From the § Department of Medicine, Division of
Endocrinology and Metabolism and Department of Cell
Biology, University of Alabama at Birmingham, Birmingham, Alabama
35294, ¶¶ Veterans Affairs Medical Center, Birmingham,
Alabama 35294, ¶ Edison Biotechnology Institute and Department of
Biomedical Sciences, College of Osteopathic Medicine, Ohio
University, Athens, Ohio 45701, ** Immunex Corp., Seattle,
Washington 98101,
Center for
Endocrinology, Metabolism, and Molecular Medicine, Department of
Medicine, Northwestern University Medical School, Chicago, Illinois
60611, and §§ Veterans Administration Chicago
Health System, Lakeside Division, Chicago, Illinois 60611
Received for publication, February 9, 2001, and in revised form, April 9, 2001
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ABSTRACT |
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Growth hormone (GH) initiates its cellular action
by properly dimerizing GH receptor (GHR). A substantial fraction of
circulating GH is complexed with a high-affinity GH-binding protein
(GHBP) that in many species can be generated by GHR proteolysis and
shedding of the receptor's ligand-binding extracellular domain. We
previously showed that this proteolysis 1) can be acutely promoted by
the phorbol ester phorbol 12-myristate 13-acetate (PMA), 2) requires a
metalloprotease activity, 3) generates both shed GHBP and a membrane-associated GHR transmembrane/cytoplasmic domain remnant, and
4) results in down-regulation of GHR abundance and GH signaling. Using
cell culture model systems, we now explore the effects of GH treatment
on inducible GHR proteolysis and GHBP shedding. In human IM-9
lymphocytes, which endogenously express GHRs, and in Chinese hamster
ovary cells heterologously expressing wild-type or cytoplasmic domain
internal deletion mutant rabbit GHRs, brief exposure to GH inhibited
PMA-induced GHR proteolysis (receptor loss and remnant accumulation) by
60-93%. PMA-induced shedding of GHBP from Chinese hamster ovary
transfectants was also inhibited by 70% in the presence of GH. The
capacity of GH to inhibit inducible GHR cleavage did not rely on
JAK2-dependent GH signaling, as evidenced by its
continued protection in JAK2-deficient Growth hormone (GH)1
acts on its target tissues by interacting with the transmembrane GH
receptor (GHR) (1, 2). In multiple species, a substantial fraction of
circulating GH is complexed with a high-affinity GH-binding protein
(GHBP) that corresponds to the ligand-binding extracellular domain of
the GHR (3-5). By virtue of its interaction with GH, GHBP may function
as either a potentiator (by delaying GH clearance) or an inhibitor (by
GH sequestration) of GH bioavailability (3, 6-9). Whereas in some
species GHBP arises as a secreted alternatively spliced form of the GHR
that lacks the transmembrane and cytoplasmic domains (10-12), GHBP in
humans, rabbits, and several other species is formed
posttranslationally by shedding of the proteolytically cleaved GHR
extracellular domain (14). As a GHBP-generating mechanism, GHR
proteolysis can also lead to down-regulation of receptor abundance and
GH signaling, thus further contributing to regulation of GH
bioavailability (13-15).
We and others have shown that GHR proteolysis and GHBP shedding can be
observed in cell culture model systems (13-24). Our work indicated
that such processing can be induced by the protein kinase C activator
phorbol 12-myristate 13-acetate (PMA) and by growth factors such as
platelet-derived growth factor and serum (13, 15). In addition, the
alkylating reagent N-ethylmaleimide can also cause GHR
proteolysis (13, 15-17, 19, 22-24). Each of these stimuli promotes
rapid GHR proteolysis that, in addition to causing GHBP shedding,
results in formation of a cell-associated receptor cytoplasmic domain
remnant (13, 15). Although the exact site of cleavage (presumably in
the proximal extracellular domain) is not yet known, GHR proteolysis
and GHBP shedding in each instance are prevented by the metalloprotease
inhibitor, Immunex Compound 3 (13-15). Our genetic reconstitution
experiments strongly suggest that the transmembrane metzincin
metalloprotease, tumor necrosis factor An additional important step in understanding the molecular
mechanism(s), physiological role(s), and regulation of GHR proteolysis is to determine the impact, if any, of GH on this process. GH initiates
intracellular signals by interacting with the cell surface transmembrane GHR to form a complex of 1:2 GH:GHR stoichiometry (28,
29). GH-induced formation of a GHR dimer is believed to be essential
for normal GH signaling; GH antagonists with mutations at GH site 2 that do not allow proper formation of receptor dimers cannot themselves
promote signaling but rather antagonize signaling by wild-type GH
(30-32). Whereas it is known that the long-standing excess GH levels
found in acromegalic patients are often associated with decreased GHBP
levels (33-35), it is unclear how this effect is exerted. Furthermore,
it may be that steady-state GHBP levels reflect tissue GHR levels (4,
36-38); thus, effects of chronic GH excess or deficiency on GHBP
levels may not solely reflect the effect of GH on the proteolytic
process itself.
Using human HepG2 cells stably transfected with the rabbit GHR,
Harrison et al. (22) noted a significant 30% decline in GHBP spontaneously released over a 24-h interval when the cells were
incubated with 100 ng/ml human or bovine GH. Amit et al. (39) also addressed the issue of the effects of GH on GHBP production and postulated an inverse relationship between GHR internalization and
proteolytic GHBP generation in a CHO-GHR reconstitution system similar
to ours. These authors suggested that the ability of GH to cause GHR
internalization and degradation may account for its ability to prevent
GHBP shedding induced in that study by N-ethylmaleimide treatment.
In this study, we examine in model tissue culture systems the acute
effects of GH on PMA-induced GHR proteolysis and GHBP shedding. Our
results present interesting mechanistic and physiological implications
concerning metalloprotease-mediated GHR processing.
Materials--
PMA, hygromycin B, and routine reagents were
purchased from Sigma Chemical Co. unless otherwise noted. Restriction
endonucleases were obtained from New England Biolabs (Beverly, MA).
Recombinant hGH was kindly provided by Eli Lilly Co. Recombinant
hGH-G120K was kindly provided by Sensus Corp. (Austin, TX).
