From the Department of Biochemistry and Molecular Biology, John
Curtin School of Medical Research, Canberra, A.C.T.2601, Australia, and
St. Vincent's Medical Research Institute, Fitzroy,
Victoria 3065, Australia
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ABSTRACT |
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Parathyroid hormone-related protein (PTHrP),
expressed in a range of tumors, has endocrine, autocrine/paracrine, and
intracrine actions, some of which relate to its ability to localize in
the nucleus. Here we show for the first time that extracellularly added
human PTHrP (amino acids 1-108) can be taken up specifically by
receptor-expressing UMR106.01 osteogenic sarcoma cells and accumulate
to quite high levels in the nucleus and nucleolus within 40 min.
Quantitation of recognition by the nuclear localization sequence
(NLS)-binding importin subunits indicated that in contrast to proteins
containing conventional NLSs, PTHrP is recognized exclusively by
importin Parathyroid hormone-related protein
(PTHrP)1 is expressed in a
range of tumors and is an endocrine agent in humoral hypercalcemia of
malignancy. It is also expressed in many normal tissues where it exerts
autocrine/paracrine or intracrine actions (1-6). The structural
similarities to parathyroid hormone (PTH) at the amino terminus of
PTHrP are sufficient to confer functions similar to those of PTH,
mediated by the shared PTH/PTHrP receptor and its ability to activate
adenylate cyclase. Although both PTH and PTHrP promote bone resorption
and reduce renal calcium excretion (1, 7), roles in the regulation of
placental calcium transport to the fetus (1, 7, 8), osteoclast
inhibition (9, 10), and the regulation of cell growth and apoptosis (2, 11, 12) have been ascribed to distinct regions of PTHrP.
We and others have recently shown that PTHrP is expressed in a cell
cycle-dependent manner (13, 14) as well as being localized to the nucleus/nucleolus at G1 (14). Regulation of the
nuclear localization of PTHrP appears to be mediated through
phosphorylation by the cyclin-dependent kinases
p33cdk2 and
p34cdc2.2 These
observations are consistent with the idea that cell
cycle-dependent regulation of nuclear localization of PTHrP
is central to the control of growth and apoptosis (2, 4). Of
significance in this regard is our observation2 that within
amino acids 61-93, PTHrP retains a putative CcN motif, originally
described for the SV40 large tumor antigen (T-ag; Refs. 16 and 17),
comprising consensus protein kinase CK2 (S61DDE and
ET78KYE-"C") and confirmed cyclin-dependent
kinase (KT85PGK-"c") phosphorylation sites in the
vicinity of a T-ag-like nuclear localization sequence
(NLS-GKKKKGK93-"N"), as well as a putative nucleolar
localization sequence (NOS-PGKRKEQEKKKRRTR108) (Fig.
1A). In the case of T-ag, the
CcN motif CK2 and cyclin-dependent kinase sites strongly
regulate NLS function and the kinetics of nuclear import (16, 17); with
respect to PTHrP, transfection studies indicate that deletion of amino
acids 87-107, removing both NLS and NOS, results in completely
cytoplasmic localization of PTHrP and concomitant impaired
PTHrP-conferred resistance to apoptosis on the part of CFK2
chondrocytes (2). Based on the observation that nuclear PTHrP effects
an increase in mitogenesis in vascular smooth muscle cells, Massfelder
et al. (5) suggested that the clusters of basic amino acids
(88-91 and 102-106) in PTHrP might be recognized by the NLS-binding
importin/karyopherin subunit complex of the cellular nuclear protein
import machinery.
and not by importin
. The sequence of PTHrP responsible
for binding was mapped to amino acids 66-94, which includes an SV40
large tumor-antigen NLS-like sequence, although sequence determinants
amino-terminal to this region were also necessary for high affinity
binding (apparent dissociation constant of ~2 nM
for importin
). Nuclear import of PTHrP was assessed in
vitro using purified components, demonstrating that importin
,
together with the GTP-binding protein Ran, was able to mediate
efficient nuclear accumulation in the absence of importin
, whereas
the addition of nuclear transport factor NTF2 reduced transport. The
polypeptide ligand PTHrP thus appears to be accumulated in the
nucleus/nucleolus through a novel, NLS-dependent nuclear import pathway independent of importin
and perhaps also of NTF2.
INTRODUCTION
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Abstract
Introduction
References
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Fig. 1.
