| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Medicine, Division of Endocrinology and Metabolism (J.J., S.J.F.), and Department of Cell Biology (R.G., Y.Z., S.J.F.), University of Alabama, and Veterans Affairs Medical Center (S.J.F.), Birmingham, Alabama 35294; Center for Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Northwestern University Medical School (C.A.B., G.B.), Chicago, Illinois 60611; and Immunex Corp. (R.A.B.), Seattle, Washington 98101
Address all correspondence and requests for reprints to: Dr. Stuart J. Frank, University of Alabama, 1530 3rd Avenue South, BDB 731, Birmingham, Alabama 35294-0012. E-mail: frank{at}endo.dom.uab.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A further level of regulation of GH action is modulation of GHR availability at GH target tissues. Down-regulation of GHRs by GH is well described, although the mechanisms underlying it are incompletely understood (9). GHR levels and/or cell surface availability can also be regulated by hormones other than GH (10, 11). The proteolytic process leading to GHBP by shedding might also be expected to regulate GHR levels, even in cell types and species that do not generate the majority of their circulating GHBP by a proteolytic mechanism.
We recently described in human IM-9 cells a phorbol ester-induced protein kinase C (PKC)-dependent shedding of GHBP that is inhibited by compound 3 (IC3; Immunex Corp., Seattle, WA), a hydroxamate-based inhibitor of metzincin metalloproteases (12). This GHBP generation is accompanied by loss of cell-associated immunoreactive GHR and accumulation of a cell-associated GHR remnant protein detected with antibodies directed at the receptor’s cytoplasmic, but not extracellular, domain. Although it is PKC dependent, the signaling pathways that mediate such phorbol 12,13-myristate acetate (PMA)-induced GHR processing are as yet unknown. It is also not known whether other stimuli promote GHR proteolysis and if GHR proteolysis can lead to receptor down-regulation.
We now explore these processes further and demonstrate that PMA-induced IC3-inhibitable GHR proteolysis and GHBP shedding can also be detected in murine 3T3-F442A and 3T3-L1 preadipocytes that endogenously express GHRs and in Chinese hamster ovary (CHO) cells stably expressing the rabbit GHR (rbGHR), although the degree of GHBP shedding is much smaller for the murine GHR than for the rabbit or human GHRs. The PMA-induced GHR proteolysis in 3T3-F442A, 3T3-L1, and CHO-rbGHR cells is significantly reduced by pretreatment with inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1), the upstream activator of mitogen-activated protein (MAP) kinases (MAPKs), suggesting involvement of the MAPK pathway in regulation of this PKC-dependent effect.
To determine whether other, possibly more physiologically relevant, stimuli induce GHR proteolysis, we also tested the effects of platelet-derived growth factor (PDGF) and serum on this process. Treatment of serum-deprived cells with PDGF (in 3T3-F442A cells) or serum (in 3T3-F442A and CHO-rbGHR cells) promotes GHR proteolysis, which is inhibited by IC3. PMA-, PDGF-, and serum-induced GHR processing is associated with substantial decreases in GH- induced activation of Janus kinase-2 (JAK2), which are also nearly completely prevented by IC3. These findings strongly suggest that inducible metalloprotease-mediated GHR proteolysis constitutes an important mechanism of receptor down-regulation and modulation of GH signaling.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cells and cell culture
3T3-F442A cells, kindly provided by Drs. H. Green (Harvard
University, Boston, MA) and C. Carter-Su (University of Michigan, Ann
Arbor, MI), and 3T3-L1 cells (a gift from Dr. R. Hardy, University of
Alabama at Birmingham, Birmingham, AL) were cultured in DMEM
(4.5 g/liter glucose; Cellgro, Inc.) supplemented with 10% calf
serum (Biofluids, Rockville, MD) and 50 µg/ml gentamicin
sulfate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from
Biofluids). CHO cells (a gift from Dr. J. Kudlow,
University of Alabama at Birmingham, Birmingham, AL) were maintained in
DMEM (1 g/liter glucose; Cellgro, Mediatech, Herndon, VA) supplemented
with 7% FBS (Biofluids) and 50 µg/ml gentamicin
sulfate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from
Biofluids). Stable transfection of CHO cells was achieved
by introducing pSX rbGHR (14) (20 µg in 3 ml DMEM in
60 x 15-mm dishes) along with 1 µg pSP65-SR
.2-HAtag-Hygro
(empty vector carrying the hygromycin resistance marker, provided by
Dr. M. Streuli, Dana-Farber Cancer Institute, Boston, MA), using
lipofectin (Life Technologies, Inc., Grand Island, NY),
selected in 500 µg/ml hygromycin B, and characterized as reported
previously (15).