Cells, Cell Culture, and Transfections--
COS-7, IM-9, and CHO
(a gift of J. Kudlow, University of Alabama at Birmingham, Birmingham,
AL) cells were maintained as described previously (13, 15, 32,
40). COS-7 cells were transiently transfected with pSX rbGHR, pSX
rbGHRdel 297-406, pSX rbGHRdel 294-498, or
pSX rbGHR1-390 (rabbit GHR mutants with deletion of
residues 297-406, 294-498, or 391-620, respectively) as described
previously (41). Stable transfection of CHO cells was achieved by
introducing either pSX rbGHR, pSX rbGHR C241S (a point mutant rabbit
GHR in which cysteine 241 is changed to serine (32)), or pSX
rbGHRdel 297-406 (20 µg of each in 3 ml of Dulbecco's
modified Eagle's medium in 60 × 15-mm dishes) along with 1 µg
of pSP65-SR
Plasmid Construction--
The pSX plasmid (a gift of Dr. J. Bonifacino (National Institutes of Health, Bethesda, MD) and Dr. K. Arai (DNAX)), which drives eukaryotic protein expression from
the SR Antibodies--
The 4G10 monoclonal anti-phosphotyrosine
antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid,
NY). The rabbit polyclonal antisera anti-GHRcyt-AL37, which
was directed against residues 271-620 of the human GHR (the entire
cytoplasmic domain (45)), and anti-GHRcyt, which was
directed against residues 317-620 of human GHR, have been described
previously (46, 47).
Anti-GHRcyt-AL47 is a rabbit serum raised against a
bacterially expressed N-terminally His-tagged fusion protein
incorporating human GHR residues 271-620. The cDNA encoding this
fusion was created by polymerase chain reaction in the pET vector
system (Novagen) (polymerase chain reaction primer sequences are
available upon request). Molecular biology techniques, cDNA
sequencing for verification of fidelity, bacterial fusion protein
expression, and preparation of Ni2+-agarose-purified fusion
protein were as described previously (41, 46-49) and followed the
manufacturer's suggestions. For the purposes of the experiments in
this study, the three anti-GHR cytoplasmic domain antibodies each
behaved similarly for immunoblotting of GHRs and remnants.
Cell Stimulation, Protein Extraction, Electrophoresis, and
Immunoblotting--
Serum starvation of COS-7 transfectants, IM-9, CHO
transfectants, GHBP Assay--
GHBP activity was measured in conditioned media
by a standard GH binding assay, as reported previously (13, 50).
Conditioned medium (1 ml) from cells treated as indicated was incubated
with freshly labeled 125I-hGH (~0.5 ng) 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. Statistical analysis was
performed by unpaired t test.
Densitometric Analysis--
Densitometric quantitation of ECL
immunoblots was performed using a video camera and the Image 1.49 program (developed by W.S. Rasband; Research Services Branch, National
Institute of Mental Health, Bethesda, MD). The fractional loss of
full-length GHRs in extracts from PMA-treated cells was estimated by
measuring the intensity of the GHR signal relative to that present in
non-PMA-treated cell extracts from the same experiment. The PMA-induced
loss of receptors determined thus in samples not pretreated with
GH was considered the maximum (100%) within that particular
experiment. PMA-induced GHR loss in the presence of GH was expressed
relative to this maximum. PMA-induced GHR remnant accumulation was
estimated by subtracting the remnant signal present in non-PMA-treated
cell extracts from that present in the PMA-treated cells. Maximum
(100%) remnant accumulation within each experiment was that arising
from PMA treatment without prior GH stimulation. PMA-induced remnant accumulation in the presence of GH was expressed relative to this maximum. As indicated when graphically shown, pooled data from several
experiments are displayed as the mean ± S.E. The significance of
differences of pooled results are estimated by unpaired t tests.
GH Inhibits PMA-induced GHR Proteolysis and GHBP
Shedding--
Proteolytic generation of the GHBP can be detected in
both cells that endogenously express GHRs and cells that heterologously express GHRs (13-24). In our previous studies with human IM-9 lymphoblasts and CHO cells stably expressing the rbGHR, we have shown
that metalloprotease-mediated GHBP shedding in response to activation
of protein kinase C with the phorbol ester PMA is associated with loss
of the full-length GHR and accumulation of cell-associated 60-68-kDa
cytoplasmic domain-containing remnant proteins (13, 15). As we observed
previously, exposure of CHO-rbGHR cells to PMA for as little as 10 min
allowed detection of receptor loss and remnant accumulation by
immunoblotting with an antiserum against the GHR cytoplasmic domain
(Fig. 1A, lane 2 versus lane
1).
Because GHBP shedding has been observed in cells that express GHR
isoforms that lack a large portion of the cytoplasmic domain (51, 52),
we tested whether this biochemical signature of inducible shedding
(receptor loss and remnant accumulation) could be detected in the
absence of the full receptor cytoplasmic domain. We previously
characterized a rbGHR mutant with an in-frame internal deletion of
residues 297-406 (rbGHRdel 297-406) that thus lacks most
of the membrane-proximal one-third of the receptor's cytoplasmic
domain but has an intact Box B1 (JAK2-binding) region (41).
rbGHRdel 297-406 binds GH normally, interacts with JAK2,
and allows GH-induced JAK2 tyrosine phosphorylation (41). As seen in
Fig. 1B, in CHO cells that stably express this mutant (CHO-rbGHRdel 297-406), the internally deleted receptor was easily detected by anti-GHRcyt at the molecular
mass noted previously. A protein of ~43 kDa, the size expected
of a remnant, was also detected. PMA treatment resulted in loss of
receptor and further accumulation of the remnant protein (lane 2 versus lane 1). In other experiments (data not shown), we also
observed PMA-induced receptor loss and remnant accumulation in COS-7
cells transiently expressing GHR mutants lacking cytoplasmic domain residues 294-498 (rbGHRdel 294-498) and 391-620
(rbGHR1-390) (both described previously in Ref. 41). Thus,
neither the full cytoplasmic domain nor any particular cytoplasmic
domain region (at least distal to the Box B1 element) is clearly
required for PMA-induced receptor proteolysis. We do not yet know if
the increased remnant present in CHO-rbGHRdel 297-406
cells even in the absence of PMA reflects an alteration in basal
protease sensitivity in this mutant; such a change in protease
specificity has been noted, for example, in mutants of the interleukin
6 receptor, another TACE substrate (53). In any case, the parallel
findings of PMA-inducible proteolysis in the wild-type and mutant
rbGHRs indicate the utility of both stably transfected CHO cell lines as model systems in the current study.
