Endocrinology, doi:10.1210/en.2007-0157
Endocrinology Vol. 148, No. 5 2282-2289
Copyright © 2007 by The Endocrine Society
Type 1 Parathyroid Hormone Receptor (PTH1R) Nuclear Trafficking: Regulation of PTH1R Nuclear-Cytoplasmic Shuttling by Importin-
/ß and Chromosomal Region Maintenance 1/Exportin 1
Bryce W. Pickard,
Anthony B. Hodsman,
Laurence J. Fraher and
Patricia H. Watson
Departments of Physiology and Pharmacology, Medicine, and Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B8
Address all correspondence and requests for reprints to: Patricia H. Watson, Ph.D., Room E4-155, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. E-mail: pwatson{at}lhrionhealth.ca.
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Abstract
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The type 1 PTH/PTH-related peptide receptor (PTH1R) is a class B G protein-coupled receptor that demonstrates immunoreactivity in the nucleus as well as cytoplasm of target cells. Our previous studies on the PTH1R have shown that it associates with the importin family of transport regulatory proteins. To investigate the role of the importins in PTH1R nuclear import, we used small interfering (si)RNA technology to knock down the expression of importin-ß in the mouse osteoblast-like cell line, MC3T3-E1. Immunofluorescence microscopy as well as ligand blotting for PTH1R in nuclear fractions of importin-ß siRNA-treated cells demonstrated a decrease in nuclear localization of the PTH1R in comparison with control cells. Under normal culture conditions, PTH1R is present in both the nucleus and cytoplasm of cells. Serum starvation favors nuclear localization of PTH1R, whereas returning cells to serum or treatment with PTH-related peptide induced its cytoplasmic localization. To address the nuclear export of PTH1R, interactions between PTH1R and chromosomal region maintenance 1 (CRM1) were investigated. PTH1R and CRM1 coimmunoprecipitated from MC3T3-E1 cells, suggesting that CRM1 and PTH1R form a complex in vivo. After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, PTH1R accumulated in the nucleus. Taken together, our studies show that PTH1R shuttles from the nucleus to the cytoplasm under normal physiological conditions and that this nuclear-cytoplasmic transport is dependent upon importin-
/ß and CRM1.
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Introduction
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THE TYPE 1 PTH/PTH-RELATED peptide (PTHrP) receptor (PTH1R) is a seven-transmembrane-spanning G protein-coupled receptor (GPCR) belonging to the class B secretin-like GPCR family. PTH1R is responsible for maintaining a variety of cellular processes primarily due to the fact that it generates signals in response to two ligands, PTH and PTHrP, and thus is widely expressed throughout various organs and tissues. In response to ligand, PTH1R has the capability to elicit downstream effects through both the Gs-mediated cAMP pathway and the Gq/11-mediated phospholipase C pathway (1, 2, 3). In addition to these signaling pathways, which have been well characterized and are common to many GPCRs, PTH1R also has demonstrated nuclear localization, which is a relatively new avenue in GPCR study. The angiotensin II type 1 receptor (AT1-R), a class A GPCR, was the first GPCR for which nuclear localization and trafficking was clearly demonstrated (4, 5). A group of class A GPCRs, the prostaglandin E2 receptors EP1, EP3, and EP4, were next characterized as nuclear signaling receptors (for review see Ref. 6). The PTH1R was the third GPCR found to traffic to the nucleus of cells and, so far, the only member of the class B GPCR subfamily (7, 8, 9). In the last 5 yr, 11 more GPCRs have been found to localize to the nucleus of cells. Although the mechanisms involved in regulating the membrane-bound signaling properties of the GPCRs are relatively well known, the knowledge of mechanisms regulating the nuclear transport and function of GPCRs is sparse.
The transport of proteins into and out of the nucleus occurs through a variety of mechanisms involving the nuclear pore complex and the interaction between a nuclear localization sequence (NLS) and members of the importin family of transport proteins (10). Proteins, such as PTH1R, containing a classical bipartite NLS (characterized by two stretches of basic residues separated by a spacer region) are targeted to the nucleus through the action of importin-ß and an adaptor, importin- (11). Importin- links the cargo protein containing an NLS to importin-ß, which docks the complex at the nuclear pore, facilitating entry of the cargo to the nucleus. To date, the only GPCR that has been shown to associate with the importins has been the PTH1R; however, several have been shown to contain a functional NLS (8, 9, 12, 13). In contrast to the signaling properties of membrane-bound PTH1R, the function of the nuclear PTH1R is currently unknown. However, we do know that it has a dynamic nuclear expression and is actively imported to, and exported from, the nucleus during specific stages of the cell cycle (9).
