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Department of Medicine, Division of Endocrinology and Metabolism (J.A., J.J., J.F.G., S.J.F.), and the Department of Cell Biology (X.Y., S.J.F.), University of Alabama, and the Veterans Affairs Medical Center (J.F.G., S.J.F.), Birmingham, Alabama 35294; the Center for Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Northwestern University Medical School (H.G.M., 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, Room 756, Diabetes Research and Education Building, UAB Station, Birmingham, Alabama 35294. E-mail: frank{at}endo.dom.uab.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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5 min) of mature GHRs (115–140
kDa) from either total cell or detergent-soluble extracts. Loss of
full-length GHRs was accompanied by accumulation of four proteins
(65–68 kDa), each reactive with the cytoplasmically directed
antiserum. The pattern of appearance of these GHR ctyoplasmic domain
proteins, the electrophoretic and immunological characteristics of
which are similar to those of a recombinant rabbit GHR mutant that
lacks the extracellular domain, was such that progressively faster
migrating forms were evident between 5–60 min of PMA exposure.
Treatment with N-ethylmaleimide (NEM; 5 mM),
an agent known to cause GHBP shedding from IM-9 cells, promoted a
similar rapid loss of full-length GHRs and an accumulation of GHR
cytoplasmic domain remnant proteins. PMA-induced, but not NEM-induced,
GHR proteolysis was blocked by the protein kinase C inhibitor,
GF109203X. Both PMA- and NEM-induced receptor proteolysis were,
however, inhibited by the metalloprotease inhibitor, Immunex Compound 3
(minimum effective concentration, 10 µM). Notably, PMA
and NEM also promoted shedding of GHBP into the conditioned medium of
the cells, as determined by a chromatographic [125I]human
GH binding assay; this GHBP shedding was also inhibited by Immunex
Compound 3. These results strongly implicate a member(s) of the
metalloprotease family as a potential GHBP-generating enzyme. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In various species, a substantial fraction of circulating GH is carried by a high affinity GH-binding protein (GHBP), the structure of which corresponds to the GHR extracellular domain (4, 5, 6). In rodents, it is believed that the GHBP is largely derived by translation of an alternatively spliced GHR messenger RNA encoding only the receptor extracellular domain and a hydrophilic tail, which replaces the transmembrane and cytoplasmic domains and facilitates the protein’s secretion (7, 8, 9). In humans and rabbits, proteolytic cleavage of the membrane-anchored receptor [either the full-length GHR or recently described truncated GHR forms (10, 11)] releases the GHR extracellular domain, which thereby becomes the GHBP (4, 5).
The mechanistic details of proteolytic shedding of the human and rabbit GHRs have remained elusive despite the establishment of cell culture models to facilitate such studies (12, 13, 14, 15, 16, 17, 18, 19, 20). Constitutive shedding of both endogenous and transfected GHRs has been observed; some degree of inhibition of proteolytic release has been achieved with EDTA and certain protease inhibitors, and a divalent metal dependence for the proteolysis has been postulated (16). Treatment of cells with sulfhydryl alkylating reagents promotes GHR shedding, although the mechanism of this effect is not clear (12, 13, 15, 19).
Using antibodies directed against both the receptor’s extracellular and cytoplasmic domains, we now demonstrate in human IM-9 cells [B lymphoblasts that homologously express human (h) GHRs] that pharmacological activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) promotes rapid proteolytic cleavage of the GHR that is similar to but distinguishable from that promoted by the sulfhydryl alkylator, N-ethylmaleimide (NEM). We also observe that constitutive, PMA-induced, and NEM-induced GHR proteolysis are all strongly inhibited by the protease inhibitor, Immunex Compound 3 (IC3). Notably, IC3 also inhibits NEM- and PMA-induced shedding of the GHBP into the conditioned medium of these cells. These results strongly implicate a member(s) of the metalloprotease family as a potential GHBP-generating enzyme.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells, cell culture, and transient transfections
IM-9 and COS-7 cells were maintained as described previously
(22). IM-9 cells were treated with PMA or NEM [or dimethylsulfoxide
(DMSO) vehicle control] after serum starvation for 16–20 h. Serum
starvation for both IM-9 and COS-7 cells was accomplished by
substituting 0.5% (wt/vol) BSA (fraction V, Boehringer Mannheim,
Indianapolis, IN) for FCS in the culture medium.
