help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Amit, T.
Right arrow Articles by Hochberg, Z.'e.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amit, T.
Right arrow Articles by Hochberg, Z.'e.
Endocrinology Vol. 140, No. 1 266-272
Copyright © 1999 by The Endocrine Society

ARTICLES

The Human Growth Hormone (GH) Receptor and Its Truncated Isoform: Sulfhydryl Group Inactivation in the Study of Receptor Internalization and GH-Binding Protein Generation

Tamar Amit, Orit Bar-Am, Florence Dastot, Moussa B. H. Youdim, Serge Amselem and Ze’ev Hochberg

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human GH receptor (hGHR) contains nine intracellular and seven extracellular cysteines, of which six are linked by disulfide bonds and one, at position 241 proximal to the membrane, is free. Recently, an alternatively spliced GHR isoform has been isolated; it encodes a truncated receptor lacking most of the cytoplasmic domain (hGHRtr). In the present study, we have examined the effect of sulfhydryl group(s) inactivation on receptor internalization and GH binding-protein (GHBP) generation from the human (h) and rabbit (rb) full-length GHR, as well as from hGHRtr and a mutant of the free extracellular cysteine (hGHRtr-C241A), expressed in Chinese hamster ovary (CHO) cells. In CHO/rbGHR and CHO/hGHR cells, permeable sulfhydryl-reactive agents, like N-ethylmaleimide (NEM) and iodacetamide (IA), inhibited GHR internalization and induced an immediate dose-dependent loss of cellular GHR, associated with a concomitant marked increase in released GHBP. In contrast, the membrane impermeable IA derivative A-484 had no effect on either GHBP release or on GHR internalization. NEM exposure of CHO cells, expressing hGHRtr, resulted in a dose-dependent increase in GHBP generation, but only a moderate decrease in cellular hGHRtr. The importance of the only unpaired cysteine in these processes was evaluated in CHO/hGHRtr-C241A cells. hGHRtr-C241A was similar to hGHRtr in its impaired internalization and enhanced GHBP release by NEM.

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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GH receptor (GHR), a member of the family of cytokine receptors, consists of 620 amino acids with a 350-amino acid cytosolic portion, a 24-amino acid transmembrane domain, and a 246-residue exoplasmic domain (1). The extracellular region contains seven cysteines, of which six are linked by disulfide bonds and one, at position 241 proximal to the membrane, is free (2). The seven extracellular cysteines are conserved in the human and rabbit sequences. Of the nine intracellular cysteines in the human receptor and eight in the rabbit receptor, seven are homologous (1). In addition to the membrane-bound form of GHR, a soluble GHBP, which corresponds to the extracellular domain of GHR, has been described (3, 4, 5, 6, 7). In mouse and rat, specific messenger RNAs for GHBP have been isolated; they are generated by alternative splicing of the primary GHR transcripts (8, 9). However, in human and rabbit, no equivalent mechanism has been observed, and GHBP is generated by proteolytic cleavage of the full-length transmembrane GHR (1, 10, 11). Indeed, GHBP is shed spontaneously from CHO cells transfected with rabbit (but not rat) GHR (rbGHR) complementary DNA (10). In addition, in human (h) cells, we and others have previously demonstrated GHBP release from IM-9 lymphocytes (11, 12) and human hepatoma Hep G2 cells (13, 14) by sulfhydryl-reactive agents, suggesting a role for sulfhydryl groups in GHR cleavage.

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfections
CHO cells stably expressing the rabbit GHR (rbGHR) were kindly provided by Drs. M. C. Postel-Vinay and P. A. Kelly (INSERM, Paris, France), who described in detail the plasmid construct and transfection procedure (10). Transfection and selection of CHO cells, stably expressing the full-length human GHR (hGHR), or the truncated hGHR (hGHRtr) have been described previously (15) and the same procedure was used to obtain CHO cells stably expressing a mutant of hGHR, where cysteine 241 was replaced by an alanine residue (hGHRtr-C241A). CHO cells were cultured in Ham’s F-12 medium supplemented with 10% (vol/vol) FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mg/liter penicillin/streptomycin/nystatin, and 10 mM HEPES buffer, pH 7.4. The sterile culture medium, FCS, and antibiotic solutions were purchased from Biological Industries (Kibbutz Beth HaEmek, Israel). Cell cultures were incubated at 37 C in a humid 5% CO2-95% air environment. Stable transfectants were selected in 500 µg/ml G418 (neomycin; Life Technologies, Grand Island, NY).

