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Department of Pharmacology, Yale University School of Medicine (M.S.L., S.S.K., P.S.D.), New Haven, Connecticut 06520; the Department of Pediatrics, Cornell University Medical College (M.P.W., J.W., R.L.), New York, New York 10021; the Department of Pediatrics, Johns Hopkins University School of Medicine (L.P.P.), Baltimore, Maryland 21287; and the Department of Pediatrics, Columbia University College of Physicians and Surgeons (J.M.G., R.L.), New York, New York 10032
Address all correspondence and requests for reprints to: Priscilla S. Dannies, Yale University School of Medicine, 333 Cedar Street, Department of Pharmacology, New Haven, Connecticut 06520-8066.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helix bundle with 2
intramolecular disulfide bridges. The mature 22-kDa GH protein accounts
for approximately 75% of circulating GH; the majority of the remaining
25% is a 20-kDa product that results from alternate splicing of the
GH1 gene, deleting amino acids 32–46 (del32–46-GH)
(1). Familial isolated GH deficiency type II (IGHDII) segregates as autosomal dominant severe short stature. Several families with this disorder have mutations in the first, fifth, or sixth base pair of the donor splice site of intron 3 of the GH1 gene (2, 3, 4). Mutations in the first and sixth base pairs have been shown to result in missplicing of messenger RNA (mRNA) and loss of exon 3, so that GH produced from this message lacks amino acids 32–71 (del32–71-GH) (2, 5, 6). These amino acids constitute the entire connecting loop between helix 1 and helix 2 (7), and without them the GH molecule cannot fold normally. The mechanisms by which this del32–71 mutant apparently suppresses the secretion of wild-type GH in individuals heterozygous for the mutation are not known.
Secretory proteins fold in the endoplasmic reticulum. Unfolded or misfolded proteins synthesized in the secretory pathway are usually retained in the endoplasmic reticulum and degraded and do not usually interfere with the folding of other proteins (8, 9, 10, 11). Human GH is a relatively small, monomeric, soluble protein, and the presence of a mutant protein that cannot fold properly would not necessarily be expected to interfere with the folding of wild-type hormone. Two examples of previously identified mutations of the GH1 gene that result in forms with aberrant folding are consistent with this expectation (12, 13). In one, the first 56 amino acids have the normal GH sequence, but a subsequent 2-bp deletion results in a frame shift and altered amino acid sequence (12). In the second, a splice site mutation results in a mRNA with an altered reading frame after the first 103 amino acids (13). These changes in amino acid sequence will alter the tertiary structure of these proteins compared with that of wild-type GH. Although these proteins cannot fold normally, the mutations are phenotypically recessive, and GH produced from the wild-type gene in heterozygotes is sufficient to support normal growth.
These two mutations suggest that the production of a protein with an inability to fold is not sufficient to suppress the production of wild-type GH. The mechanisms previously proposed for autosomal dominant deficiencies related to protein folding are the toxic accumulation of misfolded or unfolded proteins, the accumulation of dysfunctional heterodimers, or a combination of the two (14, 15, 16). We investigated whether expressing del32–71-GH in pituitary cells exerts a dominant negative effect on the accumulation of wild-type hormone and whether these mechanisms play a role in such suppression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell transfections
GH4C1 cells were
incubated in a 1:1 mixture of Ham’s nutrient F-10, and DMEM plus 15%
horse serum with 1 nM estradiol, 5 nM epidermal
growth factor, and 300 nM insulin and transiently
transfected using Superfect (QIAGEN, Chatsworth, CA),
using conditions determined to give maximum secretion of transfected
hormones in GH4C1 cells: 1
µg total plasmid DNA plus 6 µl Superfect/50,000 cells for 3 h.
COS, CHO, and AtT20 cells were transfected using the same
conditions.
Hormone assays
Plates were collected 1 day after transfection and assayed for
mutant and wild-type GH by SDS-PAGE and then immunoblotting or were
treated first with 400 µCi Express 35S Protein
Labeling Mix (NEN Life Science Products, Boston, MA), and
then collected and assayed for labeled hormone by immunoprecipitation
followed by SDS-gel electrophoresis (18). In some experiments,
transfected cells were incubated with serum-free medium for 3 h or
more, and the medium (1.5 ml) was concentrated by centrifugation using
Ultrafree-MC Filters (Millipore Corp., Bedford, MA) to a
volume of 20 µl followed by SDS-PAGE and then immunoblotting the
entire amount of concentrated medium. The antibody used for both assays
was antihuman GH GH-IC-3 antiserum from the National Hormone and
Pituitary Program, NIDDK, NICHHD, the USDA, and Dr. A. F. Parlow.