GH binding causes dimerization of the GHR, activation of the
GHR-associated JAK2 tyrosine kinase, and internalization of the receptor (reviewed in Refs. 1 and 2). We showed previously that GHR
proteolysis acutely affects receptor abundance (13-15) and can thereby
impact upon GHR activation (15); we next explored whether GH
stimulation could influence receptor proteolysis. As seen in Fig. 1,
A and B, treatment with GH (500 ng/ml) for 5 min did not acutely promote GHR proteolysis (loss of GHR with accumulation of remnant) in CHO-rbGHR or CHO-rbGHRdel 297-406 cells
(lane 3 versus lane 1). However, GH
exposure did substantially diminish the ability of PMA to cause
receptor proteolysis in both cell types. This was apparent in comparing
the abundance of either remaining full-length GHRs or GHR remnants
accumulated in response to PMA in the presence and absence of GH (Figs.
1, A and B, lane 4 versus lane 2). When several
such experiments were analyzed by densitometry, relative PMA-induced
loss of full-length GHRs declined on average by 66% and 93% in
GH-treated CHO-rbGHR and CHO-rbGHRdel 297-406 cells,
respectively (Fig. 1, C and D, top panels).
Remnant abundance induced by PMA was diminished on average by 79% and
62% in these cells when exposed to GH before the PMA (Fig. 1,
C and D, bottom panels). As indicated in Fig. 1,
C and D, each of these differences was
statistically significant.
The concentration dependence of the ability of GH to prevent
PMA-induced GHR cleavage was examined in the experiment in Fig. 2, in which protection from proteolysis
is compared with GH-induced GHR disulfide linkage. We have shown
previously that GH induces the concentration- and
time-dependent appearance of a high molecular weight form
of the GHR detected by immunoblotting only under nonreducing conditions
(32, 40, 47). CHO-rbGHRdel 297-406 cells were treated with
0-500 ng/ml GH for 5 min before exposure to PMA for 10 min. Cell
extracts were divided into two fractions; proteins were resolved by
SDS-PAGE under either nonreducing (Fig. 2A) or reducing
(data not shown) conditions. Similar to our previous data using IM-9
cells, GH promoted the appearance of the disulfide-linked (dsl) form of rbGHRdel 297-406 when evaluated
by anti-GHRcyt immunoblotting of nonreduced detergent cell
extracts (Fig. 2A). The GH concentration dependence of the
relative abundance of the disulfide-linked form of rbGHRdel
297-406 (lanes 1, 3, 5, and 7 of Fig.
2A) is displayed graphically in Fig. 2B, left
panel. The subsequent treatment with PMA did not markedly affect
the abundance of this GH-induced disulfide-linked form. In contrast, in
the absence of GH, PMA did cause loss (roughly a 17.5% decrease in
abundance by densitometry) of the non-disulfide-linked rbGHRdel
297-406 form (Fig. 2A, lane 2 versus lane
1; graphically displayed in Fig. 2B, middle panel). GH
treatment prevented this PMA-induced loss in a
concentration-dependent manner (progressively less
fractional loss in the presence of 50 and 100 ng/ml GH
versus 0 ng/ml GH, and no loss in the presence of 500 ng/ml
GH; Fig. 2B, middle panel). We also assessed by densitometry
the GH concentration dependence of inhibition of PMA-induced
rbGHRdel 207-406 remnant formation in the reduced extracts
of this same experiment. As seen in Fig. 2B, the profiles of
GH inhibition of PMA-induced loss of non-disulfide-linked rbGHRdel
297-406 (middle panel) and appearance of the
rbGHRdel 297-406 remnant (right panel) are
similar and inversely mirror the profile of GH-induced receptor
disulfide linkage (left panel). The appearance of this
disulfide-linked form of the GHR requires GH-induced GHR dimerization,
and thus it reflects the degree to which GH engages and dimerizes its
receptor (32). These results suggest very similar concentration
dependences for the GH-induced appearance of the disulfide-linked
(engaged and dimerized) GHR and for the ability of GH to prevent
PMA-induced GHR proteolysis. At high GH concentration, no further
GH-induced GHR disulfide linkage is observed (32, 47), and the
protective effect of GH approaches its maximum. This presumably
reflects the lack of availability (perhaps reflective of their
subcellular localization) of remaining (nondimerized) GHRs for either
dimerization or proteolysis. In other experiments with IM-9 and
CHO-rbGHR cells, we observed that the minimum effective concentration
for both effects was roughly 25 ng/ml (data not shown).
We also tested whether the acute inhibitory effect of GH on PMA-induced
GHR proteolysis would be reflected in measurement of the shed GHBP from
the CHO stable transfectants. Because the assay relies on the ability
of GHBP to bind 125I-hGH, the presence of high
concentrations of unlabeled GH in the tissue culture supernatant
complicates GHBP determination; thus, we tested the ability of a modest
GH concentration, 50 ng/ml, to affect GHBP generation (Fig.
3). Cells were treated with PMA for 25 min after pretreatment for 5 min with GH or vehicle control. Supernatants were harvested and rendered equivalent in GH concentration before assaying GHBP levels. As seen in Fig. 3, inclusion of GH during
the period of PMA stimulation led to a statistically significant 70 ± 11% (mean ± S.E.) fall in GHBP shedding, in
correspondence to the findings for immunological measures of GHR
proteolysis in Figs. 1 and 2.
GH Inhibition of PMA-induced GHR Proteolysis Does Not Require
JAK2-dependent GH Signaling--
Having observed acute
inhibition of both PMA-induced GHR proteolysis and GHBP shedding by GH,
we next addressed possible mechanisms to account for this effect.
