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Life Science Laboratories (M.W., H.U., M.I., B.T., N.N., M.H.) and Computational Science Laboratory (S.B., E.T.) Mitsui Chemicals, Inc. Mobara, Chiba 297, Japan Institute of Biological Science (Y.H.) Mitsui Pharmaceuticals, Inc. Mobara, Chiba 297 Japan
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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5–10% of
pituitary hGH (1, 2). It differs from 22K-hGH in its deleted region
corresponding to amino acid residues E32-Q46 of 22K-hGH and arises from
the same gene (hGH-N) as 22K-hGH by alternative mRNA splicing (3, 4). Human GH exerts a wide variety of effects, which are generally classified as somatogenic or lactogenic (5). The former activity is induced via hGH receptor (hGHR) (6), the latter via human PRL receptor (7). Regarding an interaction of 22K-hGH with hGHR, considerable information has been accumulated. There are two binding sites called site 1 and site 2 in 22K-hGH (8) whose locations have already been characterized by alanine-scanning mutagenesis (9, 10) and an x-ray structural analysis (11). According to the previous gel filtration study, at a 1:2 (10 µM:20 µM) ratio of 22K-hGH to the recombinant hGH-binding protein (hGH-BP), which corresponds to the extracellular domain of hGHR (12), all proteins chromatographed as a single 1:2 (hGH:hGH-BP) complex. However, when the ratio was greater than 1:2, a 1:1 complex and a free 22K-hGH appeared in addition to a 1:2 complex (8). This finding was consistent with the result of the cell proliferation assay performed using mouse FDC-P1 cells expressing a hybrid receptor of hGHR and the murine G-CSF receptor (13). In the assay, 22K-hGH showed a bell-shaped dose-response curve, i.e. it induced cell proliferation at low concentrations but self-antagonized at high concentrations.
Based on these findings, a sequential dimerization model for an activation of hGHR by 22K-hGH has been proposed (10, 13, 14). At low concentrations, 22K-hGH binds with hGHR first at site 1 and subsequently at site 2 to produce an active 1:2 complex. In contrast, at high concentrations where an excess of 22K-hGH exists relative to hGHR, 22K-hGH produces an inactive 1:1 complex by binding preferentially at site 1, thereby behaving as an antagonist. On the other hand, the stoichiometry of hGH-BP complexes with 22K-hGH in human plasma has been reported to differ from that of cell surface hGHR. Most 22K-hGH exists as a 1:1 complex with hGH-BP prevailing in plasma (0.35–2 nM), even though virtually all 22K-hGH is captured in a 1:2 complex by cell surface hGHR (60 nM–6.7 µM) (15). However, it is not clear whether these models are adaptable for 20K-hGH.
In the present study, Ba/F3-hGHR cell proliferation and gel filtration assay were performed to examine the stoichiometry of complexes of 20K-hGH with cell surface hGHR and hGH-BP prevailing in human plasma. Next we predicted the conformational difference between 20K- and 22K-hGH by computer-aided homology modeling. Our results demonstrate that 20K-hGH forms a 1:2 complex as efficiently as 22K-hGH, but that 20K-hGH has difficulty in forming a 1:1 complex, which might be explained partly by a conformational change occurring in its site 1 region.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) and found that both isoforms showed similar potencies except at 5
µM, where the self-inhibitory effect of 20K-hGH was
significantly weaker (P < 0.005). In view of a
sequential dimerization model, it was speculated that 20K-hGH might
have difficulty in generating a 1:1 complex with hGHR even though it
could generate a 1:2 complex to the same extent as 22K-hGH.
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); however,
only a slight reduction occurred in 20K-hGH at 0.05 and 0.25
nM (P < 0.05, Fig. 2B). The maximal
inhibition (49.2% inhibition) was observed at 0.05 nM in
22K-hGH, whereas the maximal inhibition (11.0%) was at 0.25
nM in 20K-hGH. When 1 nM hGH-BP was added, a
similar result was obtained (data not shown). This finding strongly
suggests that 20K-hGH may be poorly captured by hGH-BP in the
physiological range.