COS-7 cells (6 x 106/dish) were transiently transfected in 10 ml DMEM medium in 100 x 20-mm dishes (Falcon, Oxnard, CA) by the calcium phosphate precipitation method as described previously (23). Each dish was transfected with 30 µg pSX rGHR or pSX rGHRdel ext, which are described below, or (for a negative control) with the pSX vector only. Serum starvation was begun 24 h after transfection and continued for 18–20 h before harvesting and detergent extraction, as described below.
Antibodies
Polyclonal rabbit anti-GHRcyt serum (24), directed
at the residue 317–620 region of the human GHR cytoplasmic domain, has
been described. The anti-GHRext mouse monoclonal antibody
(IgG1 
) was raised against a glutathione-S-transferase
(GST) fusion protein incorporating residues 1–245 of the rabbit (r)
GHR (2) [GST/rGHR-(1–245)] and screened by ELISA at the University
of Alabama Multipurpose Arthritis Center Hybridoma Facility (Dr. M.
Accaviti). [Induction and purification of the fusion protein have been
described previously (25).] Anti-GHRext reacted by ELISA
specifically with GST/rGHR-(1–245) and not with GST. Similarly,
immunoblotting of thrombin-cleaved GST/rGHR-(1–245) and
GST/hGHR-(271–620) fusions indicated that anti-GHRext
reacted with rGHR-(1–245), but not with hGHR-(271–620) or GST; this
pattern of reactivity was identical to that of mAb 263, a well
characterized, commercially available (Agen) monoclonal antibody to the
GHR extracellular domain (not shown). Anti-GHRext was
purified from mouse ascites using the Affi-Gel Protein A MAPS II Kit
(Bio-Rad, Richmond, CA) according to the manufacturer’s
suggestions.
Plasmid construction
The pSX plasmid (a gift from Dr. J. Bonifacino, NIH, and
Dr. K. Arai, DNAX) drives eukaryotic protein expression from the SR
promoter, which is composed of the simian virus 40 early promoter and
the R-U5 segment of the human T cell leukemia virus-1 long terminal
repeat (26). The generation of pSX and the ligation into it at the
XbaI and KpnI sites of the rGHR complementary DNA
(cDNA) (2) (pBS.RGHR1.1, a gift from Dr. W. Wood, Genentech, South San
Francisco, CA) to generate the expression plasmid, pSX rGHR, has been
previously described (25). The cDNA encoding the rGHRdel
ext mutant [in-frame internal deletion of residues 1–247 of
the rGHR with preservation of the predicted signal sequence (residues
-18 to -1)] was generated by PCR using the pBS.RGHR 1.1 plasmid as a
template. The 5'-primer corresponded to a sequence in the Bluescript
plasmid 5' to the multiple cloning site. The 3'-primer was an
oligonucleotide 36-mer, which 5' to 3' corresponded antisense to bases
encoding residues 249 and 248 (a naturally occurring and unique
NcoI restriction site) and -1 to -8 of the rabbit GHR (2).
This PCR product was ligated into the NotI (from the pBS
multiple cloning site) and NcoI sites of pBS.RGHR1.1 after
removal from that plasmid of the NotI-NcoI
fragment that encoded all rabbit GHR residues amino-terminal to residue
248. The resultant plasmid, pBS rGHRdel ext, was subjected
to dideoxy-DNA sequencing to ensure the presence of the in-frame
internal deletion mutation and the fidelity of the remaining sequences
exposed to PCR amplification. The cDNA region encoding rGHRdel
ext was then ligated into pSX using the XbaI and
KpnI sites to generate the expression plasmid, pSX
rGHRdel ext.
Cell stimulation, protein extraction, and immunoprecipitation
Serum-starved IM-9 cells were resuspended at 25–50 x
106 cells/ml in binding buffer [BB; consisting of 25
mM Tris-HCl (pH 7.4), 120 mM NaCl, 5
mM KCl, 1.2 mM MgCl2, 0.01%
(wt/vol) BSA, and 1 mM dextrose]. After a 15-min
preincubation at the specified temperature for equilibration, PMA, NEM,
or their vehicle, DMSO (each added at a 1:100 to 1:200 dilution), was
added at the indicated final concentrations. For time-course
experiments, PMA, NEM, and DMSO were added such that all stimulations
ended simultaneously. After gentle agitation to ensure adequate mixing,
cells were incubated in a gently shaking water bath for the indicated
durations. For inhibitor experiments, GF109203X or IC3 (each
solubilized in DMSO) was added at a 1:100 dilution to the indicated
final concentrations, generally during the 15-min preincubation period,
unless otherwise indicated.