The antiserum was used at a dilution of 1:1000 for immunoblots and
1:400 for immunoprecipitation. The amount of antiserum bound to GH in
the immunoblots was assayed by
[125I]protein A binding, and both immunoblots
and [35S]GH were quantified using a
Molecular Imager (Bio-Rad Laboratories, Inc.,
Richmond, CA).
Concentrations of human GH and human PRL in the medium were assayed by RIA with the reagents provided by the National Hormone and Pituitary Program and Dr. A. F. Parlow. At the dilution of the antisera used for RIAs, there was no cross-reaction with rat GH or rat PRL from GH4C1 cells.
RNA extraction and Northern analysis were carried out as previously described (18).
Quantitative comparisons of hormone synthesis or accumulation were always made using cells that were transfected and assayed in the same experiment. The values in the text are the mean ± SE of three or more independent experiments unless otherwise indicated. The mean of two experiments is given ± the range.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In rat pituitary GH4C1
cells transfected with wild-type GH alone, human GH was detected by
immunoblotting after electrophoresis (Fig. 1A
, lanes 2 and 6), and there was little
or no cross-reaction with rat GH produced by
GH4C1 cells at the
dilutions of antiserum used (Fig. 1A, lanes 1 and 5). Cells transfected
with del32–71-GH alone contained little or no immunoreactive product
at 17.5 kDa, the predicted size of the del32–71 mutant protein (Fig. 1A, lane 3). Cells transfected with the same total amount of DNA, as a
mixture of half wild-type and half del32–71-GH, would be expected to
contain 50% as much wild-type GH if coexpression of del32–71-GH did
not affect the accumulation of wild-type GH. The amount of
intracellular wild-type GH in cells transfected with the mixture of
wild-type GH and del32–71-GH was consistently less than 50% (Fig. 1A, lane 4), averaging 11 ± 2.7% of the intracellular GH in cells
transfected with wild-type GH alone.
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, lane
7) and did not decrease the accumulation of wild-type hormone (Fig. 1A, lane 8). The average accumulation of wild-type GH in cells transfected
with the mixture of wild-type and del32–46-GH was 64 ± 9.8% of
that in cells transfected with wild-type alone. The average
accumulation of del32–46-GH in cells transfected with the mixture of
wild-type and del32–46-GH was 44 ± 10.0% of that in cells
transfected with del32–46-GH alone. We also examined the effect of cotransfecting wild-type GH and vector either with no insert or with cDNA for human PRL as an insert. The average accumulation of wild-type GH protein in cells transfected with the mixture of wild-type protein and vector with no insert was 55 ± 4% (n = 2) of that in cells transfected with wild-type protein alone, and the average accumulation of wild-type GH in cells transfected with the mixture of wild-type GH and human PRL was 58 ± 4% (n = 2) of that in cells transfected with wild-type GH alone. Therefore, the suppression of wild-type GH accumulation by coexpression in GH4C1 cells is specific to del32–71-GH.
Secretion of human GH from transfected
GH4C1 cells was assessed by
immunoblots of concentrates of serum-free medium after several hours of
incubation with the transfected cells.
GH4C1 cells secreted
wild-type GH (Fig. 1B, lanes 2 and 6) and del32–46-GH (Fig. 1B, lanes
7 and 8), and there was no immunoreactivity at the 17.5-kDa position
expected for del32–71-GH (Fig. 1B, lane 3). We also measured by RIA
the amount of human GH immunoreactivity accumulated in serum-containing
medium during the 24-h incubation after transfection. No immunoreactive
product accumulated in the medium of
GH4C1 cells transfected
with del32–71-GH alone. In those experiments with transfected
GH4C1 cells in which
intracellular GH levels assayed by immunoblots were compared with
secretion over 24 h by RIA, the results achieved with the two
different methods were similar. Therefore, coexpression of
wild-type and del32–71-GH suppressed both secretion and intracellular
accumulation of wild-type GH.