Nuclear-cytoplasmic shuttling plays an important role in the regulation of various cellular processes such as gene expression and proliferation (reviewed in Ref. 14). The most common mechanism of nuclear export of proteins involves a nuclear export signal (NES), a characteristic pattern of hydrophobic amino acids that is specifically recognized by the nuclear export receptor, chromosomal region maintenance 1 (CRM1)/exportin 1 (15). To facilitate nuclear export, CRM1 associates with the NES of the cargo protein through an association involving RanGTP, which results in the translocation of the complex from the nucleus to the cytoplasm. In contrast to several GPCRs containing an NLS, to date, no GPCR has had a functional NES identified.
Although the presence of PTH1R in the nucleus and its association with importin-2 and -ß has been clearly established, the functional relevance of this association and the physiological conditions under which nuclear trafficking occurs are unclear (9). In this study, we demonstrate that the mechanism controlling the nuclear import of PTH1R is that of importin-/ß. In addition, we show that nuclear export is mediated by CRM1. We also show that PTH1R subcellular distribution changes under normal physiological conditions controlled by the presence or absence of growth factors, suggesting that its regulation is required for the proper function of the cell. This research brings new insights into the mechanism controlling the trafficking and functions of nuclear PTH1R and may have broader implications for our understanding of GPCR nuclear localization.
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Materials and Methods
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Cell culture
MC3T3-E1 mouse nontransformed osteoblasts were cultured in -MEM containing penicillin (10 U/ml), streptomycin (10 µg/ml), and 10% fetal bovine serum (FBS) (Invitrogen, Burlington, Ontario, Canada) in a 5% CO2 in air, humidified atmosphere. All cells used for experiments were between passages three and ten. For serum starvation, MC3T3-E1 cells (cultured under normal conditions) were washed with -MEM containing 0.1% FBS and then incubated for 24 h in -MEM containing 0.1% FBS. The effects of serum on the cells were investigated by reintroducing 10% serum for 1 h. For PTHrP stimulation, cells were starved in -MEM containing 0.1% FBS for 24 h and then stimulated with human PTHrP (100 ng/ml) in 0.1% FBS for 60 min at 37 C. Leptomycin B (LMB) (Sigma Chemical Co., St. Louis, MO) was used at a concentration of 20 nM. Cells were either incubated in the presence of LMB or the equivalent amount of 70% ethanol as a vehicle control for 3 h at 37 C before cell treatments.
Small interfering (si)RNA sequences
Prevalidated small interfering RNA molecules (siRNA) corresponding to importin-ß, as well as siRNA negative control no. 1 (catalog item 4611) were purchased from Ambion (Austin, TX). The siRNA sequences targeting importin-ß correspond to the coding region of exon 11 (catalog item 16704 siRNA ID 158323) and exon 4 (catalog item 16704 siRNA ID 158321).
siRNA transfection
Optimization of starting cell number and siPORT NeoFX transfection agent amount was done initially (data not shown). Importin-ß siRNA concentration was optimized through various incubation times and starting concentrations. The most effective combination was a siRNA concentration of 90 nM with 5 µl transfection reagent per well of a six-well plate. Transfections were performed using the MC3T3-E1 cell line with siPORT NeoFX as per manufacturer’s instructions (Ambion; catalog item 4511). Briefly, MC3T3-E1 cells were trypsinized and diluted to a concentration of 1 x 105 cells/ml. Transfection complexes were formed by mixing the appropriate amount of siRNA and transfection reagent and incubating at room temperature for 10 min. After the incubation, 1.5 x 105 cells were overlaid onto the siRNA complexes in each well of the plate. Medium was replaced 24 h after the transfection to ensure cell viability. After media replacement, cells were cultured under serum starved conditions (-MEM containing 0.1% FBS) for 24 h to induce nuclear accumulation of PTH1R. Cells were assayed for the knockdown of importin-ß 72 h after transfection by Western blot. From previous optimization experiments, we found that 72 h of culture was the optimal time to assess importin-ß siRNA transfection effectiveness.
