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Department of Pharmacology, Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel (T.A., O.B.A., M.B.H., Z.H.); INSERM U468, Hopital Henri Mondor, Creteil, France (F.D., S.A.); and Department of Pediatrics (Z.H.), Rambam Medical Center, Haifa, Israel
Address all correspondence and requests for reprints to: Dr. Ze’ev Hochberg, Faculty of Medicine, POB 9649, Haifa 31096, Israel. E-mail: repzeev{at}tx.technion.ac.il
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Taken together, these data suggest that intracellular sulfhydryl groups, within membranal endocytic vesicles, that do not belong to the GHR molecule, are involved in receptor internalization and GHBP generation. In addition, the present study demonstrates that despite impaired hGHR internalization/down-regulation, the inducible release of GHBP was not affected, further suggesting that GHR endocytosis is not a prerequisite for GHBP generation.
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Introduction |
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Recently, an alternatively spliced form of hGHR was demonstrated to encode a truncated isoform of hGHR (hGHRtr) and to regulate GHBP generation (15, 16, 17). Functional studies confirmed that while hGHRtr was inactive by itself, it could act as a dominant negative regulator of the full-length receptor (16, 18). Using CHO cells, stably transfected with hGHR or with hGHRtr, we have reported recently that in contrast to hGHR, hGHRtr failed to internalize, is relatively fixed at the cell membrane, but generates large amounts of soluble GHBP (17). In the present study, we have examined the effect of sulfhydryl group(s) inactivation on the induction of GHBP generation from the truncated isoform (hGHRtr) and a mutant of the free extracellular cysteine (hGHRtr-C241A), compared with the naturally occurring full-length rbGHR and hGHR, expressed in CHO cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Drug treatment
Iodoacetamide (IA), N-ethylmaleimide (NEM),
iodoacetic acid (IAA),p-chloromercuriphenylsulfonic acid (PCMBS) were
obtained from Sigma Chemical Co. (St. Louis, MO). The
membrane impermeable thiol blocker A-484 (4-acetamido-4'-(iodoacetyl)
amino) stilbene-2,2' disulfonic acid) was obtained from Molecular Probes, Inc. (Eugene, OR).
Binding assays
Recombinant authentic hGH (a kind gift from Bio-Technology
General, Rehovot, Israel) was radiolabeled with 125I-Na
(Nuclear Research Center-Negev, Beersheva, Israel) by the chloramine-T
method. The specific activity of 125I-hGH ranged from
70–80 µCi/µg.
Confluent cells were incubated with 125I-hGH (2 ng) in the absence (total binding) or presence (nonspecific binding) of 2 µg hGH in a final volume of 200 µl binding buffer containing 10 mM phosphate (PO4) buffer, 1% BSA, and 30 mM MgCl2, pH 7.4, for 20 h at 4 C. After removal of the binding buffer, cell monolayers were washed three times with ice-cold 10 mM PBS, pH 7.4.
Cell-bound activity was measured in a multiwell 
-counter. All
determinations were carried out in triplicate. Specific binding was
expressed as a percentage of the total radioactivity added, and data
were normalized to 200 µg cellular protein. The protein concentration
was determined by the method of Lowry et al. (19). The mean
protein content was approximately 250 µg protein/106
cells.
GH internalization
Surface-bound radiolabeled ligand was differentiated from
internalized ligand using an acid extraction procedure as previously
reported (20). Briefly, after washing with PBS, cell surface-bound
radioactivity was removed by incubating the cells with 500 µl 10
mM PO4 containing 50 mM HCl, pH 3,
for 1 min at 4 C. The fraction containing the internalized,
acid-resistant ligand was lysed with 0.1% SDS and counted in a
multiwell -counter. Internalized GHR was calculated as a percentage
of the total cell-associated radioactivity. Cell surface receptor was
estimated by incubation of cells at 4 C for 20 h with
125I-hGH.
Determination of secreted GHBP
Conditioned media of confluent cells were centrifuged at
3,000 x g (20 min, 4 C) to remove cell debris, and the
cleared supernatants were concentrated 10-fold by lyophilization. To
ascertain removal of all cell debris, medium that was ultracentrifuged
at 100,000 x g (60 min, 4 C) yielded similar binding
results. GHBP, released into the medium during incubation, was measured
by specific binding of 125I-hGH, as previously described
(21). After incubation, bound and free hormones were separated by
adding 1 ml dextran-coated charcoal (4% Norit-A, 0.4% dextran T-70)
in 10 mM phosphate buffer, pH 7.4. Specific binding was
expressed as a percentage of the total radioactivity incubated, and
data were normalized to 200 µg cellular protein.