A, the human PTHrP amino acid sequence
is indicated in the single-letter amino acid code, with the putative
NLS underlined, nucleolar localization sequence
underlined and italicized, and the CcN motif
phosphorylation sites in bold type. The phosphorylation site
serine/threonines are numbered (14). B, the minimal importin
-binding region of PTHrP (established in this study) is compared
with that from TCPTP (19), as well as to similar sequences from GAL4
(20) and HIV-1 Rev (17). The carboxyl-terminal basic cluster is
underlined, conserved/similar sequences are denoted in
bold type, and predicted
-helical and
-turn
(dotted line) structural elements (Robson-Garnier) are
boxed.
In the present study we use recombinant and synthetic PTHrP peptides
encompassing various regions of PTHrP to demonstrate for the first time
that PTHrP can be internalized by receptor-expressing UMR106.01 cells
and localize rapidly in the nucleus and nucleolus. In addition we show
that in contrast to conventional NLS-containing proteins, PTHrP is
recognized with high affinity by the and not the
subunit of the
importin heterodimer. We map the specific region of PTHrP recognized by
importin
and show that it alone, in concert with the GTP-binding
protein Ran, can mediate nuclear accumulation of PTHrP in a
reconstituted nuclear import assay system. We conclude that PTHrP
localizes in the nucleus through a novel import pathway, possibly
shared by other importin
-recognized proteins such as HIV Rev (18),
the T-cell protein tyrosine phosphatase (TCPTP; Ref. 19), and the yeast
transcription factor GAL4 (20).
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MATERIALS AND METHODS |
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Chemicals and Peptides-- Reagents were from the sources previously described (14, 20-25).2 Peptides PTHrP 1-34, 50-69, 66-86, 66-94, 67-84, 67-86, 67-94, and 83-108 were synthesized using the Merrifield solid phase procedure and purified and characterized as described (26). Recombinant PTHrP 1-108 and 35-141 were expressed in Escherichia coli and purified as described previously (27). Peptide and protein concentrations were determined by quantitative amino acid analysis following acid hydrolysis. Bacterially expressed human NTF2 (28) was provided by Bryce Paschal (University of Virginia, Charlottesville, VA).
Cell Culture-- Cells of the HTC hepatoma tissue culture and UMR106.01 osteoblast lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (16, 21).
Fluorescent Labeling-- PTHrP 1-108 was conjugated to Oregon GreenTM (FITC) (Molecular Probes, Eugene OR), whereby 10 mg/ml PTHrP 1-108 in 0.1 M sodium bicarbonate, pH 9.0, was incubated with a one-tenth volume of 10 mg/ml FITC (in Me2SO) for 1 h at room temperature in the dark. Unbound chromophore was removed by passage through a PD-10 column (Pharmacia Biotech AB, Uppsala, Sweden) using phosphate-buffered saline, pH 7.0, or 0.1 M acetic acid, pH 2.0. Fractions of the eluant containing >0.1 mg/ml protein were pooled, and the concentration of the pooled fraction was determined to be 627 nM by radioimmunoassay (29), with the PTH activity/mol determined by assaying cAMP production in UMR106.01 cells (27, 30), being equivalent to that of either PTH 1-34 or unlabeled PTHrP 1-108.
Intact Cell Uptake of PTHrP (1-108)-- UMR106.01 cells were grown on 25-mm diameter round coverslips to 50-70% confluence, prior to transfer into an open perfusion microincubator at 37 °C. Incubations were performed in Tyrode's Salt Solution (Sigma, Sigma-Aldrich Pty. Ltd., Castlehill, NSW, Australia). FITC-PTHrP 1-108 was added at a final concentration of 2.5 nM, and the cells were observed over time. For competition, cells were preincubated for 15 min in medium containing 10 µg/ml PTHrP 1-108 before the addition of FITC-PTHrP 1-108. Visualization was performed by confocal laser scanning microscopy. Identical detector and laser settings were used for all samples within each experiment. Quantitation was performed using the NIH Image 1.68 software (21).
Expression of Glutathione S-Transferase Fusion Proteins--
The
NLS-binding mouse (m - PTAC58 and PTAC97) and yeast (y - karyopherin - Kap60 and Kap95) importin (Imp) and
subunits were expressed as
glutathione S-transferase (GST) fusion proteins and purified
as previously (22-24, 31). GST-free mImp
and yImp
were prepared
by thrombin cleavage (22, 31). Human Ran was similarly expressed as a
GST fusion protein, and GST-free Ran was prepared by thrombin cleavage
and loaded with GDP, as described (32, 33). Protein concentrations were
determined using the dye binding assay of Bradford (34), with bovine
serum albumin as a standard.
