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Original Studies |
Department of Pharmacology (T.A., T.B., M.B.H.Y., Z.H.), Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel; INSERM U468 (F.D., S.A.), Hopital Henri Mondor, 94010 Creteil, France; and Department of Pediatrics (Z.H.), Rambam Medical Center, Haifa, 31096 Israel
Address all correspondence and requests for reprints to: Dr. Ze’ev Hochberg, Faculty of Medicine, Technion, P.O. Box 9649, Haifa 31096, Israel. E-mail: rprzeev{at}tx.technion.ac.il
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Abstract |
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100 kDa, which would indicate the hGHRtr
to be in molecular mass form of about 80 kDa. When compared with
CHO/hGHR, CHO/hGHRtr cells secreted higher amounts of soluble
GH-binding protein (GHBP). In contrast to CHO/hGHR cells, CHO/hGHRtr
cells did not exhibit any GH-induced receptor down-regulation, and
internalization was markedly reduced. Analysis of the constitutive
turnover of cellular hGHR and soluble GHBP showed that incubation of
CHO/hGHR cells with cycloheximide caused parallel disappearance of hGHR
and GHBP. This contrasted with the stability of GHRtr, which showed no
decline after cycloheximide treatment for up to 4 h, suggesting
that the bulk GHRtr and GHBP may be derived from preformed proteins.
Thus, in contrast to hGHR, hGHRtr is fixed at the cell membrane; it
undergoes minimal internalization, no down-regulation by hGH, no
constitutive turnover for as long as 4 h, but increased capacity
to generate a soluble GHBP. Because hGHRtr failed to undergo
ligand-induced internalization, the source of the continuous,
undisturbed GHBP released into the medium may be from an intracellular
storage pool. The relative abundance of these two hGHR isoforms,
through regulation of splicing, could be of critical importance in
modulating the biological effects of GH. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Upon GH binding to cell surface GHR, a single molecule of GH is bound by a dimer of GHR (2), leading to receptor internalization and down-regulation and, finally, processing by one of two pathways: a small rapid, nondegradative pathway, which results in recycling or exocytosis, and a slower, lysosomal degradative pathway (3). Cell-surface GHRs turn over rapidly and constitutively and are not replaced in the absence of protein synthesis (3, 4). In addition, like many cytokine receptors in its family, GHR exists in a soluble form of a GH-binding protein (GHBP) that corresponds to the extracellular domain of GHR (5, 6).
In humans, 2 isoforms of the human GHR (hGHR) messenger RNA (mRNA) have been identified: exon 3 can be either retained or deleted, leading to an in-frame deletion of 22 amino acids within the extracellular domain of the receptor, with no apparent impact on binding isotherms or on signal transduction. (7, 8, 9). An alternatively spliced form in the cytoplasmic domain of hGHR (hGHRtr) has recently been described in several human tissues. hGHRtr mRNA is identical in sequence to hGHR, except for a 26-bp deletion, leading to the creation of a stop codon at position 280, thereby truncating 97.5% of the intracellular domain of hGHR (10). As shown by transient expression studies, when compared with hGHR, hGHRtr showed a significantly increased capacity to generate a soluble GHBP (10, 11).
The present study was undertaken to further characterize this new, naturally occurring hGHRtr isoform. To this aim, Chinese hamster ovary (CHO) cells were stably transfected with expression vectors encoding hGHR or hGHRtr and tested for GH-binding properties, GH-mediated internalization, and down-regulation and GHR turnover, in relation to GHBP generation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To obtain cell lines overexpressing the two GHR constructs, CHO cells were cotransfected with a neomycin resistance plasmid and phGHR or phGHRtr corresponding to the previously described SV40 promoter-based expression vector pECE (12) carrying the full-length hGHR complementary DNA sequence or hGHRtr, respectively (10). Transfections were performed at 60% confluence by the LipofectAmine method (Life Technologies, Grand Island, NY) in Iscove’s medium, and selection in 500 µg/mL G418 was carried out for 2 weeks before subcloning survivors. Stably transfected cells (designated CHO/hGHR and CHO/hGHRtr) and nontransfected CHO cells were maintained at 37 C in Ham’s F-12 medium, supplemented with 10% FCS (Kibbutz Beth-Haemek, Israel) and processed for mRNA analysis, binding, and cross-linking studies. Nontransfected CHO cells exhibit no GH binding, as previously reported (4).
RNA Isolation and Northern blot analysis
Total cellular RNA was isolated in parallel from nontransfected and GHR-overexpressing CHO cells and transferred by capillary blotting onto Hybond nylon membranes (Amersham, Aylesburg, UK). Filters were hybridized with the hGHR complementary DNA probe labeled by random hexanucleotide priming (Life Technologies), as previously described (13). The relative abundance of RNA in each lane was judged to be similar by comparing ethidium bromide-staining intensity of the ribosomal bands. For further confirmation, the blots were hybridized with a probe for an endogenous housekeeping gene, ß-actin (data not shown).
GH-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.
