Proparathyroid Hormone-related Protein Is Associated with the Chaperone Protein BiP and Undergoes Proteasome-mediated Degradation*

Parathyroid hormone-related peptide (PTHrP) is an important causal factor for hypercalcemia associated with malignancy. In addition to the endocrine functions attributed to secretory forms of the peptide, PTHrP also plays a local role as a mediator of cellular growth and differentiation presumably at least in part through intracellular pathways. In studying the post-translational regulation of PTHrP, we observed that PTHrP was conjugated to multiple ubiquitin moieties. We report here that the proteasome is responsible for the degradation of the endoplasmic reticulum-associated precursor, pro-PTHrP. Cells expressing prepro-PTHrP and exposed to lactacystin accumulate pro-PTHrP assessed by anti-pro specific antibodies. Brefeldin A-treated cells also accumulate pro-PTHrP suggesting that degradation does not occur in the endoplasmic reticulum (ER) lumen. Subcellular fractionation of both lactacystin and brefeldin A-treated cells indicated that accumulated pro-PTHrP resides in microsomal fractions with a portion of the protein exposed to the cytosolic side of the ER membrane as assessed by protease protection experiments. Immunoprecipitation and Western blot analysis identified pro-PTHrP in association with the ER molecular chaperone protein BiP. We conclude that pro-PTHrP from the ER can gain access to the cytoplasmic side of the ER membrane where it can undergo ubiquitination and degradation by the proteasome.

Parathyroid hormone-related protein (PTHrP) 1 is a secretory factor that is responsible for hypercalcemia in patients with cancer (1). There is amino acid homology within the first 13 amino acids of the parathyroid hormone (PTH). This limited homology is sufficient for PTHrP to bind to a common PTH/ PTHrP receptor and to share many of the biological properties of PTH (2,3). Unlike PTH, PTHrP is widely expressed in a variety of fetal and adult tissues where it is thought to regulate cellular proliferation and differentiation (4 -9). In addition, PTHrP plays a critical role in normal skeletal development as has recently been emphasized by gene deletion experiments in mice (10,11) and overexpression of PTHrP targeted to chondrocytes (12). Thus, PTHrP is proposed to enhance the proliferation of chondrocytes and delay their terminal differentiation (13). This action is most likely in large part because of the secretion of PTHrP from normal proliferating and maturing chondrocytes. PTHrP also inhibits apoptosis in vitro (14) and in vivo (15). The ability of overexpressed PTHrP to inhibit apoptosis was shown to depend on nuclear localization of PTHrP (14) and induction of the apoptotic inhibitor, Bcl-2 (12).
Taken together, these studies suggest an intracellular function for PTHrP distinct from the classical signal transduction cascades linked to the PTH/PTHrP receptor. PTHrP is synthesized as a prepro protein that encodes a mature 141-amino acid protein (in the rat) that is post-translationally cleaved to generate the bioactive peptide 1-36 as well as other poorly characterized fragments. While studying the post-translational regulation of PTHrP, we showed that full-length endogenously overexpressed PTHrP was degraded by the ubiquitin-dependent proteolytic system (16). The ubiquitin proteolytic pathway is responsible for the degradation of misfolded or aberrant polypeptides in the cytosol and nucleus (17). In addition to aberrant proteins, many normal short-lived proteins are also degraded, indicating that this system plays a role in regulated protein degradation (18). Ubiquitin-dependent degradation is a reaction cascade that involves the conjugation of multiple ubiquitin moieties to a target protein through the action of three enzymes (E1, E2, and E3) followed by degradation through a multicatalytic protease, known as the 26 S proteasome. In several reports, however, proteasomal degradation of proteins was shown not to be dependent on ubiquitin conjugation (19 -23).
The degradation of proteins that are destined to be secreted or plasma membrane-bound can occur in the endoplasmic reticulum (ER). ER-associated degradation is proposed to function, at least in part, as a quality control mechanism to ensure that only correctly folded, processed, and completely assembled proteins exit this compartment for further transport through the secretory pathway (24). However, the biochemical mechanisms and proteases that mediate this ER degradation remain unclear.
