[CrossRef][Web of Science][Medline]
6. Boivin B, Chevalier D, Villeneuve LR, Rousseau E, Allen BG. Functional endothelin receptors are present on nuclei in cardiac ventricular myocytes. J Biol Chem 278: 29153–29163, 2003.
7. Carpenter G. Nuclear localization and possible functions of receptor tyrosine kinases. Curr Opin Cell Biol 15: 143–148, 2003.[CrossRef][Web of Science][Medline]
8. Cook JL, Mills SJ, Naquin R, Alam J, Re RN. Nuclear accumulation of the AT1 receptor in a rat vascular smooth muscle cell line: effects upon signal transduction and cellular proliferation. J Mol Cell Cardiol 40: 696–707, 2006.[CrossRef][Web of Science][Medline]
9. Cook JL, Re R, Alam J, Hart M, Zhang Z. Intracellular angiotensin II fusion protein alters AT1 receptor fusion protein distribution and activates CREB. J Mol Cell Cardiol 36: 75–90, 2004.[CrossRef][Web of Science][Medline]
10. De Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T. International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev 52: 415–472, 2000.
11. Ebinu JO, Yankner BA. A RIP tide in neuronal signal transduction. Neuron 34: 499–502, 2002.[CrossRef][Web of Science][Medline]
12. Fierens F, Vanderheyden PM, De Backer JP, Vauquelin G. Binding of the antagonist [3H]candesartan to angiotensin II AT1 receptor-transfected [correction of tranfected] Chinese hamster ovary cells. Eur J Pharmacol 367: 413–422, 1999.[CrossRef][Web of Science][Medline]
13. Fortini ME. Gamma-secretase-mediated proteolysis in cell-surface-receptor signalling. Nat Rev Mol Cell Biol 3: 673–684, 2002.[CrossRef][Web of Science][Medline]
14. Gonchar Y, Pang L, Malitschek B, Bettler B, Burkhalter A. Subcellular localization of GABAB receptor subunits in rat visual cortex. J Comp Neurol 431: 182–197, 2001.[CrossRef][Web of Science][Medline]
15. Gotlieb AI, Heggeness MH, Ash JF, Singer SJ. Mechanochemical proteins, cell motility and cell-cell contacts: the localization of mechanochemical proteins inside cultured cells at the edge of an in vitro "wound." J Cell Physiol 100: 563–578, 1979.[CrossRef][Web of Science][Medline]
16. Grantcharova E, Furkert J, Reusch HP, Krell HW, Papsdorf G, Beyermann M, Schulein R, Rosenthal W, Oksche A. The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent process. J Biol Chem 277: 43933–43941, 2002.
17. Gustafsson T, Andersson P, Arnqvist HJ. Different inhibitory actions of IGFBP-1, -2 and -4 on IGF-I effects in vascular smooth muscle cells. J Endocrinol 161: 245–253, 1999.[Abstract]
18. Harrison-Bernard LM, Imig JD, Carmines PK. Renal AT1 receptor protein expression during the early stage of diabetes mellitus. Int J Exp Diabetes Res 3: 97–108, 2002.[CrossRef][Medline]
19. Hunyady L, Baukal AJ, Gaborik Z, Olivares-Reyes JA, Bor M, Szaszak M, Lodge R, Catt KJ, Balla T. Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor. J Cell Biol 157: 1211–1222, 2002.
20. Hunyady L, Zhang M, Jagadeesh G, Bor M, Balla T, Catt KJ. Dependence of agonist activation on a conserved apolar residue in the third intracellular loop of the AT1 angiotensin receptor. Proc Natl Acad Sci USA 93: 10040–10045, 1996.
21. Kambe T, Kinjyo N, Hiruki H, Kubo T. Basal transcriptional regulation of rat AT1 angiotensin II receptor gene expression. Clin Exp Pharmacol Physiol 31: 96–100, 2004.[CrossRef][Web of Science][Medline]
22. Kimberly WT, Zheng JB, Guenette SY, Selkoe DJ. The intracellular domain of the beta-amyloid precursor protein is stabilized by Fe65 and translocates to the nucleus in a notch-like manner. J Biol Chem 276: 40288–40292, 2001.
23. Kinoshita A, Shah T, Tangredi MM, Strickland DK, Hyman BT. The intracellular domain of the low density lipoprotein receptor-related protein modulates transactivation mediated by amyloid precursor protein and Fe65. J Biol Chem 278: 41182–41188, 2003.
24. Le MT, De Backer JP, Hunyady L, Vanderheyden PM, Vauquelin G. Ligand binding and functional properties of human angiotensin AT1 receptors in transiently and stably expressed CHO-K1 cells. Eur J Pharmacol 513: 35–45, 2005.[CrossRef][Web of Science][Medline]
25. Lin SY, Makino K, Xia W, Matin A, Wen Y, Kwong KY, Bourguignon L, Hung MC. Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nat Cell Biol 3: 802–808, 2001.[CrossRef][Web of Science][Medline]
26. Maloney JA, Tsygankova O, Szot A, Yang L, Li Q, Williamson JR. Differential translocation of protein kinase C isozymes by phorbol esters, EGF, and ANG II in rat liver WB cells. Am J Physiol Cell Physiol 274: C974–C982, 1998.
27. Merjan AJ, Kanashiro CA, Krieger JE, Han SW, Paiva AC. Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells. Braz J Med Biol Res 34: 1175–1183, 2001.[Web of Science][Medline]
28. Miserey-Lenkei S, Parnot C, Bardin S, Corvol P, Clauser E. Constitutive internalization of constitutively active agiotensin II AT1A receptor mutants is blocked by inverse agonists. J Biol Chem 277: 5891–5901, 2002.
