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Endocrinology Vol. 142, No. 2 694-703
Copyright © 2001 by The Endocrine Society

ARTICLES

Nuclear Forms of Parathyroid Hormone-Related Peptide Are Translated from Non-AUG Start Sites Downstream from the Initiator Methionine1

M. T. Audrey Nguyen2, Bin He and Andrew C. Karaplis3

Division of Endocrinology, Department of Medicine, Sir Mortimer B. Davis-Jewish General Hospital and Lady Davis Institute for Medical Research, McGill University, Montréal, Canada H3T 1E2

Address all correspondence and requests for reprints to: Andrew C. Karaplis, Lady Davis Institute for Medical Research, 3755 Côte-Ste-Catherine Road, Montréal, Québec, Canada, H3T 1E2. E-mail: akarapli{at}ldi.jgh.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH-related peptide (PTHrP), normally a secreted protein, exerts at least part of its biological functions via intracrine actions at the level of the cell nucleus/nucleolus. To gain insight into the mechanism whereby this peptide accesses the cytosol for subsequent nuclear import, we characterized its nuclear forms. In transfected COS-1 cells, three nuclear PTHrP species were produced, which were larger than the mature form of the protein but smaller than prepro-PTHrP and comprised both the amino- and carboxyl-terminal regions of the peptide. This suggested that nuclear PTHrP proteins contain part of the prepropeptide and likely arise from alternate initiation of translation at downstream non-AUG codons within the signal sequence. Transient expression of two PTHrP forms, one in which the unique initiator ATG was mutated to a noninitiator ATC codon, and another encompassing an engineered N-linked glycosylation site, generated peptides of size comparable to nuclear PTHrP proteins. These were inefficiently targeted to the ER, bypassed ER transit, and exclusively localized to nucleoli. Using a polyclonal antiserum against the pro-region of PTHrP, nuclear PTHrP species were shown to harbor the propeptide sequence. Hence, our data argue that nuclear PTHrP forms result from translation initiated at alternate, internal codons. This may constitute the first example of translation initiation at downstream non-AUG codons in a mammalian protein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH-RELATED PEPTIDE (PTHrP) was initially characterized as a secreted, tumor-derived agent responsible for hypercalcemia in patients with malignancy. It is structurally related to PTH, the main regulator of calcium homeostasis, particularly in its first 13 amino acids (1). In addition to its production by tumors, PTHrP is widely expressed, albeit at low basal levels, in a variety of embryonic and adult tissues.

The biological roles attributed to PTHrP are numerous and diverse, including skeletal and mammary gland development, modeling of the vasculature, blood pressure regulation, placental calcium transport, and lactation (1, 2). For the most part, PTHrP elicits its autocrine/paracrine actions by binding via its N-terminal region [amino acids (aa) 1–34] to the G protein-coupled, cell-surface type 1 PTH or PTH/PTHrP receptor, and triggering downstream signal transduction cascades (3). Moreover, we and others have shown that PTHrP could promote some of its functions independently of amino-terminal activity and cell-surface receptor interaction, specifically by localizing to the cell nucleus/nucleolus (4, 5, 6, 7). This translocation requires the functional, mid-region, bipartite nuclear localization sequence (NLS) comprising aa 87–107 of the mature protein (4). Several cellular effects have been associated with PTHrP nuclear import in vitro, such as enhanced survival of serum-deprived CFK2 chondrocytes undergoing apoptosis (4), modulation of chondrocyte proliferation, and differentiation (5), G1 cell cycle arrest in HaCaT keratinocytes (6), and cell cycle activation in cultured A-10 vascular smooth muscle cells (7). Endogenous PTHrP has also been localized in situ to the dense fibrillar component of nucleoli (4), a subnuclear structure involved in ribosomal (r)RNA gene transcription. While it is not yet known how nuclear/nucleolar PTHrP brings about its intracrine actions, the peptide was recently reported to bind homopolymeric and total cellular RNAs (8), suggesting its potential involvement in regulating RNA metabolism.

A number of secreted growth factors have previously been observed to localize to nuclei and nucleoli (9, 10, 11) to mediate specific biological functions. In most cases, they enter the nucleus from the cytoplasmic compartment, following internalization of ligand-receptor complexes. In other instances, alternative splicing and alternate translation initiation generate cytoplasmic forms of secretory proteins that are directed to the nucleus (11).

Import of proteins in the nuclear compartment is a selective and timed process that necessitates the interplay between the NLS and components of the nuclear pore complex (12). Reconstituted import assays indicate that, in contrast to most NLS-containing proteins, PTHrP is recognized exclusively by importin ß and not importin {alpha}, which, together with Ran-GTP, mediates PTHrP nuclear transport (13). The timing of this event is linked to the cell cycle (6, 7) and regulated by phosphorylation. PTHrP is a substrate for p34cdc2 kinase in vitro (14), and mutations of residue Thr85 in PTHrP have been associated with alterations in the cytosolic/nuclear distribution of the peptide (15).