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In the micromolar level study, hGH-BP concentration was fixed at 1.8
µM. Mixtures of hGH with hGH-BP at various ratios
corresponding to 5:1, 2:1, 1:1, 0.5:1, and 0.25:1 were separated on
TSK-G2000 SWXL column (
7.8 x 300 mm) and detected on a
fluorometer (excitation: 280 nm; emission: 340 nm). When 22K-hGH was
used as a control (Fig. 3A), only a
single peak was detected at a retention time (RT) of 7.4 min at a 0.5:1
ratio, and two other peaks (RT 7.8 and 9.0 min) appeared at ratios
greater than 0.5:1. In view of the previous report (14), these three
peaks were considered to correspond to a 1:2 complex (RT 7.4 min), a
1:1 complex (RT 7.8 min), and free 22K-hGH (RT 9.0 min), respectively.
These peaks were also identified by determination of molecular size of
cross-linked mixtures subjected to SDS-PAGE (data not shown). At a
0.25:1 ratio, a peak at RT of 9.0 min was considered to represent an
excess of free hGH-BP. In contrast, in 20K-hGH (Fig. 3B), only two
peaks representing a 1:2 complex (RT 7.5 min) and free 20K-hGH (RT 9.3
min) were detected at ratios exceeding 0.5:1, but no peak of a 1:1
complex was detected even under the condition where a 1:1 complex
predominates in 22K-hGH. This finding is consistent with the weaker
self-inhibition of 20K-hGH seen in Fig. 1
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-1, a single peak of a 1:2 complex
of 22K-hGH with hGH-BP (150 nM:300 nM) was
detected. It was replaced by the peak of a 1:1 complex as the
concentrations decreased (see Figs. 4A-2,3,4 and 5) consistent with a
previous report (15). However, in 20K-hGH, the peak area of a 1:2
complex was gradually diminished and finally disappeared without an
appearance of a detectable peak of a 1:1 complex (see Figs. 4B-1,2,3,4
and 5), which agrees with the data in Fig. 2.
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10
mm x 450 mm) using 22K-hGH as a control. The quantification of
22K-hGH or 20K-hGH in each fraction was performed by the EIA described
above. As shown in Fig. 5, two peaks were
detected in 22K-hGH. The first peak was considered to correspond to the
previously reported 1:1 complex, and the second peak corresponded to
free 22K-hGH (15). In contrast, no 1:1 complex peak of 20K-hGH was
detected. This result directly indicates that 20K-hGH can be poorly
captured by human plasma hGH-BP.
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distance between two
clusters (I32-E36 and E43-K47) of exo-1,4 ß-D-glycanase
best fits that between the two clusters (T27-F31 and C38-S42) adjacent
to the N32-L37 in 20K-hGH (18) (Fig. 6A).
Root mean square deviation was only 1.24 Å. Then we built up a 20K-hGH
model assuming the structural variable region would be restricted to
the region of these six residues. The stability of this model was
assessed by 50 psec molecular dynamics simulation surrounded by 7 Å
explicit water, where it was demonstrated that only the loop region
moved whereas the four helices remained an initial coordinate (data not
shown). Next we built up a 1:2 complex model of 20K-hGH with hGH-BP by
substituting 22K-hGH of the 1:2 complex model for 20K-hGH, followed by
molecular minimization (Fig. 6B). A conformational difference between
20K- and 22K-hGH was seen only in the loop (E30-S47 in 20K-hGH; E30-S62
in 22K-hGH) connecting helix 1 with helix 2. The contact surface area
of 20K-hGH with hGH-BP 1 was calculated to be 1150 Å2,
which was approximately 200 Å2 smaller than that of
22K-hGH (1350 Å2). In contrast, the contact surface area
of 20K-hGH with hGH-BP 2 was unchanged. Next, we estimated hydrophobic
interactive regions in the interface between 22K-hGH and hGH-BP 1 based
on the previously reported crystal structure (11). Consequently, we
found two regions by searching hydrophobic residues that interact with
each other within 5 Å: one is composed of W104 (hGH-BP 1), W169