Antibodies
The rabbit polyclonal sera,
anti-GHRcyt-AL37 (directed at residues 271–620
of the hGHR (the entire cytoplasmic domain)),
anti-GHRcyt (directed at residues 317–620 of
hGHR), and anti-JAK2AL33 (directed at residues
746-1129 of murine JAK2), have been described, as has the procedure for
affinity purification of anti-GHRcyt-AL37
(16, 17). Anti-MAPK affinity-purified rabbit antibody
[directed at residues 333–367 of rat extracellular signal-regulated
kinase 1 (ERK1); recognizes both ERK1 and ERK2], antiphosphotyrosine
(APT) monoclonal antibody 4G10 (both from Upstate Biotechnology, Inc., Lake Placid, NY) and antiphospho-MAPK affinity-purified
rabbit antibody (recognizing the dually phosphorylated
Thr183 and Tyr185 residues
that correspond to the active forms of ERK1 and ERK2; Promega Corp.) were all purchased commercially.
Plasmid construction
The pSX plasmid (a gift from Dr. J. Bonifacino, NIH, and
Dr. K. Arai, DNAX, Palo Alto, CA), which drives eukaryotic
protein expression from the SR promoter (composed of the simian
virus 40 early promoter and the R-U5 segment of the human T cell
lymphotrophic virus-1 long terminal repeat), has been described
previously (18). Preparation of the rbGHR complementary
DNA (a gift from W. Wood, Genentech, Inc., South San
Francisco, CA) and its ligation into the pSX vector have been described
previously (14, 19).
Inhibitor pretreatment, cell stimulation, protein extraction,
electrophoresis, and immunoblotting
Serum starvation of 3T3-F442A cells and CHO transfectants was
accomplished by substitution of 0.5% (wt/vol) BSA (fraction V,
Roche Molecular Biochemicals, Indianapolis, IN) for serum
in their respective culture media for 16–20 h before experiments.
Pretreatment and stimulation were carried out at 37 C in serum-free
medium. GF109203X (0.5 µM), PD98059 (100
µM), U0126 (10 µM), IC3 (50
µM), or dimethylsulfoxide (DMSO; as a vehicle control)
were incubated with serum-starved cells for 15, 30, 30, or 15 min,
respectively, before treatment with PMA (1 µg/ml, unless otherwise
noted), NEM (5 mM), GH, PDGF-BB, calf serum (at the
indicated concentrations), or vehicle controls. The inhibitors and
stimulators were diluted from DMSO-dissolved stock solutions
(GF109203X, 1 mM; PD98059, 50 mM; U0126, 10
mM; IC3, 10 mM; PMA, 1 mg/ml; NEM, 1
M), 10 mM acetic acid stock solution (PDGF-BB,
1 mg/ml), or undiluted calf serum. Stimulations were performed at 37 C.
Details of the treatment protocol have been described previously
(14, 15). Briefly, cells were stimulated in confluent
60 x 15-mm dishes (or, for immunoprecipitation experiments,
100 x 20-mm dishes; Falcon, Becton Dickinson & Co.,
Franklin Lakes, NJ) in serum starvation medium. Stimulations were
terminated by washing the cells once with and then harvesting by
scraping in ice-cold PBS in the presence of 0.4 mM sodium
orthovanadate (PBS-vanadate). Pelleted cells were collected by brief
centrifugation. For each cell type, pelleted cells were solubilized for
15 min at 4 C in fusion lysis buffer [1% (vol/vol) Triton X-100, 150
mM NaCl, 10% (vol/vol) glycerol, 50 mM
Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1
mM PMSF, 1 mM sodium orthovanadate, 10
mM benzamidine, and 10 µg/ml aprotinin] as indicated.
After centrifugation at 15,000 x g for 15 min at 4 C,
the detergent extracts were either subjected to immunoprecipitation or
directly electrophoresed under reducing conditions, as indicated below.
In some experiments harvested cells were solubilized directly in
reducing SDS-PAGE sample buffer, as indicated, before
electrophoresis.
Anti-GHRcyt-AL37 and anti-JAK2AL33 immunoprecipitations were performed as previously described (15, 16). Resolution of proteins under reducing conditions by SDS-PAGE, Western transfer of proteins, and blocking of Hybond-ECL (Amersham Pharmacia Biotech, Arlington Heights, IL) with 2% BSA were performed as previously described (14, 15, 20). Immunoblotting with affinity-purified anti-GHRcyt-AL37 and anti-GHRcyt, anti-JAK2AL33 (1:1000), 4G10 (1:1000), anti-MAPK (1:1000), and antiphospho-MAPK (1:5000) with horseradish peroxidase-conjugated antirabbit or antimouse secondary antibodies (1:2000) and ECL detection reagents (all from Amersham Pharmacia Biotech) and stripping and reprobing of blots were accomplished according to the manufacturer’s suggestions.