Recently, several studies have suggested a role for the proteasome in the degradation of ER proteins (25). Whereas proteasomes have never been localized within the lumen of the ER, they have been reported to stud the cytosolic face of the ER membrane (26). Thus, ER retained forms of mutant integral membrane proteins, such as cystic fibrosis transmembrane regulator (27, 28) and 3-hydroxy-3-methylglutaryl-CoA reduc-tase (20,29), that are most likely degraded by the proteasome on the cytosolic side of the ER membrane. Proteasomal degradation of mutant ER lumenal proteins, such as prepro ␣-factor (19), ␣ 1 -1 antitrypsin (21,23), and carboxypeptidase Y (30,31), may also occur. Finally, we previously reported proteasomal degradation of PTHrP, a nonmutant secretory protein (16), and others have recently reported proteasomal degradation of apolipoprotein B, another nonmutant protein (32)(33)(34).
It has been proposed that lumenal proteins may be degraded by the proteasome after reverse transport from the ER back into the cytoplasm. This has been demonstrated for both soluble (19,23) and membrane-bound proteins (35,36). Reverse transport to the cytoplasm likely occurs through the Sec61containing translocon (30,37,38) and may involve the lumenal chaperone, BiP. Alternatively, co-translational degradation by the cytosolic proteasome has also been described (32,33).
In this study we have extended our previous observation that PTHrP can be ubiquitinated (16) and can be degraded by the proteasome. We thus assessed whether ubiquitination is a prerequisite for proteasome-mediated degradation of PTHrP, whether the PTHrP form serving as a substrate for proteasome degradation has already entered the ER, whether the degradation occurs in the cytoplasm or the ER, and whether PTHrP in the ER is associated with a chaperone.

MATERIALS AND METHODS
Materials-Lactacystin was purchased from E. J. Corey (Harvard University, Cambridge, MA) and prepared as a 10 mM (1000ϫ) stock solution in water. Brefeldin A was obtained from Sigma and dissolved in methanol. The protease inhibitors, aprotinin and leupeptin, were obtained from Boehringer Mannheim. Phenylmethylsulfonyl fluoride and N-ethylmaleimide were from Sigma. The anti-human PTHrP-(38 -64) monoclonal antibody-1 was from Oncogene Research Products (Cambridge, MA), and the rat anti-grp78 (BiP) monoclonal antibody was purchased from StressGen Biotechnologies (Victoria, Canada). Polyclonal antiserum to pro-PTHrP was raised in a rabbit using a synthetic peptide corresponding to amino acids Ϫ12 to ϩ1 conjugated to methylated bovine serum albumin. Secondary horseradish peroxidaseconjugated anti-mouse and anti-rabbit antibodies were purchased from Bio-Rad.
Cells and Cell Culture-The Chinese hamster lung E36 and ts20 cell lines were kindly provided by William Dunn (University of Florida Health Science Center). The cells were cultured in Dulbecco's modified Eagle's medium (plus 4.5 g/liter glucose; Life Technologies, Inc.) and supplemented with 10% fetal calf serum and antibiotic-antimycotic (Life Technologies, Inc.) in a humidified atmosphere at 30°C with 5% CO 2 . Stable transfection of prepro-PTHrP or a leaderless form of PTHrP (⌬P) in E36 and ts20 cells was with Lipofectin (Life Technologies, Inc.) and the expression plasmids CMV PTHrP-His 6 and CMV ⌬P-His 6 (16). As a control, cells were transfected with pRC/CMV vector alone. Fortyeight hours after transfection, cells were treated with 1 mg/ml G418 (Life Technologies, Inc.) for approximately 1 week. Clonal colonies were isolated, expanded, and maintained in medium containing 0.4 mg/ml G418. Clonal cell lines were screened for PTHrP production by Northern blotting.