29. Modrall JG, Nanamori M, Sadoshima J, Barnhart DC, Stanley JC, Neubig RR. ANG II type 1 receptor downregulation does not require receptor endocytosis or G protein coupling. Am J Physiol Cell Physiol 281: C801–C809, 2001.
30. Morel G, Gallego R, Boulanger L, Pintos E, Garcia-Caballero T, Gaudreau P. Restricted presence of the growth hormone-releasing hormone receptor to somatotropes in rat and human pituitaries. Neuroendocrinology 70: 128–136, 1999.[CrossRef][Web of Science][Medline]
31. Myers JM, Martins GG, Ostrowski J, Stachowiak MK. Nuclear trafficking of FGFR1: a role for the transmembrane domain. J Cell Biochem 88: 1273–1291, 2003.[CrossRef][Web of Science][Medline]
32. Nehring RB, Horikawa HP, El Far O, Kneussel M, Brandstatter JH, Stamm S, Wischmeyer E, Betz H, Karschin A. The metabotropic GABAB receptor directly interacts with the activating transcription factor 4. J Biol Chem 275: 35185–35191, 2000.
33. Ohyama K, Yamano Y, Chaki S, Kondo T, Inagami T. Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis. Biochem Biophys Res Commun 189: 677–683, 1992.[CrossRef][Web of Science][Medline]
34. Perkinton MS, Standen CL, Lau KF, Kesavapany S, Byers HL, Ward M, McLoughlin DM, Miller CC. The c-Abl tyrosine kinase phosphorylates the Fe65 adaptor protein to stimulate Fe65/amyloid precursor protein nuclear signaling. J Biol Chem 279: 22084–22091, 2004.
35. Prieto M, Dipp S, Meleg-Smith S, El-Dahr SS. Ureteric bud derivatives express angiotensinogen and AT1 receptors. Physiol Genomics 6: 29–37, 2001.
36. Savoia C, Tabet F, Yao G, Schiffrin EL, Touyz RM. Negative regulation of RhoA/Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats. J Hypertens 23: 1037–1045, 2005.[Web of Science][Medline]
37. Senbonmatsu T, Saito T, Landon EJ, Watanabe O, Price E Jr, Roberts RL, Imboden H, Fitzgerald TG, Gaffney FA, Inagami T. A novel angiotensin II type 2 receptor signaling pathway: possible role in cardiac hypertrophy. EMBO J 22: 6471–6482, 2003.[CrossRef][Web of Science][Medline]
38. Seol KC, Kim SJ. Nuclear matrix association of insulin receptor and IRS-1 by insulin in osteoblast-like UMR-106 cells. Biochem Biophys Res Commun 306: 898–904, 2003.[CrossRef][Web of Science][Medline]
39. Sherrod M, Liu X, Zhang X, Sigmund CD. Nuclear localization of angiotensinogen in astrocytes. Am J Physiol Regul Integr Comp Physiol 288: R539–R546, 2005.
40. Stachowiak MK, Fang X, Myers JM, Dunham SM, Berezney R, Maher PA, Stachowiak EK. Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forward-and-gate" signaling module that controls cell growth and differentiation. J Cell Biochem 90: 662–691, 2003.[CrossRef][Web of Science][Medline]
41. Tan DS, Cook A, Chew SL. Nucleolar localization of an isoform of the IGF-I precursor. BMC Cell Biol 3: 17, 2002.[CrossRef][Medline]
42. Thekkumkara TJ, Du J, Dostal DE, Motel TJ, Thomas WG, Baker KM. Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: rapid desensitization by angiotensin II. Mol Cell Biochem 146: 79–89, 1995.[CrossRef][Web of Science][Medline]
43. Thekkumkara TJ, Du J, Zwaagstra C, Conrad KM, Krupinski J, Baker KM. A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells. Mol Cell Biochem 152: 77–86, 1995.[Web of Science][Medline]
44. Waugh MG, Hsuan JJ. EGF receptors as transcription factors: ridiculous or sublime? Nat Cell Biol 3: E209–E211, 2001.[CrossRef][Web of Science][Medline]
45. Wells A, Marti U. Signalling shortcuts: cell-surface receptors in the nucleus? Nat Rev Mol Cell Biol 3: 697–702, 2002.[CrossRef][Web of Science][Medline]
46. Wu A, Sciacca L, Baserga R. Nuclear translocation of insulin receptor substrate-1 by the insulin receptor in mouse embryo fibroblasts. J Cell Physiol 195: 453–460, 2003.[CrossRef][Web of Science][Medline]
47. Zeng C, Luo Y, Asico LD, Hopfer U, Eisner GM, Felder RA, Jose PA. Perturbation of D1 dopamine and AT1 receptor interaction in spontaneously hypertensive rats. Hypertension 42: 787–792, 2003.
This article has been cited by other articles:
|
|
 |
|
  J. L. Cook, R. N. Re, D. L. deHaro, J. M. Abadie, M. Peters, and J. Alam The Trafficking Protein GABARAP Binds to and Enhances Plasma Membrane Expression and Function of the Angiotensin II Type 1 Receptor Circ. Res., June 20, 2008; 102(12): 1539 - 1547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Re Intracellular renin-angiotensin system: the tip of the intracrine physiology iceberg Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H905 - H906. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