(hGH-BP 1), P61 (22K-hGH), F176 (22K-hGH), and I179 (22K-hGH), while
the other is composed of two pairs of interaction between W76 (hGH-BP
1) and P48 (22K-hGH), and between P106 (hGH-BP 1) and L45 (22K-hGH). In
20K-hGH, the latter region was found to be quite changed due to the
deleted 15 residues. Accordingly, P48 (22K-hGH) seemed to be replaced
by L37 (20K-hGH), and L45 (22K-hGH) was replaced by P33 (20K-hGH), as
shown in Fig. 6C. Furthermore, in previous work, four salt bridges
between 22K-hGH and hGH-BP 1 such as K41 (22K-hGH) or R167 (22K-hGH)
and E127 (hGH-BP 1), D171 (22K-hGH) and R43 (hGH-BP 1), E56 (22K-hGH)
and R71 (hGH-BP 1), and R64 (22K-hGH) and D164 (hGH-BP 1) (11, 19),
were also demonstrated. However, the 20K-hGH model could not form one
salt bridge among them due to the lack of K41 (22K-hGH). Nevertheless,
R152 (20K-hGH) next to E127 (hGH-BP1) is likely to replace K41
(22K-hGH). From these data, we can predict that site 1 binding affinity
of 20K-hGH could be reduced even though three residues (P33 and L37 in
hydrophobic interaction; R152 in salt bridge) in 20K-hGH can partially
compensate for a deficit of 200 Å contact surface area.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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).
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The slight but significant inhibition of 20K-hGH bioactivity by 20K-hGH itself at 5 µM and by hGH-BP at 1 and 10 nM strongly suggests that 20K-hGH does form a 1:1 complex with hGHR/hGH-BP even if the amount might be quite low. A previous report has also shown that the recombinant hGH-BP at less than 10 nM competed significantly for the binding of [125I]20K-hGH to the IM-9 cells, but less than that of [125I]22K-hGH (22). These findings disagree with the result of our gel filtration study in which no clear peak of 20K-hGH 1:1 complex was detected. The inconsistency can be explained by the possibility that the amount of the resulting 1:1 complex of 20K-hGH may be too insignificant to be recognized as a single peak by detection systems such as fluorometry and EIA.
Gel filtration analysis using human plasma has provided direct evidence that 20K-hGH forms a 1:1 complex poorly with hGH-BP prevailing in human plasma while 22K-hGH forms it efficiently. We have obtained similar results in other samples of human and rat plasma (our unpublished data). Baumann et al. (23) have reported that 26–31% of pituitary-derived 20K-hGH, although less than that of 22K-hGH (39–59%), bound to human plasma hGH-BP. We speculate that such a higher binding potency of the pituitary-derived 20K-hGH might result from contamination with 22K-hGH and other impurities in natural 20K-hGH preparations.
The poor interaction of 20K-hGH with circulating hGH-BP may be associated with its plasma clearance rate, which still remains controversial. Earlier work demonstrated that pituitary-derived 20K-hGH had a much slower clearance rate than 22K-hGH in the rat (24); in contrast, another group reported contradictory data that recombinant methionylated 20K-hGH was cleared at a faster rate than 22K-hGH when injected with recombinant hGH-BP into the guinea pig (25). Furthermore, it was reported that 20K- and 22K-hGH had similar plasma half-lives in the mouse (26). Conclusive evidence in the human must await the results of clinical testing. However, our unpublished work suggests that plasma half-life of our recombinant 20K-hGH is slightly longer in the rat when injected intravenously.
As another possibility, 20K-hGH may exert a stronger in vivo effect than 22K-hGH on some tissues that release hGH-BP. Recently, human abdominal fat tissues have been reported to determine hGH-BP level in healthy nonobese adults (27). Therefore, abdominal fat seems to be one of the most likely candidates for the tissues on which 20K-hGH may exert a stronger effect than 22K-hGH.