Heat Treatment and Inhibitor Treatment of Cells-For heat shock experiments, E36 and ts20 cells stably expressing prepro-PTHrP were incubated for 18 h at either 30 or 40°C in T25 flasks with the caps sealed shut. Cell extracts were prepared by first rinsing the cells with cold phosphate-buffered saline and then lysing the cells in 8 M urea, 0.1 M NaPO 4 , and 0.01 M Tris-Cl, pH 7.4. Cell lysates were centrifuged at 14,000 ϫ g for 10 min at 4°C, and the supernatants were kept at Ϫ70°C. Protein concentrations were determined by the Bradford assay (Bio-Rad), and equal amounts of total cell protein were analyzed by immunoblotting.
For inhibitor-treated experiments, cells were plated on Petri dishes. The next day, fresh medium was added that contained either 10 M lactacystin or 10 g/ml brefeldin A, or both. Cells were treated for 18 h, and cell extracts were prepared as described above or subjected to subcellular fractionation. Subcellular Fractionation-Subcellular fractionation was essentially as described (36). Briefly, cells (ϳ10 7 ) were rinsed in phosphate-buffered saline, then in homogenization buffer (0.25 M sucrose, 10 mM triethanolamine, 10 mM acetic acid, and 1 mM EDTA, pH 7.4) containing a protease inhibitor mixture of leupeptin (10 g/ml), aprotinin (10 g/ml), phenylmethylsulfonyl fluoride (1 mM), and N-ethylmaleimide (5 mM), and then scraped and resuspended in 800 l of homogenization buffer. Cells were homogenized on ice using a Dounce homogenizer (30 strokes) with a tight fitting pestle (Thomas, pestle type A). The homogenate was spun at 1000 ϫ g for 10 min, which yielded the 1000 ϫ g pellet. The supernatant was spun at 10,000 ϫ g for 30 min, which yielded the 10,000 ϫ g pellet. Finally, the supernatant was subjected to 100,000 ϫ g centrifugation for 60 min, providing the 100,000 ϫ g pellet and the 100,000 ϫ g supernatant (cytosol). Pellets were resuspended in homogenization buffer, and all fractions were analyzed by immunoblotting.
Protease protection experiments were performed on fresh aliquots of the 100,000 ϫ g pellets resuspended in homogenization buffer. Aliquots were incubated for 30 min on ice with trypsin (100 g/ml) in the presence or absence of 0.1% Triton X-100. The reaction was stopped by the addition of phenylmethylsulfonyl fluoride (2 mM) for 10 min on ice. The samples were analyzed by SDS-PAGE and immunoblotting.
Immunoprecipitation-Control and inhibitor-treated cells were lysed on ice for 10 min in lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40) plus the protease inhibitor mixture as described above. Cell lysates were spun at 1000 ϫ g for 10 min. For ATP-depleted extracts, 5 units/ml hexokinase and 10 mM 2-deoxyglucose were added fresh to the lysis buffer. In the case where ATP was present, the lysis buffer was supplemented with 1 mM ATP. 50 g of total cell protein was incubated with either 1 g of anti-BiP antibody or 1 g of anti-PTHrP antibody overnight at 4°C. Following a 1-h incubation with protein A-Sepharose, the beads were washed 3 times with buffer containing 50 mM Tris-Cl (pH 7.5), 400 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate and once with 20 mM Tris-Cl (pH 7.5). The immunoprecipitates were resolved on SDS-PAGE and immunoblotted with the antibodies as described above.