Cellular GH binding
Confluent cells were incubated with [125I]hGH (1
ng) in the absence (total binding) or presence (nonspecific binding) of
1 µg hGH in a final vol of 200 µL binding buffer containing 10
mmol/L PO4 buffer, 1% BSA, and 30 mmol/L MgCl2
(pH 7.4) for 90 min at 30 C or for 20 h at 4 C. After removal of
the binding buffer, cell monolayers were washed three times with
ice-cold 10 mmol/L 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, determined by the Lowry method. The affinity constant was
calculated from incubation with increasing concentrations of unlabeled
hGH, according to Scatchard, using the Ligand program (14).
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 (4). Briefly, binding of [125I]hGH (1 ng) was performed in a final vol of 270 µL binding buffer, with or without 1 µg hGH for 20 h at 4 C. Free and bound [125I]hGH were separated by adding 1 mL dextran-coated charcoal (0.2% Dextran T-70 and 2% Norit-A charcoal) in 10 mmol/L PO4 buffer, pH 7.4. After incubation on ice and centrifugation, the radioactivity was measured in the supernatant. Specific binding was expressed as a percentage of the total radioactivity incubated, and data were normalized to 200 µg cellular protein, so that cellular GHR and medium GHBP for each well were jointly normalized.
GH internalization and down-regulation
Surface-bound radiolabeled ligand was differentiated from
internalized ligand using an acid extraction procedure (15). Briefly,
after washing with PBS, cell surface-bound radioactivity was removed by
incubation of cells with 500 µL 10 mmol/L PO4 containing
50 mmol/L 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. Down-regulation was measured by incubating
cells with different concentrations of hGH in serum-free medium for 90
min at 30 C. The cells were placed on ice and associated hGH was
removed by acid wash, as described (15). Residuals GHRs at the cell
surface were measured by [125I]hGH binding for 20 h
at 4 C.
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 mmol/L disuccinimidyl suberate (Pierce Chemical Co., Rockford, IL), freshly dissolved in dimethylsulfoxide for 1 h at 4 C. Cells were homogenized in ice-cold 10 mmol/L Tris containing 300 mmol/L 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 mmol/L ethylenediamine tetraacetate, 3.2 µmol/L aprotinin, 2 mmol/L phenylmethylsulfonyfluoride, 10 µg/mL leupeptin, and 10 mmol/L benzamidine (Sigma Chemical Co. St. Louis, MO). Samples were dissolved in an equal volume of 2-fold concentrated Laemmli sample buffer, boiled for 3 min, and equal amounts of protein were subjected to 10% SDS-PAGE. After drying, autoradiography was performed using Kodak X-omat AR film (Sigma Chemical Co).
Statistical analysis
Experiments were repeated at least three times, and the data were analyzed by ANOVA, followed by Student’s t test.
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Results |
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Cross-linking of [125I]hGH to CHO/hGHR and
CHO/hGHRtr cells is shown in Fig. 1
. In
both cases, specific complexes were displaced in the presence of an
excess unlabeled hGH. For the full-length hGHR, we observed two
cross-linked specific bands with apparent Mr of 150 kDa and 120 kDa.
Subtraction of the molecular mass of hGH yields Mr values of 130 kDa
and 100 kDa, that may represent the native hGHR and a degradation
fragment or a GHBP dimer, respectively. CHO cells expressing hGHRtr
revealed a majored specific complex with an apparent Mr value of 100
kDa, which would indicate the hGHRtr to be, in molecular mass form,
about 80 kDa, after accounting for the hormone component. The larger
band of about 170 kDa could represent a dimeric form of the
receptor.
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Fig. 2 shows the levels of cellular
GHR and secreted soluble GHBP in CHO cells transfected with hGHR or
hGHRtr. The amount of secreted GHBP by CHO/hGHRtr cells was
significantly higher than that generated by CHO/hGHR (2.8 ±
0.1-fold and 4.2 ± 0.7-fold for 1 h and 4 h,
respectively). Scatchard analysis revealed a single-component,
high-affinity constant of 8.3 x 109
mol/L-1 and 4.3 x 109
mol/L-1 for hGHR and hGHRtr, respectively.
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We examined the ability of CHO/hGHRtr cells to internalize
[125I]hGH, compared with the full-length hGHR. CHO/hGHR
showed rapid internalization of [125I]hGH, which reached
an apparent equilibrium within 30 min, when around 60% of the total
specific binding was localized intracellularly (Fig. 3). In contrast, in CHO/hGHRtr cells, hGH
internalization was markedly reduced with only 10% of the total
specifically bound hGH internalized by 1 h.
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In addition to internalization studies, we further examined
GH-induced down-regulation of hGHRtr, compared with hGHR. As shown in
Fig. 4, CHO/hGHR cells showed a
dose-dependent down-regulation of GHR. After 90 min incubation with 100
and 5,000 ng/mL hGH, followed by an acid wash to expose occupied
binding sites, 50% and 70%, respectively, of specific GHRs were
down regulated. In contrast, the apparent up-regulation of hGHRtr,
observed with increasing hGH concentrations up to 1,000–5,000 ng/mL
(P < 0.05 vs. control) and declined with
50,000 ng/mL. The control experiment, in the absence of an acid wash,
verifies an initial binding to cell surface receptors of both cell
lines.