Protein preparation and Western blotting
Briefly, whole-cell protein was isolated following a standard protocol (16). The total protein content was determined using the BCA protein assay kit (Pierce, Rockford, IL), and samples were normalized accordingly. Protein (50 µg/lane) was separated on a 10% SDS-PAGE gel, transferred overnight to nitrocellulose, and probed with an antibody to importin-ß (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA; sc-1863). To ensure specificity of importin-ß knockdown, protein from cells treated with importin-ß siRNA were also probed with importin-1 (1:250) (Santa Cruz; sc-6918) or -2 (1:250) (Santa Cruz; sc-6917) antibodies. An antibody to lamin A/C (1:250) (Santa Cruz; sc-6215) was used for all loading controls. Densitometry of band intensities was performed using GeneTools (Syngene, Frederick, MD). Western blot data were analyzed by one-way ANOVA, and results were considered significant at P < 0.01.
Immunofluorescence
Immunofluorescence was performed as described (17). Cells were treated for antigen retrieval with 0.1% Triton X-100/PBS for 20 min followed by an overnight incubation with primary antibody at a dilution of 1:100. A polyclonal antirabbit PTH1R antibody raised against a peptide mapping near the amino terminus was used to visualize PTH1R (Covance, Princeton, NJ; PRB-620P). This antibody has been well characterized and used extensively in our lab for these and other published results (7, 8, 9). Detection of the primary antibody was done using the Alexa Fluor 488 donkey antirabbit IgG (Molecular Probes, Eugene, OR; A21207) at a dilution of 1:250 with an incubation time of 45 min. After the addition of the secondary antibody, the cells were washed in PBS after which nuclei were stained using 4'6-diamidino-2-phenylindole (DAPI) according to manufacturer’s instructions (Molecular Probes; D21490). Controls in which primary antibody was replaced with nonimmune serum were routinely used.
Subcellular fractionation and ligand blot analysis
To further confirm the decrease in nuclear accumulation of PTH1R due to the importin-ß knockdown, subcellular fractions were isolated from siRNA-treated cells using the Calbiochem (La Jolla, CA) ProteoExtract Subcellular Proteome Extraction Kit according to manufacturer’s recommended conditions (catalog no. 539790). Briefly, cells were transfected with importin-ß or negative control siRNA as previously described. Through a series of washes and extraction buffers provided by the manufacturer, the membrane/organelle, nucleic protein, and cytoskeletal matrix protein fractions of the cells were isolated. The protein content of all fractions were determined using the BCA protein assay kit (Pierce), and samples were normalized accordingly. Fractions from the control siRNA as well as importin-ß transfections were separated on a 10% SDS-PAGE gel, transferred overnight to nitrocellulose, and probed with biotinylated PTH (1–84) (ECL protein biotinylation module from Amersham, Arlington Heights, IL) for the presence of PTH1R. An antibody to lamin A/C (1:250) (Santa Cruz; sc-6215) was used as a loading control for all nuclear fractions. An antibody to pan-cadherin (1:200) (Santa Cruz; sc-1499) was used for membrane/organelle fraction loading controls. An antibody to pan-cytokeratin (1:500) (Santa Cruz; sc-17843) was used for cytoskeletal matrix protein fraction loading controls.
Immunoprecipitation and ligand blot analysis
Immunoprecipitations were performed using the Amersham immunoprecipitation kit following the manufacturer’s recommended protocol. Five micrograms of a goat polyclonal antibody raised against a peptide mapping near the carboxy terminus was used for the immunoprecipitation of PTH1R (Santa Cruz; sc-12778). Five micrograms of a goat polyclonal antibody raised against a peptide mapping at the carboxy terminus was used for the immunoprecipitation of CRM1 (Santa Cruz; sc-7825). Briefly, whole-cell protein was extracted from random cycling MC3T3-E1 cells using a high-salt buffer (500 mM NaCl; 1% Nonidet P-40; 50 mM Tris (pH 8.0); and 1 mM phenylmethylsulfonyl fluoride), total protein content was determined using the BCA protein assay kit (Pierce, Rockford, IL), and samples were normalized accordingly. Antibody was added, and samples were incubated overnight at 4 C. Fifty microliters of protein A Sepharose beads were added, and the mixture was incubated for 1 h at 4 C. After a series of washes with high-salt buffer, the immunocomplex was eluted from the Sepharose by heating at 95 C for 3 min in the presence of Laemmli buffer. The immunoprecipitates were separated on a 10% SDS-PAGE gel, transferred overnight to nitrocellulose, and probed with biotinylated PTH (1–84) (ECL protein biotinylation module from Amersham) for the presence of PTH1R. Mock immunoprecipitations were also carried out in the absence of primary antibody and using an anti-GFP antibody (Santa Cruz; sc-4304).
