Type 1 Parathyroid Hormone Receptor (PTH1R) Nuclear Trafficking ...

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Endocrinology 147(7):3326 –3332 Copyright © 2006 by The Endocrine Society doi: 10.1210/en.2005-1408

Type 1 Parathyroid Hormone Receptor (PTH1R) Nuclear Trafficking: Association of PTH1R with Importin ␣1 and ␤ 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 Previous studies have shown that the type 1 PTH receptor (PTH1R), a class B G protein-coupled receptor, appears in the nucleus of target cells. Through immunofluorescence and deconvolution microscopy, we demonstrate that PTH1R, importin ␣1, and importin ␤ are present within the nucleus and cytoplasm of osteoblast-like cell lines with the nuclear PTH1R being restricted to the nucleoplasm. Immunofluorescence studies showed that nuclear accumulation of PTH1R was associated with specific stages of the cell cycle. Using immunoprecipitation and affinity chromatography, we show that the PTH1R forms a complex with the importin family of transport molecules. Total cell protein from osteoblast-like cells was immunoprecipitated with antibodies for PTH1R, importin ␣1, or importin ␤. When the immunoprecipitates were separated

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HE TYPE 1 PTH/PTH-RELATED peptide receptor (PTH1R) is a seven-transmembrane-spanning G proteincoupled receptor (GPCR) belonging to the class B secretinlike GPCR family. The PTH1R has two distinct ligands, PTH and PTH-related peptide (PTHrP), to which it responds through a number of signaling pathways. PTHrP is ubiquitously expressed and is involved in a number of cellular processes including development, growth and differentiation, and migration (1). The very prominent role PTHrP plays in the regulation of embryonic development can be seen with the PTHrP ⫺/⫺ mouse, which dies at or just before birth (2). In contrast to the wide range of PTHrP functions, PTH is primarily responsible for the maintenance of serum calcium levels through its actions on the kidney and the control of bone remodeling. How the PTH1R responds in a very specific fashion to two different ligands can be attributed to various signal control mechanisms. For example, PTH binding to PTH1R activates both the G␣s-mediated cAMP pathway and the G␣q/11-mediated phospholipase C pathway (3–5). Partial signal response is possible after the activation of the PTH1R with truncated synthetic PTH peptides. Nterminally truncated PTH analogs such as PTH (3–34) and PTH (7–34) bind to PTH1R with high affinity but elicit First Published Online March 30, 2006 Abbreviations: DAPI, 4⬘,6-Diamidino-2-phenylindole; GPCR, G protein-coupled receptor; NLS, nuclear localization sequence; PTH1R, type 1 PTH receptor; PTHrP, PTH-related peptide; RIPA, radioimmunoprecipitation buffer. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

and subsequently exposed to biotinylated PTH (1– 84) a single band was present on the gel at 66.3 kDa, corresponding to the PTH1R. To confirm the interaction between PTH1R and both importin ␣1 and ␤, the complex was purified from total cell protein of osteoblast-like cells using a PTH-linked affinity chromatography column. Using an anti-importin ␣1 antibody, Western blots detected importin ␣1 at 58 kDa in the purified sample. Also, using an anti-importin ␤ antibody, Western blots detected importin ␤ at 94 kDa. These results indicate that the importins were associated with the PTH1R at the time of the purification. In conclusion, we show that the PTH1R forms a complex with the transport regulatory proteins, importin ␣1 and importin ␤, and that nuclear PTH1R is associated with the nucleoplasm. (Endocrinology 147: 3326 –3332, 2006)

little or no increase in cAMP accumulation (6). N-terminal fragments, such as PTH (1–31), retain their full binding affinity and activate only the adenylate cyclase pathway as measured by cAMP accumulation (7). Another mechanism of signal propagation and control is through internalization of the receptor. After the binding of either PTH or PTHrP, PTH1R is internalized via the well known mechanism common to all GPCRs, the formation of a clathrin-coated pit, involving ␤-arrestin2 (8). At first, the internalization of the receptor was associated with desensitization and dampening of the receptor response to ligand (9). However, recent studies have shown that the arrestins, which were once thought to be involved only in the formation of the clathrin-coated pit, can actually propagate signal by acting as adapters for Srcfamily tyrosine kinases and as receptor-regulated scaffolds for several ERK, c-Jun N-terminal kinase, and p38 MAPK modules (10). Expanding on the concept of several levels of PTH1R signaling is the discovery of PTH1R, and several other GPCRs, in the nucleus of target cells. Studies on the nuclear localization of PTH1R in cultured osteoblast-like cells (ROS 17/2.8, UMR-106, MC3T3-E1, and SaOS-2) demonstrate immunoreactivity for PTH1R both in the nucleus and cytoplasm (11). The nuclear translocation of PTH1R is yet another mechanism used by a single receptor with a wide variety of roles in the body. As of yet, evidence for GPCR translocation to the nucleus is relatively sparse. Examples of these receptors include angiotension II AT1 receptor, prostaglandin E2 receptors EP3 and EP4, and receptors for apelin and bradykinin B2, all of which belong to the class A rhodopsin-like family of GPCRs (12–14). In addition to the class A receptors, functional