Whereas it is not known with certainty in which subcellular locale
induced GHR proteolysis takes place, it is believed that a
substantial fraction occurs at the cell surface (4, 5). Strongly
supporting this contention is the finding that a naturally occurring
membrane-anchored GHR splice variant that lacks most of the receptor
cytoplasmic domain (and is thus heavily localized to the cell surface)
yields ample quantities of GHBP (51, 52). In addition, experiments with
surface trypsinization and membrane-impermeable sulfhydryl blockers
point to the cell surface as the site of GHBP generation (20, 22). In
principle, the ability of GH to inhibit PMA-induced GHR proteolysis
could therefore reside in the ability of GH to cause the receptor to traffic away from the cell surface and/or to a subcellular location that protects it. However, the results with CHO-rbGHRdel
297-406 (Fig. 1C) suggest that this is not the
explanation. GH-induced and constitutive GHR internalization is
dependent on the presence of a critical phenylalanine (rat Phe-346,
corresponding to rbGHR residue 327) (54), which resides in a region of
the receptor that is internally deleted in rbGHRdel
297-406. This receptor, like other mutant GHRs truncated to
eliminate this region (54, 55), is likely not significantly
internalized in response to GH.
Another possible explanation for GH inhibition of GHR proteolysis is
that GH-induced signaling either directly inhibits activation of the
GHR-cleaving enzyme or impairs PMA's induction of protein kinase C
activity, which is apparently required for induced GHR cleavage (13,
15). In either case, such GH-induced signaling would presumably begin
with activation of JAK2. To test this possibility, we used the human
fibrosarcoma cell line GH Inhibition of PMA-induced GHR Proteolysis Corresponds to the
Ability of GH to Dimerize the Receptor--
The data presented in
Figs. 1-4 strongly suggest that the acute inhibition of inducible GHR
proteolysis and GHBP shedding by GH could not be explained by either
GH-induced GHR internalization or signaling. We next considered whether
GH binding and/or GH-induced receptor dimerization could account for
this inhibition. The GH antagonist, G120K (which is similar to the
previously reported G120R), is mutated at site 2 of the human GH
molecule; it thus binds normally to the GHR via the nonmutated G120K
site 1 region but fails to properly dimerize the receptor because its
mutated site 2 region inadequately binds to a second GHR monomer
(30-32). We used G120K to explore whether GH binding versus
productive GH-induced GHR dimerization is required for GH inhibition of
PMA-induced GHR proteolysis.
In the experiment shown in Fig.
5A, human IM-9 cells were
exposed to PMA or its vehicle for 10 min after a 5-min pretreatment with either vehicle, GH, G120K, or the combination of G120K and GH at a
3:1 ratio, and PMA-induced GHR loss and remnant accumulation were
assessed by immunoblotting. As expected, GH substantially inhibited
PMA-induced GHR proteolysis (loss of GHR and appearance of remnant;
lanes 3 and 4 versus lanes
1 and 2), consistent with the data shown in Figs. 1, 2,
and 4. Notably, an equivalent concentration of G120K, despite its
normal ability to bind to GHR monomers, did not inhibit PMA-induced GHR
proteolysis (lanes 5 and 6 versus lanes 1 and 2). Furthermore, when in the presence
of a 3-fold excess of G120K, GH lost its ability to block inducible GHR
proteolysis (lanes 7 and 8 versus
lanes 3 and 4). We have shown previously that a
2-fold or greater excess of the G120K antagonist markedly reduces the
ability of GH to cause GHR proper dimerization in these cells (32).
This was again demonstrated in Fig. 5B (lane 7 versus lane 3), in which the cell extracts from
the same experiment were resolved under nonreducing conditions. This
blot also shows the correlation between the ability of GH to induce GHR
disulfide linkage and its inhibition of GHR proteolysis (lane 4 versus lanes 2, 6, and 8). Similar findings
were obtained when either CHO-rbGHR (data not shown) or
CHO-rbGHRdel 297-406 were used. Fig. 5C shows
the densitometrically measured pattern of PMA-induced remnant
accumulation in the absence or presence of GH, G120K, or the 3:1
G120K:GH ratio in CHO-rbGHRdel 297-406 cells. These
experiments strongly suggest that the ability of GH to properly dimerize the GHR, rather than simply its ability to bind the receptor via site 1, confers to the GHR a relative resistance to PMA-induced proteolytic cleavage.
Although GH-induced GHR disulfide linkage reflects GHR dimerization,
and dimerization is required for disulfide linkage to occur, we
demonstrated previously that noncovalent GHR dimerization is induced by
GH even in cells that express a mutant GHR incapable of undergoing
disulfide linkage (32). Mutation of cysteine 241, which lies in the
membrane-proximal extracellular domain, to serine (C241S) was shown to
abrogate GH-induced GHR disulfide linkage but leave intact GH-induced
noncovalent dimerization of the GHR, JAK2 activation, and acute GHR,
JAK2, and STAT5 tyrosine phosphorylation (32). To determine whether GH
inhibition of PMA-induced GHR proteolysis is due to GHR dimerization
versus GHR disulfide linkage, we used our previously
characterized (32) CHO-rbGHR C241S cells. GH pretreatment afforded
protection from PMA-induced proteolysis to the rbGHR C241S mutant (Fig.
6, lane 4 versus lane 2),
similar to our findings with the wild-type rbGHR and rbGHRdel
297-406 in Figs. 1, 2, 4, and 5. These results suggest that it
is the ability of GH to cause receptor dimerization rather than
receptor disulfide linkage that allows inhibition of PMA-induced GHR
proteolysis.
The proteolytic cleavage of the GHR with shedding of its
extracellular domain is one established mechanism that is used by various species to generate soluble circulating GHBP. Whereas an end
product achieved (GHBP) is functionally the same, proteolysis is a
fundamentally different process than GHBP generation by alternative splicing of a common GHR mRNA precursor, the dominant GHBP
production mechanism used by rodents (10-12). Both the alternative
splicing and proteolysis mechanisms of GHBP generation could, in
principle, allow for the observed general correlation that may exist
between circulating GHBP levels and tissue GHR abundance (4, 36-38). However, in contrast to splicing, factors that cause increased GHBP by proteolysis might be expected to acutely diminish rather than
increase GHR abundance. Indeed, in tissue culture model systems, we
have observed just such a down-regulation of GHR abundance associated
with metalloprotease-mediated proteolytic GHBP generation in several
cell types in response to treatment with PMA,
N-ethylmaleimide, and serum (13-15). In the setting of GHR
proteolysis induced by PMA, platelet-derived growth factor, and serum,
this receptor down-regulation was accompanied by diminished GH
signaling (15). Additionally, we have shown that GHR proteolysis in
response to these stimuli generates a cell-associated receptor
cytoplasmic domain-containing remnant protein (13, 15); although we
have no evidence as yet of a role for it, such a cytoplasmic
domain-containing fragment has been shown to affect signaling in other
receptor systems (57, 58). These observations have suggested to us that, in addition to generating soluble GHBP, inducible GHR proteolysis may act in various ways to affect GHR function and GH signaling.