RESULTS
Lactacystin Causes Accumulation of PTHrP-We have previously shown that the proteasome inhibitor, MG-132, caused the accumulation of ubiquitinated prepro-PTHrP when PTHrP was translated in vitro in reticulocyte lysate, expressed in transiently transfected COS-7 cells, and expressed in stably transfected E36 Chinese hamster lung fibroblasts (16). Here, we assess proteasome-mediated degradation of PTHrP using the highly specific and irreversible proteasome inhibitor, lactacystin (40). Lactacystin at 10 M induced the accumulation of PTHrP in stably transfected E36 cells as assessed by immunoblotting total cell lysates with a monoclonal anti-PTHrP antibody (Fig. 1, compare lane 4 to lane 3). PTHrP expressed in E36 cells but lacked the secretory prepro leader sequence (⌬P) and thus expressed in the cytosol, which also accumulated in the presence of lactacystin (Fig. 1, compare lane 2 to lane 1). As we have previously shown, anti-PTHrP-(38 -64) monoclonal antibody cannot identify the higher molecular weight ubiquitin-PTHrP conjugates (16). Western blot analysis of prepro-PTHrP revealed a doublet (Fig. 1, lanes 3 and 4). Based on the slower migration of this doublet compared with ⌬P, we propose that these two bands represent precursor forms of mature PTHrP containing pre and/or pro sequences (see Fig. 3). The faster migrating bands appear to represent carboxyl-terminal cleavages or partially degraded intermediates.
Ubiquitin Conjugation to PTHrP Is a Prerequisite for Its Degradation-To address whether ubiquitin conjugation to PTHrP is important for its degradation, we expressed prepro-PTHrP in ts20 cells, which are derived from E36 cells and are temperature-sensitive for the ubiquitin-conjugating enzyme, E1 (39). Thus, at the permissive temperature of 30°C, the E1 enzyme is active whereas at the nonpermissive temperature of 40°C, E1 is inactivated and substrate proteins accumulate because they cannot be ubiquitinated. Prepro-PTHrP degradation was investigated in E36 and ts20 cells at both 30 and 40°C. Following incubation at the respective temperatures for 18 h, cell lysates were analyzed by Western blotting of equal amounts of total protein using a monoclonal antibody against PTHrP. As shown in Fig. 2, ts20 cells accumulate PTHrP at 40°C. This result confirms that ubiquitin conjugation to PTHrP is a necessary prerequisite for its degradation.
Effect of Brefeldin A-The in vivo finding that a specific inhibitor of the proteasome limited the degradation of prepro-PTHrP ( Fig. 1), along with the result that an E1 mutant cell line was deficient in the degradation of a wild-type lumenal secretory protein, prepro-PTHrP (Fig. 2), demonstrates that the ubiquitin-dependent proteolytic system is involved in the degradation of this mammalian secretory hormone. To determine the subcellular site for proteasomal degradation of PTHrP, we tested the effects of brefeldin A (BFA), a fungal metabolite that is able to specifically block protein transport from the ER to the Golgi and has been used extensively to characterize the subcellular site of ER-associated degradation (41). Treatment of E36 cells expressing prepro-PTHrP with 10 g/ml BFA for 18 h resulted in a slight accumulation of PTHrP immunoreactive protein when compared with control-treated cells (Fig. 3A, compare lane 5 to lane 3). BFA treatment also inhibited the secretion of PTHrP in the medium detected by radioimmunoassay by 65%, consistent with impaired transport of protein out of the ER. However, although the intracellular accumulation of PTHrP with BFA was not as great as with lactacystin alone (Fig. 3A, lane 4), the fact that PTHrP accumulated in the presence of BFA suggests that PTHrP degradation does not occur by proteases active in the lumen of the ER.
In the presence of both BFA and lactacystin there is a much greater accumulation of PTHrP than with each inhibitor alone (Fig. 3A, lane 6). This additive effect suggests that when PTHrP is prevented by BFA from leaving the ER, it becomes more available for proteasomal degradation.
Characterization of PTHrP Isoforms That Are Stabilized by Lactacystin-Western blot analysis of inhibitor-induced accumulation of prepro-PTHrP revealed two bands (Figs. 1-3A). To confirm that these bands were precursor forms of PTHrP, we immunoblotted inhibitor-treated samples with a polyclonal antiserum raised against a peptide encoding the pro region (Ϫ12 to ϩ1). The antiserum is specific as it does not recognize PTHrP that is lacking the prepro leader sequence (⌬P; Fig. 3B, lane 2). This antiserum did recognize two bands, i.e. prepro-PTHrP and pro-PTHrP, which both contain pro sequences (Fig. 3B, lanes  3-6). The faster migrating band of the doublet must be pro-PTHrP, but the slower migrating band may be prepro-PTHrP or a post-translationally modified form of pro-PTHrP. These results therefore show that the pro form of PTHrP is accumulated in the presence of lactacystin (Fig. 3B, lane 4 versus lane  3), indicating that pro-PTHrP serves as a substrate for proteasomal degradation.