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Pickard et al. • Association of PTH1R with Importin ␣1 and ␤

metabotropic glutamate receptors, mGlu5, a class C family member, has also been found to have nuclear localization (15). Advances in the study of nuclear GPCRs have revealed that the distribution within the nucleus can be unique to the receptor. For example, the apelin, angiotensin AT1 and AT2, bradykinin B2, and lysophosphatidate LP1 receptors have been localized within the nucleoplasm (14, 16, 17). In contrast, some GPCRs have been observed to have a perinuclear pattern. This can be observed for the prostaglandin E2 receptors and metabotropic glutamate, mGlu5, receptor, which are localized to nuclear membranes (13, 15). The transport of proteins into and out of the nucleus occurs through a variety of mechanisms involving the nuclear pore complex via the interaction of a nuclear localization sequence (NLS) and members of the importin family (18). There are several types of NLSs that are identified by their individual sequence of primarily basic amino acids and their location within the protein. Proteins 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 ␤ and an adaptor importin ␣ (19). Importin ␣ links the NLS-containing protein to importin ␤, which docks the complex at the nuclear pore complex facilitating entry of the complex to the nucleus. In mammals, importin ␤ 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 (NPI-1) (19, 20). The subgroups of importin ␣ are grouped based on sequence similarity with the three groups sharing an 85% sequence similarity with most differences occurring outside their NLS binding motif. Our interest in the PTH1R stems from the fact that its ligands are capable of exerting both catabolic and anabolic actions in bone and little is yet understood of the mechanisms underlying its anabolic activity. The classic physiological role of PTH and PTH1R is to stimulate bone turnover with a relative increase in bone resorption, a phenomenon readily apparent in individuals with excess PTH or chronic hyperparathyroidism (21). However, intermittent therapy with PTH is now widely accepted as a bone anabolic strategy in the treatment of severe osteoporosis where it not only stimulates bone remodeling but also initiates de novo trabecular bone growth on previously quiescent surfaces (22, 23). This is an interesting contrast in function in that the traditionally understood role of PTH does not suggest any ability to initiate new bone formation. It is unclear at this time what signaling pathway is responsible for the anabolic actions of PTH, although numerous molecules have been implicated. These include IGF-I, TGF␤1, and TGF␤2 as well as osteoprotegrin and receptor-activated nuclear factor-␬B ligand (24 – 29). It has also been suggested that cAMP generation is linked to the anabolic effect of PTH because the carboxyl-terminal truncated analog, PTH (1–31), is highly anabolic to bone in the ovariectomized rat and yet does not appear to stimulate the phospholipase C-protein kinase C pathway (7, 30). Therefore, the translocation of the PTH1R to the nucleus provides a new and interesting aspect of PTH1R physiology and could provide insight into the duality of PTH action on bone metabolism based on the mode of administration. This research brings new insights into nuclear PTH1R and may have