Computer-aided homology modeling helps us to speculate the molecular mechanism by which 20K-hGH poorly forms 1:1 complex with hGHR/hGH-BP while it can efficiently form a 1:2 complex. The calculated 20K-hGH model has shown that the 15-residue deletion causes a conformational change exclusively in the loop between helix 1 and helix 2, which leads to reduction of the contact surface area of its site 1 with hGH-BP 1 by 200 Å2 even though three residues (P33, L37, and R52) can compensate for the deficit to some extent, suggesting that the site 1 affinity of 20K-hGH might be reduced relative to that of 22K-hGH. Baumann et al. (28) reported that pituitary-derived 20K-hGH interacted weakly (Ka = 1.2 x 107 M-1) with human plasma binding component compared with 22K-hGH (Ka = 2–3 x 108 M-1). Hansen et al. also presented similar data from direct binding to 0.6 nM recombinant hGH-BP deriving from human IM-9 lymphocytes (Kd = 18.2 nM in 20K-hGH; Kd = 2.8 nM in 22K-hGH) (22). These previous data support the speculation that site 1 affinity of 20K-hGH might be reduced. Other notable evidence comes from the previously reported gel filtration assay using a genetically engineered 22K-hGH mutant (K172A/F176A) that was decreased in site 1 affinity (14). The mutant dimerized recombinant hGH-BP to the same extent as 22K-hGH at the concentrations of 110 nM 22K-hGH and 360 nM hGH-BP; however, significant dissociation of the mutant complex occurred when the complexes were diluted. Finally, mutant complex was totally dissociated at 1 nM, where 22K-hGH exists predominantly as a 1:1 complex. The dissociation pattern of K172A/F176A, which is very similar to that of 20K-hGH, indicates that a reduction in site 1 affinity would have a remarkable effect on the generation of a 1:1 complex. However, the bioactivity of K172/F176A evaluated in cell proliferation assay was more than 1000 times lower than that of 22K-hGH (13); therefore, the bioactivity of 20K-hGH should be distinquished from that of K172A/F176A. Earlier simulation showed that a reduction in site 1 affinity of 22K-hGH would lead to a shift in the dose-response curves to the right (29), but this cannot be adaptable to 20K-hGH. We speculate the reason as follows. The simulation is valid on the assumption that the site 2 affinity is kept constant, but as one possibility, a conformation of 20K-hGH/hGHR 1 complex may be more advantageous to hGHR 2 binding than 22K-hGH/hGHR 1 complex. Indeed, the binding affinities for hGHR expressed on Ba/F3 cells are the same between 20K- and 22K-hGH (0.38 ± 0.075 nM and 0.40 ± 0.039 nM, respectively) regardless of its putative reduction of 20K-hGH site 1 affinity. Since virtually all 22K-hGH is demonstrated to form a 1:2 complex with cell surface hGHR, the binding affinity of 20K-hGH for the cell surface hGHR is likely to represent its competence to dimerize the receptor, while the affinity for hGH-BP in a physiological range seems to represent its site 1 affinity only.
We also note the recent report that conformational changes after hGHR dimerization are required in hGHR for hGH signaling (30), which raise the possibility that a putatively unique conformational change in hGHR induced by 20K-hGH may result in a unique 20K-hGH signaling. Additional experiments, such as an x-ray structural analysis on 20K-hGH, will be needed to validate this hypothesis and are now being investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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EIA
Anti-22K-hGH and anti-20K-hGH polyclonal antibodies were
prepared from the serum of rabbits immunized with recombinant 22K-hGH
and 20K-hGH, respectively. Anti-22K-hGH monoclonal antibody (Lot
A36020047P) was purchased from BiosPacific Inc. (Emeryville, CA).
Anti-20K-hGH monoclonal antibody (NOREF>D05) not recognizing 22K-hGH was
prepared in our laboratory (Y. Hashimoto, I. Ikeda, M. Ikeda, Y.
Takahashi, M. Hosaka, H. Uchida, N. Kono, H. Fukui, T. Makino, M.