In this study, we have investigated the mechanism whereby PTHrP, normally a secreted protein, gains access to the cytosol for subsequent targeting to the nucleus/nucleolus. We report that alternate initiation of translation constitutes one pathway for relocating PTHrP to the cytosolic compartment and show that nuclear forms of PTHrP arise from translation starting at alternate codons downstream from the initiator methionine, within the signal sequence. This may constitute the first example of a mammalian protein being translated from downstream, non-AUG start sites.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of plasmids
All PTHrP plasmid constructs were derived from the plasmid (p)PTHrP/1 comprised of the 1.2-kb, full-length, rat (r)PTHrP cDNA (clone rPLPm 10) inserted in the EcoRI polylinker site of the mammalian expression vector pcDNA1.1 (Invitrogen, Carlsbad, CA). p{Delta}-36+1/1, which encodes the mature form of PTHrP lacking the prepro-sequence, was constructed as described earlier (4). A plasmid encoding human (h)PTH cDNA in pcDNA1.1 (pPTH) was also used as negative control in transfection and immunoblotting experiments.

pATC/2 was generated by mutating the initiator ATG codon (aa -36) of PTHrP cDNA in pGEM2 (pPTHrP/2; Promega Corp., Madison, WI), to an ATC codon (boldface) with the Chameleon Double-Stranded, Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using oligonucleotides 5'-C GGC ACG ATC CTG CGG AGG-3' and 5'-GGT TTC TTA GTC GTG AGG TGG CAC TTT TCG G-3', as mutagenesis and selection (SW3) primers, respectively. ATC-PTHrP cDNA was inserted into pcDNA1.1 by restriction digest of pATC/2 with EcoRI, yielding pATC/1.

pG/1 encompassing an engineered N-linked glycosylation consensus sequence (59Asn-Gly-Ser61), where the original Phe59 codon was substituted (boldface), was obtained in a similar manner, using the SW3 oligonucleotide and the mutagenesis primer 5'-CCT GTG CGG AAT GGG TCA GAT G-3'.

To facilitate protein immunodetection, PTHrP cDNA was tagged at the carboxyl-terminal with an epitope derived from the influenza virus hemagglutinin (HA) protein. pPTHrP/HA was generated by PCR amplification, using pPTHrP/1 as template DNA, a sequence-specific sense primer (PLP3), 5'-G TAC AAA GAG CAG CCA CTC-3', and a C-terminally extended antisense oligonucleotide (HA-tag), 5'-GAG TTA GCT GGC GTA GTC GGG CAC GTC GTA GGG GTA ATG CGT CCT TGA GCT GGG-3'. The latter encodes the last six amino acids of PTHrP (italicized) without a stop codon, followed by the ten amino acid residues of the HA tag (YPYDVPDYAS) and a termination codon (boldface). The amplified product was ligated into the pCR2.1 vector (Invitrogen, CA) and digested with SmaI/XbaI. The resulting fragment was inserted back into pPTHrP/1.

p{Delta}-36+1/HA, pATC/HA and pG/HA were obtained by digesting p{Delta}-36+1/1, pATC/1 and pG/1 with HindIII/SmaI and substituting each of these fragments into the original pPTHrP/HA. These four constructs are depicted in Fig. 1AGo. All constructs were verified by sequencing (GenAlytic, Guelph, Ontario, Canada).



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  		Figure 1. A, Schematic representation of the PTHrP cDNA constructs used and subcellular localization of the recombinant proteins. Depicted are wild-type PTHrP, the two mutant forms (one lacking the initiator methionine, the other encompassing an engineered consensus N-linked glysocyslation site, Y), and the prepro-less, mature form of the protein. The empty box represents PTHrP prepropeptide; the gray box, the mature form of PTHrP; and the black box, the HA tag. B, Microscopic examination of nuclei preparations. The upper and lower panels show a typical sample of purified nuclei stained with the nuclear Hoechst 33258 dye and examined under phase-contrast microscopy, respectively. C, Immunoblotting of total (t), cytoplasmic (c) and nuclear (n) protein fractions using a monoclonal Golgi- specific (anti-58K, upper panel) or histone H1 antibody (anti-H1, lower panel). A band of ~56 kDa (arrowhead) indicated that Golgi-specific 58K protein was present in (t) and (c) lysates but absent from the (n) extract, whereas a doublet at ~32–33 kDa (arrowheads) corresponding to wild-type and hyperphosphorylated histone H1 forms was present in both (t) and (n) but not in the (c) fraction. This confirmed that nuclear and cytoplasmic preparations were free of protein contaminants of Golgi and nuclear origin, respectively.

 
Cell culture
COS-1 cells were cultured in DMEM supplemented with 10% FCS (Life Technologies, Inc., Rockville, MD) in a humidified atmosphere at 37 C, with 5% CO2. Cells were transiently transfected by either the calcium phosphate or DEAE-Dextran (Amersham Pharmacia Biotech, Inc., Baie d’Urfé, Québec, Canada) method.

Cell lysis and subcellular fractionation
Our procedure was partially derived from the method of Blobel and Potter (16), with modifications and adaptations. Transiently transfected COS-1 cells were harvested and lyzed in ice-cold, low-detergent, sucrose buffer (10 mM Tris-HCl, pH 8.0, 250 mM sucrose, 2 mM MgCl2, 1 mM CaCl2, 1% TritonX-100) containing a mixture of protease inhibitors, aprotinin, bestatin, E-64, leupeptin, pefabloc, and pepstatin, at concentrations suggested by the manufacturer (Roche Diagnostics, Laval, Québec, Canada). Following disruption with a Dounce homogenizer (tight pestle), lysates were centrifuged at 1, 000 x g at 4 C, and supernatants constituting crude cytoplasmic protein fractions (c) were collected. Pellets, which are crude nuclei preparations, were resuspended in 50 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM MgCl2, 250 mM sucrose, to which 2 volumes of the same buffer containing 2.3 M sucrose were subsequently added, to a final sucrose concentration of approximately 1.62 M. Nuclei suspensions were layered atop a cushion of 2.3 M sucrose and subjected to ultracentrifugation at 120,000 x g at 4 C. Recovered nuclei were lyzed in 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.4% SDS plus protease inhibitors, and sonicated to yield pure nuclear protein fractions (n). As for total-cell extracts (t), they were prepared by lyzing and sonicating COS-1 cells in cold 20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 M EDTA, and 0.5 mM DTT, with protease inhibitors. All protein concentrations were estimated using the Bio-Rad Laboratories, Inc. Dc Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA).