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Results
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Results shown are representative of at least one osteoblast-like cell line with at least three independently repeated experiments.
Knockdown of importin-ß expression results in decreased nuclear PTH1R accumulation
To investigate the role of the importins in the nuclear transport of PTH1R, importin-ß expression was selectively knocked down using siRNA. In response to decreased levels of importin-ß, the nuclear accumulation of PTH1R was decreased as assessed by immunofluorescence microscopy (Fig. 1A
) and subcellular fractionation (Fig. 1B). Control siRNA cells can be observed to have high levels of nuclear PTH1R immunoreactivity with relatively lower levels in the cytoplasm. In contrast, importin-ß siRNA-treated cells show little PTH1R fluorescence in the nucleus and readily apparent fluorescence in the cytoplasm. Nuclear protein lysate from control and importin-ß siRNA-treated cells was isolated from whole-cell protein lysate and separated by 10% SDS-PAGE and probed with biotinylated PTH (1–84) for the presence of PTH1R (Fig. 1B). A decrease in nuclear PTH1R can be observed in importin-ß siRNA-treated cells compared with control siRNA-treated cells. The amount of PTH1R protein in the cytoskeletal and membrane fractions were unaffected by the siRNA treatment.
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FIG. 1. Decrease in PTH1R nuclear localization in response to importin-ß knockdown. A, Immunofluorescence of MC3T3-E1 cells transfected with either importin-ß siRNA or control siRNA. Cells were fixed and subjected to immunofluorescence staining for PTH1R and counterstained with DAPI to visualize DNA. In all panels, PTH1R staining is in green and DAPI staining is in blue. Control siRNA cells can be observed to have high levels of nuclear PTH1R immunoreactivity with relatively lower levels in the cytoplasm. In contrast, importin-ß siRNA-treated cells show a decreased level of PTH1R in the nucleus and a relatively higher amount in the cytoplasm. Solid arrows highlight nuclei that are positive for PTH1R. Open arrows indicate nuclei that show less nuclear PTH1R. Scale bar, 20 µm. B, Cell fractionation and ligand blotting of MC3T3-E1 cells transfected with either importin-ß siRNA or control siRNA. Membrane, cytoskeletal, and nuclear protein fractions from control and treated cells were isolated from and separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with biotinylated PTH (1–84) for the presence of PTH1R. A decrease in nuclear PTH1R can be observed in importin-ß siRNA-treated cells compared with control siRNA-treated cells. Membrane-bound and cytoskeletal PTH1R show no change in protein levels. An antibody against pan-cadherin, pan-cytokeratin, and lamin A/C were used as loading controls for membrane, cytoskeletal, and nuclear fractions, respectively.
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Transfection of importin-ß siRNA into MC3T3-E1 cells resulted in a specific reduction of importin-ß expression as determined by Western blot (Fig. 2, A and B). Control siRNA was transfected into MC3T3-E1 cells following the same experimental conditions and did not alter the level of importin-ß expression. As shown in Fig. 3, the level of importin-ß decreased to 62% of normal levels as compared with control siRNA-treated cells (P < 0.01). To ensure that the siRNA is specific to importin-ß and that no secondary effects were observed, the levels of other importin family members were also assessed, namely importin-1 and -2 (Fig. 2C). The expression of importin-1 and -2 did not change in response to importin-ß siRNA showing a selective knockdown of importin-ß with no cross-reactivity to other importin family members.
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FIG. 3. Quantification of importin-ß knockdown. Densitometry analysis of importin-ß protein levels were performed using GeneTools (Syngene). Importin-ß protein levels are expressed as a percentage of control importin-ß protein. Data are presented as mean ± SEM of six independent experiments. *, P < 0.01.