Stimulations were terminated, and IM-9 cells were collected by centrifugation (800 x g for 1 min at 4 C) and aspiration of the BB. Cell pellets were either processed directly or snap-frozen in liquid nitrogen and stored at -80 C before processing (no difference in results was noted regardless of whether cell pellets were first frozen). Detergent cell extracts were generated by resuspending the cell pellet at 100 x 106 cells/ml in a lysis buffer consisting of 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 phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, and 10 µg/ml aprotinin and incubating for 15 min at 4 C. After centrifugation at 15,000 x g for 15 min at 4 C, the detergent-soluble supernatant was collected and mixed with an equal volume of twice concentrated (2 x) SDS sample buffer [1 x sample buffer contains 1% (wt/vol) SDS, 50 mM Tris-HCl (pH 6.8), 10% (wt/vol) glycerol, and 3% (vol/vol) 2-mercaptoethanol] and boiled before SDS-PAGE. Total cell extract was prepared as previously described (27); in brief, stimulation of cells in BB at 25 x 106 cells/ml was terminated by the addition of an equal volume of boiling 2 x sample buffer. The resulting mixture was clarified by boiling and vortexing before SDS-PAGE. COS-7 detergent cell extracts were prepared as described for IM-9 cells, except that the cells were collected by scraping and centrifugation in ice-cold PBS before addition of the lysis buffer.
For immunoprecipitation of the hGHR with the monoclonal anti-GHRext antibody, 0.6 µg purified antibody was added to detergent cell extract and tumbled on a rotator at 4 C for 2 h before the addition of protein G-Sepharose (Pharmacia, Piscataway, NJ) to adsorb immune complexes. After extensive washing with lysis buffer, SDS sample buffer eluates were resolved by SDS-PAGE, as described below.
Electrophoresis and immunoblotting
Resolution of proteins in immunoprecipitates, detergent cell
extracts, and total cell extracts (prepared as above) under reducing
conditions by SDS-PAGE, Western transfer of proteins, and blocking of
nitrocellulose membranes (Hybond-ECL, Amersham, Arlington Heights, IL)
with 2% BSA were performed as previously described (22, 28).
Anti-GHRcyt serum was affinity purified (or used without
purification (1:2000)) and used for immunoblotting as previously
described (27). Horseradish-peroxidase-conjugated antirabbit secondary
antibodies (1:1500) and ECL detection reagents (all from Amersham) were
used for detection of specifically recognized hGHRs according to the
manufacturer’s suggestions.
GHBP assay
GHBP activity was measured in conditioned medium by a
standardized GH binding assay (29). Briefly, conditioned medium from
IM-9 cells treated as indicated in Fig. 6
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. Nonspecific binding under these conditions is
about 1.5% and was subtracted. The coefficient of variation for the
assay is 7.5%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Full-length
receptors present in these immunoprecipitates were detected after
SDS-PAGE by immunoblotting with an antibody directed at the cytoplasmic
domain (anti-GHRcyt). Significant loss of full-length hGHRs
was detected after as little as 5 min of treatment with PMA [1 µg/ml
(1.6 µM) at 30 C].
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). In addition, this analysis revealed concurrent PMA-induced
accumulation of a group of anti-GHRcyt-reactive proteins of
65–68 kDa (Fig. 1B, arrowheads) that were not detected in
anti-GHRext precipitates (compare Fig. 1B with Fig. 1A).
The pattern of appearance of these GHR cytoplasmic domain-containing
proteins was such that progressively faster migrating forms were
evident between 5–60 min of PMA exposure; the predominant (and fastest
migrating) form present at 60 min migrated identically to a protein
present, but less abundant, in the sample exposed to the DMSO vehicle
only (compare the proteins indicated by the lowest arrowhead
in lanes 1 and 4). Neither the hGHR nor the 65- to 68-kDa proteins were
detected when a nonimmune serum was used for immunoblotting (data not
shown).