Deglycosylation of protein fractions
Cytoplasmic fractions were treated with Peptide: N-glycosidase F (PGNase F) to remove carbohydrate residues from proteins, according to the manufacturer’s recommendations (New England Biolabs, Inc., Beverly, MA).

Microscopic examination of nuclei
Purified nuclei were incubated in the above sucrose lysis buffer, with or without Hoechst 33258 dye (Sigma-Aldrich Corp., Oakville, Ontario, Canada), following the manufacturer’s recommendations. Subsequently, they were dispersed on coverslips, mounted in 70% glycerol, and photographed using an Olympus BH21 microscope (Olympus America, Inc., Melville, NY).

In vitro transcription and translation
pPTHrP/HA, p{Delta}-36+1/HA and pATC/HA were linearized with XbaI, and 5 µg of DNA were used in each T7 RNA polymerase-driven transcription reaction (Promega Corp., WI), according to the manufacturer’s instructions. One microgram of mRNA was translated per 40 µl-reaction, which contained nuclease-treated rabbit reticulocyte lysate (Promega Corp., WI) and [3H]leucine (ICN Pharmaceuticals, Inc., Costa Mesa, CA), with or without canine microsomal membranes (Promega Corp., WI), for 1 h, at 30 C. Proteins were fractionated by SDS-PAGE on 15–20% gradients. Gels were treated with fluorographic autoradiography enhancer (En3Hance, NEN Life Science Products, Boston, MA), dried and exposed to BioMax MR film (Eastman Kodak Co., Rochester, NY), at -80 C.

Indirect immunofluorescence
Transiently transfected COS-1 cells were plated onto coverslips 18–24 h before study. After fixation and permeabilization in cold methanol:acetone (1:1), cells were blocked in PBS/1% EGTA/2% goat serum (Sigma-Aldrich Corp.), and incubated with primary polyclonal rabbit antiserum diluted in 2% goat serum/PBST (PBS+0.2% Triton X-100). Following incubation with goat antirabbit fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Sigma-Aldrich Corp.), cells were washed with PBST, mounted in 70% glycerol, and examined under a Leitz Aristoplan microscope (Leica Corp., Heerbrugg, Switzerland).

SDS-PAGE and immunoblotting
Aliquots of total-cell (t), cytoplasmic (c), and nuclear (n) extracts were fractionated by SDS-PAGE on 15–20% gradient gels. Proteins were transferred onto supported nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH), blocked in TBS buffer (10 mM Tris-HCl, 50 mM NaCl, 0.1% Tween 20) containing powdered milk and BSA, and incubated first with the primary antibody, and then with the appropriate horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich Corp.). Antigen-antibody complexes were detected using the BM Chemiluminescence Blotting Substrate (POD) Kit (Roche Molecular Biochemicals).

Antibodies
Monoclonal antibodies recognizing an epitope on the microtubule-binding, peripheral, Golgi membrane protein 58K (anti-58K), the histone H1 (anti-H1), the HA tag (YPYDVPDYA) (anti-HA) were purchased from Sigma-Aldrich Corp. (1:1000), Upstate Biotechnology, Inc. (Lake Placid, NY) (1 µg/ml, clone AE-4) and Roche Molecular Biochemicals (0.4 µg/ml, clone 12CA5), respectively. Polyclonal antisera to PTHrP 1–34 (anti-PTHrP 1–34) and PTHrP pro-region (anti-pro), gifts from D. Goltzman (McGill University, Montréal, Québec, Canada), were raised in rabbit, against synthetic peptides comprising amino acids 1 to 34 and -12 to +1 (i.e. the last six amino acid residues of the signal sequence and the six amino acids of the propeptide, plus the first amino acid of the mature protein) of PTHrP, respectively (1:1000 dilution).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nuclei and nuclear protein preparations
To gain insight into the mechanism whereby PTHrP, normally a secreted protein, could access the cell cytosol for subsequent nuclear translocation, we first characterized the PTHrP species within the nuclear compartment. Toward this goal, we set out to establish a suitable methodology for disrupting cells, purifying and lyzing nuclei, and recovering cytoplasmic fractions, which ensured the segregation of nuclear PTHrP peptides from those of cytosolic/cytoplasmic origin. Our protocol was partially derived from the nuclei isolation method of Blobel and Potter (16). COS-1 cells were lyzed in low-detergent sucrose buffer, homogenized, and centrifuged to yield crude cytoplasmic protein extracts (supernatant) and nuclei (pellet) that were subsequently processed as described in the Materials and Methods section. In a typical preparation, as presented in Fig. 1BGo, we identified recovered nuclei using nuclear Hoechst 33258 stain, and verified the absence of contaminating organelles by phase-contrast microscopy.

To further ascertain that fractions prepared from isolated nuclei were free of Golgi contaminants, we analyzed nuclear protein lysates by SDS-PAGE and Western blotting using a Golgi-specific antibody, anti-58K (17). A band corresponding to this Golgi protein at ~56 kDa (arrowhead) was detected in both total-cell (t) and cytoplasmic (c) (Fig. 1CGo, upper panel, lanes 1 and 2) but not nuclear (n) extracts (lane 3). Furthermore, in Fig. 1CGo, lower panel, the cytoplasmic lysates were shown to be free of nuclear contamination, using a monoclonal antibody to nuclear histone H1 protein, anti-H1 (18). The doublet detected at ~32–33 kDa (double arrowhead), due to the hyperphosphorylation of H1, was present in (t) and (n), but not (c) fractions (lanes 1, 3 vs. 2). This confirmed that the purification procedure precluded protein cross-contamination between nucleus and cytosol/cytoplasm. Thus, nuclear protein fractions prepared according to our protocol were amenable for the characterization of nuclear PTHrP.