Subcellular Fractionation and Protease Protection Experiments-To search for and characterize the subcellular localization of PTHrP degradation, we fractionated control and lactacystin-treated cells into soluble and particulate fractions by differential centrifugation. PTHrP localized almost exclusively to the particulate fractions, and lactacystin increased PTHrP accumulation in these fractions (Fig. 4A). PTHrP in the pellet of the 1000 ϫ g spin appeared to represent retention of the protein in incompletely lysed cells (Fig. 4A, lanes 2 and 3). PTHrP in the 10,000 ϫ g pellet is consistent with its reported nuclear localization (14), although the 10,000 ϫ g pellet also contains microsomal vesicles (36). PTHrP accumulation in both the 10,000 and 100,000 ϫ g pellets is therefore compatible with its association with microsomal vesicles.
The species of PTHrP accumulated in the presence of lactacystin in microsomal fractions also reacted with the pro antiserum (Fig. 4B, lane 1), suggesting that pro-PTHrP must have entered the ER. Furthermore, a faint but distinct band of pro-PTHrP was also observed in the cytosolic fraction (Fig. 4B,  lane 4).
We then used protease protection assays to determine the site of pro-PTHrP accumulation associated with microsomal vesicles. Treatment of the microsomal membrane fraction with trypsin resulted in a substantial disappearance of pro-PTHrP, whereas the heat shock protein 70 analogue, BiP, which is localized in the lumen of the ER, remained resistant to trypsin treatment (Fig. 4C). The additional treatment of the membrane fraction with Triton X-100 rendered pro-PTHrP fully and BiP partially susceptible to trypsin digestion (Fig. 4C). The BiP control suggests that the vesicles were intact during the trypsin treatment. Therefore, a significant portion of pro-PTHrP did not reside within the lumen of the ER but was adherent to the cytosolic face of the ER membrane. Consequently, a portion of pro-PTHrP became accessible to the cytosolic face of the ER membrane, enabling it to be recognized for proteasomal degradation.
Pro-PTHrP Interacts with the Chaperone Protein BiP-Because chaperonins play important roles in many ER-associated functions, we tested whether the chaperone protein, BiP, could play a role as an intermediate in the ubiquitin-dependent proteolytic process. Because BiP has been well characterized to retain partially folded, misfolded, and unassembled proteins in the ER, it is proposed that it participates in the quality control of proteins in the ER (42,43). BiP is an ATP-binding protein and dissociates from protein substrates to which it is bound in an ATP-dependent manner (44,45). To test for an interaction between PTHrP and BiP, we immunoprecipitated either BiP (Fig. 5A) or PTHrP (Fig. 5B) from control or lactacystin-treated cells in the presence or absence of ATP. The immunoprecipitates were then subjected to Western blot analysis with either anti-PTHrP (Fig. 5A) or anti-BiP (Fig. 5B) antibody. In Fig. 5A, PTHrP was detected in BiP immunoprecipitates (lane 2 versus lane 1), and this reaction was augmented by lactacystin (lane 3 versus lane 2). Lane 6 represents total PTHrP in lactacystintreated cells. Based on the comparisons of lanes 3 and 6, at least 60% of PTHrP that can be degraded by the proteasome is bound to BiP. In the presence of ATP, the BiP-PTHrP complex was dissociated, and therefore no PTHrP immunoreactivity was observed in the BiP immunoprecipitate (lanes 4 and 5). No  3 versus lane 2). In the presence of ATP, the PTHrP-BiP complex was dissociated and therefore no BiP immunoreactivity was observed in the PTHrP immunoprecipitate (lanes 4 and 5). No BiP immunoreactivity was detected in the vector control (lane 1). Fig. 5 shows that BiP interacts with a component of PTHrP in an ATP-dependent manner, the PTHrP species co-migrates with pro-PTHrP, and proteasome inhibition can augment the complex. DISCUSSION In the present study, we have used prepro-PTHrP-transfected cells to study the proteasomal degradation of a secretory peptide. First, we confirmed that degradation was occurring by the proteasome by using the highly specific proteasomal inhibitor, lactacystin (Fig. 1). Proteasomal degradation of secretory proteins has been shown to be ubiquitin-dependent (28,31,34) and -independent (20,23). Here we show that proteasomal degradation of prepro-PTHrP was ubiquitin-dependent by using a cell line that is temperature sensitive for the activity of the ubiquitin-activating enzyme, E1 (Fig. 2). In our studies, both prepro-PTHrP and the leaderless protein (⌬P) were degraded by the proteasome (Figs. 1 and 3). Consequently, it seems likely that the degradation signal resides in the mature region of the protein and not in the leader sequence.