Honjo, submitted) according to the standard method reported by
Köhler and Milstein (31). Briefly, BALB/c mice were immunized
with recombinant 20K-hGH using Freund’s complete adjuvant (DIFCO
Laboratories, Detroit, MI), and the spleen cells were fused with
the P3X63Ag8 myeloma cell line (American Type Culture Collection,
Rockville, MD) using polyethylene glycol 4000 (GIBCO BRL, Gaithersburg,
MD). Hybridoma clones were screened by measuring the binding of their
generating monoclonal antibodies (50 ng/well) to 20K-hGH and 22K-hGH
immobilized to microtiter plates (0.5 ng/well) via polyclonal
antibodies. Finally, a monoclonal antibody (D05) that preferentially
bound to 20K-hGH was selected. Data from BIAcore (Pharmacia
Biosensor, Tokyo, Japan) showed that D05 had an apparent Kd
for 20K-hGH of 280 pM and a cross-reactivity with 22K-hGH
less than 0.1%. The sandwich EIA for quantitating 20K- or 22K-hGH was
performed by utilizing a fully automated EIA analyzer (QUARTUS; Mitsui
Pharmaceuticals, Inc., Tokyo, Japan). In the assay, the monoclonal
antibody (D05 for measuring 20K-hGH or A36020047P for 22K-hGH)
immobilized to magnetic particles was used as the first antibody, and
the anti-20K-hGH or the anti-22K-hGH polyclonal antibody labeled with
horseradish peroxidase was used as the second antibody.
Tetramethylbenzidine was used as a chromogen.
Plasmids
The mammalian expression plasmid pCXN2, which was a derivative
of pCXN plasmid containing chicken ß-actin promoter and
neomycin-resistant gene (32), was generously provided by Prof. J.
Miyazaki (Osaka University). The construction of the pCXN2 containing
hGHR cDNA (pCXN2-hGHR) was described previously (33). The construction
of Escherichia coli expression plasmid pGHR30 containing
coding region of the extracellular domain of hGHR (hGH-BP) was as
follows. The hGH-BP cDNA fragment was amplified by PCR using hGHR cDNA
as a template and was inserted into pGHR10 plasmid (17) in place of
20K-hGH cDNA under the downstream of Bacillus
amyloliquefaciens neutral protease promoter and the modified
neutral protease signal sequence.
Recombinant hGH-BP
The E. coli strain W3110 harboring pGHR30, which
secreted hGH-BP into its periplasmic space, was constructed in this
laboratory. It was grown at 30 C for 24 h in modified LB medium
(20 g/liter polypepton, 10 g/liter yeast extract, 10 g/liter glycerol,
10 mg/liter tetracyclin and was adjusted to pH 7.0 with KOH). Cells
were harvested by centrifugation, and the periplasmic fraction was
prepared by osmotic shock (34). Solid ammonium sulfate was added to 277
g/liter, and the precipitate was collected by centrifugation at
10,000 x g for 30 min. The pellet was resuspended in
PBS containing 1 mM PMSF, then dialyzed against the same
buffer. The dialysate was applied to an 22K-hGH affinity column. The
column was washed with PBS and then eluted with PBS containing 4
M MgCl2. The peak fractions were collected and
dialyzed with 20 mM Tris-HCl (pH 7.0) at 4 C overnight and
applied to a Mono Q column (HR 10/10). The column was washed with 20
mM bis-Tris propane-HCl (pH 7.0) and eluted with a linear
gradient of 0–0.1 M NaCl. The protein finally obtained was
verified in its purity by SDS-PAGE, molecular size by Western analysis,
and amino acid sequence on protein sequencer model PSQ-1 (Shimadzu,
Kyoto, Japan).
Receptor Expression
Ba/F3 cells were purchased from RIKEN Cell Bank (Ibaraki,
Japan). Ba/F3 cells were maintained in culture medium (RPMI-1640
supplemented with 10% FCS, 50 µM 2-mercaptoethanol, 50
µg/ml streptomycin sulfate, 50 U/ml penicillin G, and 1 ng/ml
recombinant mouse IL-3 (R & D Systems Inc., Minneapolis, MN). Fifty
micrograms of pCXN2-hGHR were transfected into 1 x
107 Ba/F3 cells by being pulsed at 200 V, 960 µFarads in
ice-cold Opti-MEM medium (GIBCO BRL). Cells expressing hGHR were
cultured in selection medium (RPMI-1640 medium containing 1 mg/ml G418,
10% FCS, 50 µM 2-mercaptoethanol, 10 nM
22K-hGH, and antibiotics). Resultant hGH-responsive cells were examined
for hGHR expression by binding assay to
[125I]22K-hGH.