ELISA-based Binding Assay-- An ELISA-based assay (22-25) was used to examine binding between importin subunits (with and without GST moieties) and NLS-containing proteins or peptides. The latter were coated onto 96-well microtiter plates and hybridized with increasing concentrations of importin subunits, and detection of bound importin-GST was performed using goat anti-GST primary and alkaline phosphatase-coupled rabbit anti-goat secondary antibodies and the substrate p-nitrophenyl phosphate (22). Absorbance measurements were performed over 90 min using a plate reader (Molecular Devices, Sunnyvale, CA), and values were corrected by subtracting absorbance both at 0 min and in wells incubated without importin (22).
In Vitro Nuclear Transport--
Nuclear import kinetics were
analyzed at the single cell level using mechanically perforated HTC
cells in conjunction with confocal laser scanning microscopy (16, 21,
23-25). Experiments were performed for 40 min at room temperature in a
5-µl volume containing 30 mg/ml bovine serum albumin, 2 mM GTP, an ATP regenerating system (0.125 mg/ml creatine
kinase, 30 mM creatine-phosphate, 2 mM ATP),
transport substrate (375 ng/ml FITC-PTHrP), and where indicated, 4 µM RanGDP, 0.15 µM NTF2, and 1 µM importin and/or
subunits. In experiments where
PTHrP peptides were tested for their ability to compete FITC-PTHrP
transport, 0.5 µM mImp
and 4 µM RanGDP
were used, together with a 100-fold molar excess of peptide. In some
experiments, reticulocyte lysate (45 mg/ml) was used in place of
purified importins/Ran/NTF2 (21, 23-25) in the absence or presence of
the nuclear envelope permeabilizing detergent CHAPS (0.025%); in the
latter case; nuclear/nucleolar accumulation can only occur through
binding to nuclear/nucleolar components (24, 25). Image analysis and
curve fitting was performed as described (23-25).
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RESULTS |
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PTHrP Localizes in the Nucleus after Endocytosis by Intact UMR106.01 Cells-- We initially tested whether PTHrP could localize in the nucleus/nucleolus subsequent to receptor-mediated endocytosis. FITC-PTHrP 1-108 was incubated with UMR106.01 osteoblast cells without or with (nonspecific binding) preincubation/coincubation with an excess of unlabeled ligand, and subcellular localization was visualized and quantitated using confocal laser scanning microscopy (Fig. 2). Nuclear/nucleolar uptake reached maximal levels (4- and over 13-fold, respectively) by about 40 min (Fig. 2B). It could be blocked by an excess of either unlabeled PTHrP 1-108 (Fig. 2) or unlabeled PTHrP 1-34 (not shown), which is responsible for PTHrP binding to the PTHrP receptor, indicating that the uptake was receptor-mediated and thus comparable with that of other nuclear localizing ligands such as interleukin-5 (35) and growth hormone (36).
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PTHrP Is Recognized Specifically by -Importins--
The ability
of PTHrP to localize in the nucleus in intact cells implied that PTHrP
may possess a functional NLS (Fig. 1A). We accordingly set
out to quantitate the binding of mouse importin subunits to purified
polypeptides and synthetic peptides encompassing various parts of PTHrP
using an ELISA-based assay (22, 23), which we have used successfully to
determine the binding affinities (apparent dissociation constants
(Kd)) of importin subunits for different NLSs (20,
24, 37). Surprisingly, we found that PTHrP 1-108 and 35-141 were
recognized with high affinity by importin
and not importin
(Fig. 3, top panels, and Table I). Maximal binding by importin
was
only about 50% that of importin
, whereas the Kd
values for PTHrP 1-108/35-141 and importin
were 3.5 and 1.8 nM, respectively, about 2 orders of magnitude lower than
the corresponding values for importin
(Table I). The unexpected
finding that PTHrP was recognized by mImp
and not Imp
was
confirmed using importin subunits from yeast (31), results clearly
indicating high affinity recognition of PTHrP 1-108/35-141 by yImp
and not yImp
(Table I and data not shown). This high affinity
recognition by importin
was in direct contrast to our observations
using the same assay system for the conventional NLS-containing
proteins T-ag (22), Rb (24), and Dorsal (37), all of which require
importin
for NLS recognition and are not directly recognized by
importin
.
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Mapping of the PTHrP NLS--
Shorter peptides were used to define
the specific region of PTHrP recognized by importin . PTHrP 50-69,
67-94, and 83-108 were initially tested with results clearly
indicating binding to 67-94 and not to either 50-69 or 83-108 (Fig.