In this report, we examined the impact of GH on the ability of PMA to
induce GHR proteolysis. We found that a brief treatment with GH
substantially inhibited subsequent PMA-induced GHR proteolysis (receptor loss/remnant accumulation and GHBP shedding) in several cell
types. The concentration dependence for the protective effect of GH was
similar to that for its promotion of GHR disulfide linkage (which is a
proxy for proper receptor dimerization (32)). By using cells expressing
a GHR mutant (CHO-rbGHRdel 297-406) and lacking expression
of JAK2 ( Relatively little is known about the effects of GH on GHBP levels.
Prolonged elevated GH levels found in acromegalics are frequently
associated with reduced circulating GHBP content (33); however, it is
difficult to interpret the significance of this observation with regard
to its mechanism(s) because of the many adaptations to long-term
elevations of GH that can occur. When investigated in normal male
children, plasma GHBP levels were shown to correlate inversely with
24-h GH secretion (mean 24-h GH concentration, the sum of GH pulse
amplitudes, the sum of GH pulse areas, interpulse mean GH
concentration, and the number of GH pulses per 24 h) (59). Among
other possibilities, this observation may suggest that a negative
influence of GH on production of GHBP may have physiological relevance.
Mullis et al. (21) studied the effects of GH on GHR mRNA
abundance, GHR mRNA transcription rate, and GHBP level in the
supernatants of HuH7 human hepatoma cells in culture. This study showed
a biphasic response, with an increase in GHR expression at low GH
levels and a decrease in GHR expression at high levels. GHBP in cell supernatants declined within the first hour of GH treatment for GH
concentrations of 50 ng/ml or less, remained constant in the presence
of 150 ng/ml GH, and increased after a 3-h incubation in 500 ng/ml GH.
These complex results led the authors to conclude that GHBP abundance
did not necessarily reflect GHR mRNA abundance and can be regulated
posttranscriptionally and can likely be regulated posttranslationally.
In our study, we examined the acute effects of GH on PMA-inducible GHR
proteolysis in cells that both endogenously (IM-9) and heterologously
(the CHO and Whereas our current data on PMA-induced (and, in data not shown,
N-ethylmaleimide-induced) GHR proteolysis are in agreement with the above-mentioned effects observed by others, we believe our
results substantially extend our knowledge in several important ways.
First, although we agree that GH-induced receptor internalization and
GHBP release are likely inversely correlated, we interpret our findings
to indicate that GHR proteolysis is not diminished because of that
internalization; rather, GHR internalization and protection from
proteolysis are both seen as being concomitants of GH engagement.
Second, GH apparently protects against GHR proteolysis without the
requirement of generating intracellular (JAK2-dependent, tyrosine phosphorylation-mediated) signaling. Finally, the capacity of
GH to dimerize its receptor appears to underlie its ability to inhibit
GHR proteolysis. These findings suggest that models of the inhibitory
effects of GH on GHR proteolysis and GHBP generation that require steps
beyond GH-induced GHR dimerization may be unduly complicated and misleading.
Proteolysis and shedding of receptors are potentially important in
regulation of various aspects of receptor function. However, to date,
there is only limited information regarding the effects of a
receptor's engagement with its cognate ligand on proteolysis of that
receptor. Two recent reports suggest situations in which ligands
promote receptor proteolysis, albeit likely by different mechanisms.
Zhou and Carpenter (60) showed in several cell lines that cleavage of
the ErbB-4 receptor tyrosine kinase was enhanced by treatment with
heregulin, an ErbB-4 ligand. That study suggested that activated ErbB-4
receptors are subject to cleavage involving an intracellular
metalloprotease during the process of ligand-dependent receptor trafficking. Dri et al. (61) demonstrated in
polymorphonuclear leukocytes that TNF- Several interesting and potentially testable questions regarding GH
signaling and mechanisms of inducible GHR proteolysis also emerge from
our work. The model presented in Fig. 7
is based on the data generated in this study and depicts some of these issues. For example, does GH inhibition of GHR proteolysis serve to
enhance GH signaling? Such a role could be envisioned if it is assumed
that, as we have observed in tissue culture (15), growth factors or
other stimuli might down-regulate GHR abundance by proteolysis. In this
instance, an already GH-dimerized receptor, either poised for or
already engaged in signaling, would be less susceptible to
downregulation (deactivation) by these other stimuli. Receptor
proteolysis generates a GHR cytoplasmic domain-containing remnant
protein with an unknown signaling significance. Could dimerization-driven protection from proteolysis also serve as a
mechanism by which GH prevents the generation of this remnant and
thereby modulates the remnant's signaling (or the remnant's inhibition of signaling by the full-length GHR)?
2A rabbit GHR cells. The GH concentration dependence for inhibition of PMA-induced GHR
proteolysis paralleled that for its promotion of receptor dimerization
(as monitored by formation of GHR disulfide linkage). Unlike GH, the GH
antagonist, G120K, which binds to but fails to properly dimerize GHRs,
alone did not protect against PMA-induced GHR proteolysis; G120K did,
however, antagonize the protective effect of GH. Our data suggest that
GH inhibits PMA-induced GHR proteolysis and GHBP shedding by inducing
GHR dimerization and that this effect does not appear to be related to
GH site 1 binding, GHR internalization, or GHR signaling. The
implications of these findings with regard to GH signaling and
GHR down-regulation are discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-converting enzyme (TACE or
ADAM-17) (25-27), can function as a GHR sheddase (14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
.2-HAtag-Hygro (an empty vector carrying the hygromycin
resistance marker; kindly provided by Dr. M. Streuli; Dana-Farber
Cancer Institute, Boston, MA) using Lipofectin (Life Technologies,
Inc.), and cells were selected in 500 µg/ml hygromycin B and
characterized as reported previously (32).