Endocrinology, July 2006, 147(7):3326 –3332

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broader implications for our understanding of the various roles of GPCRs, especially those of the class 2 secretin-like family, in health and disease. Although the presence of PTH1R in the nucleus has been clearly established, the mechanism mediating the translocation as well as the precise spatial location within the nucleus was, until now, unclear. Here, using three independent lines of evidence, we identify the importins as a family of transport molecules that associate with the PTH1R. We also show that the expression of nuclear PTH1R changes based on the time point of the cell cycle. In addition, we show that PTH1R localizes to the nucleoplasm and does not associate with the nuclear membrane. Materials and Methods Cell culture ROS 17/2.8 rat osteosarcoma cells were a gift from Dr. S. J. Dixon (University of Western Ontario); MC3T3-E1 mouse nontransformed osteoblasts were a gift from Dr. M. Underhill (University of Western Ontario); and SaOS-2 human osteosarcoma cells were purchased from the American Type Culture Collection (Manassas, VA). All cell lines were cultured in ␣-MEM containing penicillin (10 U/ml), streptomycin (10 ␮g/ml), and 15% fetal calf serum (Invitrogen, Burlington, Ontario, Canada) in a 5% CO2 in air, humidified atmosphere. For immunofluorescence and deconvolution studies, cells were grown on 22-mm2 glass coverslips. Cells were grown to 60 – 80% confluence before fixation in 4% paraformaldehyde in PBS for 30 min. For the coimmunoprecipitation and affinity chromatography studies, cells were grown in T-75 flasks (Sarstedt, Montreal, Quebec, Canada) and whole-cell protein was extracted using a modified radioimmunoprecipitation buffer (RIPA) following a standard protocol (31). For the cell cycle studies, nuclear morphology was used to determine approximate stages of the cell cycle.

Characterization of antibodies Whole-cell protein was extracted from wild-type random cycling MC3T3-E1 cells using RIPA following a standard protocol (32). The total protein content was determined using BCA protein assay kit (Pierce, Rockford, IL), and samples were normalized accordingly. Whole protein samples were separated on an 8% SDS-PAGE gel, transferred to nitrocellulose, and probed with antibodies to either PTH1R (1:100) (Covance, Princeton, NJ; PRB-620P), importin ␣1 (1:100) (BD Biosciences, Franklin Lakes, NJ; 43020), importin ␣1 (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA; sc-6197), or importin ␤1 (1:100) (Santa Cruz Biotechnology; sc-1863) (Fig. 1). Mock immunoprecipitations were carried out in the absence of primary antibody following the standard protocol (Amersham Pharmacia Biotech, Piscataway, NJ). The immunoprecipitates were then separated on an 8% SDS-PAGE gel, transferred to nitrocellulose, and probed as above, using whole-cell lysate as a positive control. These Western blots were done in conjunction with the aforementioned blots for reference to a positive control lane. The Western blots were negative, indicating a stringent wash procedure and an antibodyspecific immunoprecipitation procedure (Fig. 1).

Immunofluorescence Immunofluorescence was performed as described (24). 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 rabbit antimouse PTH1R antibody raised against a peptide mapping near the amino terminus was used to visualize PTH1R (Covance; PRB-620P). A polyclonal mouse antihuman importin ␣1 (BD Biosciences; 43020) and an importin ␤ antibody (Santa Cruz Biotechnology; sc-1863) were used to visualize the importins. Detection of the PTH1R primary antibody was done using the Alexa Fluor 488 donkey antirabbit IgG (Molecular Probes, Eugene, OR; A21207). Detection of importin ␣1 was done using Alexa Fluor 594 donkey antimouse IgG (Molecular Probes; A21203), whereas detection of importin ␤1 was done using Alexa Fluor 594 donkey antigoat IgG (Molecular Probes;

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FIG. 1. Western blot analysis of PTH1R, importin ␣1- and ␤-specific antibodies. Whole-cell lysate from random cycling MC3T3-E1 cells was subjected to Western blot analysis (WB) as described in Materials and Methods. For the PTH1R antibody, a specific band was observed at 66.5 kDa; for both importin ␣1 antibodies used, a specific band was observed at 58 kDa; and for the importin ␤ antibody, a specific band was observed at 97 kDa. For all of the antibodies used, the resultant Western blots were free of background with the absence of nonspecific bands illustrating the specificity of the antibodies used. To demonstrate the specificity of the immunoprecipitations (IP), primary antibody was omitted and the procedure was followed as described in Materials and Methods. The resultant immunoprecipitates were subsequently analyzed by Western blot using the antibodies used in the immunoprecipitation experiments. For each antibody used, the Western blot showed no nonspecific background bands, showing the integrity of the immunoprecipitation procedure. A11058). All secondary antibodies were used 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 instructions from the manufacturer (Molecular Probes; D21490). Controls in which primary antibody was replaced with nonimmune serum were routinely used. Deconvoluted images were obtained using the SoftWoRx 2.50 software (Applied Precision, Mississauga, Ontario, Canada).