3, middle panels, and Table I). The latter, although
encompassing the NOS together with the NLS (Fig. 1A),
maximally bound only about 40% the amount of importin
bound by
67-94, with a Kd well above (over 300-fold in the
case of mImp
) that of the latter (Fig. 3, middle panels;
Table I; and data not shown), implying that residues 95-108 were not
were required for high affinity binding, whereas sequences
amino-terminal to the NLS (KKKKGK93) clearly were. Of other
peptides tested, PTHrP 66-94, 66-86, 67-84, and 67-86, only 66-94
bound importin
(Kd of around 2 nM;
Fig. 3, bottom panels, and Table I), consistent with this.
The fact that the Kd values for recognition of PTHrP
66-94 by importin
were significantly smaller (3-fold in the case
of mImp
) than those for 67-94 and entirely comparable with those
for PTHrP 1-108 (Table I) implied that Arg66 was involved
in binding to importin
. Based on the results for the various
peptides, the importin
binding site was concluded to comprise PTHrP
amino acids 66-94.
Nuclear Import of PTHrP 1-108 Can Be Reconstituted in Vitro Using
Importin and Ran--
To test whether nuclear import of PTHrP
1-108 could be mediated by importin
in the absence of importin
, we tested the ability of mImp
to mediate nuclear import in our
previously described reconstituted in vitro system (16,
21)3 in the absence or
presence of purified RanGDP and NTF2. In the absence of importins,
nuclear entry but not accumulation of PTHrP 1-108 (about 12.5 kDa) was
evident, in direct contrast to the strong accumulation in the
presence of mImp
together with Ran (Fig.
4 and Table
II). Maximal levels of nuclear
accumulation (Fn/cmax) were as high
as 8-fold those in the cytoplasm, half-maximal accumulation being
achieved within about 5 min (Fig. 4B; see Table II for
pooled data). mImp
could not substitute for mImp
in mediating nuclear accumulation (Fn/cmax of about 1.5), whereas the
combination of
and
was not as efficient as Imp
alone but
more efficient than Imp
alone (Fn/cmax of about 3.5; see
Table II). The inclusion of NTF2 in combination with Imp
and Ran
increased the import rate (half-maximal accumulation at 1.8 min; see
Table II) but reduced maximal transport by over 50%, resulting in an
Fn/cmax of about 2.5. It was
concluded that Imp
and Ran were sufficient to mediate nuclear import
of PTHrP 1-108 and that in contrast to the nuclear import of
proteins containing conventional NLSs such as
T-ag,3,4 neither Imp
nor
NTF2 were required. That PTHrP nuclear import could be mediated by
Imp
in the absence of Imp
was confirmed using the importin
subunits from yeast (Table II and data not shown), yImp
clearly
being able to mediate PTHrP 1-108 nuclear import in the presence of
RanGDP, and in the absence of yImp
, although not as efficiently as
mImp
(Table II and results not shown).
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To demonstrate formally that residues 66-94 were responsible for
nuclear transport of PTHrP 1-108, we performed competition experiments
using the different PTHrP peptides characterized above for importin recognition. Suboptimal conditions for in vitro transport
were used (see "Materials and Methods") to enable an inhibitory
effect of a 100-fold excess of peptide on nuclear accumulation of
FITC-PTHrP 1-108 to be assessed. Of the peptides/polypeptides tested,
PTHrP 1-108 and 67-94 inhibited maximal accumulation by over 80%
(Fig. 5). In contrast, PTHrP 67-84,
83-108, and 50-69, all of which do not encompass the complete NLS,
had negligible or minor effects, inhibiting transport maximally by less
than 30% (Fig. 5). It was concluded that PTHrP amino acids 67-94 are indeed responsible for the nuclear localization of PTHrP 1-108, consistent with the in vivo finding that deletion of PTHrP
residues 87-107 yields exclusively cytoplasmically localized PTHrP,
which exhibits impaired signaling (2, 5).
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Nuclear import measurements were also performed using cytosol in place
of purified subunits, rapid nucleolar as well as nuclear accumulation
being evident (Fig. 4A, bottom left panel).