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Analysis of the turnover of cellular GHR and soluble GHBP in
CHO/hGHR and CHO/hGHRtr cells was performed after inhibition of protein
synthesis by cycloheximide. Confluent cells were incubated with
cycloheximide (20 µg/mL) for 1–4 h before measuring
[125I]hGH binding to cells (Fig. 5A) and medium (Fig. 5B). In CHO/hGHR
cells, cycloheximide caused a time-dependent decrease in GHR and GHBP,
with a half-time of about 2 h and 3 h, respectively. In
contrast, in CHO/hGHRtr cells, cycloheximide had no effect on GHR and
GHBP levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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The present study shows that hGHRtr misses completely both constitutive and ligand-regulated turnover. Cell-surface GHRs turn over rapidly, under both ligand-regulation and constitutively in various cell types, and are not replaced after blocking of protein synthesis (3, 4). Consistent with our earlier observations (4) and with the present results, incubation of CHO/hGHR cells with cycloheximide caused a parallel disappearance of GHR and GHBP, with t1/2 values that are comparable with those reported earlier (3, 4). This turnover time is in marked contrast to the relative stability of hGHRtr, which shows no measurable decline after cycloheximide treatment up to a 4-h test period, suggesting that the bulk GHR and GHBP may be derived from preformed proteins. For the rat GHR, it has been shown that the domain(s) involved in GH internalization is located in the cytoplasmic domain region between positions 294 and 454 (16). Furthermore, a region of the rat GHR between amino acids 319 and 380 has been reported to be essential for GH-induced internalization, with a single phenylalanine (Phe 346) being critical (17). It was recently shown that ubiquination of the cytoplasmic domain is essential for endocytosis and degradation of the GHR (19), which may also fit with the markedly reduced ability of GH internalization of hGHRtr.
We have previously reported on the synchronization of GHR turnover with GH pulsatility (20). hGHRtr adds a new dimension to this concept. Whereas hGHR (in response to a hGH pulse) internalizes, degrades, or recycles and restores itself at the cell surface (to receive the next GH pulse), hGHRt would remain fixed at the cell membrane, either occupied with a preload of hGH, guarding leftover ligand for a next hGHR emergence, or receiving a ligand during the refractory period.
Thus, the intracellular processing of hGHRtr is significantly different from the usual model suggested for cellular processing of GH and GHR, in which, upon GH binding, the [GH-GHR] complexes are rapidly internalized, the majority of the internalized GH and GHR are degraded, and rapid receptor synthesis is required to maintain GH binding capacity of the cell (21). Moreover, because the hGHRtr failed to undergo ligand-induced internalization, the source of the continuous, undisturbed, soluble GHBP released into the medium may be from an intracellular storage pool. This is consistent with the observation that in CHO/hGHRtr cells, cycloheximide did not affect GHR and GHBP levels, and with earlier findings suggesting that cycloheximide does not interfere with externalization of receptors from the intracellular compartment (22). The receptor expressed on the cell surface may not be the source of the GHBP released through the action of an exoprotease, but rather, GHBP may originate from within the cell of a receptor protein that perhaps is generated at an early posttranslational state.
The present results suggest that internalization/down-regulation are not required for GHBP generation and that these may be inversely related processes. It is speculated that because internalization is a path for GHR degradation, it may result in rapid receptor clearance, leading to cellular desensitization (23). The absence of this attenuation mechanism in CHO/hGHRtr cells could be compensated for by the enhancement of GHBP generation. Indeed, GHBP competes with GHR for the ligand, which results in an inhibition of receptor binding and consequent GH action in vitro (24, 25).
The existence of hGHRtr (a naturally occurring isoform of hGHR that fails to internalize and to undergo GH-induced down-regulation, but fully retains the capacity to generate GHBP) may reflect the different functions associated with the relative abundance of hGHR and hGHRtr in different cell types. The relative abundance of hGHR and hGHRtr differ among tissues (10), implying a regulated process. Under such conditions, alternative splicing may be regulated to favor one of these species over the other. GHBP generation may be favored in conditions when alternative splicing deviates towards hGHRtr. Interestingly, hGHRtr mRNA is favorably expressed in placenta and mammary gland (10), where estrogen activity is obvious, and indeed, estrogens have been suggested to increase serum GHBP (26). This principle may apply to other regulators of GHR, such as GH itself. In vitro studies performed in 293 cells have suggested that heterodimerization of the two isoforms could result in eventual negative regulation of GHR by GHRtr (11). The relative abundance of the two hGHR isoforms, which, as documented in the present study, is regulated at the plasma membrane, could be of critical importance in modulating the biological effects of GH.
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Footnotes |
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2 Incumbant of the E.S.P.E.-Henning Anderson Award, 1997, for this
work.
Received May 2, 1997.
Revised July 16, 1997.
Accepted July 23, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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