Immunoprecipitation and ligand blot analysis Immunoprecipitations were performed using the Amersham immunoprecipitation kit following the manufacturer’s recommended protocol. Random cycling wild-type MC3T3-E1 cells were used for the assay. Five micrograms of a goat polyclonal antibody raised against a peptide mapping at the carboxy terminus of either importin ␣1 (Santa Cruz Biotechnology; sc-6197) or importin ␤ (Santa Cruz Biotechnology; sc1863) was used for the immunoprecipitation of the importins, and a rabbit polyclonal antibody raised against a peptide mapping near the amino terminus was used for the immunoprecipitation of the PTH1R (Covance; PRB-620P). Whole-cell protein was extracted as previously described, total protein content was determined using the BCA protein assay kit (Pierce), and samples were normalized accordingly. Antibody was added, and samples were incubated for 1 h 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 RIPA, the immunocomplex was eluted from the Sepharose by heating at 95 C for 3 min in Laemmli buffer. The immunoprecipitates were then separated on a 3–13% gradient 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.

Affinity chromatography and Western blot analysis A PTH affinity column was generated using the UltraLink immobilization kit according to the manufacturer’s instructions (Pierce). Briefly, 50 ␮g PTH (1– 84) was dissolved in carbonate coupling buffer and added to the UltraLink Biosupport medium. The gel-PTH mixture was incubated for 1 h at room temperature with mixing. The mixture was then washed with PBS, and quenching buffer of 3.0 m ethanolamine was added and allowed to incubate for 2.5 h at room temperature. After the incubation, the column was then washed with PBS followed by wash solution (1.0 m NaCl). The column was stored at 4 C until use. Random


cycling wild-type ROS 17/2.8 cells were used for the assay. Whole-cell protein was extracted as previously described with total protein content determined using the BCA protein assay kit (Pierce), and samples were normalized accordingly before application to the column. Unbound protein was washed out of the column using PBS until the UV280 reached a value of less than 0.01. These washes ensured that all of the nonspecific protein was washed out of the column, ensuring the eluted protein was previously bound to the column. PTH1R and any associated proteins were eluted using 0.1 m glycine-HCl (pH 2.5) and collected into tubes containing 120 ␮l of 1m Tris-HCl (pH 8.8) per 1-ml fraction. Fractions were measured for protein content, and the peak, which corresponded to eluted PTH1R from the column, was collected for additional analysis. Purified PTH1R samples were separated on an 8% SDS-PAGE gel, transferred to nitrocellulose, and probed with antibodies to PTH1R (1:100) (BabCo; PRB-620P), importin ␣1 (1:100) (BD Biosciences; 43020), or importin ␤ (1:100) (Santa Cruz Biotechnology; sc-1863) to examine interactions between PTH1R and the importins.

Results

Results shown are representative of two or more osteoblast-like cell lines with three independently repeated experiments. Characterization of PTH1R, importin ␣1, and importin ␤ antibodies

To confirm the specificity of the antibodies used in this study, Western blots were performed using whole-cell lysate of MC3T3-E1 cells (Fig. 1). For each of the antibodies used, a single band was present on the Western blot corresponding to the expected size: PTH1R, 66.3 kDa; importin ␣1, 58 kDa; and importin ␤, 94 kDa. These results also demonstrate that the antibodies used specifically recognize the individual importins and that no cross-reactivity occurs. Also, to confirm the specificity of the immunoprecipitations, the procedure was carried out in the absence of primary antibody. The immunoprecipitates were then used in a Western blot, and no bands were visible. This demonstrates the specificity of the antibodies and rigor of the procedure. Immunofluorescent staining of osteoblast-like cells for PTH1R, importin ␣1, and importin ␤

Immunofluorescent staining of osteoblast-like cells reveals the presence of PTH1R, importin ␣1, and importin ␤ in both the cytoplasm and nucleus (Fig. 2). In Fig. 2, A and D, PTH1R can be observed in both the cytoplasm and the nucleus. The nuclear PTH1R seems to have different levels of fluorescence with some cells showing less PTH1R than others. This might suggest that nuclear PTH1R is not simply present or absent in the nucleus but can be differentially regulated. The importins, both ␣1 and ␤, can be observed to be localized in both the cytoplasm and the nucleus, similar to the PTH1R; however, because the importins are involved in the shuttling a variety of molecules to the nucleus, nuclear fluorescence is greater than that of the PTH1R (Fig. 2, B and E). The presence of the importins in these cell lines suggests that they are involved in the regulation of PTH1R translocation to the nucleus. However, the importins are involved in a wide variety of cellular processes involving nuclear translocation, so these results do not necessarily indicate an association. Additional biochemical evidence was used to substantiate the interaction. Nuclear localization of GPCRs studied thus far have revealed localization to either the nucleoplasm or