Maximal levels of accumulation in the nucleus and nucleolus
(Fnu/cmax) were 3.4 and 6.9, respectively, with half-maximal levels being achieved in 1.0 and 1.6 min, respectively (Table II). This strong nucleolar accumulation of
PTHrP observed in the presence of cytosol was in contrast to the
results using importins and Ran/NTF2 (Fig. 4A, Table II, and
data not shown), where neither accumulation in nor exclusion from the
nucleolus was observed (Fig. 4A, top right panel,
and data not shown), indicating that nucleolar entry but not
accumulation above the nucleoplasmic level was occurring. The results
for high nucleolar accumulation in the presence of cytosol implied that
factors additional to Imp and Ran may be required to mediate
nucleolar accumulation. Nuclear/nucleolar accumulation in the presence
of cytosol was also observed in the presence of the nuclear
envelope-permeabilizing detergent CHAPS (Fig. 4A,
bottom right panel, and Table II). Because under these conditions nuclear accumulation occurs exclusively through binding in
the nucleus (24, 25), this implied that PTHrP 1-108 was capable of
binding to nuclear and nucleolar components, in stark contrast to
conventional NLS-containing proteins such as T-ag and Rb in this
experimental system (24). This binding was not dependent on the
presence of either exogenous cytosol or ATP (Table II).
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DISCUSSION |
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A number of recent reports have established that there are
multiple signal-dependent nuclear transport pathways
additional to that mediated by the importin /
heterodimer. These
include those mediated exclusively by importin
-related homologs
such as transportin or exportin where no importin
subunit homolog is involved (39-42) and those where soluble cytosolic receptors appear
not to be required at all (25, 43, 44). It has also recently been shown
that importin
, in the absence of importin
, can mediate nuclear
import of U snRNPs (41), as well as recognize NLS-containing proteins
such as HIV-1 Rev (18), GAL4 (20), and TCPTP (19), implying that
importin
may be able to function independently as a nuclear import
receptor. The findings of the present study not only establish that
PTHrP is similar to the above proteins in being recognized by importin
alone but also demonstrate conclusively for the first time that
importin
, in concert with Ran, is sufficient to mediate nuclear
import of PTHrP in the absence of importin
. Clearly, although the
details are not fully understood, novel NLS-dependent
nuclear import pathways exclusively mediated by importin
exist that
are presumably analogous to those mediated by transportin and other
importin
homologs (39). Based on the results here showing that NTF2
is able to reduce importin
/Ran-mediated transport in our
reconstituted system, it appears that, in contrast to the more
conventional importin
/
-mediated pathways (Refs. 28, and 45; see
also Refs. 33 and 46),3 NTF2 may be a negative regulator of
this pathway, as has been described for in vivo nuclear
import/export (47). Interestingly, importin
and Ran did not appear
to be sufficient to effect nucleolar accumulation of PTHrP in
vitro, which was, however, observed in vitro in the
presence of cytosol (Fig. 4A, bottom panels), and in intact cells subsequent to endocytosis of ligand (Fig. 2), implying
that other factors may be required. Based on the results of Tiganis
et al. (19) where both importin
and a 116-kDa protein (p116) appear to recognize the NLS-containing region of TCPTP, it could
be speculated that p116 may be an additional component of the pathway
shared by proteins recognized exclusively by importin
. Since
extracellularly added PTHrP can be targeted to the nucleus subsequent
to receptor-mediated endocytosis (see also below), the potential
importance of this novel nuclear import pathway should not be underestimated.
This study defines the 29-amino acid PTHrP sequence (amino acids
66-94) that confers high affinity binding to importin as the
region specific for targeting PTHrP to the nucleus. The data indicate
that although a consensus bipartite NLS sequence can be identified in
PTHrP comprising the clusters of basic amino acids 88-91 and 102-106
(2, 5), the region for importin binding and nuclear targeting appears
to require only amino acids 66-94. That it is directly responsible for
the nuclear transport of PTHrP is supported by in vivo
studies (2, 5) and the peptide competition experiments in this study
(Fig. 5). Comparison of PTHrP amino acids 66-94 with the importin
-recognized NLS of TCPTP (20), shown in Fig. 1B,
indicates a shared carboxyl-terminal cluster of five basic amino acids,
as well as a number of other conserved/similar residues (shown in
bold type), and predicted secondary structure where an
amino-terminal
-helix of 11-15 residues (including (N/Q)KV(E/Q))
flanks a
turn, adjacent to the basic cluster. Significantly,
although the minimal sequences for importin
-binding on the part of
Rev and GAL4 have not been determined, similar sequences within these
proteins can be identified (Fig. 1B). Clearly, although it
contains a carboxyl-terminal cluster of basic residues similar to the
T-ag NLS, the importin
binding NLS is quite distinct from importin
-recognized NLSs (17, 48); site-directed mutagenesis should enable
the key sequence elements of the PTHrP NLS to be defined more
precisely. Our preliminary characterization4 of the region
of importin
responsible for binding PTHrP indicates that, as for
TCPTP (19), amino acids 380-643 are involved, meaning that binding is
mediated by a region of importin
distinct from that recognizing
importin
.