The same pattern of acute PMA-induced receptor loss and corresponding
appearance of 65- to 68-kDa anti-GHRcyt-reactive proteins
observed in detergent-soluble extracts was noted when cells were
directly solubilized in boiling SDS-PAGE sample buffer (thus generating
total cell extracts) before electrophoresis and anti-GHRcyt
immunoblotting (Fig. 1C). This finding indicated that PMA appears to
cause a loss of total cell full-length hGHRs rather than a
redistribution of receptors to a detergent-insoluble pool, such as we
recently reported to occur in these cells in response to hGH (27).
Quantitative analysis of the kinetics of this PMA-induced loss of total
cell hGHRs is presented in Fig. 1D, in which the pooled results of
several experiments such as that shown in Fig. 1C are displayed. At 37
C, 1 µg/ml PMA promoted loss of hGHRs with a t1/2 of
about 5 min. In separate experiments (data not shown), as little as
0.01–0.1 µg/ml PMA induced receptor loss and the corresponding
appearance of 65- to 68-kDa anti-GHRcyt-reactive
proteins.
The apparent conversion of full-length GHRs into a discrete set of smaller proteins immunologically identified as including the receptor cytoplasmic domain made us consider that this process might represent a constitutive discrete proteolytic cleavage of the transmembrane-anchored receptor that is enhanced by PMA. Such a process would be analogous to that observed for some other receptors and procytokines (32, 33), in which the extracellular domain is released (shed) into the medium and a transmembrane/cytoplasmic domain remnant is thereby generated. As the constitutive proteolysis of the hGHR and rabbit GHR involved in GHBP generation is believed to occur at or near the receptor extracellular-transmembrane domain junction (13, 15), we tested whether a recombinantly derived rGHR remnant would share properties with the remnant generated by expression of the full-length rGHR.
As indicated in Fig. 2A, we generated a
cDNA encoding a mutant receptor, designated rGHRdel ext, in
which the rGHR extracellular domain is deleted, but the wild-type rGHR
transmembrane and cytoplasmic domains remain. COS-7 cells were
transiently transfected with expression plasmids encoding the wild-type
rGHR, rGHRdel ext, or (as a negative control) no insert
(Fig. 2B, lanes 1–3). Detergent extracts of each pool of transfected
cells were resolved by SDS-PAGE and immunoblotted with
anti-GHRcyt. Bands at approximately 115 and 66 kDa were
detected in the resolved extract of cells transfected with wild-type
rGHR, but not in that of cells transfected with the empty vector
(compare lanes 1 and 3). As we have previously observed (28), the
115-kDa band is consistent with the mature, fully glycosylated
full-length rGHR, whereas the 66-kDa anti-GHRcyt-reactive
band, constitutively detected in these cells, is analogous to the
protein(s) of similar Mr in the IM-9 cells described above.
Notably, rGHRdel ext (lane 2), which had the expected
specific anti-GHRcyt reactivity, migrated nearly
identically to the 66-kDa protein constitutively generated in the
rGHR-expressing cells. Thus, this 66-kDa protein and, by inference,
those observed basally and in response to PMA in IM-9 cells, have
electrophoretic and immunologic characteristics consistent with a GHR
transmembrane/cytoplasmic domain remnant.
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, treatment
with NEM (5 mM) for only 5 min induced the loss of
full-length hGHRs detectable in total cell extracts and led to the
appearance of a remnant similar to those observed with PMA treatment.
In other experiments (not shown), the minimum effective NEM
concentration to promote this processing was 0.5 mM.
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, compare
lanes 5–8 with lanes 1–4). Notably, pretreatment with GF109203X under
the same conditions had no inhibitory effect on NEM’s ability to
induce rapid receptor loss and remnant appearance (Fig. 3C, compare
lanes 5–8 with lanes 1–4). Thus, although both PMA and NEM promote
apparently similar hGHR proteolytic processing, NEM may be working
through a PKC-independent pathway.