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Control of PTH1R nuclear-cytoplasmic shuttling
To gain more insight into the control of PTH1R nuclear-cytoplasmic shuttling, we examined the cellular conditions under which shuttling takes place. Under normal cell culture conditions (control, -MEM containing 10% FBS), PTH1R displays both nuclear and cytoplasmic localization; however, it is predominately present in the cytoplasm (Fig. 4A, left panel). When cells are cultured under conditions of serum starvation for 24 h (starved, -MEM containing 0.1% FBS) PTH1R localizes to the nucleus of all cells with a marked decrease in cytoplasmic localization (Fig. 4A, middle panel). When the cells are subsequently returned to normal serum conditions (serum) after serum starvation, nuclear export of PTH1R is observed with the majority of PTH1R fluorescence being restricted to the cytoplasm (Fig. 4A, right panel). These data would suggest that a component of FBS contributes to the regulation of PTH1R subcellular localization. One of the components of FBS is a variety of growth factors possibly including PTHrP; because PTHrP is one of the ligands of PTH1R, we investigated the addition of exogenous PTHrP to the cell culture after serum starvation. Stimulation of serum-starved cells with PTHrP resulted in a time-dependent decrease in nuclear PTH1R fluorescence and an associated increase in cytoplasmic fluorescence (Fig. 4B). There was no apparent change in the total cellular amount of PTH1R as assessed by Western blot analysis (Fig. 4C). Taken together, these results demonstrate that PTH1R subcellular distribution changes under normal physiological conditions, suggesting that its regulation is required for the proper function of the cell.
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FIG. 4. Normal physiological conditions regulate PTH1R nuclear-cytoplasmic shuttling. A, MC3T3-E1 cells were cultured under three different conditions: control-MEM containing 10% FBS, starved-MEM containing 0.1% FBS for 24 h, and serum starved for 24 h at which time returned to control conditions. Cells were fixed and subjected to immunofluorescence staining for PTH1R; in all panels, PTH1R staining is in green. PTH1R localizes to both the nucleus and cytoplasm of MC3T3-E1 cells under control culture conditions (far left panel). Under conditions of serum starvation, PTH1R localizes to the nucleus with minimal staining being observed in the cytoplasm (middle panel). When cells are maintained under starved conditions for 24 h and then stimulated with 10% serum for 1 h, PTH1R localizes to the cytoplasm with minimal staining being observed in the nucleus. Solid arrows highlight nuclei that are positive for PTH1R. Open arrows indicate nuclei that show less nuclear PTH1R. Scale bar, 20 µm. B, MC3T3-E1 cells were cultured under serum-starved conditions for 24 h and stimulated by PTHrP (100 ng/ml) for the indicated time points. As the treatment time of PTHrP increases, the nuclear localization of PTH1R decreases and an increase in PTH1R cytoplasmic localization is observed. Solid arrows highlight nuclei that are positive for PTH1R. Open arrows indicate nuclei that show less nuclear PTH1R. Scale bar, 20 µm. C, Whole-cell protein lysate was isolated from PTHrP-treated cells and separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with biotinylated PTH (1–84) for the presence of PTH1R. We observed no change in the protein levels of PTH1R for the treatment points examined. An antibody against lamin A/C was used as a loading control.
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Prediction of a PTH1R NES
The nuclear export of proteins by CRM1 is NES dependent; therefore, we searched for an NES in the PTH1R using the NES prediction server, NetNES (18). Analysis of the human PTH1R sequence (NCBI accession no. NM_000316) revealed only two residues, leucine 261 and leucine 416, with an NES motif score more than 0.5. Inspection of the residues surrounding these found that none of them fit the generally accepted consensus sequence for an NES (LX1–3LX2–4LXL), most notably they did not contain a core LXL region (18, 19). Additional sequence analysis using the core region and the critical hydrophobic residues as a start point found that amino acids 201–211 fit the consensus sequence for an NES (Table 1). Because the NLS for PTH1R is highly conserved among species, we then looked to see whether the NES sequence was conserved over mouse/rat (NCBI accession number NM_008970/NCBI accession number NM_020073), opossum (NCBI accession number M_74445), and pig (NCBI accession number NM_214382) sequences as a step in the validation of the NES (Table 1). We found that this possible NES was highly conserved between all species examined, adding weight to the contention that this is a functional NES.