2A cells (a human fibrosarcoma cell line that does not express JAK2
protein or mRNA (42); a gift of Dr. G. Stark, Cleveland Clinic
Foundation, Cleveland, OH) were maintained in Dulbecco's modified
Eagle's medium (1 g/liter glucose) (Cellgro, Inc.) supplemented with
7% fetal bovine serum (Biofluids, Rockville, MD) and 50 µg/ml gentamicin sulfate, 100 units/ml penicillin, and 100 µg/ml
streptomycin (all from Biofluids). Stable transfection of
2A cells
was achieved by introducing pSX rbGHR (20 µg in 3 ml of Dulbecco's
modified Eagle's medium in 60 × 15-mm dishes), along with 1 µg
of pSP65-SR
.2-HAtag-Hygro using Lipofectin according to the
manufacturer's protocol. Transfected cells were grown in complete
Dulbecco's modified Eagle's growth medium for 48 h. After
dilution, clones were negatively selected in medium supplemented with
500 µg/ml hygromycin B and screened for GHR expression by anti-GHR
immunoblotting (untransfected
2A cells express no detectable GHR
(Fig. 4)).
promoter (43), has been described previously (32).
Preparation of the rbGHR cDNA (a kind gift of W. Wood; Genentech,
Inc., South San Francisco, CA) and the cDNAs encoding rbGHR
C241S, rbGHRdel 297-406, rbGHRdel 294-498,
and rbGHR1-390 and their ligation into pSX have been
described previously (32, 41, 44).
2A, and
2A transfectants was accomplished by
substitution of 0.5% (w/v) bovine serum albumin (fraction V; Roche
Molecular Biochemicals) for serum in their respective culture media for 16-20 h before experiments. Unless otherwise noted, stimulations were
performed at 37 °C. Details of the hGH (at indicated concentrations) and PMA (at 1 µg/ml) treatment protocols have been described
previously (14, 15, 32, 40). Briefly, for IM-9 cells, cells were stimulated in suspension at 10 million cells/ml in binding buffer (BB).
Stimulations were terminated, and cells were collected by centrifugation (800 × g for 1 min at 4 °C) and
aspiration of the BB. COS-7 transfectants, CHO transfectants,
2A
cells, and
2A transfectants were stimulated in confluent 150 × 20-mm dishes (Falcon) in BB. Stimulations were terminated by washing
the cells once and harvesting by scraping in ice-cold
phosphate-buffered saline in the presence of 0.4 mM sodium
orthovanadate. 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 (FLB), as indicated. After
centrifugation at 15,000 × g for 15 min at 4 °C,
the detergent extracts were electrophoresed under nonreducing or
reducing conditions, as indicated. Resolution of proteins under
reducing or nonreducing conditions by SDS-PAGE, Western transfer of
proteins, and blocking of Hybond-ECL (Amersham Pharmacia Biotech) with
2% bovine serum albumin were performed as described previously (32).
Immunoblotting with antibodies 4G10 (1:2000),
anti-GHRcyt-AL37 (1:1000), anti-GHRcyt
(1:1000), or anti-GHRAL47 (1:1000) with horseradish
peroxidase-conjugated anti-mouse or anti-rabbit 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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
GH inhibits PMA-induced
proteolysis of GHRs in CHO-rbGHR and CHO-rbGHRdel 297-406
cells. A and B, serum-starved CHO-rbGHR
(A) or CHO-rbGHRdel 297-406 (B)
cells at confluence in a 100 × 20-mm dish were exposed to PMA (+)
or the DMSO vehicle ( ) for 10 min after pretreatment for 5 min with
or without GH (500 ng/ml, as indicated). Detergent extracts (the
equivalent of one-half of a dish of cells/sample) were resolved by
SDS-PAGE and immunoblotted with anti-GHR cytoplasmic domain antibodies,
as indicated. The full-length GHR and the GHR remnant protein that
appears in response to PMA are indicated by a bracket and an
arrow, respectively. C and D,
densitometric analysis of the effect of GH pretreatment (
) on
PMA-induced loss of GHR (top panels) and accumulation of
remnant (bottom panels) in comparison with no GH
pretreatment (
) for CHO-rbGHR (C) and CHO-rbGHRdel
297-406 (D) cells. See "Experimental Procedures"
for a description of the densitometric analysis. Mean ± S.E. is
shown for several determinations. CHO-rbGHR, n = 4;
CHO-rbGHRdel 297-406, n = 5. For samples
treated with PMA in the presence versus absence of GH: *,
p < 0.05; **, p < 0.02.
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Fig. 2.
Concentration dependence of GH-induced
receptor disulfide linkage and GH inhibition of PMA-induced receptor
proteolysis in CHO-rbGHRdel 297-406 cells.
A, serum-starved CHO-rbGHRdel 297-406 cells,
prepared as described in the legend for Fig. 1A, were
exposed to PMA (+) or the DMSO vehicle ( ) for 10 min after
pretreatment for 5 min with or without GH (at the indicated
concentrations). Detergent extracts were resolved by SDS-PAGE under
nonreducing (top panel) and reducing (bottom
panel) conditions and immunoblotted with anti-GHRcyt
or anti-GHRcyt-AL47, as indicated. The disulfide-linked
(dsl) and non-disulfide-linked receptors present for each
treatment condition in the top panel are
bracketed. The positions of the receptor and the remnant
resolved under reducing conditions in the bottom panel are
indicated by a bracket and an arrow,
respectively. B, densitometric analysis of the immunoblot in
A. Left panel, abundance of GH-induced
disulfide-linked GHRdel 297-406 formed in response to 50, 100, and 500 ng/ml GH (maximum accumulation (100%) was seen in the
presence of 500 ng/ml GH). Middle panel, fractional
PMA-induced loss of non-disulfide-linked GHRdel 297-406
caused by PMA in the presence of 0, 50, 100, and 500 ng/ml GH
(expressed for each GH concentration as the loss of signal caused by
PMA divided by the signal measured for the non-PMA-treated sample).
Right panel, accumulation of remnant induced by PMA in the
presence of 0, 50, 100, and 500 ng/ml GH (maximum accumulation (100%)
was seen in the presence of 0 ng/ml GH). The left and
middle panels reflect the nonreducing immunoblot; the
right panel reflects the reducing immunoblot. The experiment
shown in A and B is representative of three such
experiments.
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Fig. 3.