The metalloprotease inhibitor, IC3, blocks PMA-enhanced,
NEM-enhanced, and constitutive hGHR proteolysis
Previous reports indicated a possible divalent metal dependence
for the constitutive GHBP shedding from IM-9 cells (16); additionally,
it has recently been determined that the PMA-enhanced proteolytic
shedding of the transmembrane form of the cytokine tumor necrosis
factor- (TNF) that yields soluble TNF is mediated by a
metalloprotease activity (34, 35). Given these observations, we
investigated whether the metalloprotease inhibitor, IC3, which blocks
several metalloprotease-mediated shedding events (21, 36, 37, 38, 39, 40), would
also be active in inhibiting the PMA- and/or NEM-enhanced hGHR
proteolytic processing that we observed in IM-9 cells.
In the experiment shown in Fig. 4A, IM-9
cells were treated at 37 C with PMA for 5, 15, or 60 min in the
presence of IC3 (50 µM; added 70, 60, or 15 min,
respectively, before the addition of PMA) or in its absence (addition
of the DMSO vehicle instead of IC3). Notably, anti-GHRcyt
immunoblotting of resolved total cell extracts indicated that the
presence of IC3 completely inhibited both the loss of full-length hGHR
and the accumulation of remnants promoted by PMA at each time point
(compare lanes 5–8 with lanes 1–4). In concentration dependence
experiments (not shown), detectable PMA-enhanced proteolytic processing
of the hGHR under these conditions was completely inhibited in the
presence of as little as 10 µM IC3. Time-course
experiments (not shown) also indicated that IC3 preincubation (
10
µM) for as little as 5 min blocked PMA-enhanced hGHR
proteolysis. IC3 dramatically inhibited NEM-enhanced hGHR processing as
well (Fig. 4B). Comparison of lanes 5–8 with lanes 1–4 shows marked
diminution in both NEM-enhanced hGHR loss and remnant generation with
IC3 treatment.
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, A and B (samples in
which the effect of IC3 alone, in the absence of NEM or PMA, can be
observed) indicates that exposure of the cells to IC3 for 75 min at 37
C resulted in a greater abundance of total cell hGHRs and a decrease in
an already low basal level of remnant(s). This effect of IC3 on
constitutive GHR proteolysis is presented quantitatively in Fig. 5, in which the results of several such
anti-GHRcyt immunoblotting determinations were
densitometrically measured and expressed as the abundance of
full-length GHR or remnant present after IC3 exposure relative to that
present after exposure to the DMSO vehicle alone. IC3 treatment
resulted, on the average, in a 26% increase in steady state, total
cell, full-length GHRs and a 33% decline in the steady state remnant
level. Thus, not only did IC3 inhibit PMA- and NEM-enhanced hGHR
proteolytic processing; this metalloprotease inhibitor also inhibited a
constitutive hGHR proteolysis that results in the loss of full-length
receptor and the accumulation of the remnant protein. [Interestingly,
similar analysis (not shown) of additional experiments such as those
shown in Fig. 3 indicated that treatment with GF109203X alone for 75
min resulted in a modest increase (16.4 ± 4.4%;
P < 0.01) in GHR abundance and a decrease (16.2
± 5.1%; P < 0.05) in remnant abundance compared with
treatment with DMSO vehicle; thus, like PMA-induced receptor
proteolysis, constitutive GHR proteolysis by an IC3-inhibitable
protease may be at least in part mediated by PKC.]
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and 5),
we investigated whether this would be reflected by inhibition of GHBP
shedding as well. IM-9 cells were incubated for 90 min at 37 C in the
presence of PMA, NEM, or the DMSO vehicle, and the conditioned media of
the cells were assayed for GHBP activity by a standardized GH binding
assay (29), as described in Materials and Methods. As shown
in Fig. 6A, PMA and NEM both induced
significant elevations in GHBP in the cell supernatants (from 3% GH
bound/350 µl conditioned medium for DMSO treatment (constitutive
shedding under these circumstances) to 16% and 24% for PMA- and
NEM-treated cells, respectively). Notably, inclusion of IC3 (50
µM) during the incubation eliminated (for PMA) or greatly
reduced (for NEM) these PMA- and NEM-induced increases in GHBP activity
in the conditioned medium (compare PMA- and NEM-treated samples to
those treated with PMA plus IC3 and NEM plus IC3). The already low
level of constitutively shed GHBP during this 2-h experiment was not
detectably diminished by IC3 treatment (compare DMSO to DMSO plus IC3
samples). Qualitatively very similar results were obtained when the
same experiment was performed at 30 C (Fig. 6B), except that the levels
of GHBP shed in response to PMA and NEM were reduced at that lower
temperature. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 µM) for 30 min] (30). It was concluded that the
PMA-induced receptor down-regulation was related to acute
internalization of the GHR, probably as a consequence of activation of
protein kinase C. However, in that study the conditioned medium of the
cells was not examined for the presence of GHBP-like activity, nor was
the presence of a smaller cell-associated receptor remnant sought.