The nuclear forms of PTHrP
To enhance immunodetection and monitoring of PTHrP, full-length and prepro-less PTHrP cDNAs were tagged with sequences encoding an epitope from the influenza virus hemagglutinin protein (HA tag). These constructs were tested by transient expression in COS-1 cells to confirm, by indirect immunofluorescence using both the HA antibody (anti-HA) and PTHrP 1–34 antiserum (anti-PTHrP 1–34), that the tag did neither interfere with the normal subcellular distribution of the peptide nor alter the bioactivity of the protein, as assessed in a cAMP bioassay (data not shown).

Total-cell (t), cytoplasmic (c), and nuclear (n) extracts were prepared from COS-1 cells transiently transfected with plasmids encoding HA-tagged full-length (pPTHrP/HA) or prepro-less rPTHrP (p{Delta}-36+1/HA), or hPTH (pPTH) as negative control, and fractionated by SDS-PAGE. Immunoblotting analysis using the monoclonal anti-HA (Fig. 2AGo) revealed three distinct forms of PTHrP protein in the nucleus (lane 3, triple arrowheads), of ~25 kDa, all of which were larger than the mature form of the peptide (lanes 4–6, single arrowhead). The predominant nuclear species were the largest and smallest ones in size. Corresponding forms were also present in total-cell and cytoplasmic preparations (lanes 1–2), but the signal intensity was weaker because PTHrP is more likely to be enriched in nuclear extracts with respect to its relative content in whole-cell and cytoplasmic lysates. Anti-HA did not recognize untagged hPTH protein in all three fractions (lanes 7–9), as expected.



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  		Figure 2. Nuclear PTHrP species. Total-cell (t), cytoplasmic (c), and nuclear (n) protein lysates from COS-1 cells transiently transfected with pPTHrP/HA (lanes 1–3), p{Delta}-36+1/HA (lanes 4–6) or pPTH (lanes 7–9) as control, were analyzed by SDS-PAGE and immunoblotting using a monoclonal antibody to the HA tag (anti-HA) (A) or a polyclonal antiserum to PTHrP 1–34 (anti-PTHrP 1–34) (B). A, COS-1 cells expressing pPTHrP/HA produced three distinct nuclear forms of PTHrP, of ~25 kDa (lane 3, triple arrowhead). The two prominent species were the highest and lowest molecular weight forms, and all nuclear PTHrP proteins were larger in size than the mature form of the peptide encoded by p{Delta}-36+1/HA (lane 6, single arrowhead). B, The specificity of anti-HA was assessed by a concurrent experiment using anti-PTHrP 1–34. Like anti-HA, anti-PTHrP 1–34 recognized the three nuclear PTHrP species (lane 3, triple arrowhead) as well as the prepro-less, mature form of the protein (lanes 4–6, arrowhead).

 
The specificity of anti-HA in detecting HA-tagged PTHrP was verified by conducting a parallel experiment using antiserum raised against residues 1 to 34 of PTHrP. As illustrated in Fig. 2BGo, PTHrP/HA and {Delta}-36+1/HA peptides detected by anti-PTHrP 1–34 (lanes 3–6), corresponded to those recognized by anti-HA, which further implied that nuclear PTHrP contained both the amino- and carboxy-terminal regions of the protein. Conversely to anti-HA, however, anti-PTHrP 1–34 displayed inconsistent ability in detecting full-length PTHrP (lanes 1–2 vs. 3), indicating that anti-HA is more suitable for immunoblotting analysis. Although anti-PTHrP 1–34 produced more background, it did not detect hPTH protein, as well.

Comparison of nuclear and in vitro translated PTHrP species
Nuclear PTHrP peptides were compared with in vitro translated protein to delineate potential mechanisms implicated in the generation of nuclear forms (Fig. 3Go). Nuclear fractions were obtained from COS-1 cells transiently expressing pPTHrP/HA (lane 3) or p{Delta}-36+1/HA (lane 4) and analyzed by SDS-PAGE and HA-immunoblotting, along with in vitro translated PTHrP standards (lanes 1–2). As shown, all nuclear PTHrP species were smaller than in vitro translated prepro-PTHrP (lane 3 vs. 2), yet larger than the mature form of the protein (lane 4, arrowhead), as observed above. Additional nonspecific bands were detected in both samples.



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  		Figure 3. Relative size of the nuclear PTHrP forms. pPTHrP/HA (lane 3) and p{Delta}-36+1/HA (lane 4) nuclear extracts, and PTHrP/HA protein transcribed and translated in rabbit reticulocyte lysate (ivt), in the presence (lane 1) or absence (lane 2) of canine microsomal membranes were fractionated by SDS-PAGE and analyzed by HA-immunoblotting. The relative size of the nuclear forms with respect to prepro- and pro-PTHrP suggested that nuclear PTHrP proteins contained part of the prepropeptide.

 
Given that nuclear PTHrP proteins were smaller in size than prepro-PTHrP and encompassed both the amino- and carboxyl-terminal ends of the peptide, we postulated that they still contained part of the prepro sequence.