Perhaps most significantly, this study demonstrates for the first time
that the polypeptide ligand PTHrP is able to accumulate rapidly in the
nucleus/nucleolus of intact UMR106.01 cells subsequent to
receptor-mediated uptake; that uptake is receptor-mediated is indicated
by the fact that accumulation can be competed by an excess of unlabeled
ligand (Fig. 2). The precise details of the pathway by which PTHrP is
able to localize in the nucleus subsequent to receptor-mediated
endocytosis are incomplete at this stage, but the speed of
nuclear/nucleolar accumulation (detectable within 35 min) implies that
a lysosomal pathway is unlikely to be involved, as is the case for
receptor-mediated uptake/nuclear localization of growth hormone (36),
interleukin-5 (35), and other polypeptide ligands (49, 50). In
analogous fashion to growth hormone and interleukin-5, it seems
possible that PTHrP is internalized, rapidly escapes from the endosomal
vesicle by an as yet undetermined mechanism (see Ref. 36), and then
undergoes initial high affinity interaction with importin (Kd of 2 nM) as well as other factors of
the cellular nuclear import machinery including RanGDP to accumulate
rapidly in the nucleus/nucleolus, with binding to nuclear components
(Fig. 4A)4 possibly contributing to the latter
as well.
Although its functional importance has been demonstrated in diverse
cell types (2, 5), the exact signaling role of nuclear targeting of
PTHrP is largely unclear at this stage. By analogy with other nuclear
targeting polypeptide ligands, possibilities include PTHrP-mediated
piggy back co-transport of the PTH/PTHrP receptor to the nucleus (shown
for interleukin-5-36), where it may activate signaling components (15,
38), or the modulation of gene transcription through binding of PTHrP,
with or without bound receptor, to nuclear factors or chromatin itself
(49, 50). The ability of PTHrP to accumulate in the nucleus/nucleolus in vitro in the absence of an intact nuclear envelope (Fig.
4A) implies that the latter is feasible, but this will of
course require confirmation using a variety of approaches.
Determination of the precise nature of signaling by nuclear PTHrP, as
well as the details of its novel nuclear import pathway, should enable
insight into the importance of nuclear targeting to the unique
endocrine, autocrine/paracrine, and intracrine roles of PTHrP in malignancy.
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ACKNOWLEDGEMENTS |
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We are indebted to Tony Tiganis for helpful discussions, Frosa Katsis for peptide synthesis and amino acid analysis, Ginny Leopold for tissue culture, Patricia Ho for performing the bioassay for PTHrP, and Bryce Paschal (University of Virginia, Charlottesville) for providing recombinant NTF2.
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FOOTNOTES |
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* This work was supported by grants from the Anti-Cancer Council of Victoria and National Health and Medical Research Council (to T. J. M. and M. T. G.) and by Chugai Pharm. Co. (Japan).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of an Anti-Cancer Council of Victoria Post-Doctoral Fellowship.
¶ To whom correspondence should be addressed: Dept. of Biochemistry & Molecular Biology, John Curtin School of Medical Research, P.O. Box 334, Canberra City, ACT 2601, Australia. Tel.: 612-62494188; Fax: 612-62490415; E-mail: David.Jans{at}anu.edu.au.
2 M. H. C. Lam, C. M. House, K. I. Mitchelhill, B. Sarcevic, A. Cures, R. Ramsay, B. E. Kemp, T. J. Martin, J. M. Moseley, and M. T.Gillespie, submitted for publication.
3 S. Hübner, H. M. S. Smith, W. Hu, C. K. Chan, H.-P. Rihs, B. M. Paschal, N. V. Raikhel, and D. A. Jans, submitted for publication.
4 M. Lam, L. J. Briggs, W. Hu, T. J. Martin, M. T. Gillespie, and D. A. Jans, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PTHrP, parathyroid hormone-related protein; PTH, parathyroid hormone; T-ag, SV40 large tumor antigen; NLS, nuclear localization sequence; NOS, nucleolar localization sequence; HIV, human immunodeficiency virus; TCPTP, T-cell protein tyrosine phosphatase; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
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