Intriguingly, however, a 55-kDa phosphoprotein (thought to be receptor
associated rather than a GHR fragment per se) was observed
to inducibly copurify with the IM-9 receptor in response to PMA
(30). Our findings correspond to some aspects of the above observations and differ in other respects. The time course of the PMA-induced loss of full-length hGHRs that we observed by anti-GHRcyt immunoblotting of anti-GHRext immunoprecipitates, detergent extracts, or total cell extracts of IM-9 cells corresponds well to that seen for PMA-induced receptor down-regulation. However, our studies do not support intracellular redistribution as the sole explanation for PMA-induced receptor down-regulation, as the loss of hGHRs we detected was observed even in total cell extracts (cellular proteins extracted by directly boiling the cells in SDS sample buffer). [This point is bolstered by our observation that although we detected a hGH-induced detergent-insoluble pool of hGHRs in IM-9 cells, we did not detect any such accumulation of detergent-insoluble receptors in response to PMA (27).] The enhanced appearance in response to PMA of anti-GHRcyt-reactive protein(s), the electrophoretic and immunoreactivity characteristics of which are consistent with their being transmembrane/cytoplasmic domain remnants, supports the idea that a significant component of PMA-induced hGHR loss in IM-9 cells is due to rapid and discrete proteolysis. The complete inhibition of PMA-induced receptor loss by the metalloprotease inhibitor IC3 furthers this conclusion.
Our studies have particular relevance with regard to understanding the mechanism(s) of GHBP generation. Although its physiological role is not known with certainty, it has been estimated that up to 50% of serum GH in the human circulation is carried by this high affinity binding protein (6). Experimental evidence exists in support of both an inhibitory and a potentiating role for the GHBP in GH signaling (41, 42, 43, 44). Evidence regarding the physiological regulation of GHBP levels is also inconclusive (4, 5). As mentioned above, the rodent GHBP is believed to be largely derived by translation of an alternatively spliced GHR message that encodes the receptor external domain, but in which the transmembrane and cytoplasmic domains are replaced by a short hydrophilic sequence (7, 8, 9). By contrast, human and rabbit GHBPs are believed to be largely derived by proteolytic shedding of the external domain of membrane-anchored GHRs, although recent studies indicate that splice variants of the hGHR message that encode membrane-anchored receptors that are prematurely truncated in the proximal cytoplasmic domain may account for some of the receptors that are shed (10, 11). It is not known whether this proteolysis occurs at the plasma membrane or at intracellular membranes.
Using the IM-9 cell, a well established cell culture model system for hGHR shedding, we observed that PMA promoted rapid proteolytic hGHR cleavage, with the resultant formation of a cell-associated transmembrane/cytoplasmic domain remnant and release into the conditioned medium of significant GHBP activity. We do not yet know whether the multiple forms of the remnant that appear with a reproducible time course during PMA treatment reflect proteolysis at several discrete, closely spaced sites or a reversible modification (such as phosphorylation) that occurs to the remnant during or just after its generation. The fact that the sulfhydryl alkylator, NEM, promoted generation of a similar remnant(s) in the process of shedding functional GHBP activity is consistent with the findings of Trivedi and Daughaday (13). We note that PKC inhibition prevented PMA-induced receptor proteolysis, whereas NEM’s effect was insensitive to this inhibition. This interesting distinction may prove useful in further understanding the nature of the enzymatic activity(s) that mediates GHBP shedding. Although we do not yet know the identity of the cleaving enzyme(s), the finding that IC3, a well characterized metalloprotease inhibitor, inhibited both PMA- and NEM-induced proteolysis and GHBP shedding may indicate that the pathways or proteins affected by these two distinct reagents may ultimately lead to activation of the same or a similar GHBP-generating enzyme(s).