Scatchard analyses
For determination of GHR and GHBP capacities and affinities
(Ka), binding to cells and media was performed as described
above with increasing concentrations of unlabeled hGH, and data were
calculated according to the method of Scatchard (22).
Affinity cross-linking
Confluent cells were incubated with 125I-hGH (10 ng)
in the absence or presence of 10 µg hGH (nonspecific) at 30 C for 90
min. Covalent cross-linking was achieved by the addition of 1
mM disuccinimidyl suberate (DSS; Pierce Chemical Co., Rockford, IL) freshly dissolved in dimethylsulfoxide (DMSO)
for 1 h at 4 C. Cells were homogenized in ice-cold 10
mM Tris containing 300 mM sucrose and protease
inhibitors, pH 7.4 (homogenization buffer), and centrifuged at
15,000 x g for 5 min. The protease inhibitors used
were 1 mM EDTA, 3.2 µM aprotonin, 2
mM phenylmethylsulfonylfluoride (PMSF), 10 µg/ml
leupeptin, and 10 mM benzamidine (Sigma Chemical Co. St. Louis, MO). Cross-linking studies with GHBP were
performed in concentrated (x 10) culture medium from confluent cells,
as previously described (21). Medium was incubated with
125I-hGH (10 ng) in the presence (nonspecific) or absence
(total) of hGH (10 µg) at 4 C for 20 h, followed by covalent
cross-linking and immunoprecipitation, by the addition of monoclonal
antibody (MAb) 263 (23) or an unrelated MAb (Anti-Brucella), kindly
provided by Dr. M. J. Waters, Queensland, Australia, at a 1:100
(vol/vol) final dilution. After incubation at 4 C for 2 h, the
immune complexes were collected on protein A-Sepharose beads, and the
pellets were washed four times with 10 mM Tris buffer, pH
7.4. Samples were dissolved in an equal volume of 2-fold concentrated
Laemmli sample buffer, boiled for 3 min, and subjected to 10%
SDS-PAGE. After drying, autoradiography was performed using Kodak X-O
mat AR film (Sigma Chemical Co.).
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Results |
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). The effect is not reversible, since
after removal of NEM, GHR did not recover for up to 3 h incubation
(data not shown). To examine the affinity constants and the
Mr of cellular GHR and GHBP following NEM treatment,
Scatchard analyzes and cross-linking studies were performed. Scatchard
analysis of 125I-hGH binding to cellular GHR and soluble
GHBP yielded linear plots. For GHR, the affinity constants
(Ka) values for untreated control cells and cells treated
with NEM (0.5 mM), were 6.81 x 109
M-1 and 7.24 x 109 M-1,
respectively. For GHBP, Ka values for control and
NEM-treated cells were also similar (1.54 x 109
M-1 and 1.04 x 109 M-1,
respectively),
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). In control, untreated cells, the
major band of membrane bound GHR-125I-hGH complex migrated
with an apparent Mr of 150 kDa, corresponding to the
expected size for GHR (
130 kDa) (Fig. 1A, lanes 1, 2, 5, and 6).
This band had decreased intensity when cells were treated with NEM 0.5
mM) (Fig. 1A, lanes 3 and 4). The additional, slightly
lower specific complex, which appeared in control samples, probably
represents a degradation fragment. Cross-linking of
125I-hGH to conditioned medium from control (Fig. 1B, lanes
4 and 5) and NEM-treated cells (Fig. 1B, lanes 6 and 7) indicated an
Mr of approximately 85 kDa, which is consistent with an
Mr of approximately 65 kDa for GHBP, after accounting for
the Mr of hGH. Similar Mr value was also
obtained for GHBP released over 24 h at 37 C incubation period
(Fig. 1B, lanes 1–3). Taken together, these results demonstrate that
NEM did not modify the affinity constants and the Mr of
cellular GHR or released GHBP.
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, lanes 8 and 9, and Fig. 2, initial incubation), whereas when
cells were initially exposed to NEM at 4 C and then washed and
subsequently incubated at 37 C with fresh medium, GHBP release markedly
increased in a time-dependent manner (Fig. 2B), in parallel with GHR
loss (Fig. 2A). Thus, these data indicate that following the initial
reaction with NEM, the continuous presence of NEM in the incubation
medium is no longer needed to promote GHBP release, suggesting an
irreversible process, which is temperature dependent.