Del32–71-GH mRNA accumulated in transfected
GH4C1 cells (Fig. 2, lane 3). Most of the mRNA that
hybridized with the probe for wild-type or del32–71-GH migrated as
high mol wt forms near the top of the gel, indicating that the
transcription termination signal in the pcDNA3 vector did not function
as effectively as the initiation signal at the CMV promoter, so that
RNA polymerase transcribes the entire vector more than once. Such high
mol wt mRNA molecules also accumulated in COS cells (not shown). The
amounts of GH mRNA were approximately the same whether cells were
transfected with wild-type, del32–71-GH, or the mixture of half
wild-type and half del32–71-GH (Fig. 2, lanes 2–4).
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). Secretion of wild-type GH
assessed by RIA in AtT20 cells transfected with half wild-type and half
del32–71-GH was 23 ± 4.7% of that secreted by cells transfected
with wild-type GH alone.
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, lane 2).
No del32–71-GH was detected in the incubation medium of COS cells
(Fig. 4B, lanes 3 and 4); this result was expected, as proteins that
cannot fold are not usually secreted from cells. Accumulation of
wild-type GH in the incubation medium of COS cells transfected with
half-wild-type and half del32–71-GH was 63 ± 6% (n = 2) of
that in the medium from cells transfected with wild-type GH alone. COS
cells, therefore, differed from the neuroendocrine cell lines,
GH4C1 and AtT20, by virtue
of intracellular accumulation of the mutant and by the absence of a
dominant negative effect of del32–71-GH on wild-type GH accumulation.
Transfection of CHO cells gave results similar to those for COS cells
(data not shown).
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, top panel, section A), using AtT20 cells because rat GH
normally made by GH4C1
cells was also precipitated at the concentrations of antiserum
necessary to immunoprecipitate human GH. Cells transfected with half
wild-type and half del32–71-GH would be expected to synthesize 50% as
much wild-type GH as cells transfected with wild-type GH alone if
coexpression of del32–71-GH did not affect the synthesis of wild-type
GH. AtT20 cells cotransfected with wild-type and del32–71-GH contained
50.1 ± 4.5% as much wild-type [35S]GH at
the end of the 7-min pulse (Fig. 6A),
indicating that synthesis of wild-type GH was not suppressed. We
assayed GH degradation by measuring the amounts of
[35S]GH that remained in the cultures 4 h
after the 7-min pulse of 35S-labeled amino acids
(Fig. 5, top panel, sections B and C). (The quantitation of
human GH in arbitrary units in the experiment shown in Fig. 5, top panel, is as follows: cells transfected with
wild-type GH alone: pulse 108; chase in cells, 30; in medium, 147; and
cells transfected with both: pulse, 62; chase in cells, 2; chase in
medium, 46.) In cells transfected with wild-type GH alone, the
[35S]GH present at the end of the 7-min pulse
was still present 4 h later in the cells and medium (Fig. 6B); in
some experiments the amount of [35S]GH even
increased, most likely because the incorporation of
35S-labeled amino acids was not completely
prevented during the 4-h chase period. In cells transfected with the
mixture of wild-type and del32–71-GH, however,
[35S]GH in the cells and medium after 4 h
was about 40% less than that present at the end of the 7-min pulse
(Fig. 6B). Therefore, wild-type GH was stable in AtT20 cells expressing
wild-type protein alone, but was degraded in cells transfected with the
mixture.
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, bottom panel, section A, lane 3). During a 40-min chase, the
amount of 35S-labeled 17.5-kDa protein decreased
and that of 35S-labeled 14.5-kDa protein
increased, consistent with the conversion of the 17.5-kDa protein to
the 14-kDa form with time (Fig. 5, bottom panel, sections A
and B, lanes 3). (The quantitation of human GH in the experiment shown
in Fig. 5, bottom panel, in arbitrary units was as follows:
cells transfected with wild-type GH alone: pulse, 169; chase in cells,
81; chase in medium, 59; cells transfected with del32–71-GH alone, the
17.5-kDa form: pulse, 116; chase, 30; and the 14-kDa form: pulse, 55;
chase, 63.)
Incorporation of 35S-labeled amino acids into
17.5- and 14-kDa forms was detected in AtT20 cells after a 30-min
incubation with 35S-labeled amino acids (Fig. 7, lane 2), and the intensities of these
bands increased when proteasome inhibitors were added during the
incubation (Fig. 7, lane 3), indicating at least some del32–71-GH is
synthesized by AtT20 cells.