Alternate initiation of translation in PTHrP mRNA
One of the mechanisms that accounts for preservation of the prepropeptide is alternate initiation of translation. This often occurs when an optimal context of translation initiation around the start codon, as defined by Kozak, is lacking (19). Examination of PTHrP nucleotide sequence surrounding the initiator AUG (Met-36) revealed that it does not rest within such a facilitating context, suggesting that this codon is not highly efficient at inducing translation. Since prepro-PTHrP cDNA does not contain additional AUGs, non-AUG translation start sites such as CUG and GUG codons are likely to be functional. Figure 4AGo shows the nucleotide sequence of rPTHrP cDNA encoding the prepro segment of the protein as well as the corresponding sequences from mouse, dog, and human cDNAs. Alternate translation may be initiated at CUGs (Leu-35, -32, -25, -22) and GUGs (Val-24, -17, -10) (boldface), with the exception of Leu-25, and Leu-35 and -25 for dog and human cDNAs, respectively. These codons are all positioned downstream from the initiator AUG, within the PTHrP signal sequence. Translation from such sites would be highly unusual, as in eukaryotic mRNAs only upstream non-AUG codons are used (19). However, in PTHrP, several alternate codons are well conserved among the various species (boxed) and lie within a favorable (CUG-35, GUG-10) or optimal (CUG-32, -25) context of translation initiation, identifying them as putative start sites. Their selection is enhanced further by the high G+C content of PTHrP prepro-sequence, which sustains the formation of multiple hairpin structures (facing arrows) that constrain ribosome attachment (20).



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  		Figure 4. Alternate initiation of translation in PTHrP mRNA. A, Nucleotide sequence of the 5' prepro-region of rat PTHrP cDNA and corresponding sequences from mouse, dog, and human. Leu and Val residues, encoded by CUG and GUG respectively, which could function as alternate start sites are shown in boldface. Those well conserved among the various mammals are boxed. Facing arrows indicate the presence of palindromic inverted repeats that support the formation of stem-loop structures. The vertical arrowhead shows the cleavage site of PTHrP signal peptide. B, Generation of non-AUG initiated PTHrP peptides. The initiator ATG in pPTHrP/HA was mutated to a noninitiator ATC, generating pATC/HA. pATC/HA along with wild-type pPTHrP/HA and prepro-less p{Delta}-36+1/HA were transcribed and translated in vitro and proteins were analyzed by SDS-PAGE and autoradiography. Low levels of two ATC species of ~25 kDa were produced (lanes 2–4, double arrowhead), which were larger in size than the mature form of the protein (lane 1, single arrowhead). Conversely to prepro-PTHrP, which was cleaved to pro-PTHrP upon addition of canine microsomal membranes (lane 6 vs. 5), prepro-ATC peptides were not cleaved to pro-ATC by membranes (lane 2 vs. 3–4), suggesting that alternate translation initiation truncates PTHrP prepropeptide and impairs its targeting to and processing in the ER.

 
We hypothesized, therefore, that PTHrP translation could be initiated at alternate sites located within the signal sequence, downstream from the initiator AUG. This would generate proteins with a truncated signal peptide that is ineffective in binding to the signal recognition particle (SRP) and targeting the nascent peptides to the ER. Consequently, these PTHrP forms would be translated and released in the cytosol to be subsequently transported to the nucleus/nucleolus on the strength of their fully functional NLS.

Translation of mutant ATC-PTHrP cDNA
To test this hypothesis, we mutated the initiator ATG in pPTHrP/HA to an ATC (pATC/HA), a codon never reported to serve as alternate codon for upstream or downstream initiation of translation, thereby allowing for the potential synthesis of non-AUG initiated PTHrP peptides. pATC/HA, pPTHrP/HA and p{Delta}-36+1/HA, were transcribed and translated in vitro in the presence of radiolabeled leucine. As illustrated in Fig. 4BGo, SDS-PAGE and autoradiographic analysis revealed that two ATC proteins of ~25 kDa were produced (double arrowhead, lane 2), albeit at low levels, which was not unexpected given that non-AUG codons are generally less efficient at initiating translation (19). Both species were larger than the mature form of PTHrP (lane 1, single arrowhead). When comparing non-AUG to AUG-initiated peptides, the large ATC species appeared to be smaller than prepro-PTHrP (lanes 2–4 vs. 5–6), and the size of the small ATC form ranged between that of prepro- and pro-PTHrP, reminiscent of the mid-size nuclear PTHrP protein (see Fig. 2AGo, lanes 1–3). Thus, translation in PTHrP mRNA was initiated at alternate codons, either in very close proximity to the start codon or further downstream to account for the observed size differences.

When canine microsomal membranes were added during translation to mimic ER membrane cleavage of the signal peptide, prepro-PTHrP was processed to yield pro-PTHrP (lane 6 vs. 5). In contrast, prepro-ATC proteins were not cleaved to pro-ATC, even with increasing amounts of membranes (lane 2 vs. 3 and 4). It appears, therefore, that alternate initiation of translation does indeed lead to the formation of a shorter signal peptide that binds poorly to SRPs in the cytosol, if at all, and fails to be identified by components of the transport system at the surface of the ER.

Non-AUG initiated PTHrP proteins localize exclusively to nucleoli
If alternate initiation of translation impairs ER targeting of PTHrP, it was anticipated that ATC peptides transiently expressed in COS-1 cells would accumulate exclusively in the nuclear compartment. To validate this, we transiently expressed pPTHrP/HA and pATC/HA in COS-1 cells and examined by indirect immunofluorescence, using PTHrP 1–34 antiserum, the subcellular distribution of recombinant proteins (Fig. 5AGo). A large number of cells expressing pPTHrP/HA demonstrated immunostaining along the secretory pathway (not shown), whereas a small proportion exhibited both secretory and nucleolar staining (left panel), as previously described (4). In contrast, pATC/HA expressing cells displayed exclusive nucleolar immunostaining (right panel), which advocated that alternate initiation of translation in PTHrP mRNA produced forms that are not secreted but released within the cytosol for nuclear import.