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, treatment of CHO/rbGHR cells with the
cell-permeable reagent iodoacetamide (IA), induced a dose-dependent
increase in GHBP released into the medium and a parallel loss of
cellular GHR, similar to that induced by the permeable NEM. The less
lipophilic iodoacetic acid (IAA) was less active, whereas the
impermeable p-chloromercuriphenylsulfonic acid (PCMBS) affected mostly
GHR loss, but not GHBP release. The membrane impermeable iodoacetamide
derivative A-484, had no effect on either GHBP release or on cellular
GHR level. These results may imply that the essential sulfhydryl
group(s), which are effective in promoting GHBP release, are located at
sites inaccessible to the less permeable sulfhydryl-reactive
agents.
Effect of sulfhydryl-reactive agents on GH internalization in
CHO/rbGHR cells
To clarify the relationship between GHBP generation and GHR
internalization, we further studied the effect of various
sulfhydryl-reactive agents, which have been shown to promote GHBP
release, on GH internalization. CHO/rbGHR cells showed internalization
of 125I-hGH, which reached equilibrium after approximately
1 h, when approximately 80% of the specifically bound GH was
localized intracellularly (Fig. 3A).
However, GH internalization was greatly reduced by NEM (0.5
mM), with only approximately 20% of the specifically bound
GH internalized within 1 h (Fig. 3). Similarly, IA and IAA
markedly inhibited GH internalization (Fig. 3B). In contrast, the
effect observed by the impermeable agents PCMBS or A-484 was not
significant (Fig. 3B).
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GHBP generation in CHO/hGHR and CHO/hGHRtr cells
We further examined the effect of NEM on the formation of soluble
GHBP in CHO cells stably expressing the truncated hGHR isoform
(hGHRtr). When compared with CHO cells expressing the full-length hGHR,
CHO/hGHRtr cells exhibited a significant increase in GHBP generation
(Fig. 4) in keeping with previous results
(15, 17), suggesting that the absence of the cytoplasmic domain may be
involved in increased release of GHBP. Testing the effect of NEM, we
found that, similar to CHO/hGHR cells, exposure of CHO/hGHRtr cells to
NEM resulted in a dose-dependent increase in GHBP generation (Fig. 5B). Cross-linking studies of
125I-hGH to the conditioned media from control CHO/hGHR or
CHO/hGHRtr and NEM-treated cells indicated a similar Mr
values for GHBP (65 kDa) (data not shown). Thus, it appears that
cytoplasmic cysteines of hGHR are not required to activate GHBP release
by NEM. However, in CHO/hGHRtr, the increase in GHBP, observed
following NEM treatment, was accompanied by only a moderate decrease in
cellular hGHRtr (Fig. 5A).
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). Also, similar to hGHRtr (17), GH
internalization in CHO/hGHRtr-C241A cells was markedly impaired, with
only approximately 10% of the total specifically bound hGH
internalized by 1 h (data not shown). Testing the effect of NEM on
CHO/hGHRtr-C241A cells, we found a marked increase in GHBP release, but
unlike hGHRtr, this was associated with a corresponding decrease in
cellular hGHRtr-C241A, which may be due to differences in the
conformation structure of the receptor (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial experiments, using CHO cells, stably transfected with the full-length rabbit or human GHR, confirmed that cell-permeable sulfhydryl-reactive agents induced marked shedding of GHBP, whereas the Ka and molecular weight did not change. In addition, cell-permeable sulfhydryl-reactive agents proved to be more effective in promoting GHBP release, suggesting that endopeptidase (s) within membranal endocytic vesicles may participate in GHR cleavage, in keeping with earlier findings in IM-9 cells (11). However, A-484 was partially active in increasing GHBP shedding in Hep G2 cells, stably transfected with rabbit GHR (14).