The potency of IC3 with regard to inhibition of PMA-induced hGHR
proteolysis in our experiments was similar to that published for other
IC3-inhibited shedding events (e.g. TNF, interleukin-6
receptor , and transforming growth factor-) that have been
intensively studied (21, 35, 36, 37, 38, 39, 40). We find it particularly notable that
constitutive hGHR proteolytic processing, in addition to that induced
by PMA, was also IC3 sensitive in IM-9 cells. Our inability to clearly
detect an inhibitory effect of IC3 on constitutive shedding similar to
that observed for its effect on constitutive proteolysis probably
reflects the limit of the sensitivity of the GHBP activity assay in
discriminating a change in the already low level of GHBP constitutively
generated during the 105-min incubation period. Nonetheless, the
biochemical findings highlight the possibility that the same enzyme(s)
might be responsible for both the dramatic pharmacological
(PMA-induced) hGHR shedding and the more subtle, but probably more
physiologically relevant, consititutive shedding exhibited by these
(and perhaps other) cells. This avenue is currently being actively
pursued. We are also intrigued by the potential functional relevance of
the basally detected and PMA- and NEM-augmented remnant protein. Given
our previous findings (22) of GH-independent association of the GHR
cytoplasmic domain with JAK2 (and, therefore, possibly with other
JAK2-associated signaling molecules), we raise the possibility that the
remnant we have identified may be capable either of GHR-specific
signaling by itself or of modulation of full-length GHR signaling in
cells in which the GHR and remnant are both present. We are actively
investigating these possibilities as well.
The recent molecular characterizations (34, 35) of an IC3-sensitive
enzyme responsible for the processing of the membrane-anchored
precursor form of TNF into the soluble TNF [the so-called
TNF-converting enzyme (TACE)] may be enlightening in understanding
the mechanism(s) of GHR shedding. TACE, a member of the ADAM subgroup
of the metzincin family of proteases (45), is a transmembrane molecule
with a zinc-dependent protease domain and a regulatory cysteine switch
in its extracellular domain (46). Although the physiological
mechanism(s) of regulation of TACE is as yet incompletely understood,
activation by sulfhydryl alkylating reagents, such as NEM, is a common
feature of this class of metalloproteases. Our findings in IM-9 cells
that PMA- and NEM-induced as well as constitutive, proteolytic cleavage
of the hGHR are inhibitable by IC3 raise the possibility that a
TACE-like enzyme might be responsible for generation of the GHBP and
suggest strategies that might be employed to identify such an
enzyme.
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Acknowledgments |
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Footnotes |
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Received October 8, 1997.
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References |
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from cells. Nature 385:729–733[CrossRef][Medline]
. Nature 385:733–736[CrossRef][Medline]
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 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]
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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]
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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]
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S. J. Frank, X. Wang, K. He, N. Yang, P. Fang, R. G. Rosenfeld, V. Hwa, T. R. Chaudhuri, L. Deng, and K. R. Zinn In Vivo Imaging of Hepatic Growth Hormone Signaling Mol. Endocrinol., November 1, 2006; 20(11): 2819 - 2830. [Abstract] [Full Text] [PDF]
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S. L. Gadd and C. V. Clevenger Ligand-Independent Dimerization of the Human Prolactin Receptor Isoforms: Functional Implications Mol. Endocrinol., November 1, 2006; 20(11): 2734 - 2746. [Abstract] [Full Text] [PDF]
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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]
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N. Gallardo, C. Arribas, M. Villar, M. Ros, J. M. Carrascosa, C. Martinez, and A. Andres ObRa and ObRe Are Differentially Expressed in Adipose Tissue in Aged Food-Restricted Rats: Effects on Circulating Soluble Leptin Receptor Levels Endocrinology, November 1, 2005; 146(11): 4934 - 4942. [Abstract] [Full Text] [PDF]
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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]
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 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]