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  		Figure 5. Expression of non-AUG-initiated PTHrP peptides in COS-1 cells. A, Indirect immunofluorescence studies in COS-1 cells transiently transfected with pPTHrP/HA and pATC/HA, using anti-PTHrP 1–34. COS-1 cells expressing full-length PTHrP displayed either secretory (not shown) or both secretory and nucleolar PTHrP staining (left panel), as reported earlier (4 ). In contrast, those expressing non-AUG initiated PTHrP exhibited immunostaining restricted to nucleoli (right panel). B, Nuclear ATC/HA species. SDS-PAGE and immunoblotting analysis using anti-HA was performed on (n) extracts prepared from COS-1 cells transiently transfected with pPTHrP/HA, pATC/HA and pATC (lanes 2, 3 and 5), and in vitro translated (ivt) PTHrP/HA and ATC/HA proteins (lanes 1 and 4). There were two ATC/HA species present in the nucleus (dots) that were specifically detected by anti-HA (lane 3 vs. 5, untagged pATC nuclear fraction) and co-migrated with in vitro translated ATC/HA peptides (lane 4). The large ATC/HA species co-migrated with the largest nuclear form of PTHrP/HA, which is smaller than prepro-PTHrP/HA (lane 1). As for the small ATC/HA protein, it migrated between the largest and smallest nuclear forms of PTHrP/HA (lane 3 vs. 2), reminiscent of the mid-size nuclear species.

 
Two non-AUG initiated PTHrP species are found in the nuclear compartment
To characterize the non-AUG-initiated PTHrP species found in the nucleus, nuclear fractions were prepared from COS-1 cells transiently transfected with pPTHrP/HA, pATC/HA, and pATC and analyzed by SDS-PAGE and Western blotting using anti-HA. In vitro translated PTHrP/HA and ATC/HA proteins were also processed as standards. As shown in Fig. 5BGo, two ATC/HA peptides were found in the nuclear (lane 3, double dots) but not the cytoplasmic lysate (data not shown), as expected, which comigrated with in vitro translated ATC/HA (lane 4). Nuclear ATC/HA forms were specifically detected by anti-HA since they were absent from untagged pATC nuclear extracts (lane 5). The large ATC/HA peptide comigrated with the largest nuclear PTHrP/HA species (lane 3 vs. 2). The small but predominant form migrated between the highest and lowest molecular weight PTHrP/HA proteins, similarly to mid-size nuclear PTHrP (see Figs. 2AGo and 3Go). This suggested that, in intact cells, at least two non-AUG downstream codons may be used to generate nuclear PTHrP proteins, one in close proximity to and another further downstream from the initiator AUG. To achieve adequate immunodetection of low levels of ATC/HA proteins, 20-fold excess of ATC/HA nuclear extract was loaded while the amount of PTHrP/HA nuclear lysate was simultaneously decreased by 2- to 5-fold. This led to visual discrepancies in the intensity and thickness of migrating bands, band distortion, and pronounced background.

The low levels of ATC/HA expression observed may reflect the reduced capacity of natural non-AUG codons to induce translation, especially without the synergistic contribution of a default start codon (19), the absence of an appropriate cellular context (activating factors or stimuli) favoring non-AUG codon usage, and/or the tight control over the synthesis of alternate PTHrP proteins that possess distinct biological properties. In addition, disparities in the production of non-AUG-initiated peptides between reticulocyte lysates and intact cells such as COS-1 cells could be ascribed to differences in translation conditions. Cell-free systems seem to be less sensitive to subtle alterations of mRNA structure than the translation machinery within the living cell.

Expression of a N-linked glycosylation mutant PTHrP cDNA
To further strengthen our proposition that nuclear PTHrP species bypass ER transit, a consensus sequence for N-linked glycosylation was engineered by site-directed mutagenesis in pPTHrP/HA (pG/HA), as described in Materials and Methods. Because N-linked glycosylation is confined to the ER, the presence of glycosylated PTHrP forms in the nucleus would imply that they had translocated to the ER lumen before nuclear transport. pG/HA was transiently transfected in COS-1 cells, and indirect immunofluorescence studies using PTHrP 1–34 antiserum were performed. Most cells displayed immunostaining in a secretory pattern (not shown), whereas some exhibited both secretory and nucleolar staining, as presented in Fig. 6AGo. This was consistent with the normal distribution pattern of PTHrP (Fig. 5AGo, left panel), confirming that this mutation did not alter the subcellular routing of the peptide.



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  		Figure 6. Expression of a PTHrP mutant cDNA encompassing a consensus N-linked glycosylation site (pG/HA) in COS-1 cells. A, Subcellular distribution of G/HA protein by indirect immunofluorescence using anti-PTHrP 1–34. Most transfected COS-1 cells exhibited secretory PTHrP immunostaining (not shown) while others displayed both secretory and nucleolar staining, confirming that this mutation did not alter the subcellular localization of the peptide. B, upper panel: the nuclear G/HA species. (n) and (c) protein fractions were prepared from COS-1 cells expressing either pPTHrP/HA (lanes 1 and 4) or pG/HA (lanes 2–3) and analyzed by SDS-PAGE and HA-immunoblotting. In the G/HA (n) lysate, anti-HA did not detect additional protein species beside the three peptides (triple arrowhead) that comigrated with the three wild-type nuclear PTHrP/HA forms. In contrast, in the G/HA (c) fraction, species of higher molecular weight than the cytoplasmic PTHrP/HA proteins were observed (small arrowheads), likely arising from glycosylation of the peptide. Lower panel: deglycosylation of cytoplasmic extracts. The G/HA and PTHrP/HA (c) fractions were subjected to PNGase F treatment. Upon addition of glycosidase, at least one of the bands corresponding to the additional G/HA cytoplasmic species was no longer present (lane 2 vs. 1, small arrowhead), confirming that some higher molecular weight forms result from glycosylation of the peptide.