We further studied the relationship between the inducible shedding of GHBP and GHR internalization, using different sulfhydryl-reactive agents that vary in their cell-permeability potency. Interestingly, cell-permeable sulfhydryl-reactive agents, like NEM and IA, also exhibited marked inhibition of GHR internalization, in keeping with reports on GHR internalization in IM-9 lymphocytes (25), insulin receptor (25), and interleukin-8 receptor (26). In contrast, cell-impermeable agents were much less (PCMBS) or even not (A-484) effective. Assuming that internalization/down-regulation processes occur subsequent to GHR dimerization (27), these results are in agreement with the inhibitory effect of NEM, but not IAA, on hGH-induced disulfide dimerization (28). It is, therefore, postulated that GHBP generation and GHR internalization are inversely related. This is also supported by our recent report demonstrating that in contrast to hGHR, hGHRtr is fixed at the cell membrane and undergoes minimal internalization, but has increased capacity to generate GHBP (17).
To explain the complex interrelationship between GHR internalization and GHBP generation, we propose the following sequence of events: upon GH binding to cell surface GHR, a single molecule of GH is bound sequentially by a dimer of GHR, leading to signal transduction, but also to receptor internalization/down-regulation. Ligand-induced internalization is associated with degradation of the [GH-GHR] complex, resulting in short-term cellular desensitization to GH. Monomeric GHRs, either unoccupied or GH-occupied, undergo proteolytic cleavage to generate GHBP. Indirect support for this model may derive from in vitro studies demonstrating that GH inhibited GHBP release (14) and from our previous review indicating that across a wide scope of comparative studies, ontogenic data, experimental systems, physiological conditions, nutritional states and disease situations the pulsatility of serum GH level is negatively correlated with GHR and GHBP (29).
We then sought to determine whether sulfhydryl alkylators, which act
intracellularly, affect cytoplasmic cysteines of GHR. Similar to
CHO/hGHR cells, GHBP release from CHO cells, expressing the short
isoform hGHRtr, was also markedly increased when these cells were
exposed to NEM. This indicates that truncation of most of the
intracellular domain of hGHR (97.5% of the cytoplasmic domain) did not
abolish NEM-induced shedding of GHBP. Thus, the cytoplasmic
domain of hGHR is not involved in the cleavage process. Yet, GHBP
release following NEM was associated by only a moderate reduction in
125I-hGH binding to cell-surface hGHRtr, apparently due to
the enhancement of cryptic receptors, resulting from the failure of
hGHRtr to internalize and its slow turnover (17). Previously, we have
reported that the cleavage process of GHBP from GHR may occur partly in
the cytoplasmic side of the plasma membrane (24). We now refine this
model to suggest the involvement of cryptic (latent) receptors that may
reside in membrane vesicles, associated with the cytoplasmic side of
the plasma membrane, as described for the receptors of insulin (30, 31), transferrin (32), tumor necrosis factor-
(TNF) (33), and
insulin-like growth factor II (34). Thus, it might be postulated that
cryptic and exposed receptors would be interchangeable (25, 26, 27, 28, 29) and
that the relative fraction of cryptic receptors is related directly to
receptor density and inversely to receptor turnover.
We further studied the role of the only free thiol group in hGHRtr, the extracellular cysteine 241 (which exists in close proximity with the cell membrane) in GHBP generation, using CHO cells, stably expressing the C241A mutation in hGHRtr (hGHRtr-C241A). Our data suggest that cysteine 241 is not essential for GH binding because C241A mutation does not interfere with the binding activity of the membrane receptor or the soluble GHBP, in keeping with an earlier observation (2). Under basal conditions, increased amount of GHBP was released by CHO/hGHRtr-C241A cells, as compared with CHO/hGHRtr cells. Similar overproduction of GHBP by hGHRtr-C241A was also obtained in several independent transient expression studies and by another mutant at position 241, in which the cysteine was replaced by serine (unpublished observations). One possible explanation is that the absence of cysteine 241 modifies the conformation of the region that includes the putative cleavage site, thereby exposing it to a proteolytic system. In addition, NEM induced GHBP generation from CHO/hGHRtr-C241A cells, indicating that cysteine 241 of the hGHR is not a target for sulfhydryl-alkylator agents. Alternative mechanisms could be suggested, such as alkylation of an intracellular free sulfhydryl group(s) of a neighboring protein, that may stabilize and maintain the GHR integrity, or on a protease that subsequently becomes activated to induce GHBP release. In support, it was recently reported that the metalloprotease inhibitor, IC3, blocked NEM-induced proteolysis and GHBP shedding (35), indicating that NEM may induce the activation of a GHBP-generating enzyme(s) of the metalloprotease family.
Received April 30, 1998.
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