GH inhibition of PMA-induced GHBP
generation. One ml of conditioned media from CHO stable
transfectants was assayed for GHBP accumulation in response to PMA (25 min) in the absence or presence of GH (50 ng/ml, starting 5 min before
PMA exposure). Data are expressed as the percentage of GHBP activity
accumulated with PMA alone. Data shown represent four experiments. *,
p < 0.005.
2A. This cell line was previously shown to
lack JAK2 at the mRNA and protein levels (42).
2A also lacks
immunologically detectable GHR protein; we stably expressed the rbGHR
in this cell by transfection to yield
2A-rbGHR (Fig.
4A, lane 2 versus
lane 1). GH stimulation of
2A-rbGHR cells caused
disulfide linkage of the transfected rbGHR (Fig. 4B),
verifying that the receptor could bind GH and become dimerized.
However, consistent with previous reports (56), GH treatment did not yield discernable tyrosine phosphorylation of cellular proteins, as
assessed by anti-phosphotyrosine immunoblotting of cellular extracts
resolved by SDS-PAGE (data not shown). This is consistent with the lack
of JAK2 in these cells. Despite their lack of JAK2,
2A-rbGHR cells
responded to PMA treatment with substantial loss of full-length GHR and
accumulation of the cell-associated receptor cytoplasmic
domain-containing remnant protein (Fig. 4C, lane 4 versus lane 3 compared with lane 2 versus lane 1). In other experiments (data not
shown), PMA-induced GHBP shedding was also easily detected in
2A-rbGHR cell supernatants. Thus, the inducible proteolysis and
shedding apparatus did not depend on the presence of JAK2. Interestingly, pretreatment with GH substantially inhibited PMA-induced GHR proteolysis in
2A-rbGHR cells (Fig. 4C, lane 4 versus lane 2), much as we observed in the CHO
transfectants and in IM-9 cells (see below). Densitometric assessment
of GH inhibition of PMA-induced remnant accumulation in several similar
experiments with
2A-rbGHR cells is graphically shown in Fig.
4D. Thus, the ability of GH to inhibit inducible GHR
proteolysis did not depend on either the presence of JAK2 or the
ability of the hormone to promote JAK2-mediated intracellular
signaling.
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Fig. 4.
GH inhibition of PMA-induced GHR proteolysis
is not dependent on JAK2. A and B,
reconstitution of JAK2- and GHR-deficient 2A cells with rbGHR.
A,
2A (lane 1) and
2A-rbGHR (lane
2) cells were detergent-solubilized, and extracts (the equivalent
of one-half of a 100 × 20-mm dish of cells/sample) were resolved
by SDS-PAGE under reducing conditions and immunoblotted with
anti-GHRcyt-AL47. The stably transfected rbGHR present in
2A-rbGHR cells is bracketed. B, serum-starved
2A-rbGHR cells at confluence (prepared as described in A)
were treated with (+) or without (
) GH (500 ng/ml) for 15 min.
Detergent extracts (the equivalent of one-half of a dish of
cells/sample) were resolved by SDS-PAGE under nonreducing conditions
and immunoblotted with anti-GHRcyt-AL47. The positions of
he disulfide-linked (dsl) and non-disulfide-linked receptors
present for each condition are bracketed. Note that GH
caused disulfide linkage of the rbGHR, confirming that receptors are
engaged and dimerized even in the absence of JAK2. C and
D, GH inhibition of PMA-induced GHR proteolysis in
2A-rbGHR cells. C, serum-starved
2A-rbGHR cells
(prepared as described in A and B) were exposed
to PMA (+) or the DMSO vehicle (
) for 10 min after pretreatment for 5 min with or without GH (500 ng/ml, as indicated). Detergent extracts
were resolved by SDS-PAGE and immunoblotted with
anti-GHRcyt-AL37. The full-length GHR and the GHR remnant
protein that appears in response to PMA are indicated by a
bracket and an arrow, respectively. D,
densitometric analysis of the effect of GH pretreatment (
) on
PMA-induced accumulation of remnant in comparison with no GH
pretreatment (
), as described in Fig. 1. Mean ± S.E. for each
is shown for n = 3 determinations. For samples treated
with PMA in the presence versus absence of GH: *,
p < 0.01.
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Fig. 5.
Effect of the GH antagonist G120K on GH
inhibition of PMA-induced receptor proteolysis. A and
B, human IM-9 cells. Serum-starved cells (5 million
cells/sample) were exposed to PMA (+) or the DMSO vehicle ( ) for 10 min after pretreatment for 5 min with or without GH (500 ng/ml) and/or
the GH antagonist G120K (500 or 1500 ng/ml, as indicated). Detergent
extracts were resolved by SDS-PAGE under reducing (A) or
nonreducing (B) conditions and immunoblotted with
anti-GHRcyt or anti-GHRcyt-AL47, as indicated.
The disulfide-linked (dsl) and non-disulfide-linked human
(hGHR) receptors present for each condition are
bracketed; the GHR remnant induced by PMA is indicated by an
arrow. The experiment shown is representative of three such
experiments. C, CHO-rbGHRdel 297-406 cells.
Serum-starved cells (prepared as described in the legend for Fig.
1B) were treated with vehicle GH, G120K, or the combination
of GH + G120K before exposure to PMA or the DMSO vehicle for 10 min, as
described in A and B. After resolution of
detergent extracts by SDS-PAGE and anti-GHRcyt
immunoblotting, the PMA-induced accumulation of remnant was assessed
densitometrically. The effect of GH, G120K, or the 3:1 G120K:GH ratio
on PMA-induced remnant accumulation in two independent experiments is
graphically displayed (mean ± the extent of the range of the
values).
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Fig. 6.
Receptor disulfide linkage is not required
for GH inhibition of PMA-induced receptor proteolysis.
Serum-starved CHO-rbGHR C241S cells (prepared as described in the
legend for Fig. 1) were exposed to PMA (+) or the DMSO vehicle ( ) for
10 min after pretreatment for 5 min with or without GH. Detergent
extracts were resolved by SDS-PAGE and immunoblotted with
anti-GHRcyt. The full-length rbGHR C241S and the remnant
protein that appears in response to PMA are indicated by a
bracket and an arrow, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2A-rbGHR), we concluded that GH engagement of the GHR
likely did not inhibit PMA-induced GHR proteolysis by either causing
receptor internalization or intracellular signaling. Furthermore,
although it can bind the GHR, the G120K antagonist itself was unable to
protect the receptor from PMA-induced proteolysis, but it did
antagonize GH's protection; thus we concluded that GH-induced GHR
dimerization, rather than simply receptor occupancy, was required to
inhibit the cleavage process. Finally, by comparing cells expressing
the wild-type receptor (CHO-rbGHR) and a receptor mutant that is
dimerized but not disulfide-linked by GH (CHO-rbGHR C241S), we
determined that covalent (disulfide-mediated) receptor dimerization was
not necessary to confer protection from inducible proteolysis.