Competitive Displacement Binding Assay
Ba/F3-hGHR cells were incubated in the culture medium described
above overnight to remove the hGH binding to the cell-surface receptor.
Cells were incubated with [125I]20K-hGH or
[125I]22K-hGH and a series of various concentrations of
cold 20K- or 22K-hGH in HEPES buffer (50 mM HEPES, pH 7.6,
150 mM NaCl, 10 mM CaCl2, 0.1%
BSA). Cells were spun down and washed with ice-cold HEPES buffer, and
their radioactivities were measured on a 
-counter.
Cell Proliferation Assay
Ba/F3 cells expressing hGHR were grown in the selection medium
to log phase (2 x 106 cells/ml) and were fasted
before assay. Cells were incubated in the assay medium (RPMI-1640
supplemented with 5% FCS, 50 µM 2-mercaptoethanol, and
antibiotics) for 4 h and were resuspended in a fresh assay buffer
at densities of 8 x 105 cells/ml. Sample solution (50
µl) and cell suspension (50 µl) were mixed together into the well
of a 96-well plate and incubated for 18 h. The measurements of
cell proliferation were achieved using a MTT assay kit (CellTiter 96
Non-Radioactive Cell Proliferation Assay, Promega) according to the
manufacturer’s protocol.
Gel Filtration Chromatography of Mixtures of 20K- or 22K-hGH with
Recombinant hGH-BP
In the micromolar level experiment, the concentration of hGH-BP
was fixed at 1.8 µM, and the ratios of hGH to hGH-BP were
varied as 5:1, 2:1, 1:1, 0.5:1, and 0.25:1. The mixtures were incubated
at 25 C for 15 min in 20 mM potassium phosphate (pH 6.8)
containing 0.05% Tween 20, and the aliquots (50 µl) were applied to
TSK-G2000 SWXL column ( 7.8 x 300 mm) and eluted with the same
buffer at 1.0 ml/min. Peaks were monitored on a fluorometer (Ex: 280
nm, Em: 340 nm).
In the nanomolar level experiment, the ratio of hGH to hGH-BP was fixed
at 1:2, and the concentrations of hGH and hGH-BP were varied as 1.5
nM:3 nM, 7.5 nM:15 nM,
15 nM:30 nM, 75 nM:150
nM, and 150 nM:300 nM. Mixtures
were incubated as described above, and the aliquots (100 µl) were
applied to TSK-G2000 SWXL column ( 7.8 x 300 mm). Eluted
samples were fractionated at 0.25 ml/tube, after which hGH
concentration in each fraction was measured by EIA using free 22K- or
20K-hGH as a standard. Therefore, the vertical scale represented the
concentration relative to free 22K- or 20K-hGH.
Gel Filtration Chromatography of Mixtures of 20K- or 22K-hGH with
Human Plasma
We modified a method previously described by Baumann et
al. (23). Fresh heparinized human plasma was obtained from a
normal 38-yr-old male volunteer. Original GH (20K- and 22K-hGH)
concentration in this plasma, estimated by EIA, was less than 0.1
ng/ml. The plasma was incubated with 20K-or 22K-hGH (final 20 ng/ml) at
37 C for 45 min. The mixtures (1 ml) were then separated by gel
filtration on 1 x 45-cm Sephadex G-100 column in 0.01
M Na phosphate buffer, pH 7.4, containing 0.14
M NaCl and 0.1% BSA at 4 C, and eluted fraction was pooled
at 0.5 ml/tube. The hGH concentration in each fraction was measured by
EIA using free 22K- or 20K-hGH as a standard.
Computer-Aided Homology Modeling
Because of some residues that could not be determined due to
disorder or were modeled as an alanine residue in the previous 1:2
complex of 22K-hGH with hGH-BP (11), we built an initial structure for
the complex as follows. First of all, undetermined residues were built
up by Insight II/Search-Loop (Biosym/MSI, San Diego, CA) (35).