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 {gamma}-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 {gamma}-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.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of sulfhydryl-reactive agents on GHBP release in CHO/rbGHR cells
Initial experiments clearly showed that treatment of CHO/rbGHR cells with NEM, a cell-permeable sulfhydryl-reactive agent, for 30 min at 30 C, resulted in a dose-dependent loss of cellular GHR and a concomitant increase in soluble GHBP, released into the medium (Table 1Go). 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),


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of various sulfhydryl-reactive agents on GHR and GHBP. Confluent CHO/rbGHR cells were incubated in the absence (control) or presence of the above sulfhydryl-reactive agents for 30 min at 30 C and 125I-hGH binding to GHR and GHBP was measured, as described in Materials and Methods. Data are represented as a percentage of control (mean ± SE; n = three independent experiments). 100% for GHR = 20.1 ± 2.5%/200 µg protein and for GHBP = 2.3 ± 0.2%/200 µg protein.

 
CHO/rbGHR cells and media were also analyzed by cross-linking of 125I-hGH with DSS, followed by SDS-PAGE (Fig. 1Go). 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. 1AGo, lanes 1, 2, 5, and 6). This band had decreased intensity when cells were treated with NEM 0.5 mM) (Fig. 1AGo, 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. 1BGo, lanes 4 and 5) and NEM-treated cells (Fig. 1BGo, 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. 1BGo, 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.



View larger version (48K):
[in this window]
[in a new window]
  		Figure 1. Effect of NEM on GHR and GHBP cross-linked to 125I-hGH. Confluent CHO/rbGHR cells were incubated in the absence [for 30 min at 30 C (A, lanes 1 and 2, and B, lanes 4 and 5) or at 4 C (A, lanes 5 and 6, and B, lane 8)], or presence of 0.5 mM NEM for 30 min at 30 C [(A, lanes 3 and 4, and B, lanes 6 and 7), or at 4 C (A, lanes 7 and 8, and B, lane 9)]. Control medium was obtained after incubation for 24 h at 37 C (B, lanes 1–3). Cells (A) or media (B) were incubated with 125I-hGH (15 ng) in the absence (A, lanes 1, 3, 5, and 7, and B, lanes 1, 2, 4, 6, 8, and 9) or presence of excess hGH (15 µg) (A, lanes 2, 4, 6, and 8 and B, lanes 3, 5, and 7). Cross-linking was carried out by the addition of 1 mM DSS, and cells were homogenized (A) or immunoprecipitated with MAb 263 (B, lanes 1, 3–9) or an unrelated MAb (anti-Brucella; B, lane 2). Samples containing equal amounts of protein were subjected to 10% SDS-PAGE and autoradiographed. The proteins of the molecular mass markers (x103) are shown on the left, and specific bands are indicated by arrows on the right.

 
The stimulatory effect of NEM on GHBP release from CHO/rbGHR cells was further studied at different temperatures: at 4 C, it was markedly reduced (Fig. 1BGo, lanes 8 and 9, and Fig. 2Go, 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. 2BGo), in parallel with GHR loss (Fig. 2AGo). 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.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. Recovery of GHR and GHBP after removal of NEM. Confluent CHO/rbGHR cells were intially incubated in the absence (control) or presence of NEM (0.5 mM) for 30 min at 4 C. after removal of NEM (see arrows), cells were reincubated at 4 C or 37 C for the indicated times. Binding data are expressed as a percentage of specific binding per 200 µg cellular protein. Data are shown as the mean ±SE (n = three independent experiments).*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control.