In contrast to the many reports that observed proteasomemediated degradation of misfolded and mutant ER-associated proteins, we show that this pathway is used also for a wild-type secretory protein, PTHrP. Another wild-type secretory protein, apolipoprotein B, has also been recently shown to be degraded by this pathway (33,34). We have excluded the possibility that ubiquitin-dependent proteasomal degradation of PTHrP is a consequence of mistargeting of an overproduced polypeptide to the cytoplasm because we identified pro-PTHrP to be a sub-strate for the proteasome (Fig. 3). Consequently, this substrate for proteasomal degradation must have entered the ER and been processed by signal peptidase. In addition, subcellular fractionation revealed that accumulated pro-PTHrP associated with microsomes consistent with the localization for a secretory protein (Fig. 4, A and B). However, BFA, which inhibits ER to Golgi transport of proteins, caused an accumulation of pro-PTHrP (Fig. 3), indicating that PTHrP degradation probably did not occur via proteases active in the lumen of the ER. In addition, we observed that a substantial amount of lactacystinstabilized microsomal pro-PTHrP was sensitive to tryptic digestion, indicating that pro-PTHrP became exposed to the cytosolic side of the ER membrane (Fig. 4C). Finally, a small but definite amount of pro-PTHrP was identified in the cytosolic fraction after lactacystin treatment. Therefore, we propose that signal-cleaved pro-PTHrP is reverse transported out of the ER to the cytoplasm where it is degraded by the proteasome in association with membranes.
Our work is consistent with previous reports of proteasomemediated degradation of lumenal ER proteins. It has been reported that proteins in yeast (19,23,31) and in mammalian cells (35,36) can be reverse transported out of the ER back into the cytosol and degraded by the proteasome. It is currently believed that the export of a misfolded or mutant secretory protein from the ER to the cytoplasm occurs through the translocation channel formed by the Sec61 protein complex (38). Recently, the yeast Sec61p protein was shown to play a direct role in reverse protein transport (30,37). These findings implicate the existing protein translocating machinery for both import and export of proteins across the ER membrane. At least one function of reverse transport may thus be to eliminate secretory proteins that fail to satisfy the ER quality control process. Nevertheless, our studies indicate that a native secreted protein, PTHrP, can also be retrotranslocated from the ER and that this interesting pathway is not used simply as a quality control mechanism for eliminating abnormal proteins.