GHBP assay
GHBP activity was measured in conditioned medium by a
standardized GH binding assay, as previously reported (12, 21). Medium from CHO transfectant cells was directly assayed,
whereas medium from 3T3-F442A cells required concentration to yield
detectable GHBP activity. In the latter case, 7 ml medium were
concentrated by ultrafiltration on Amicon Centricon 10 devices to a
final volume of approximately 100 µl. GHBP recovery under those
conditions is more than 95%. Conditioned medium from cells treated as
indicated was incubated either undiluted (CHO transfectant cells; 1 ml)
or after concentration (3T3-F442A cells; final incubation volume, 150
µl) with freshly labeled [125I]hGH (
0.5
ng, 15 µl) for 45 min at 37 C. Bound GH was then immediately
separated from free GH by gel chromatography on a Sephadex G-100 column
at 4 C. The fraction of GH bound was determined by peak integration. In
experiments in which treatment with PMA or PMA plus inhibitors was
tested, the GHBP present in negative control (DMSO-treated) samples,
which was minimal, was subtracted. Statistical analysis was performed
with unpaired t test or ANOVA, followed by Newman-Keuls
test, as appropriate. P < 0.05 was accepted as
significant.
Densitometric analysis
Densitometry of ECL immunoblots was performed using a solid
state video camera (Sony-77, Sony Corp.) and a 28-mm MicroNikkor
lens over a light box of variable intensity (Northern Light Precision
890, Imaging Research, Inc., Toronto, Canada).
Quantification was performed using a Macintosh II-based image analysis
program (Image 1.49, developed by W. S. Rasband, Research Services
Branch, NIMH, Bethesda, MD). The fraction of full-length GHRs remaining
in extracts from PMA-, PDGF-, or calf serum-treated cells was estimated
for each condition by measuring by densitometry the intensity of the
specifically detected GHR signal relative to that signal present within
the same experiment in extracts from unstimulated cells. Relative
specific JAK2 tyrosine phosphorylation was estimated by normalizing the
densitometric signal for tyrosine phosphorylation (APT immunoblot) of
immunoprecipitated JAK2 by the abundance of JAK2
(anti-JAK2AL33 immunoblot) in that precipitate;
this ratio for GH-stimulated samples without prior pretreatment was
considered equal to 100%. As indicated when graphically shown, pooled
data from several experiments are displayed as the mean ±
SEM. The significance of differences of pooled results was
estimated using unpaired t tests.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Despite evidence that GHR proteolysis does not account for a
significant portion of the circulating GHBP in rodents
(4), we wondered whether PMA might also promote GHR
proteolysis in murine cell lines. The 3T3-F442A fibroblast expresses
immunologically detectable GHRs and displays robust biochemical
(e.g. cellular tyrosine phosphorylation) and functional
(e.g. initiation of adipocyte differentiation) responses
upon GH treatment (14, 23, 24), and hence was selected as
a study model. We treated 3T3-F442A cells with PMA for 30 min, after
which detergent-soluble proteins were extracted and resolved by
SDS-PAGE (Fig. 1A
). Immunoblotting with
anti-GHRcyt-AL37, our previously described
antibody to the GHR cytoplasmic domain (15, 16), revealed
that PMA acutely promoted substantial loss of the full-length GHR
(bracketed protein in lane 1 vs. lane 2). This loss of GHR
was accompanied by increased abundance of an
anti-GHRcyt-AL37-reactive protein of roughly 65
kDa (arrowhead), which, by analogy to our previous findings
in IM-9 cells, we refer to as a GHR remnant protein. This same pattern
of GHR loss and remnant accumulation in response to PMA was also
specifically detectable when detergent-extracted proteins from the
3T3-F442A cells were first immunoprecipitated with
anti-GHRcyt-AL37 before SDS-PAGE and
anti-GHRcyt-AL37 immunoblotting (not
shown).
|
, lanes 1–3, and is graphically displayed in Fig. 1C, in which the
abundance of remaining GHR detectable by
anti-GHRcyt-AL37 immunoblotting in several
experiments was densitometrically determined. The profile of this rapid
and substantial PMA-induced loss of immunologically detectable GHR
(>60% loss in 15 min) is quite similar to the inhibitory effect of
PMA on [125I]GH binding and GH-induced JAK2
tyrosine phosphorylation observed by King et al. previously
in these same cells (25). PMA-induced GHR proteolysis was
also observed in 3T3-L1 preadipocytes with a time course similar to
that seen for 3T3-F442A cells (Fig. 3A and data not shown) and was seen
in both cell types regardless of whether detergent-soluble extracts (as
in Fig. 1) or total cell extracts (directly lysed in 1% SDS-containing
PAGE buffer; not shown) were examined.
|
, lanes 4–6
vs. 1–3). Murine GHR proteolysis induced by PMA was also
prevented when the hydroxamate-based inhibitor of metalloproteases,
IC3, was present. IC3 strongly inhibited both the PMA-induced loss of
full-length GHR and the corresponding appearance of the cytoplasmic
domain-containing GHR remnant (Fig. 1A, lanes 3 and 4 vs.
lanes 1 and 2).