 
Nuclear and cytoplasmic fractions were prepared from COS-1 cells expressing pG/HA and pPTHrP/HA and compared by SDS-PAGE and HA-immunoblotting analysis (Fig. 6BGo). In the nucleus, anti-HA detected only three G/HA species (upper panel, lane 2, triple arrowhead), which comigrated with the three wild-type nuclear PTHrP/HA forms (lane 1), indicating that they had not undergone N-linked glycosylation. In contrast, anti-HA recognized at least two additional PTHrP peptides of higher molecular weight in pG/HA but not pPTHrP/HA cytoplasmic lysate (lane 3 vs. 4, small arrowheads).

To confirm that these larger species resulted from glycosylation of the protein, cytoplasmic fractions were subjected to PNGase F treatment, followed by HA-immunoblotting. This analysis showed higher background than usual, possibly due to altered buffer composition of the samples. Nevertheless, as illustrated in Fig. 6BGo, lower panel, when glycosidase enzyme was added to the G/HA cytoplasmic fraction, at least one of the higher protein bands disappeared (lane 2 vs. 1, small arrowhead), indicating that some of these species were indeed glycosylated forms of PTHrP. These findings are consistent with our contention that nuclear forms of PTHrP bypass ER transit and add further support for alternate initiation of translation.

Nuclear PTHrP species contain the propeptide
If nuclear PTHrP proteins arise from alternate initiation of translation at internal sites within the signal sequence, it was predicted that they contain the propeptide. To examine this possibility, nuclear fractions from COS-1 cells expressing pPTHrP/HA and p{Delta}-36+1/HA were immunoblotted with anti-pro, an antiserum raised against a synthetic peptide comprising the last six amino acids of the presequence (RSVEGL), the six amino acids of the entire proregion (GRRLKR), and the first amino acid (A) of the mature protein. As shown in Fig. 7AGo, anti-pro specifically recognized two proteins in pPTHrP/HA nuclear extract (lane 2, double arrowhead), which were absent from p{Delta}-36+1/HA nuclear lysate (lane 1). To identify these species as PTHrP, a parallel immunoblot was performed using anti-HA. Anti-HA detected two peptides that comigrated with those recognized by anti-pro, confirming that they were indeed nuclear forms of PTHrP (lane 3 vs. 2). Anti-HA also detected {Delta}-36+1/HA protein in the nuclear preparation, unlike anti-pro, since this PTHrP form possesses the HA tag but lacks the prepropeptide sequence.



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  		Figure 7. Nuclear PTHrP peptides contain the propeptide. A, Nuclear fractions of COS-1 cells transiently transfected with pPTHrP/HA and p{Delta}-36+1/HA were analyzed by SDS-PAGE and immunoblotting using either anti-pro, a polyclonal antiserum raised against the pro-region of PTHrP (-12 to +1) (lanes 1–2), or anti-HA (lanes 3–4). Anti-pro specifically detected two protein species in pPTHrP/HA nuclear extract (double arrowhead) that were absent from control p{Delta}-36+1/HA nuclear fraction. The proteins recognized by anti-pro comigrated with those specifically detected by anti-HA (lane 2 vs. 3), identifying them as nuclear PTHrP species. B, Immunofluorescence studies in COS-1 cells transiently expressing pPTHrP/HA (upper row) or p{Delta}-36+1/HA (lower row) using anti-pro (left column) or anti-PTHrP 1–34 (right column). In the upper row, full-length PTHrP, which displayed dual subcellular distribution (secretory and nucleolar), was detected by both antisera, in contrast to the mature form of the peptide, which was only recognized by anti-PTHrP 1–34 but not anti-pro.

 
These results were corroborated by indirect immunofluorescence studies using anti-pro (left column, Fig. 7BGo) and anti-PTHrP 1–34 (right column). COS-1 cells expressing full-length PTHrP displayed either secretory (not shown) or secretory and nucleolar immunostaining (pPTHrP/HA, upper row) with both antisera, proving that nuclear PTHrP species encompass the PTHrP propeptide, in concordance with our hypothesis. COS-1 cells expressing the mature form of the peptide ({Delta}-36+1/HA, lower row) exhibited nucleolar immunostaining with anti-PTHrP 1–34 but not with anti-pro, as anticipated.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we present compelling evidence that nuclear forms of PTHrP arise from alternate initiation of translation at downstream, non-AUG codons within the signal sequence. This generates PTHrP species with a truncated signal peptide that is inefficient in targeting the proteins to the ER, thereby enabling them to be translated and released within the cytosol for subsequent nuclear import. Such a process gives rise to PTHrP peptides that 1) contain the propeptide, 2) bypass ER transit, and 3) localize exclusively to the cell nucleus/nucleolus, as shown here.