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FIG. 2. Immunofluorescence localization of PTH1R and importin ␣1 and ␤ in MC3T3-E1 cells. Cells were fixed and subjected to immunofluorescence staining with either a combination of PTH1R and importin ␣1 antibodies (A and B, respectively) or with a combination of PTH1R and importin ␤ antibodies (D and E, respectively). The cells were counterstained with DAPI showing the location of the nuclei for orientation (C and F). Both importin ␣1 and importin ␤ can be observed with PTH1R in both the cytoplasm and the nucleus of the cells. The presence of both importins with PTH1R suggests an involvement of the importins in the regulation of PTH1R nuclear transport. Cells were also analyzed by deconvolution microscopy, demonstrating that nuclear PTH1R was localized to the nucleoplasm and not associated with the nuclear membrane (G and H). Controls in which primary antibody was replaced with nonimmune serum are also shown (I). Scale bars, 10 ␮m.

nuclear membrane; in no instance has the GPCR been localized to both. Through the use of deconvolution microscopy, we observed PTH1R localized to the nucleoplasm of these osteoblast-like cells (Fig. 2, G and H). No PTH1R is observed in either cell line to be associated with the nuclear membrane. Representative images of the MC3T3-E1 cell line are presented in Fig. 2. Purification of the PTH1R/importin complex using affinity chromatography

To confirm the interaction between PTH1R and both importin ␣1 and importin ␤, the complex was purified out of total cell protein using affinity chromatography. PTH1R was purified out of total cell protein from wild-type random cycling osteoblast-like cells. Western blots showed that the purified PTH1R protein samples contained PTH1R at 66.3 kDa as well as both importin ␣1 at 58 kDa and importin ␤ at 94 kDa, indicating that the importins were associated with the PTH1R at the time of the purification (Fig. 3). Representative results from ROS 17/2.8 cells are shown in Fig. 3.

FIG. 3. Western blots of purified PTH1R from total cell protein of ROS 17/2.8 cells. Cell extract was purified on a PTH (1– 84)-linked chromatography column. Whole-cell extract was passed through the column, and nonspecific protein was washed from the column with several washes of PBS. PTH1R and the associated proteins were then released from the column, and samples were separated on an 8% SDSPAGE gel, transferred to nitrocellulose, and probed with antibodies to PTH1R or importin ␣1 or ␤. Wholecell extract was also run as a positive control. The Western blots show that the purified PTH1R samples contain both importin ␣ (58 kDa) and importin ␤ (94 kDa), indicating that the importins were attached to the PTH1R at the time of purification.

Coimmunoprecipitation of PTH1R with importin ␣1 and ␤

The interaction between PTH1R and both importin ␣2 and ␤1 was further examined by coimmunoprecipitation and ligand blot analysis. Total cell protein from random cycling wild-type osteoblast-like cells was immunoprecipitated with antibodies for PTH1R, importin ␣1, or importin ␤. After the immunoprecipitates were separated, blotted, and subsequently exposed to biotinylated PTH (1– 84), a single band was present on the gel at 66.3 kDa corresponding to the PTH1R (Fig. 4). The immunoprecipitations demonstrate the interaction between PTH1R and importin ␣1 as well as the interaction between PTH1R and importin ␤ because PTH1R will coimmunoprecipitate with both importin ␣1 and ␤. Representative results from MC3T3-E1 cells are shown in Fig. 4. Altered nuclear PTH1R in association with cell cycle progression

The examination of random cycling MC3T3-E1 cells revealed a distinct pattern of PTH1R nuclear localization during the cell cycle. During the early interphase (G0/G1, S, and

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FIG. 4. Ligand blot of PTH1R in cultured mouse osteoblast, MC3T3-E1 cells. Cells were homogenized and immunoprecipitated with antibodies against PTH1R, importin ␣1, or importin ␤. Immunoprecipitates (IP) were separated by 3–13% gradient SDS-PAGE, transferred to nitrocellulose, and probed with biotinylated PTH (1– 84) for the presence of PTH1R. Whole-cell extract was used as a positive control. The ligand blot picked up a specific band for the PTH1R at 66.3 kDa in each of the immunoprecipitates, indicating an interaction between PTH1R and both the importins. The control lane also contains three bands in addition to the PTH1R, which are most likely other biotin-binding proteins in the cell extract.