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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]
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 S. R. Edmondson, S. P. Thumiger, G. A. Werther, and C. J. Wraight Epidermal Homeostasis: The Role of the Growth Hormone and Insulin-Like Growth Factor Systems Endocr. Rev., December 1, 2003; 24(6): 737 - 764. [Abstract] [Full Text] [PDF]
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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]
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R. Graichen, J. Sandstedt, E. L. K. Goh, O. G. P. Isaksson, J. Tornell, and P. E. Lobie The Growth Hormone-binding Protein Is a Location-dependent Cytokine Receptor Transcriptional Enhancer J. Biol. Chem., February 14, 2003; 278(8): 6346 - 6354. [Abstract] [Full Text] [PDF]
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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]
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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]
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M. Maamra, M. Bidlingmaier, M.-C. Postel-Vinay, Z. Wu, C. J. Strasburger, and R. J. M. Ross Generation of Human Soluble Leptin Receptor by Proteolytic Cleavage of Membrane-Anchored Receptors Endocrinology, October 1, 2001; 142(10): 4389 - 4393. [Abstract] [Full Text] [PDF]
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R. Guan, Y. Zhang, J. Jiang, C. A. Baumann, R. A. Black, G. Baumann, and S. J. Frank Phorbol Ester- and Growth Factor-Induced Growth Hormone (GH) Receptor Proteolysis and GH-Binding Protein Shedding: Relationship to GH Receptor Down-Regulation Endocrinology, March 1, 2001; 142(3): 1137 - 1147. [Abstract] [Full Text] [PDF]
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Y. Zhang, J. Jiang, R. A. Black, G. Baumann, and S. J. Frank Tumor Necrosis Factor-{{alpha}} Converting Enzyme (TACE) Is a Growth Hormone Binding Protein (GHBP) Sheddase: The Metalloprotease TACE/ADAM-17 Is Critical for (PMA-Induced) GH Receptor Proteolysis and GHBP Generation Endocrinology, December 1, 2000; 141(12): 4342 - 4348. [Abstract] [Full Text] [PDF]
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 M. Ballesteros, K.-C. Leung, R. J. M. Ross, T. P. Iismaa, and K. K. Y. Ho Distribution and Abundance of Messenger Ribonucleic Acid for Growth Hormone Receptor Isoforms in Human Tissues J. Clin. Endocrinol. Metab., August 1, 2000; 85(8): 2865 - 2871. [Abstract] [Full Text]
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P. Reddy, J. L. Slack, R. Davis, D. P. Cerretti, C. J. Kozlosky, R. A. Blanton, D. Shows, J. J. Peschon, and R. A. Black Functional Analysis of the Domain Structure of Tumor Necrosis Factor-alpha Converting Enzyme J. Biol. Chem., May 5, 2000; 275(19): 14608 - 14614. [Abstract] [Full Text] [PDF]
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T. Amit, M. B. H. Youdim, and Z.'e. Hochberg Does Serum Growth Hormone (GH) Binding Protein Reflect Human GH Receptor Function? J. Clin. Endocrinol. Metab., March 1, 2000; 85(3): 927 - 932. [Abstract] [Full Text]
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L. Rui, S. F. Archer, L. S. Argetsinger, and C. Carter-Su Platelet-derived Growth Factor and Lysophosphatidic Acid Inhibit Growth Hormone Binding and Signaling via a Protein Kinase C-dependent Pathway J. Biol. Chem., January 28, 2000; 275(4): 2885 - 2892. [Abstract] [Full Text] [PDF]
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Y. Zhang, J. Jiang, J. J. Kopchick, and S. J. Frank Disulfide Linkage of Growth Hormone (GH) Receptors (GHR) Reflects GH-induced GHR Dimerization. ASSOCIATION OF JAK2 WITH THE GHR IS ENHANCED BY RECEPTOR DIMERIZATION J. Biol. Chem., November 12, 1999; 274(46): 33072 - 33084. [Abstract] [Full Text] [PDF]
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 C. L. Chaffin and R. L. Stouffer Expression of Matrix Metalloproteinases and Their Tissue Inhibitor Messenger Ribonucleic Acids in Macaque Periovulatory Granulosa Cells: Time Course and Steroid Regulation Biol Reprod, July 1, 1999; 61(1): 14 - 21. [Abstract] [Full Text]
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T. Amit, O. Bar-Am, F. Dastot, M. B. H. Youdim, S. Amselem, and Z.'e. Hochberg The Human Growth Hormone (GH) Receptor and Its Truncated Isoform: Sulfhydryl Group Inactivation in the Study of Receptor Internalization and GH-Binding Protein Generation Endocrinology, January 1, 1999; 140(1): 266 - 272. [Abstract] [Full Text]
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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]
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