2A transfectants) express GHRs and found similar
results in each, likely nullifying any confounding effects of changes
in GHR gene expression that might be induced by GH. Furthermore, we
opted not to measure GHBP in the cell supernatants in situations in
which >50 ng/ml GH was present because of the inherent imprecision of
such measurements. Although we did indeed detect inhibition of GHBP
shedding induced by PMA in the presence of 50 ng/ml GH, we chose
instead in all other experiments to monitor GHR proteolysis
biochemically by tracking GHR loss and GHR remnant accumulation by
anti-GHRcyt immunoblotting. Our previous work (13, 15) and
that presented herein have indicated the validity of this approach.
promoted proteolysis of both
TNF receptors (TNF-R55 and TNF-R75) by virtue of engagement of the
TNF-R55 receptor. In that case, TNF-
apparently promotes activation
of a metalloprotease (likely TACE) that can then shed the extracellular
domain of both engaged and nonengaged surface receptors. Our current
findings of GH's dimerization-dependent and activation- and
internalization-independent inhibition of GHR proteolysis stand in
contrast to the stimulatory effects of heregulin and TNF-
. This
suggests substantial receptor-specific differences in the effect of
ligands on receptor cleavage processes. It will thus be important to
discern whether the metalloprotease-mediated cleavage of other
receptors will be positively or negatively regulated by their ligands.
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Fig. 7.
GH-induced GHR dimerization protects against
inducible receptor proteolysis and GHBP shedding. This model
depicts the findings in this study and their implications for GHR
down-regulation and signaling. In both A and B,
the unliganded GHR is seen as a monomer (or perhaps a loosely
associated dimer; A, 1 and B, 1). In the absence
of GH, the transmembrane metalloprotease (likely TACE) in the vicinity
of the GHR, activated by an inducer (data not shown), gains access to
the receptor cleavage site in the near extracellular domain (A,
2). Metalloprotease-mediated processing yields shed GHBP and the
membrane-associated GHR remnant that includes the receptor
transmembrane and cytoplasmic domains (A, 3). When cells are
exposed to GH before the addition of the metalloprotease inducer, GHRs
undergo GH-induced dimerization; activation of associated JAK2
molecules results in tyrosine phosphorylation of the GHR and JAK2
(B, 2). As a consequence of GH-induced GHR dimerization,
metalloprotease activation in this case does not result in GHR
proteolysis (B, 3). The situation in A would be
the same if the GH antagonist alone were present, binding via site 1 but not dimerizing the GHR, or if both GH antagonist and GH (at a ratio
of 3:1 or greater) were present. In concert with our previous data
(13-15), this model predicts that unliganded GHRs are more susceptible
to metalloprotease-mediated heterologous down-regulation of receptor
abundance, GHBP, and remnant generation and desensitization to GH
stimulation. GHRs that were already engaged, dimerized, and activated,
in contrast, would not be targets of such metalloprotease-mediated
heterologous desensitization.
We recently identified the transmembrane metalloprotease TACE as a GHR
sheddase capable of mediating PMA-induced GHBP shedding and GHR
down-regulation (14). Our current data allow us to speculate that
receptor dimerization, which is known to involve apposition of the stem
regions at the GHR extracellular face (28, 29), might prevent GHR
proteolysis by causing a steric or conformational hindrance of TACE
access to or activity at that dimerization interface. Conversely, it
may be predicted that the GHR monomer is a better sheddase substrate
than the dimer. Given the SDS-PAGE migration-based estimated sizes of
the shed GHBP (39, 62, 63) and the GHR remnant (13, 15), this
dimerization interface region is also likely to contain the locus of
inducible GHR proteolysis. It will be interesting and important to test
the dimerization sensitivity of interaction of this site(s) with TACE
or other GHR sheddases that might be identified to further understand
the structural features underlying their catalysis. As a practical
matter, our results predict that proteolytic site mapping studies may
be facilitated by using monomeric rather than dimerized GHRs as the substrate.
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ACKNOWLEDGEMENTS |
---|
We appreciate helpful conversations with Drs. L. Liang, S.-O. Kim, X. Wang, J. Kudlow, A. Paterson, E. Chin, G. Fuller, E. Benveniste, J. Baker, and J. Messina and the generous provision of reagents by those named in the text.
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FOOTNOTES |
---|
* This work was supported in part by VA Merit Review awards (to S. J. F. and G. B.), grants from the National Science Foundation and the Northwestern Memorial Foundation (to G. B.), National Institutes of Health Grant DK46395 (to S. J. F.), and the State of Ohio Eminent Scholar Program, which includes a grant from Milton and Lawrence Goll, and by Sensus Corp. (to J. J. K.).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.
To whom correspondence should be addressed: University
of Alabama at Birmingham, 1530 3rd Ave. South, BDB 861, Birmingham, AL 35294-0012. Tel.: 205-934-9877; Fax: 205-934-4389;
E-mail: frank@ endo.dom.uab.edu.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M101281200
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ABBREVIATIONS |
---|
The abbreviations used are:
GH, growth hormone;
GHBP, GH-binding protein;
GHR, GH receptor;
PMA, phorbol 12-myristate
13-acetate;
CHO, Chinese hamster ovary;
rb, rabbit;
TACE, tumor
necrosis factor -converting enzyme;
hGH, human GH;
BB, binding
buffer (25 mM Tris-HCl, pH 7.4, 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 0.1% (w/v)
bovine serum albumin, and 1 mM dextrose);
FLB, fusion lysis
buffer (1% (v/v) Triton X-100, 150 mM NaCl, 10% (v/v)
glycerol, 50 mM Tris-HCl, pH 8.0, 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, and
10 µg/ml aprotinin);
SDS-PAGE, SDS-polyacrylamide gel
electrophoresis;
TNF, tumor necrosis factor.
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