G2 phases) stage of the cell cycle, PTH1R immunoreactivity is abundant in the nucleus (Fig 5, A–C). As the cell cycle progresses into prophase (Fig. 5, D–F) and metaphase (Fig. 5., G–I), PTH1R nuclear immunoreactivity decreases to a point at which it almost becomes undetectable. During anaphase, PTH1R nuclear immunoreactivity remains decreased (Fig. 5, J–L). PTH1R nuclear immunoreactivity remains decreased until the late phases of telophase when nuclear PTH1R immunoreactivity returns to an expression pattern similar to that observed during interphase (Fig. 5, M–O). Discussion

Our results provide a number of novel insights into the signaling mechanisms of the PTH1R. We show for the first time that the PTH1R forms a complex with the transport regulatory proteins, importin ␣1 and importin ␤. In mammals, importin ␤ 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 (NPI-1) (19, 20). Among all proteins that are imported by the ␣/␤ system, several have been systems where all three subfamilies are involved in nuclear transport. For example, the nuclear import of mammalian circadian clock component (mCRY2), a mammalian cryptochrome, and Duplin, a ␤-catenin-binding protein, can be regulated by all three subfamilies (32, 33). In contrast, there are proteins regulated by specific subfamilies. This is the case for the small GTPase Ran (RCC1) where nuclear import is dependent on importin ␣3 and the other importins are not able to localize RCC1 to the nucleus (34). Another example is that of STAT1, latent cytoplasmic transcription factor, where nuclear import is regulated by the ␣5 subfamily (35). It is a possibility that either or both of the two remaining families of importin ␣ can regulate the nuclear translocation of the PTH1R. However, because of the limited availability of commercial antibodies for importin ␣3 and ␣5 suitable for immunoprecipitation, ␣1 was chosen for this


study. Studies are currently underway to determine what role the remaining two members play in the nuclear localization of the PTH1R. Specifically, does importin ␣1 alone regulate the nuclear localization as is the case for RCC1 or do all three importin ␣ molecules display functional redundancy as is the case for mCRY2. To our knowledge, this is the first evidence of the importins interacting with a GPCR. It will be interesting to see as the study of nuclear GPCRs advances whether all nuclear GPCRs rely on the importins or whether there are other mechanisms involved. Additional studies on this interaction will also focus on the specific area of the receptor that binds with importin ␣1 and ␤. Based on previous studies on other receptors that localize to the nucleus through the action of the importins, we would predict that the importins interact with an NLS of the PTH1R (19). We have identified a potential bipartite NLS on the Cterminal domain of the PTH1R, and studies are currently underway to confirm its involvement in importin-mediated nuclear localization (11). The examination of z-sections obtained through deconvolution microscopy suggests that nuclear PTH1R is restricted to the nucleoplasm and is not incorporated in the nuclear membrane. This report adds to the modest but growing list of nuclear GPCRs that are associated with the nucleoplasm including angiotension II AT1 receptor, receptors for apelin, bradykinin B2, and lysophosphatidate receptor 1 (14 –17). Not all GPCRs that traffic to the nucleus have an association with the nucleoplasm; some are localized to the nuclear envelope such as prostaglandin E2 receptors EP3 and EP4 and metabotropic glutamate receptor mGlu5 (13, 15). The fact that not all nuclear GPCRs share a common spatial distribution within the nucleus, i.e. all either associated with the nucleoplasm or with the nuclear membrane, suggests that they may have very distinct mechanisms of action in the nucleus as determined by spatial localization. For example, it has been suggested that the AT1 receptor may interact directly with DNA to regulate transcription of known AT1 signaling genes involved in the neuromodulatory actions of angiotensin II (12). Along the same lines of evidence, the lysophosphatidate receptor 1, which is also associated with the nucleoplasm, could be involved in the direct regulation of proinflammatory gene expression through genes such as iNOS and COX-2 (17). In contrast to these receptors associated with nucleoplasm are the receptors restricted to the nuclear membrane. Receptors such as mGlu5 and endothelin have been shown to increase nuclear Ca2⫹ concentration, which in turn can affect gene transcription (36, 37). With the observation that PTH1R is localized to the nucleoplasm, we would hypothesize that it would function similarly to other GPCRs associated with the nucleoplasm and that this function could involve the direct contact to DNA or other transcriptional regulating machinery. Also in support of this hypothesis is the observation that PTH1R is present in the nucleus only during certain stages of the cell cycle. The stages that have PTH1R in the nucleus are stages where the DNA is open to transcriptional activity, specifically interphase (G0/G1, S, and G2 phases) and telophase. During cell cycle stages where DNA is compact and transcriptional events are minimal such as prophase, metaphase, and anaphase, PTH1R nuclear localization is at a