 
We further studied the effect of various sulfhydryl-reactive agents, which vary in their cell permeability, on GHBP release from CHO/rbGHR cells. As summarized in Table 1Go, 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. 3AGo). 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. 3Go). Similarly, IA and IAA markedly inhibited GH internalization (Fig. 3BGo). In contrast, the effect observed by the impermeable agents PCMBS or A-484 was not significant (Fig. 3BGo).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. Regulation of GH internalization by sulfhydryl-reactive agents. Confluent CHO/rbGHR cells were incubated with 125I-hGH (2 ng) for 2 h at 4 C. Subsequently, the cells were washed and incubated in the absence (control) or presence of NEM (0.15 mM), IA (20 mM), IAA (20 mM), PCMBS (1 mM) or A-484 (0.15 mM). After various incubation times at 37 C, cells were washed with acidic buffer and solubilized to determine intracellular radioactivity. A, Data are expressed as a percentage of total specific cell-associated binding, from a representative experiment that was repeated three times. B, Data are expressed as a percentage of the control values. Results are shown as the mean ± SE (n = three independent experiments). **, P < 0.01; ***, P < 0.001 vs. control. Control values expressed as a percentage of internalization are: 59.7 ± 4% and 76.6 ± 3.8% for 30 and 60 min, respectively.

 
These results may suggest the involvement of one or more intracellular sulfhydryl groups in efficient GHR internalization and an inverse relation of internalization and GHBP release.

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. 4Go) 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. 5BGo). 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. 5AGo).



View larger version (40K):
[in this window]
[in a new window]
  		Figure 4. Time course of GHBP release. Confluent CHO/hGHR, CHO/hGHRtr, or CHO/hGHRtr-C241A cells were incubated with fresh Ham’s F-12 medium containing 10% FCS, for 0.5, 1, and 3 h and 125I-hGH specific binding to cells and media was determined, as described in Material and Methods. Results are expressed as the ratio GHBP to GHR. ***, P < 0.001 vs. CHO/hGHR; +++, P < 0.001 vs. CHO/hGHRtr.

 


View larger version (18K):
[in this window]
[in a new window]
  		Figure 5. Effect of NEM on GHR and GHBP in CHO transfected cell lines. Confluent CHO/rbGHR, CHO/hGHR, or CHO/hGHRtr cells were incubated in the absence (control) or presence of NEM for 30 min at 30 C and 125I-hGH binding to cells (A) and media (B) was measured. Binding data are expressed as a percentage of specific binding per 200 µg cellular protein, and each point is the mean ± SE of three independent experiments. *, P < 0.05; ***, P < 0.001 vs. control.

 
GHBP generation and GH internalization in CHO/hGHRtr-C241A cells
The importance of the only unpaired cysteine 241 (at the extracellular region of hGHR) in GHBP shedding was evaluated in CHO cells, stably transfected with a mutant of hGHRtr, where cysteine 241 was substituted by alanine, hGHRtr-C241A. CHO cells expressing hGHRtr-C241A retained a similar Ka for hGH, compared with hGHR and hGHRtr (Ka = 3.74 ± 0.92 x 109 M-1, 5.15 ± 1.59 x 109 M-1 and 3.34 ± 0.58 x 109 M-1, respectively). The Bmax values were: 1.12 ± 0.57 x 10-10, 0.78 ± 0.06 x 10-10 and 4.45 ± 0.34 x 10-10 M, respectively. CHO/hGHRtr-C241A cells generated a high level of soluble GHBP (Fig. 4Go). 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. 6Go).



View larger version (39K):
[in this window]
[in a new window]
  		Figure 6. Effect of NEM on GHR and GHBP in CHO/hGHRtr-C241A. Confluent CHO/hGHRtr-C241A cells were incubated in the absence (control) or presence of 0.15 or 0.5 mM NEM for 30 min at 30 C, and 125I-hGH binding to cells and media was measured. Data are represented as a percentage of the value in control, untreated cells, and shown as the mean ±SE (n = 4 independent experiments). 100% for GHR = 9.34 ± 0.37%/200 µg protein and for GHBP = 8.84 ± 0.66%/200 µg protein. ***, P < 0.001 vs. respective control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In human and rabbit, GHBP is generated by proteolytic cleavage of the extracellular domain of GHR (1, 10, 11): GHBP has been shown to be released from CHO cells, transfected with rabbit or human GHR complementary DNA (10, 15, 17, 21, 24) as well as from IM-9 lymphocytes and Hep G2 cells by iodoacetamide or NEM (11, 12, 13, 14), suggesting a role for sulfhydryl group(s) in GHBP shedding. The present study was undertaken to examine the mechanism(s) by which sulfhydryl-reactive agents induce GHBP generation, by comparing their effects on the full-length hGHR with the hGHR truncated isoform (hGHRtr) and the hGHRtr-C241A mutant form of hGHRtr. In addition, we have analyzed the relationship between GH-induced GHR internalization and the inducible shedding of GHBP, using different sulfhydryl-reactive agents that vary in their cell-permeability potency.