Second, residues modeled as an alanine residue were replaced by
authentic ones. Third, the side chains of these changed residues were
optimized by Insight II/AutoRotamer (35). Homology model was built by
using Insight II/Homology (36). All dynamics and minimization
calculations were performed with Discover program (35), being the CVFF
forcefield employed with a distance-dependent dielectric. Structural
illustrations were created with the Insight II program (35). The
surface area calculations were conducted by using the algorithm of Lee
and Richards (37) with a rolling sphere of radius 1.4 Å. A loop
homology was searched from Brookhaven protein database (PDB).
Statistics
Statistical significance was assessed with paired t
tests, using Stat View 4.0 (Abacus Concepts, Inc., Berkeley, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 12, 1997. Revision received October 6, 1997. Accepted for publication October 24, 1997.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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M. S. Lee, M. P. Wajnrajch, S. S. Kim, L. P. Plotnick, J. Wang, J. M. Gertner, R. L. Leibel, and P. S. Dannies Autosomal Dominant Growth Hormone (GH) Deficiency Type II: The Del32-71-GH Deletion Mutant Suppresses Secretion of Wild-Type GH Endocrinology, March 1, 2000; 141(3): 883 - 890. [Abstract] [Full Text] [PDF]
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Y. Hashimoto, T. Kamioka, M. Hosaka, K. Mabuchi, A. Mizuchi, Y. Shimazaki, M. Tsunoo, and T. Tanaka Exogenous 20K Growth Hormone (GH) Suppresses Endogenous 22K GH Secretion in Normal Men J. Clin. Endocrinol. Metab., February 1, 2000; 85(2): 601 - 606. [Abstract] [Full Text]
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B. Tsunekawa, M. Wada, M. Ikeda, H. Uchida, N. Naito, and M. Honjo The 20-Kilodalton (kDa) Human Growth Hormone (hGH) Differs from the 22-kDa hGH in the Effect on the Human Prolactin Receptor Endocrinology, September 1, 1999; 140(9): 3909 - 3918. [Abstract] [Full Text]
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M. Ishikawa, S. Yokoya, K. Tachibana, Y. Hasegawa, T. Yasuda, E. Tokuhiro, Y. Hashimoto, and T. Tanaka Serum Levels of 20-Kilodalton Human Growth Hormone (GH) Are Parallel Those of 22-Kilodalton Human GH in Normal and Short Children J. Clin. Endocrinol. Metab., January 1, 1999; 84(1): 98 - 104. [Abstract] [Full Text]
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 B. Tsunekawa, M. Wada, M. Ikeda, S. Banba, H. Kamachi, E. Tanaka, and M. Honjo The Binding between the Stem Regions of Human Growth Hormone (GH) Receptor Compensates for the Weaker Site 1 Binding of 20-kDa Human GH (hGH) than That of 22-kDa hGH J. Biol. Chem., May 19, 2000; 275(21): 15652 - 15656. [Abstract] [Full Text] [PDF]
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M. S. Lee, Y. L. Zhu, J. E. Chang, and P. S. Dannies Acquisition of Lubrol Insolubility, a Common Step for Growth Hormone and Prolactin in the Secretory Pathway of Neuroendocrine Cells J. Biol. Chem., January 5, 2001; 276(1): 715 - 721. [Abstract] [Full Text] [PDF]
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). Cell number was determined by MTT assay. Each data point
represents the mean of triplicate wells, and error bars indicate
SD. Asterisk denotes a significant
difference between 20K- and 22K-hGH (**, P <
0.005).
) of 10
nM recombinant hGH-BP. B, Proliferation of Ba/F3-hGHR cells
caused by a series of concentrations of 20K-hGH in the absence (
) of 10 nM recombinant hGH-BP. Each data point
represents the mean of triplicate wells, and error bars indicate
SD. Asterisk denotes a significant
difference from control value in the absence of hGH-BP (*,
P < 0.05; **, P < 0.005).