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). In
GH4C1 cells transfected
with equal amounts of human PRL and wild-type GH, the mean ratio of
human GH to human PRL in the incubation medium was 1.03 ± 0.19,
indicating that the PRL and GH vectors induce similar levels of protein
expression. GH4C1 cells
produced as much human PRL when cotransfected with del32–71-GH as they
did when cotransfected with wild-type GH (Fig. 8), indicating that
coexpressing del32–71-GH did not suppress the production of PRL.
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-helices, but also
removes cysteine 53. Consequently, the normal disulfide bond with
cysteine 165 cannot form, leaving an unpaired sulfhydryl group. An
unpaired sulfhydryl group, however, is not necessary for the dominant
negative effect on accumulation of wild-type GH, because
del32–71,C165A-GH, which has an alanine substituted for the unpaired
cysteine, also prevents the accumulation of wild-type GH in
GH4C1 cells (Fig. 3). The
results obtained with the various mutants in the different cell lines
are summarized in Table 1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Autosomal dominant deficiencies in PTH, vasopressin, and GH have been identified that are attributable to defects in protein folding (2, 3, 5, 6, 15, 20, 21). In PTH, the mutation results in a single amino acid substitution, and in vasopressin, the mutations include single amino acid substitutions, a single amino acid deletion, and a prematurely terminated polypeptide chain (15, 20, 21). The mechanism for autosomal dominant vasopressin deficiency has been explored by stably transfecting cells with vasopressin mutants. The mutant proteins are retained in the endoplasmic reticulum, consistent with a lack of proper folding (15, 20), and in some conditions accumulation of the mutants is toxic to the cells (15). Neuro2A cells can be induced to resemble postmitotic neurons; under inducing conditions, clones of Neuro2A cells expressing mutant vasopressins die, unlike the clone expressing wild-type vasopressin (15). These results support the explanation that cell-toxic effects of misfolded proteins cause dominant negative genetic phenomena.
Toxic accumulation of misfolded proteins or formation of dysfunctional oligomers have also been proposed as explanations for autosomal dominant GH deficiency, particularly in cases in which mutations in the GH1 gene result in deletion of exon 3 (13, 15, 22). GH is active as a monomer, and dysfunctional aggregates have been proposed to form through intermolecular disulfide bond formation. We demonstrated that transfecting del32–71-GH with wild-type GH in neuroendocrine cells suppresses the accumulation and secretion of wild-type hormone by a mechanism that differs from either cell-toxic accumulation or dysfunctional oligomers. Transfecting del32–71-GH did not cause general toxic effects in neuroendocrine cells over the timespan of these experiments, because PRL production was not affected by del32–71-GH cotransfections in GH4C1 cells, and wild-type GH synthesis was not suppressed by del32–71-GH cotransfection in AtT20 cells. Del32–71-GH did not accumulate in either cell type, and its synthesis was barely detectable, so it cannot form appreciable amounts of aggregates with wild-type GH to cause suppression. On the contrary, in COS cells in which the mutant did accumulate, there was no suppression of wild-type GH accumulation. No higher mol wt forms of GH were detected in immunoblots of GH4C1 cell extracts run under nonreducing conditions (not shown). Such higher mol wt aggregates would be expected if intermolecular disulfide bonds had formed in wild-type GH. In addition, the del32–71-GH mutant that lacks a sulfhydryl group (del32–71,C165A-GH) was incapable of suppressing wild-type GH accumulation, indicating that the unpaired sulfhydryl group is not necessary for the effect.
The reduced accumulation of wild-type GH caused by del32–71-GH occurs because the stability of wild-type GH is decreased. Synthesis of wild-type GH (measured by 35S-labeled amino acid incorporation) in AtT20 cells cotransfected with wild-type and del32–71-GH was 50% of that in cells transfected with wild-type GH alone, but accumulation of wild-type GH in the cotransfected cultures was 23% of that in cells transfected with wild-type GH alone. The difference between synthesis and accumulation is due to the decreased stability of wild-type GH that we measured in cells cotransfected with del32–71-GH. Decreased stability of wild-type GH without accumulation of the mutant isoform (del32–71-GH) has not been previously described as a mechanism for dominant negative effects of a misfolded protein.