Coimmunoprecipitation of PTH1R with CRM1
To confirm that PTHrP-induced nuclear-cytoplasmic shuttling is in fact regulated nuclear export and to further define the mechanism involved, we examined the role of CRM1 in this processes. For CRM1 to be involved in the nuclear export of PTH1R it must form a complex with PTH1R at an NES. Total cell protein from random-cycling MC3T3-E1 cells was immunoprecipitated with antibodies against PTH1R or CRM1. After the immunoprecipitates were separated, transferred, and subsequently exposed to biotinylated PTH (1–84), a single band was present on the ligand blot at 66.3 kDa corresponding to the PTH1R (Fig. 5). No band was present in samples immunoprecipitated with a control anti-GFP antibody, indicating a stringent wash procedure and an antibody-specific immunoprecipitation procedure. The coimmunoprecipitation of PTH1R with CRM1 suggests that the mechanism of PTH1R export from the nucleus in response to PTHrP is mediated by CRM1.
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FIG. 5. Coimmunoprecipitation of PTH1R with CRM1. MC3T3-E1 cells were homogenized and immunoprecipitated with antibodies against PTH1R or CRM1. Immunoprecipitates (IP) were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with biotinylated PTH (1–84) for the presence of PTH1R. The ligand blot picked up a specific band for the PTH1R in the CRM1 immunoprecipitate, indicating an interaction between PTH1R and CRM1.
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Leptomycin B inhibits PTHrP-induced PTH1R nuclear export
To further confirm the role of CRM1 in PTH1R nuclear export, LMB, a known inhibitor of CRM1-mediated nuclear export, was used to disrupt PTH1R export (20). Cells were cultured under serum-starved conditions for 24 h at which time they were exposed to PTHrP for 60 min either in the presence or absence of LMB (Fig. 6). In response to PTHrP treatment, PTH1R nuclear localization decreased compared with serum-starved conditions (Fig. 6, top left and right panels). In cells treated with LMB, PTH1R nuclear export in response to PTHrP was blocked (Fig. 6, bottom left and right panels). These results show that PTH1R is actively exported from the nucleus through a CRM1-dependent mechanism.
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FIG. 6. Leptomycin B inhibits PTHrP-induced PTH1R nuclear export. MC3T3-E1 cells were maintained under serum-starved conditions for 24 h at which time they were cultured for another 3 h in the presence of 20 nM LMB (+LMB) or in the absence of LMB (vehicle) at 37 C. Cells were fixed and subjected to immunofluorescence staining for PTH1R; in all panels, PTH1R staining is in green. In response to PTHrP treatment, PTH1R nuclear localization decreases compared with serum-starved conditions (top left and right panels). In cells treated with LMB, PTH1R cytoplasmic accumulation was blocked, resulting in cells with continued PTH1R nuclear localization after PTHrP treatment compared with both control cells and untreated PTHrP-stimulated cells (bottom left and right panels). Solid arrows highlight nuclei that are positive for PTH1R. Open arrows indicate nuclei that show less nuclear PTH1R. Scale bar, 20 µm.
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Discussion
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Our studies provide a number of novel insights into the signaling mechanisms of the PTH1R. We previously showed that the PTH1R forms a complex with the transport regulatory proteins importin-1 and importin-ß (9). In the studies reported here, we show that this association is functional and necessary for the import of PTH1R to the nucleus. For proteins such as the PTH1R that are transported to the nucleus by the importins through an interaction with a bipartite NLS, importin-ß works through an adaptor protein, importin-, to target the complex to the nuclear pore complex where it then enters the nucleus. In mammals, importin-ß constitutes a single gene family, whereas importin- is a multigene family and can be classified into three subgroups: 1 (Rch1), 3 (Qip-1), and 5 (NP-1) (11, 21). Two characteristics of this process make importin-ß a convenient and effective target to block nuclear import by the importins in our cell system, first and foremost being that through a bipartite NLS, importin- cannot function without importin-ß. Therefore, knocking down importin-ß renders importin- nonfunctional in our cell system. Second, because importin-ß is a single gene family and cannot display functional redundancy as importin- can, we know that the nuclear import of PTH1R cannot occur through a different importin-ß. Therefore, based on a specific knockdown of importin-ß, we have been able to show that the mechanism controlling the nuclear import of PTH1R is through its interaction with importin-/ß. The question remains as to what role individual importins 1, 3, and 5 play in the nuclear import of PTH1R. Specifically, do all three importin- subgroups play a role in the regulation of the nuclear import of PTH1R, as is the case for the nuclear import of mammalian circadian clock component, or does one specific subgroup regulate the nuclear import, which is the case for the small GTPase Ran where nuclear import is dependent on importin-3 (22, 23). Although the discovery of a functional relationship between the PTH1R and the importins has been an important step in elucidating a mechanism for nuclear GPCR localization, it most likely is not the exclusive mechanism for the translocation of other GPCRs to the nucleus. Of the relatively small number of nuclear GPCRs discovered thus far, there are several diverse suggested mechanisms of action and spatial distributions within the nucleus. For example, receptors such as the angiotensin II type 1 receptor and the leukotriene D4 receptor contain a functional NLS and are known to target to the nucleus from the plasma membrane in response to ligand stimulation (5, 12, 13). For these receptors, a mechanism of localization for which the importins play a prominent role is possible. In contrast, endothelin receptor subtypes A and B have a perinuclear distribution, and evidence suggests that this receptor localizes to the nuclear membrane via de novo synthesis and retrograde transport; therefore, an association with the importins would likely not be necessary (24).
The nuclear localization of PTH1R varies depending upon the stage of the cell cycle (9). To examine the mechanism behind the nuclear export of PTH1R, the most common mechanism of nuclear export CRM1/exportin1 was investigated. In this study we showed that the export of PTH1R from the nucleus is mediated by CRM1. Our conclusion is based on three observations. First, we identified a possible NES in the PTH1R that fits the consensus sequence for an NES and is conserved among several species examined. However, additional biochemical studies are needed to confirm that the NES identified here is in fact functional and the only NES present in the protein. In other studies of NES-containing proteins it has been shown that multiple NES motifs can be present in a single protein and that sequences can vary significantly from the generally accepted consensus (18). For example, BRCA1 contains two NES motifs capable of directing nuclear export of the protein (25). Second, PTH1R and CRM1 coimmunoprecipitate from MC3T3-E1 cells, suggesting that CRM1 and PTH1R form a complex in vivo, thus providing a means for nuclear export of PTH1R. Third, LMB treatment resulted in accumulation of PTH1R in the nucleus after the induction of nuclear export, suggesting that LMB blocked the association of PTH1R with CRM1, effectively preventing the nuclear export of PTH1R. Taken together, these studies show that the nuclear export of PTH1R occurs through a CRM1-dependent mechanism.
To investigate the control of nuclear export, we examined the culture conditions in which PTH1R nuclear trafficking occurs. We found that in the presence of serum or PTHrP, PTH1R was actively exported from the nucleus. In contrast, during conditions of serum starvation, PTH1R was abundant in the nucleus. Additional studies will be undertaken to determine what specific factors in serum trigger the nuclear export of PTH1R. More specifically, will PTH1R be exported from the nucleus in response to other growth factors that may be present in the serum such as IGF and/or TGF-ß, or is this a mechanism solely dependent upon PTHrP? This result was a surprise finding because we would expect the presence of ligand to trigger nuclear import, rather than export, as is observed for other nuclear GPCRs such as the angiotensin II type 1 receptor (5). The active nuclear transport occurring under normal physiological conditions suggests that the nuclear localization of PTH1R is physiologically relevant and is potentially a critical factor in cellular function. In summary, our data show that PTH1R shuttles from the nucleus to the cytoplasm under normal physiological conditions and that this nuclear-cytoplasmic transport is dependent upon importin-/ß for import to the nucleus and CRM1/exportin1 for export to the cytoplasm. This research brings new insights into the function of nuclear PTH1R and may have broader implications for our further understanding of what role nuclear GPCRs play in normal cellular processes.
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Footnotes
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This work was supported by the Canadian Institutes of Health Research (Grant MOP-64228).
Disclosure Statement: B.P., L.F., A.H., and P.W. have nothing to declare.
First Published Online February 22, 2007
Abbreviations: CRM1,Chromosomal region maintenance 1; FBS, fetal bovine serum; GPCR, G protein-coupled receptor; LMB, leptomycin B; NES, nuclear export signal; NLS, nuclear localization sequence; PTH1R, type 1 PTH/PTH-related peptide receptor; si, small interfering.
Received February 2, 2007.
Accepted for publication February 13, 2007.
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Abstract
Introduction