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-{alpha} (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.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Benzel WJ, Barnard R, Waters MJ, Wood WI 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537–543[CrossRef][Medline]
  2. Fuh G, Mulkerrin MG, Bass S, McFarland N, Brochier M, Bourell JH, Light DR, Wells JA 1990 The human growth hormone receptor. Secretion from Escherichia coli and disulfole bonding pattern of the extracellular binding domain. J Biol Chem 265:3111–3115[Abstract/Free Full Text]
  3. Barnard R, Waters MJ 1997 The serum growth hormone binding protein: pregnant with possibilities. J Endocrinol 153:1–14[Abstract/Free Full Text]
  4. Baumann G, Stolar MW, Amburn K, Barasano CP, Devries BC 1986 A specific growth hormone-binding protein in human plasma: initial characterization. J Clin Endocrinol Metab 62:134–141[Abstract]
  5. Herington AC, Ymer S, Stevenson J 1986 Identification and characterization of specific binding proteins for growth hormone in normal human sera. J Clin Invest 77:1817–1823
  6. Ymer S, Herington AC 1985 Evidence for the specific binding of growth hormone to a receptor-like protein in rabbit serum. Mol Cell Endocrinol 41:153–163[CrossRef][Medline]
  7. Amit T, Barkey RJ, Bick T, Hertz P, Youdim MBH, Hochberg Z 1990 Identification of growth hormone-binding protein in rat serum. Mol Cell Endocrinol 70:197–202.[CrossRef][Medline]
  8. Smith WC, Kuniyosh J, Talamantes F 1989 Mouse serum growth hormone (GH) binding protein has GH receptor extracellular and substituted transmembrane domains. Mol Endocrinol 3:984–990[CrossRef][Medline]
  9. Baumbach WR, Horner DL, Logan JS 1989 The growth hormone-binding protein in rat serum is an alternative spliced form of the growth hormone receptor. Genes Dev 3:1199–1205[Abstract/Free Full Text]
  10. Sotiropoulos A, Goujon L, Simonin G, Kelly PA, Postel-Vinay MC, Finidori J 1993 Evidence for generation of the growth hormone-binding protein through proteolysis of the growth hormone membrane receptor. Endocrinology 132:1863–1865[Abstract]
  11. Trivedi B, Daughaday WH 1988 Release of growth hormone binding protein from IM-9 lymphocytes by endopeptidase is dependent on sulfhydryl group inactivation. Endocrinology 123:2201–2206[Abstract]
  12. Massa G, Ilondo M, Vanderschneren-Lodeweyckx M 1993 Comparison between the human serum growth hormone-binding protein and the water-soluble growth hormone-binding site released from IM-9 lymphocytes. Acta Endocrinol (Copenh) 129:559–564[Medline]
  13. Amit T, Hacham H, Dally O, Barkey RJ, Hochberg Z 1994 The Hep G2 cell line in the study of growth hormone receptor/binding protein. Mol Cell Endocrinol 101:29–36[CrossRef][Medline]
  14. Harrison SM, Barnard R, Ho KY, Rajkovic I, Waters MJ 1995 Control of growth hormone (GH) binding protein release from human hepatoma cells expressing full-length GH receptor. Endocrinology 136:651–659[Abstract]
  15. Dastot F, Sobrier M-L, Duquesnoy P, Duriez B, Goossens M, Amselem S 1996 Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein. Proc Natl Acad Sci USA 93:10723–10728[Abstract/Free Full Text]
  16. Ross RJM, Esposito N, Shen XY, Von Lane S, Chew SL, Dobson PRM, Postel-Vinay M-C, Finidori J 1997 A short isoform of the human growth receptor functions as a dominant negative inhibitor of the full-length receptor and generates large amounts of binding protein. Mol Endocrinol 11:265–273[Abstract/Free Full Text]
  17. Amit T, Bergman T, Dastot F, Youdim MBH, Amselem S, Hochberg Z 1997 A membrane-fixed, truncated isoform of the human growth hormone receptor. J Clin Endocrinol Metab 82:3813–3817[Abstract/Free Full Text]