The lack of accumulation of del32–71-GH could reflect low rates of synthesis, rapid degradation, or both. In general, proteins in the endoplasmic reticulum that do not fold properly are rapidly degraded (11). An important mechanism for such degradation is transport of the proteins back across the membrane of the endoplasmic reticulum to the cytosol, where they are cleaved by proteasomes (23, 24, 25). The mechanisms of such protein degradation are not fully elucidated, but it has been shown that proteins need not be completely transported into the endoplasmic reticulum, and possibly not even completely synthesized, for reverse transport and degradation to occur (26). The simplest explanation for the lack of accumulation of del32–71-GH is that the mutant protein is synthesized, but degraded immediately thereafter. The presence of mRNA for del32–71-GH in amounts similar to those of the mRNA for wild-type GH and the ability of proteasome inhibitors to enhance [35S]del32–71-GH accumulation are consistent with this explanation.
Binder et al. measured GH secretion of Epstein-Barr virus-transformed lymphocytes derived from an individual heterozygous for the del32–71-GH mutation and from two control subjects (22). The transformed lymphocytes from the heterozygote secreted as much human GH as one of the controls, although the other control secreted 10 times more than either of the others, so that the dominant negative effect of del32–71-GH was not apparent in lymphocytes. The apparent lack of effect in lymphocytes may reflect the tissue specificity that we found. Using transient transfections, we found dominant suppression of wild-type GH by del32–71-GH in neuroendocrine cells, but not in the other cell types that we tested.
Mutations in the GH1 gene that result in deletion of exon 3
are an apparent cause of autosomal dominant GH deficiency and severe
growth failure, because these mutations are present in all affected
members of several families with this deficiency (3, 13, 22, 27). The
data presented here support this genetic evidence by demonstrating that
del32–71-GH suppresses intracellular accumulation and secretion of
wild-type GH in transfected cultures. The decreased intracellular
stability of wild-type GH may provide a mechanism for IGHDII associated
with mutations that cause synthesis of this deletion mutant. The
decreased stability of wild-type GH is apparent within 1 day of
cotransfection with del32–71-GH and does not exclude the possibility
that other mechanisms may also reduce the production of wild-type
hormone over a longer time. Several cellular responses to unfolded
proteins have been characterized, including increased transcription of
genes for chaperones that have an unfolded protein response element
upstream of their promoters (28, 29), activation of transcription
factor NF-
B (30, 31), and phosphorylation of eukaryotic initiation
factor-2 (32, 33). Phosphorylation of initiation factor-2 decreases
overall protein synthesis. Such a general mechanism does not appear to
be triggered in neuroendocrine cells by del32–71-GH, because PRL
production is not reduced in
GH4C1 cells, and wild-type
GH synthesis is not suppressed in AtT20 cells. Synthesis of the mutant
may, however, activate other characterized or as yet unrecognized
pathways to interfere chronically with GH mRNA accumulation or
somatotrope development in addition to the direct suppression of GH
protein accumulation reported here.
Cells that express the del32–71-GH mutant are capable of synthesizing wild-type GH, but are rendered less capable of a subsequent step in the secretion of GH, so that some of the newly synthesized wild-type hormone is degraded, rather than released. The del32–71-GH mutant may directly compete with wild-type hormone for a protein, such as a chaperone, necessary for the correct folding or transport of the wild-type GH. Alternatively, expressing this mutant protein may induce changes in the secretory pathway of neuroendocrine cells that result in decreased ability to fold or transport wild-type hormone. An obvious difference in the secretory pathways of GH4C1 and AtT20 vs. COS and CHO cells is the former’s ability to store hormone. CHO and COS cells export proteins without storing them, and the two neuroendocrine cell lines store concentrated hormones in secretory granules. The mechanisms by which neuroendocrine cells concentrate proteins for storage are not known (34, 35). It is possible that differences in the responses of neuroendocrine and other cells to the expression of del32–71-GH are caused by cell-specific differences in the handling of protein hormones in the secretory pathway that are necessary for this concentration.
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Acknowledgments |
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Footnotes |
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Received September 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. Trends Biochem Sci 22:63–67[CrossRef][Medline]
B by
ER stress requires both Ca2+ and reactive oxygen
intermediates as messengers. FEBS Lett 392:129–136[CrossRef][Medline]
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32–71, vector
containing human del32–71-GH; 