To confirm that the PMA-induced IC3-inhibitable GHR proteolysis in
3T3-F442A cells occurred during the period of incubation rather than
adventitiously after cell harvesting and lysis, we measured the content
of GHBP present in the cell supernatants collected after PMA treatment
periods and before cell harvesting. It would be expected that GHR
proteolysis in these cells, by analogy to that described by us and
others in human cells (12, 26, 27, 28, 29), might be accompanied
by release from the cells into the supernatant of shed GHR
extracellular domain that maintains the capacity to bind GH. As
graphically displayed in Fig. 1D, PMA treatment for as little as 15 min
promoted release of GHBP from 3T3-F442A cells. Although the abundance
of the GHBP shed from 3T3-F442A cells was low and only detectable after
50-fold concentration of medium (expressed in this case as percentage
of GH bound per 150 µl of a 50-fold concentrate of an original 7 ml
cell supernatant2), its
specifically detectable appearance mirrored the pattern of PMA-induced
loss of immunologically detectable GHRs (Fig. 1, D vs. C).
Moreover, this release, like GHR proteolysis, was nearly completely
(94%) inhibited by IC3 (Fig. 1, D and A). Although we do not interpret
our data to indicate that GHR proteolysis contributes quantitatively to
the pool of GHBP found in the rodent circulation (4),
these results clearly indicate that PMA-induced
metalloprotease-mediated GHR cleavage and GHBP shedding can occur in
murine cells.
To study GHR proteolysis and GHBP shedding further, we generated
another model system by stably expressing the rabbit (rb) GHR in
Chinese hamster ovary (CHO) cells. As shown by others, CHO cells
express no endogenous GHR and have proven to be a useful vehicle for
GHR expression and studies of GH signaling and GHR trafficking
(15, 30, 31). Immunoprecipitation and immunoblotting of
rbGHRs from serum-starved CHO-rbGHR cells revealed the fully
glycosylated (bracket) and incompletely glycosylated
(arrow) forms of the transfected receptor (Fig. 2A, lane 1), as we have previously shown
in COS-7 cells transfected with the rbGHR (32). As found
in 3T3-F442A cells, PMA treatment acutely promoted substantial loss of
the GHR and corresponding accumulation of an
anti-GHRcyt-AL37-reactive lower
Mr protein (arrowhead) of roughly 68
kDa (Fig. 2A, lane 2). Although we do not yet know the precise
composition of this lower form, it migrates similarly to a recombinant
version of the rbGHR that lacks the receptor extracellular domain
(rbGHRdel ext), which we have previously
characterized and referred to as the GHR remnant (12).
Also, as we observed in 3T3-F442A cells, both the PMA-induced loss of
GHR and the accumulation of cell-associated remnant were inhibited by
pretreatment of the cells with either GF109203X (not shown) or IC3
(Fig. 2A, lane 3). In concert with these results, analysis of the cell
supernatants revealed easily detectable PMA-induced shedding of GHBP
that was inhibited by over 80% by IC3 (Fig. 2B; see Footnote 1). Thus,
our previous observations and those presented herein indicate a
susceptibility to PKC- and metalloprotease-mediated GHR proteolysis and
GHBP shedding among several species of GHRs and cell types.
|
, pretreatment of both 3T3-L1
(upper left panel) and 3T3-F442A (upper
right panel) fibroblasts with PD98059 (100
µM) partially inhibited the PMA (1
µg/ml)-induced GHR loss and remnant accumulation observed with
anti-GHRcyt-AL37 immunoblotting of cell extracts
(lanes 1–3 and 5–7). The partial nature of inhibition by PD98059 is
highlighted by comparison to the more complete inhibition rendered by
GF109203X (lanes 4 and 8). In these same samples, PD98059 and GF109203X
conferred a very similar and nearly complete degree of inhibition of
PMA-induced activation of the MAPKs, ERK1 and ERK2 (as assessed in the
left and right middle panels by
immunoblotting with a state-specific antibody (antiphospho-MAPK) that
specifically recognizes the phosphorylated threonine 183 and tyrosine
185 residues in the MAPK molecule that correlate to its enzymatic
activation). Yet in this and other such experiments, densitometric
analysis of anti-GHRcyt-AL37 immunoblots revealed
that PMA-induced loss of GHR was inhibited, on the average, by 56% by
PD98059 compared with 94% by GF109203X; similarly, the PMA-induced
increase in remnant abundance was diminished by 39% by PD98059
vs. 85% by GF109203X. The differences in the inhibition of PMA-induced receptor proteolysis relative to MAPK activation state could not be accounted for by a change in the abundance of ERK1 and ERK2 (bottom panel, lanes 1–4 and 5–8) in any of the samples. In other experiments with 3T3-F442A cells, acute incubation with PD98059 or GF109203X had no effect on cell viability or on the ability of the cells to respond to GH stimulation with JAK2 activation (41); thus, we think it unlikely that the effects of each inhibitor on PMA-induced GHR proteolysis can be attributed to drug toxicity. We also tested the effect of a distinct MEK1 inhibitor, U0126 (42), and found that, similarly to PD98059, U0126 partially inhibited the PMA-induced loss of GHR and accumulation of remnant in 3T3-F442A cells (data not shown).
We also examined the effect of MEK1 inhibition on PMA-induced GHBP
shedding. We measured GHBP in the conditioned medium of CHO-rbGHR cells
treated with PMA after pretreatment in the presence or absence of
PD98059 (Fig. 3B). Again, pretreatment with PD98059 resulted in a
significant, but partial (54.8 ± 6.3%) reduction of GHBP shed
into the medium in response to PMA. Thus, inhibitors of MEK1 partially
inhibited both PMA-induced GHR proteolysis and GHBP shedding, further
substantiating the linkage between these two phenomena and indicating
that at least part of the PMA-induced GHR processing in 3T3-F442A and
CHO-rbGHR cells is probably attributable to MEK1, ERK(s), or other
MEK1-dependent kinase(s).
As we and others have previously demonstrated, the sulfhydryl
alkylating reagent, NEM, also promotes GHR proteolysis and GHBP
shedding (12, 26, 27, 43). Notably, we have previously
shown that NEM- and PMA-induced GHR processing was inhibited by IC3,
but that, unlike PMA, NEM-induced receptor proteolysis was not affected
by inhibition of PKC activity. The two stimuli were thus proposed to
achieve activation of a common protease activity by potentially
distinct pathways (12). We extended this analysis by
testing NEM’s effects on GHR proteolysis in 3T3-F442A cells (Fig. 3, C
and D). Acute treatment with NEM (5 mM, 5 min) promoted
substantial loss of anti-GHRcyt-AL37
immunoblottable GHR and an increase in detectable remnant protein (Fig. 3C, lane 2 vs. lane 1). Interestingly, unlike the findings
for PMA-induced GHR proteolysis (Fig. 3A), NEM-induced GHR proteolysis
in 3T3-F442A cells was unaffected by pretreatment with either the MEK
inhibitor (PD98059) or the PKC inhibitor (GF109203X; Fig. 3C, lanes 3
and 4 vs. lanes 1 and 2). However, consistent with our
previous findings in IM-9 cells, NEM-induced GHR proteolysis was
inhibited by IC3 (lane 5 vs. lane 2). Further, the
NEM-induced proteolysis was not associated with activation of MAPK, as
shown in the antiphospho-MAPK blot in Fig. 3D. These findings strongly
support the proposition that NEM and PMA operate via distinct
mechanisms to promote GHR cleavage.
PDGF and serum induce IC3-inhibitable GHR proteolysis
Our results to date indicate that GHR proteolysis and shedding of
the receptor’s extracellular domain can be detected in several cell
types in response to pharmacological activation of PKC with phorbol
ester or in response to sulfhydryl alkylators. To further understand
other potential regulators of this process, we tested whether growth
factor stimulation might also activate GHR proteolysis; such stimuli
have been recently implicated in the shedding of several other cell
surface proteins (44, 45, 46). PDGF, in particular, was
recently shown to diminish surface GHR expression in 3T3-F442A cells,
although the mechanism(s) of this receptor loss remains uncertain
(47). Our treatment of 3T3-F442A cells with PDGF-BB (40
ng/ml) for 30 min led to substantial loss of GHR abundance, as assessed
by anti-GHRcyt-AL37 immunoblotting (Fig. 4A, upper panel,
lane 2 vs. lane 1). Densitometric evaluation indicated that
PDGF induced, on the average, a 56% decrease in GHR abundance (Fig. 4A, lower panel), consistent with the degree of loss of
surface GHR observed by Rui et al. (46) in
similar experiments. This GHR loss was associated with substantial
generation of the GHR remnant protein (Fig. 4A, upper panel,
lane 2 vs. 1). Notably, both the loss of GHR and the
accumulation of remnant induced by PDGF were blocked by IC3
(upper panel, lane 3 vs. lanes 1 and 2, and
lower panel), strongly suggesting that the PDGF-induced GHR
loss was caused by receptor proteolysis. Similarly, treatment of
serum-starved cells with 10% calf serum also caused GHR loss (36%)
and remnant accumulation, both of which were prevented by pretreatment
with IC3 (Fig. 4B).
|
) in serum-starved
CHO-rbGHR cells were easily detected in response to treatment for 60
min with 10% calf serum. The serum-induced GHBP shedding was inhibited
80% by inclusion of IC3 during the treatment period. These data
provide the first evidence of which we are aware that a
nonpharmacological stimulus can elicit GHBP formation by
proteolysis.
GHR proteolysis contributes to PMA- and growth factor-induced
down-regulation of GHR signaling
Down-regulation of GHR signaling by PMA and PDGF has been
described previously (25, 47, 48) and, as indicated above,
has been hypothesized to be accounted for by loss of GHRs through
degradative or redistributive mechanisms. We tested whether the induced
proteolysis of the GHR that we observed in response to PMA, PDGF, or
calf serum affected GH-induced signaling. 3T3-F442A cells (Fig. 5A) and CHO-rbGHR cells (Fig. 5B) were
pretreated with PMA, PMA plus IC3, or vehicle alone for 15 min before
treatment with either GH alone (50 ng/ml) or its vehicle for an
additional 15 min. GH-induced JAK2 tyrosine phosphorylation was then
assessed by anti-JAK2AL33 immunoprecipitation and
antiphosphotyrosine immunoblotting (Fig. 5, A and B, upper
panels). JAK2 abundance in each sample was normalized by
reprobing of the blots with anti-JAK2AL33
(middle panels). A ratio of tyrosine-phosphorylated
JAK2/total JAK2 was determined for each condition in several such
experiments (lower panels). GH-induced JAK2 activation was
reduced significantly (by >74% in both cell types) by pretreatment
with PMA (Fig. 5, A and B, lanes 1–3). Notably, inclusion of IC3 with
PMA nearly completely reversed PMA’s inhibition of GH-induced JAK2
tyrosine phosphorylation (lane 4 vs. lanes 2 and 3). These
results strongly support the conclusion that PMA down-regulation of GHR
abundance by metalloprotease-mediated proteolysis results in inhibition
of GH- induced JAK2 activation.
|
) or
calf serum (Fig. 5D). In each instance, growth factor pretreatment of
3T3-F442A cells significantly reduced GH-induced JAK2 activation (lane
3 vs. lane 2; approximately 50% reduction for PDGF and 37%
reduction for calf serum). Inclusion of IC3 nearly completely inhibited
PDGF- and serum-induced down-regulation of GH stimulation of JAK2
tyrosine phosphorylation (Fig. 5, C and D, lane 4 vs. lanes
2 and 3), just as seen for PMA-, PDGF-, and serum-induced
down-regulation of GHR abundance and for PMA-induced down-regulation of
GH-induced JAK2 activation. Neither PDGF nor calf serum alone had any
effect on JAK2 tyrosine phosphorylation (not shown). These data are
important in that they suggest that certain growth factors can
down-regulate both GHR abundance and GHR signaling by a
metalloprotease-mediated proteolytic mechanism. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Proteolysis of the GHR has largely been considered in the context of being the principal mechanism accounting for the generation by shedding of circulating GHBP in humans and rabbits (7, 49). This is in contrast to mice and rats, in which the vast majority of circulating GHBP is believed to be derived from an alternatively spliced GHR that lacks the transmembrane and cytoplasmic domains and, by virtue of replacement of the transmembrane domain with a hydrophilic peptide, is thus secreted (2, 3, 4). Our findings that 3T3-F442A, a GH-responsive murine cell line, can be induced to proteolyze its GHR and release a GHBP (albeit at very low abundance) are notable in this regard. Others (50) have recently observed that the monkey GHR, when overexpressed transiently, spontaneously releases GHBP (presumably by proteolytic shedding) into the medium, but that, in addition to a full-length GHR mRNA, monkey tissues express a spliced mRNA that encodes a GHBP like that found in rodents. These findings were taken as evidence for coexistence within a single species of the two mechanisms for GHBP generation.
Although we were able to detect shed murine GHBP in concert with biochemical evidence for GHR proteolysis (loss of GHR, accumulation of GHR remnant) without overexpressing the GHR, we interpret our results cautiously regarding their implications for mechanisms of GHBP generation. PMA-induced GHBP concentrations in murine cell medium are at least 50-fold lower than those in medium from CHO transfectants or IM-9 cells. Our findings therefore do not constitute a refutation of the view that rodent circulating GHBP is largely derived from the product of the alternatively spliced mRNA referred to above, nor do they indicate that shedding contributes significantly to the circulating GHBP pool in the rodent under usual conditions. However, our findings using both 3T3-F442A and CHO-rbGHR cells in concert with our previous results (12) are important in that they indicate that GHRs in various species are susceptible to processing by proteolytic machinery with some common features. Assessment of the relative degree to which rodent and nonrodent GHRs can serve as targets of this machinery will await studies that carefully compare the expression of each receptor in various cell lines.
Our current results provide insights into the pathway(s)
involved in the regulation of GHR proteolysis and GHBP shedding and the
physiological roles of this receptor processing. Using two independent
cell lines of different species (mouse and hamster), we found that a
process that culminates in IC3-inhibitable GHR proteolysis can be
activated by PMA via a pathway dependent on PKC activation and at least
partially dependent on MEK1 activation (that is, it is partially
inhibited by two distinct MEK1 inhibitors). This finding of a
susceptibility of GHR proteolysis to MEK1 inhibitors is in concert with
the recent findings of Gechtman et al. (44),
Desdouits-Magnen et al. (45), and Fan and
Derynck (46) for the shedding of heparin-binding epidermal
growth factor (44), the secretion of the soluble Alzheimer
amyloid precursor protein (45), and the shedding of
transforming growth factor- and some other surface proteins
(46). In those studies, using both inhibitors and
dominant-negative approaches, the MAPK pathway was also implicated in
stimulation of metalloprotease-mediated proteolysis and shedding. Our
observation that PMA-induced GHR proteolysis is partially, rather than
completely, sensitive to MEK1 inhibition raises the interesting
possibility that pathways downstream of both PKC and MEK1 may each
contribute to PMA-induced receptor proteolysis to some degree and may
therefore be points of differential regulation of GHR processing. This
is a worthwhile area for future investigation.
Our findings that NEM can induce GHR proteolysis in 3T3-F442A cells and CHO-rbGHR cells (not shown) complement our previously reported results with IM-9 cells (12). In each system, NEM-induced GHR proteolysis and/or GHBP shedding, although sensitive to IC3, is insensitive to inhibitors of PKC and MEK1. This strongly suggests that the pathway(s) by which PMA leads to metalloprotease-dependent GHR processing is different from that engaged by NEM. Future studies will be required to determine which protease(s) is being regulated by each stimulus and whether both types of stimuli are acting at a point of convergence of proteolysis-promoting pathways or, for example, whether one stimulus (e.g. PMA) is acting as a metalloprotease activator and the other (e.g. NEM) is working to make the substrate (GHR) more susceptible to cleavage. Although we cannot yet decipher this situation, our findings argue that studies examining GHR proteolysis and GHBP shedding using only NEM as an inducer should be interpreted with caution with respect to their generality.
A major implication of our current work relates to mechanisms of GHR down-regulation and modulation of GH signaling on the basis of receptor availability. Although PMA- and PDGF-mediated down-regulation of GHR abundance and signaling have been reported, the work described herein is the first to establish that a substantial fraction of such down-regulation can be attributed to GHR proteolysis. We also observe that serum itself, when applied to serum-starved cells in culture, can down-regulate GH signaling and diminish receptor abundance via metalloprotease-mediated proteolysis. Further, we establish in CHO-rbGHR cells that GHBP shedding can be experimentally detected in response to serum stimulation. These observations that PDGF and serum stimulation can elicit GHR proteolysis and GHBP shedding will serve as the framework to explore the degree to which other physiologically relevant stimuli can regulate these processes and similarly affect GHR signaling. It will be interesting and important to determine whether it is the generation of the shed GHBP, the decrease in GHR abundance, and/or the generation of the GHR transmembrane/cytoplasmic domain remnant that account for the diminished capacity of GH to elicit JAK2 tyrosine phosphorylation after receptor proteolysis occurs. Such studies will require identification of the enzyme(s) that catalyzes GHR cleavage, determination of the cleavage site, and isolation of model systems in which cleavage can be prevented so as to test which component (increase in soluble GHBP, receptor loss, or remnant accumulation) is responsible for the effects on full-length receptor signaling.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Estimates of molar concentrations of GHBP in
conditioned medium after PMA treatment, based on measurements of
dissociation rates during gel filtration (8 51 ) and
calculation of bound GH fractions based on the law of mass action using
a Kd of 10-9 (52 53 ), yield mean values of 5.6 pM for 3T3-F442A
medium and 360 pM for CHO-rbGHR medium, an approximately
65-fold difference. The mean GHBP concentration in IM-9
cell-conditioned medium after PMA treatment (12 ) is
550 pM, 100-fold greater than that in 3T3-F442A
medium.
Received October 6, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
from cells. Nature 385:729–733[CrossRef][Medline]
.
Nature 385:733–736[CrossRef][Medline]
by
the metalloprotease Kuzbanian. Science 283:91–94 and other transmembrane proteins is induced by receptor
tyrosine kinase activation and MAP kinase signaling cascades. EMBO J 18:6962–6972[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  X. Wang, J. Jiang, J. Warram, G. Baumann, Y. Gan, R. K. Menon, L. A. Denson, K. R. Zinn, and S. J. Frank Endotoxin-Induced Proteolytic Reduction in Hepatic Growth Hormone (GH) Receptor: A Novel Mechanism for GH Insensitivity Mol. Endocrinol., June 1, 2008; 22(6): 1427 - 1437. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Loesch, L. Deng, X. Wang, K. He, J. Jiang, and S. J. Frank Endoplasmic Reticulum-Associated Degradation of Growth Hormone Receptor in Janus Kinase 2-Deficient Cells Endocrinology, December 1, 2007; 148(12): 5955 - 5965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Yang, X. Wang, J. Jiang, and S. J. Frank Role of the Growth Hormone (GH) Receptor Transmembrane Domain in Receptor Predimerization and GH-Induced Activation Mol. Endocrinol., July 1, 2007; 21(7): 1642 - 1655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Deng, K. He, X. Wang, N. Yang, C. Thangavel, J. Jiang, S. Y. Fuchs, and S. J. Frank Determinants of Growth Hormone Receptor Down-Regulation Mol. Endocrinol., July 1, 2007; 21(7): 1537 - 1551. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Loesch, L. Deng, J. W. Cowan, X. Wang, K. He, J. Jiang, R. A. Black, and S. J. Frank Janus Kinase 2 Influences Growth Hormone Receptor Metalloproteolysis Endocrinology, June 1, 2006; 147(6): 2839 - 2849. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. He, K. Loesch, J. W. Cowan, X. Li, L. Deng, X. Wang, J. Jiang, and S. J. Frank Janus Kinase 2 Enhances the Stability of the Mature Growth Hormone Receptor Endocrinology, November 1, 2005; 146(11): 4755 - 4765. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Cowan, X. Wang, R. Guan, K. He, J. Jiang, G. Baumann, R. A. Black, M. S. Wolfe, and S. J. Frank Growth Hormone Receptor Is a Target for Presenilin-dependent {gamma}-Secretase Cleavage J. Biol. Chem., May 13, 2005; 280(19): 19331 - 19342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Jiang, X. Wang, K. He, X. Li, C. Chen, P. P. Sayeski, M. J. Waters, and S. J. Frank A Conformationally Sensitive GHR [Growth Hormone (GH) Receptor] Antibody: Impact on GH Signaling and GHR Proteolysis Mol. Endocrinol., December 1, 2004; 18(12): 2981 - 2996. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Huang, S.-O. Kim, N. Yang, J. Jiang, and S. J. Frank Physical and Functional Interaction of Growth Hormone and Insulin-Like Growth Factor-I Signaling Elements Mol. Endocrinol., June 1, 2004; 18(6): 1471 - 1485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Yang, Y. Huang, J. Jiang, and S. J. Frank Caveolar and Lipid Raft Localization of the Growth Hormone Receptor and Its Signaling Elements: IMPACT ON GROWTH HORMONE SIGNALING J. Biol. Chem., May 14, 2004; 279(20): 20898 - 20905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wang, K. He, M. Gerhart, J. Jiang, R. J. Paxton, R. K. Menon, R. A. Black, G. Baumann, and S. J. Frank Reduced Proteolysis of Rabbit Growth Hormone (GH) Receptor Substituted with Mouse GH Receptor Cleavage Site Mol. Endocrinol., October 1, 2003; 17(10): 1931 - 1943. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Huang, S.-O. Kim, J. Jiang, and S. J. Frank Growth Hormone-induced Phosphorylation of Epidermal Growth Factor (EGF) Receptor in 3T3-F442A Cells: MODULATION OF EGF-INDUCED TRAFFICKING AND SIGNALING J. Biol. Chem., May 23, 2003; 278(21): 18902 - 18913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wang, K. He, M. Gerhart, Y. Huang, J. Jiang, R. J. Paxton, S. Yang, C. Lu, R. K. Menon, R. A. Black, et al. Metalloprotease-mediated GH Receptor Proteolysis and GHBP Shedding. DETERMINATION OF EXTRACELLULAR DOMAIN STEM REGION CLEAVAGE SITE J. Biol. Chem., December 20, 2002; 277(52): 50510 - 50519. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. van Kerkhof, M. Smeets, and G. J. Strous The Ubiquitin-Proteasome Pathway Regulates the Availability of the GH Receptor Endocrinology, April 1, 2002; 143(4): 1243 - 1252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Beauloye, B. Willems, V. de Coninck, S. J. Frank, M. Edery, and J.-P. Thissen Impairment of Liver GH Receptor Signaling by Fasting Endocrinology, March 1, 2002; 143(3): 792 - 800. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhang, R. Guan, J. Jiang, J. J. Kopchick, R. A. Black, G. Baumann, and S. J. Frank Growth Hormone (GH)-induced Dimerization Inhibits Phorbol Ester-stimulated GH Receptor Proteolysis J. Biol. Chem., June 29, 2001; 276(27): 24565 - 24573. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Y. Shvartsman, M. P. Hagan, A. Yacoub, P. Dent, H. S. Wiley, and D. A. Lauffenburger Autocrine loops with positive feedback enable context-dependent cell signaling Am J Physiol Cell Physiol, March 1, 2002; 282(3): C545 - C559. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