Chaperonins are important for several ER functions and have been implicated in ER-associated degradation. Thus, calnexin appears involved in the ER degradation of mutant ␣ 1antitrypsin and prepro ␣ factor (19,21). Calnexin, however, has been shown to interact exclusively with N-linked glycosylated proteins (46), and because PTHrP does not undergo such a modification it seems unlikely to interact with this peptide. One of the most abundant and best characterized of the ER chaperones, BiP, has been shown to play a critical role as a chaperone in protein folding, in the regulation of ER degradation by retaining immature and misfolded proteins in the ER, and in the retrieval of incompletely folded proteins from the Golgi to the ER (43). BiP interacts with numerous proteins during early stages of folding to keep them in a translocationcompetent form. Thus, BiP has been shown to be required for both co-translational (47) and post-translational protein translocation (48,49). This involves an interaction between BiP and Sec63p, an integral membrane protein that exists in a multisubunit complex containing Sec61p, Sec62p, Sec71p, and Sec72p (47,48,50). Furthermore, it has recently been demonstrated that BiP can also mediate retrograde transport of a protein from the ER to the cytosol for proteasome degradation (30). Having demonstrated proteasome-mediated degradation of PTHrP, we therefore assessed whether PTHrP would bind BiP.
In our study pro-PTHrP was indeed found to bind BiP. Anti-BiP antibodies immunoprecipitated BiP in association with PTHrP from ATP-depleted extracts (Fig. 5A), and in the reciprocal experiment anti-PTHrP antibodies immunoprecipitated PTHrP in a complex with BiP (Fig. 5B). Both immunoprecipi- tates were dissociated in vitro on incubation with ATP. The accumulation of protein caused by lactacystin reflects pro-PTHrP levels, because lactacystin does not accumulate BiP, as assessed by direct Western blotting of lactacystin-treated extracts with anti-BiP antibodies (data not shown). Therefore, although BiP may conceivably play other roles in shepherding PTHrP through the secretory pathway, we suggest, in analogy with its function vis à vis other proteins, that BiP functions as an intermediate in providing access for pro-PTHrP from the ER to the cytoplasmic side of the ER membrane for proteasomal degradation.
We attempted to investigate the timing of the interaction of PTHrP with BiP and the degradation of the protein using pulse-labeled cells and subsequent immunoprecipitation with anti-PTHrP and anti-BiP antibodies from cell lysate prepared at various time points during the chase. Although theoretically straightforward, we found this experiment to be fraught with technical difficulties. As PTHrP contains only one methionine, the initiator methionine, which is subsequently lost during cleavage of the signal peptide, labeling with [ 35 S]methionine was not feasible. Incorporation of [ 3 H]leucine after a 10-min pulse was too low to detect immunoprecipitated bands. In addition, the monoclonal antibody to PTHrP, although quite specific for Western blotting, was not highly efficient at immunoprecipitation. Thus these limitations precluded performing pulse-chase studies.
Our data indicate that pro-PTHrP may not have been fully translocated to the lumenal side of the translocon in light of the fact that it was sensitive to digestion by exogenously added trypsin. Thus it may not have been completely dissociated from the Sec61 channel prior to interaction with BiP. It has been postulated that BiP, together with Sec63p and ATP, functions as a molecular motor to reel a precursor out of the pore and into the lumen of the ER (48). It is possible that prolonged association of the amino-terminal region of PTHrP with BiP can slow the co-translational movement of nascent PTHrP through the translocon, disrupt the ribosome-translocon junction, and permit cytoplasmic exposure of the carboxyl region of the nascent PTHrP molecule for ubiquitination and degradation by the proteasome. A similar mechanism has been postulated for apolipoprotein B (33). Alternatively, the ATP-mediated release from BiP of PTHrP may effect its translocation back to the cytoplasm through the Sec61 membrane channel. Reverse transport may require prolonged association with BiP, which mediates its return to the cytoplasmic side of the ER membrane where it can be, in part or in whole, ubiquitinated by ERassociated ubiquitin-conjugating enzymes and degraded by the proteasome.
In addition to its role as a secreted peptide in modulating calcium homeostasis in cancer, PTHrP plays a critical role in normal cell growth and differentiation and can localize to the nucleus, which may be the site of at least some of its actions. Regulated ER translocation to the cytoplasm and degradation by the proteasome may therefore be the cellular mechanism that dictates the amount of intracellular versus secreted PTHrP that is required for its diverse biological functions.