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FIG. 5. Immunofluorescence localization of PTH1R in MC3T3-E1 during the cell cycle. Cells were fixed and subjected to immunofluorescence staining for PTH1R and counterstained with DAPI to visualize DNA. Nuclear morphology was used to determine approximate cell cycle stage. For all panels, PTH1R staining is in green (A, D, G, J, and M) and DAPI staining is blue (B, E, H, K, and N); overlaid images are also present (C, F, I, L, and O). During interphase (G0/G1, S, and G2 phases), PTH1R staining is very prominent in the nucleus (A–C). As the cell cycle progresses into prophase (as observed by the condensation of DNA), nuclear PTH1R immunoreactivity decreases in comparison with that observed during interphase (D–F). As chromosomes align during metaphase, nuclear PTH1R is almost undetectable in the cell (G–I). Nuclear PTH1R immunoreactivity remains decreased during anaphase (J–L) and then begins to reappear during telophase (M–O). For negative antibody controls, refer to Fig 2. Solid arrows indicate nuclei that are positive for PTH1R. Open arrows indicate nuclei that show less nuclear PTH1R. Scale bar, 10 ␮m.

minimum. These results, in conjunction with the association of PTH1R with the nucleoplasm, strongly suggest a role for nuclear PTH1R to directly interact with DNA thus directly regulating genes, potentially those involved in bone metabolism. Additional research into the nuclear function of PTH1R is currently underway, particularly the PTH1R interaction with chromatin. In addition to the spatial distribution of the nuclear GPCR leading to a distinct mechanism of action from membrane GPCRs, there is also evidence for a common mechanism for nuclear GPCRs in that components of the traditional GPCR signaling pathways are present in the nucleus. These classical GPCR effector molecules include heterotrimeric G proteins (Gi␣1, Gi␣2, and Gs␣), adenylate cyclase, phospholipase A2, and phospholipase C (38 – 41). This evidence would suggest nuclear GPCRs can continue to signal in the nucleus in ways similar to that of membranebound forms. However, this mechanism seems unlikely for the PTH1R because we find no PTH1R immunoreactivity in the nuclear membrane. Nuclear trafficking of GPCRs is known to occur both in the presence and absence of ligand. For example, AT1 receptor has been shown to have both ligand-dependent and ligandindependent nuclear localizing capabilities (12, 14). Studies

to determine whether the nuclear localization of the PTH1R is dependent on the presence of ligand or whether it can occur through a ligand-independent mechanism are currently underway. Ligand dependence has the potential to be a complicated mechanism because the PTH1R has two ligands to which it responds, PTH and PTHrP. In addition, these ligands have several bioactive peptides, namely PTH (1–31 and 1–34) and PTHrP (7–34), in addition to others that also stimulate the PTH1R resulting in the activation of distinct signaling cascades (6, 7, 22). The determination that PTH1R forms a complex with the importin family of transport molecules was a necessary first step in understanding the trafficking of the PTH1R to the nucleus. In addition, the observation that nuclear PTH1R is associated with the nucleoplasm and is present during phases of the cell cycle when chromatin is accessible provides additional information on the potential functional activity of the PTH1R. Future studies of PTH1R nuclear import in conjunction with studies examining intermittent PTH therapy may provide insight into how one molecule can exert such apparently different responses depending on its mode of administration. The study of PTH1R nuclear localization can provide valuable insight into GPCR nuclear function in gen-

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eral, thus potentially expanding the understanding of the roles GPCRs play in health and disease.

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Acknowledgments

19.

We gratefully acknowledge Dr. Bing Siang Gan for use of the deconvolution microscope facility and Becky McGirr for technical assistance.

20.

Received November 7, 2005. Accepted March 23, 2006. Address all correspondence and requests for reprints to: Patricia H. Watson, Ph.D., Room G443, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. E-mail: pwatson@ lhrionhealth.ca. Funding for this project was provided by the Canadian Institute of Health Research Grant MOP-64228. B.P., L.F., A.H., and P.W. have nothing to declare.

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