Although a rare event, initiation of translation at alternate non-AUG codons has been documented for a number of viral, bacterial, yeast, plant, and animal mRNAs. In addition to translating from the initiator AUG codon, if present, these transcripts generally initiate translation at upstream ACG (19, 21), AUU (22, 23), CUG (19, 24, 25, 26, 27), or GUG (28, 29, 30, 31) codons. Usage of alternate initiator codons in natural mRNAs can be explained in part by the absence of an optimal context for translation initiation in the vicinity of the start codon (19), which favors use of other sites that conform to these structural requirements. Initiation from such codons is further enhanced by the presence of judiciously positioned downstream stem-loop structures of sufficient stability and strength (-19 kcal/mol, calculated as the Gibbs free energy of formation) (19, 20). The strongest facilitation effect is seen when the hairpin is separated from the preceding initiator codon by 12 to 15 nucleotides.

In PTHrP cDNA, the absence of additional Met residues implies that alternate initiation of translation takes place at non-AUG codons, likely at CUGs (Leu-35, -32, -25, -22) and GUGs (Val-24, -17, -10). Furthermore, the high G+C content in PTHrP prepro-sequence (about 67–68%) sustains the formation of multiple base-paired structures, at least one of which (Gibbs free energy of formation, 15.2 kcal/mol) is located 15 nucleotides downstream from the putative start codon Leu-32, and could contribute to the stabilization of translation initiation complexes at this site (32). It is of interest to note that all these CUGs and GUGs are found downstream from the unique initiator AUG (Met-36). Unlike utilization of upstream non-AUG codons in alternate translation initiation, evidence for downstream non-AUG usage is very limited. Two members of a small group of prokaryotic caseinolytic proteases, the Clp family, stand as the unique examples of such an event (33, 34). Multiple yeast, mouse, and human Clp homologs have been identified so far (35), but it is not yet known whether downstream CUG and GUG translation initiation sites are effective in generating Clp/HSP100 eukaryotic variants, as suggested by the presence of distinct family members harboring amino-terminal truncations. In addition to the limitations imposed by transcript structure, cellular factors have been proposed to dictate initiator codon selection, although details of such processes remain sketchy (30, 36, 37, 38, 39).

The production of more than one peptide from a single transcript implies that both AUG- and non AUG-initiated proteins fulfill unique biological roles. In many cases, alternate translation initiation has profound consequences on the intracellular routing of proteins, thereby endowing them with dual actions in influencing cell behavior (10, 24, 25, 27, 40). In some instances, alternate species exert a role in controlling the translation of their AUG-initiated counterparts and/or their activity (25, 33, 34). Therefore, institution of alternate initiator codons in protein translation may not be solely designed for the generation of protein diversity, but also for control over the expression of that diversity. As for PTHrP, it is not known whether the alternate species regulate the synthesis or functionality of the AUG-initiated forms.

Recent findings by Amizuka et al. (41) further support our hypothesis of alternate, internal initiation of translation in PTHrP mRNA. Our data, together with their mutagenesis studies, argue that PTHrP uses alternative codons other than the four CUGs (-35, -32, -25, -22) in the signal sequence, in agreement with our proposal that downstream GUGs may act as translation start sites. Nevertheless, this conclusion does not exclude the possibility that other mechanisms may operate to provide PTHrP with access to the cytosol, under specific conditions (42), such as reinternalization (13, 43) and ER retrograde translocation (44, 45). Characterizing the nuclear PTHrP species at the amino acid level will thus undoubtedly shed light on alternate mechanisms employed by PTHrP to access the cytosol for subsequent nuclear import. This approach, however, has been hampered by numerous technical difficulties in isolating sufficient amounts of nuclear PTHrP protein for amino acid microsequencing.

Whereas the biological importance of PTHrP targeting to the nucleus/nucleolus is not clearly understood, the nucleolar distribution of PTHrP to the dense fibrillar component (4), both in vitro and in situ, implies that PTHrP could alter cellular activities by modulating ribosomal gene transcription. PTHrP, for example, may influence the activity of RNA polymerase I, as shown for other factors (10, 46, 47, 48). Alternatively, the protein may play a role in pre-rRNA processing and ribosomal assembly. The diffuse nucleoplasmic distribution of PTHrP in A-10 vascular smooth muscle cells (7) further suggests that PTHrP could have other intranuclear actions such as in DNA replication or gene transcription. Recently, in knockout mice, a role in vascular invasion of the growth plate has been proposed for PTHrP, an effect not mediated by the classical N-terminal, type 1 PTH receptor (49). This may well arise as a consequence of PTHrP’s nuclear actions or from its interaction with other receptors, although it remains to be proven.

In summary, the results presented here indicate that some nuclear forms of PTHrP result from alternate initiation of translation within the signal sequence, downstream from the initiator methionine codon. This would lead to the formation of a shorter signal peptide that binds ineffectively to SRPs, if at all, and fails to target PTHrP proteins to the ER for secretion. Such truncated prepro-PTHrP species would remain in the cytosol and be translocated to the nucleus/nucleolus on the strength of their functional NLS. Our findings may also constitute the first example of translation initiation at alternate, non-AUG downstream sites in a mammalian protein. This may serve as a novel mechanism for cellular control of protein trafficking and subcellular distribution.


   Acknowledgments
 
We thank D. Goltzman for the PTHrP antisera, and J. Th’ng for his invaluable advice.


   Footnotes
 
1 This work was supported by the Canadian Arthritis Network and the Medical Research Council (MRC) of Canada. Back

2 Recipient of Fonds pour la Formation de Chercheurs et l’Aide à la Recherche/Fonds de la Recherche en Santé du Québec and MRC Doctoral Scholarships. Back

3 MRC Scientist. Back

Received July 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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