  18. Ayling RM, Ross R, Towner P, Von Lane S, Finidori J, Moutonssamy S, Buchanan CR, Clayton PE, Norman MR 1997 A dominant-negative mutation of the growth hormone receptor causes familial short stature. Nat Genet 16:13–14[CrossRef][Medline]
  19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265:275[Free Full Text]
  20. Roupas P, Herington AC 1987 Receptor-mediated endocytosis and degradative processing of growth hormone by rat adipocytes in primary culture. Endocrinology 120:215:2158–2165
  21. Bick T, Amit T, Manzur M, Bar-Am O, Youdim MBY, Hochberg Z 1996 Regulation of cellular rabbit growth hormone (GH) receptor and GH-binding protein generation in vitro. Endocrinology 147:3977–3985
  22. Scatchard G 1949 The interaction of proteins for small molecules and ions. Ann NY Acad Sci 51:660–672[CrossRef]
  23. Barnard R, Budensen PG, Rylatt DB, Waters MJ 1985 Evidence from the use of monoclonal antibody probes for structural heterogeneity of growth hormone receptor. Biochemistry 231:459–468
  24. Amit T, Bick T, Youdim MBH, Hochberg Z 1996 In search of the cellular site of growth hormone (GH)-binding protein cleavage from the rabbit GH receptor. Endocrinology 137:3986–3991[Abstract]
  25. Sonne O 1988 Receptor-mediated endocytosis and degradation of insulin. Physiol Rev 68:1129–1195[Free Full Text]
  26. Samanta AK, Dutta S, Ali E 1993 Modification of sulfhydryl groups of interleukin-8 (IL-8) receptor impairs binding of IL-8 and IL-8-mediated chemotactic response of human polymorphonuclear neutrophils. J Biol Chem 268:6147–6153[Abstract/Free Full Text]
  27. Ilondo MM, Damholt AB, Cunningham BA, Wells JA, De Meyts P, Shymko RM 1944 Receptor dimerization determines the effects of growth hormone in primary rat adipocytes and cultured human IM-9 lymphocytes. Endocrinology 134:2397–2403[Abstract]
  28. Frank SJ, Gilliland G, Van Epps C 1994 Treatment of IM-9 cells with human growth hormone (GH) promotes rapid disulfide linkage of the GH receptor. Endocrinology 135:148–156[Abstract]
  29. Hochberg Z, Phillip M, Youdim MBH, Amit T 1993 Regulation of the growth hormone (GH) receptor and GH-binding protein by GH pulsatility. Metabolism 42:1617–1623[CrossRef][Medline]
  30. Eriksson JW, Lonnroth P, Wesslau C, Smith U 1997 Insulin promotes and cyclic adenosine 3',5'-monophosphate impairs functional insertion of insulin receptors in the plasma membrane of rat adipocytes: evidence for opposing effects of tyrosine and serine/threonine phosphorylation. Endocrinology 138:607–612[Abstract/Free Full Text]
  31. Torossian K, Freedman D, Fantus IG 1988 Vanadate down-regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes. J Biol Chem 263:9353–9359[Abstract/Free Full Text]
  32. Tanaka H, Teitelbaum SL 1990 Vitamin D regulates transferrin receptor expression by bone marrow macrophage precursors. J Cell Physiol 145:303–309[CrossRef][Medline]
  33. Han HM, Kolhatkar AA, Marino MW, Manchester KM, Donner DB 1990 Identification, characterization, and homologous up-regulation of latent (cryptic) receptors for tumor necrosis factor-{alpha} in rat liver plasma membranes. J Biol Chem 265:18590–18594[Abstract/Free Full Text]
  34. Yu ZW, Wickman A, Eriksson JW 1996 Cryptic receptors for insulin-like growth factor II in the plasma membrane of rat adipocytes—a possible link to cellular insulin resistance. Biochim Biophys Acta 1282:57–62[Medline]
  35. Alele J, Jiang J, Goldsmith JF, Yang X, Maheshwari HG, Black RA, Baumann G, Frank SJ 1998 Blockade of growth hormone receptor shedding by a metalloprotease inhibitor. Endocrinology 139:1927–1935AU: Define footnote.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Amit, T.
Right arrow Articles by Hochberg, Z.'e.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amit, T.
Right arrow Articles by Hochberg, Z.'e.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals