From the
Substitution of arginine for glycine at position 120 in native
22-kDa human growth hormone (hGH) results in an analogue, G120R, which
is unable to dimerize the GH receptor and is widely used to probe the
molecular mechanism of action of hGH. When acting on human GH
receptors, G120R antagonizes several biological effects of hGH, but is
itself inactive as an agonist. It has been reported that this mutant
also antagonizes hGH activation of the rat or human prolactin (PRL)
receptor in cell-based assays, with no agonist activity. We have now
tested this mutant in a sensitive MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-ESTA
(eluted stain assay) bioassay using rat PRL receptors in the Nb2 cell
line. We confirm that G120R acts as an efficient antagonist of native
hGH, but show that it can also act as an agonist to generate
intracellular signals leading to metabolic activation and proliferation
of Nb2 cells. We have demonstrated an unusual sensitivity to the
presence of zinc (Zn
The third
High resolution functional
(8, 9) and structural
(10) analyses have shown that there are two receptor-binding
sites on hGH, called sites 1 and 2. Site 1 governs the initial binding
of hGH to its receptor. This is followed by recruitment of a second
growth hormone receptor molecule from the lipid matrix of the plasma
membrane, which binds to site 2 on hGH. The consequent formation of
receptor homodimers then initiates intracellular signaling. The third
It has been suggested that activation of PRL receptors by
hGH is also due to a two-step sequential dimerization process
(9, 11) . Using alanine-scanning mutagenesis, Cunningham
and Wells
(12) demonstrated that hGH contains a set of
functional determinants for binding to the hPRL receptor which
overlapped those of site 1 for binding to the hGH receptor. These sites
are similar, but not identical. Intriguingly, the site for binding to
the hPRL receptor contains three ligands for coordinating zinc
(Zn
Fuh et
al. (11) compared the effects of G120R and wild-type hGH
on two different cell systems, which expressed different forms of the
prolactin receptor. One was the Nb2 rat lymphoma cell which expressed
an intermediate length lactogenic receptor. As might have been
anticipated from work with the somatogenic growth hormone receptor,
they reported that G120R failed to stimulate
[
In recent in vivo studies
The
concentration of zinc in the ``zinc-free'' assay medium was 2
µ
M, as previously determined by atomic absorption
spectroscopy
(20) .
The cells were
incubated for 96 h under the conditions described above. Their reducing
activity was then assayed by adding 10 µl of MTT solution (5 mg/ml
in phosphate-buffered saline). After a further 40-min incubation
period, during which time activated cells reduced the yellow MTT salt
to its purple formazan, the stain was eluted into the medium by the
addition of 50 µl of acidified Triton X-100 (10% Triton X-100 in
0.09
M HCl). Bioassay responses of the 96 wells were
quantified with a Bio-Rad microtiter plate reader (scanning in 30 s)
reading optical densities at a test wavelength of 595 nm and a
reference wavelength of 655 nm. The determinations for all experiments
were made on triplicate microcultures and the results expressed as
means and standard deviations.
Using the MTT-ESTA bioassay, we have confirmed several of the
properties of the G120R analogue of hGH, which have been reported in
other systems. For example, we have confirmed its ability to antagonize
hGH and that this inhibition is potentiated by Zn
In contrast to an earlier report
(11) , we found that binding
of G120R to the lactogenic receptors located on the surface of Nb2
cells resulted in cell activation. We have characterized this
stimulation in detail, using the high throughput MTT-ESTA bioassay
system. We have also confirmed the agonist actions of G120R with a
conventional system based upon direct cell counts (Fig. 2) and
also two other response systems, namely one using an alternative
tetrazolium salt and one based upon increased uptake of
[
Thus,
according to the two-site sequential receptor dimerization model, which
has been well established for hGH interacting with somatogenic
receptors, our findings would indicate that although binding of G120R
to site 2 may be compromised, it can interact with lactogenic receptors
and stimulate target cells, if the analogue is present at sufficiently
high concentrations. Our finding that this results in a bell-shaped
dose-response relationship (Fig. 1) would be consistent with the
biological responses being linked to dimer formation, as predicted from
a computerized simulation of the dimerization model
(7) .
Moreover, the restricted concentration range of the plateau for G120R,
compared with the wild-type hGH (Fig. 1), would be consistent
with the formation of less stable dimeric complexes by G120R
(11) . However, our findings are at variance with some of the
other conclusions which Ilondo et al. (7) made from
their mathematical simulation. They predicted that mutagenesis at site
1 would result in a shift of the whole bioactivity curve to the right,
but with the maximum magnitude of the response being unaffected. In
contrast, they concluded that site 2 mutants would exhibit lowered or
absent magnitudes of response. Clearly, G120R, which is a site 2
mutant, gave a result (Fig. 1) which closely resembled that
anticipated for the analogues which have been subjected to mutagenesis
at site 1. It therefore behaved in a similar manner to the site 1
double mutant, K172A/F176A, when acting on Nb2 cells
(11) .
From our own work, and that of Fuh et al. (11) , we
conclude that the important difference between K172A/F176A and G120R
when acting as lactogenic agonists is their altered sensitivities to
added Zn
In conclusion, we have characterized the agonist and
antagonist properties of the G120R analogue of hGH when acting on the
intermediate length lactogenic receptor as encountered in the Nb2
cells. The finding that this analogue stimulates in this system
enhances its importance as a probe for deciphering the molecular
mechanism of growth hormone action via different receptors. Our
findings are equally important for in vivo studies. It has
proved difficult to establish significant hGH antagonism in vivo in the rat, and it was the suspicion of mild PRL agonist activity
that prompted this study. The present results confirm that PRL agonist
activity occurs at far lower doses than those necessary to antagonize
hGH at the rat Nb2 PRL receptor and that in vivo studies in
which G120R is used as a specific GH antagonist in the rat may need to
be interpreted with caution.
We are grateful to Genentech Inc. for their kind gift
of the recombinant human GHBP.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
). In the absence of added
Zn
, G120R shows weak but full agonist activity in the
bioassay, and this can be blocked by co-incubation with recombinant
hGH-binding protein. G120R can therefore be utilized to discriminate
between the molecular mechanisms of hGH interactions with its
somatogenic and lactogenic receptors. Future studies with G120R in the
rat may need to take account of its significant agonist effects on PRL
receptors.
-helix of human growth hormone (hGH)
(
)
appears to be especially important for its biological
activity
(1) . Changes in amino acid residues in this region
have particularly marked effects upon the potencies of the resulting
hormone analogues
(2, 3, 4) . Kopchick and
co-workers
(5) have demonstrated that a glycine residue at
position 120 in hGH is critical in this respect. This amino acid, with
its minimal, nonpolar side chain, is thought to create a
``cleft'' in the middle of the third
-helix. As a
consequence, its substitution by an amino acid which has a bulky
resonance-stabilized basic side chain, such as arginine, generates an
analogue (G120R) with greatly altered biological effectiveness. For
example, G120R fails to stimulate when tested in systems as diverse as
hGH receptor down-regulation and the induction of the tyrosine
phosphorylation of proteins in IM-9 lymphocytes
(5, 6, 7) and lipogenesis in rat adipocytes
(7) . In all of
these systems, which function via a GH receptor, G120R consistently
antagonizes stimulation by wild-type hGH. In addition, it leads to
stunted growth in transgenic mice which express G120R
(4) .
-helix and the N terminus of hGH have been demonstrated to form
binding site 2
(9, 10) . As a consequence, the analogue
G120R has compromised site 2, but not site 1, function. Although G120R
can bind normally via site 1 to the first receptor, it is thought that
recruitment of the second receptor, and hence receptor dimerization, is
blocked by the arginine substitution at the critical position in site
2. This would account for both the failure of G120R to act as an
agonist and also its ability to antagonize wild-type hGH. The general
validity of this scheme has been established by a number of other
mutations based on this two-site hypothesis. Furthermore, prolactin
receptor antagonism has also been shown for G120R in cell lines
expressing hPRL receptors and in Nb2 cells expressing a rat PRL
receptor.
) which are important for the binding of hGH to
the hPRL receptor but not for its binding to the hGH receptor
(13) . The existence and nature of a second hPRL
receptor-binding site on hGH has yet to be established.
H]thymidine uptake by the Nb2 cells and that
high concentrations of the analogue antagonized the stimulation
achieved with wild-type hGH.
(
)
it has proved difficult to
demonstrate the antagonist activity of G120R on growth in the rat and
the data was compatible with a weak but significant agonist activity in
the absence of GH, via rat PRL receptors. This would seem to be at
variance with the data of Fuh et al. (11) , who
reported only antagonist activity on rat PRL receptors. To resolve this
inconsistency, we took advantage of a recently developed highly
sensitive and precise bioassay based on Nb2 cells. This colorimetric
bioassay, which is both sensitive and precise, utilizes as its end
point the bioreduction of the tetrazolium salt MTT to its intensely
colored formazan by hormonally activated cells. This integrated
response reflects both increases in cell numbers and, also, metabolic
activation of individual cells by the lactogenic hormone
(14, 15, 16) . We now report that G120R
stimulates Nb2 cells in the MTT-ESTA bioassay.
(
)
We have characterized its actions, both as an agonist and
an antagonist, and shown that discrepancies with previous reports may
be explained by the unexpected sensitivity of the G120R/rPRL receptor
interaction to zinc (Zn
).
Materials
Recombinant G120R hGH was prepared as
described previously
(5) . Recombinant human growth
hormone-binding protein (rhGHBP), which represents the extracellular
hormone-binding domain
(17) was a gift from Genentech, Inc.
(South San Francisco, CA). The rhGHBP is nonglycosylated and has an
affinity for 22-kDa hGH comparable with that of the naturally occurring
high affinity hGHBP
(18) . Human pituitary hGH (IS 80/505) was
provided by the National Institute of Biological Standards and Controls
(Blanche Lane, South Mimms, Herts, United Kingdom). The suppliers of
80/505 state that analysis on polyacrylamide gel (pH 7.8) indicated a
predominant band of 22-kDa hGH, about 5% 20-kDa and small amounts of
``slow,'' dimer and desamido hGH, as well as trace amounts of
several other proteins
(19) . Zinc chloride (ZnCl)
was purchased from Sigma Ltd. (Poole, Dorset, UK), as was
3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
RPMI 1640,
L-glutamine, penicillin, and streptomycin were
purchased from Life Technologies, Inc. UK Ltd. Horse serum and fetal
calf serum were purchased from Tissue Culture Services UK Ltd.
Cell Culture
Rat Nb2 lymphoma cells were routinely
grown in suspension culture in RPMI medium containing 50 units
penicillin/ml, 50 µg streptomycin/ml, 2 10
M
L-glutamine, 10% fetal calf serum, and 10%
horse serum. Before commitment to bioassay, the Nb2 cells were
maintained as stock cultures for 24 h in 1% as opposed to 10% fetal
calf serum. All cultures were incubated at 37 °C in an atmosphere
of 5% CO
, 95% air throughout this study.
Bioassay
The ESTA is based upon the colorimetric
reduction of a yellow tetrazolium salt, MTT, to a purple formazan by
Nb2 cells
(14) . Dilution of the appropriate hormone standard
and the zinc chloride were prepared in assay medium consisting of RPMI
1640, 50 units/ml penicillin, 50 µg/ml streptomycin, 2
10
M
L-glutamine, and 10% horse
serum. The Nb2 cells were suspended in this assay medium at a density
of 4
10
cells/ml. The cell suspension (50
µl/well) was transferred to a 96-well microtiter plate (Nunc code 1
67008A; Kamstrup, Denmark (supplied by Life Technologies, Inc.)). The
hormone (hGH (IS 80/505) or G120R) was added to the wells at the
required concentrations as an additional 50 µl prepared in assay
medium. Zinc chloride or the rhGHBP were added at the appropriate
concentrations in a volume of 20 µl, as was G120R in the experiment
to assess its antagonistic properties on IS 80/505. Experimental
controls replaced the hormones, rhGHBP, or ZnCl
with assay
medium. All concentrations were expressed as final concentrations of
added zinc, rhGHBP, or hormone in the wells.
Conventional Nb2 Cell Bioassay
The bioassay was
performed as described above. However, instead of the addition of MTT
to determine response, cell number was determined using a Coulter
counter.
Units of Concentration
All concentrations are
expressed in molar terms. These have been calculated using the
following molecular weights: pituitary-derived hGH (IS 80/505) and
recombinant G120R, 22 kDa; rhGHBP, 28 kDa. The specific bioactivity of
the human pituitary GH preparation (IS 80/505) was 2.6 IU/mg.
The Effect of the hGH Analogue G120R on Nb2
Cells
In the absence of added Zn, dose-related
stimulation of Nb2 cells by G120R was observed over the concentration
range 0.28-142 n
M, with an EC
of 1.27
n
M (Fig. 1). A plateau maximal response was observed,
but at the highest concentrations, self-inhibition was observed
(self-IC
= 523 n
M). Within the same
experiment, IS 80/505 stimulated Nb2 cells over the concentration range
0.9-8.6
10
p
M, with an EC
of 10 p
M. Although the potency of G120R as an agonist
was 100-fold less than that of IS 80/505 as measured at the EC
the ascending limbs of the two dose-response relationships were
parallel, and the same maximal response was obtained. At high
concentrations of IS 80/505 (>17.3 µ
M), self-inhibition
was also observed, as demonstrated previously by Fuh et al. (11) . The self-IC
was 25.5 µ
M.
Hence, the potency of G120R as a self-antagonist was 49-fold greater
than that of IS 80/505. The plateau in response to IS 80/505 extended
over concentration ranges of 5 orders of magnitude, whereas that for
G120R was more restricted, approximating to 2 orders of magnitude
(Fig. 1).
Figure 1:
Stimulation of Nb2 cells by the hGH
analogue, G120R, and pituitary-derived hGH (IS 80/505) compared in the
same ESTA. Increasing doses of G120R (0.068-1136 n
M
[]) and pituitary-derived hGH (IS 80/505: 0.9
p
M to 35 µ
M (
)) were added to Nb2 cells. The
bioassay was performed as described under ``Experimental
Procedures,'' and the results are expressed as optical density
100 and are the means ± S.D. obtained from triplicate
wells. Both preparations were tested on the same microtiter plate. The
horizontal line represents the control optical density in the
presence of assay medium ( AM) only.
The stimulatory effect of G120R on Nb2 cells in the
MTT-ESTA system was accompanied by increases in cell numbers
(Fig. 2). Doses of G120R and IS 80/505 which provoked the same
maximum responses in the MTT-ESTA bioassay (Fig. 2 A)
increased Nb2 cell numbers equivalently (Fig. 2 B). This
stimulatory effect was also confirmed using an alternative tetrazolium
salt system (Promega cell titer 96 Aqueous kit) and cell proliferation
as assessed by increased uptake of [H]thymidine
(not shown). These confirmatory experiments were performed using a
different batch of Nb2 cells. The Effect of Zinc [Zn
] on G120R Stimulation
of Nb2 Cells-Stimulation of Nb2 cells by G120R was shown to be
unusually sensitive to the addition of Zn
(Fig. 3). When added over the concentration range
0.78-400 µ
M, Zn
inhibited the
stimulation of Nb2 cells by a dose of G120R which provoked near-maximum
stimulation in the MTT-ESTA bioassay in the absence of added
Zn
(2.4 n
M). The stimulation was abolished
with 50 µ
M added Zn
(51.6
µ
M, including endogenous Zn
), and the
IC
for this was 6.25 µ
M added Zn
(7.9 µ
M including endogenous Zn
).
Within the same experiment, we confirmed our previous report
(20) that 50 µ
M added Zn
slightly
increases the stimulation by a low dose of IS 80/505 (9 p
M)
observed in this bioassay. As before, higher doses of Zn
inhibited the stimulation with 80/505.
Figure 2:
Stimulation of Nb2 cells by the hGH
analogue, G120R, and pituitary-derived hGH (IS 80/505): a comparison
between the ESTA bioassay and direct cell counts. Nb2 cells were
stimulated with 2.8 n
M G120R and 0.11 n
M hGH as for a
standard ESTA bioassay, except that parallel triplicate wells were also
set up in the same microtiter plate and used to determine changes in
cell numbers. The ESTA bioassay was performed as described under
``Experimental Procedures,'' and the results are expressed as
optical density 100 for the MTT-ESTA assay ( A) or cell
number
10
/ml for the parallel wells ( B).
Both are expressed as the means ± S.D. obtained from triplicate
wells. The results are compared with the assay medium controls in the
absence of added lactogen.
Figure 3:
Effect of increasing concentrations of
Zn on stimulation of Nb2 cells by G120R and the
pituitary-derived preparation of hGH (IS 80/505) in the ESTA bioassay.
Increasing doses of Zn
(0.78-400
µ
M) were added to Nb2 cells in the presence of a constant
dose of either G120R (2.4 n
M (⊡ ) or the
pituitary-derived preparation of hGH (IS 80/505 (
)). The
bioassay was performed as described under ``Experimental
Procedures,'' and the results are expressed as optical density
100 and are the means ± S.D. obtained from triplicate
wells. The horizontal lines represent the effect of lactogen
in the absence of added Zn
. 50 µ
M
Zn
added to assay medium in the absence of lactogen
had no effect on Nb2 cells (assay medium ( AM) ±
Zn
). The entire bioasay was performed within a single
microtiter plate.
Zninhibition of stimulation of Nb2 cells by G120R was due to a
reduced maximum magnitude of response and not due to a shift of the
dose-response relationship to the right (Fig. 4). The addition of
20 µ
M Zn
would be expected to have only
a minimal effect upon stimulation of Nb2 cells with IS 80/505 (see
Fig. 3
and also Ref. 20), but it reduced the plateau response
achieved with G120R by approximately 85%.
Figure 4:
Stimulation of Nb2 cells by the hGH
analogue, G120R, and pituitary-derived hGH (IRP 80/505) in the presence
of 20 µ
M ZnClcompared in the same ESTA.
Increasing doses of G120R (0.068-1136 n
M (
)) and
pituitary-derived hGH (IS 80/505: 0.9 p
M to 35 µ
M
(
)) were added to Nb2 cells in the presence of 20 µ
M
Zn
. The bioassay was performed as described under
``Experimental Procedures,'' and the results are expressed as
optical density
100 and are the means ± S.D. obtained
from triplicate wells. The entire experiment was performed within a
single microtiter plate. The horizontal line represents the
control optical density in the presence of assay medium, with the only
additional component being Zn
(20
µ
M).
The Effect of G120R on Nb2 Cells Stimulated with IRP
80/505
G120R antagonized IS 80/505 stimulation of Nb2 cells
(Fig. 5). Using a dose of IS 80/505 (0.91 n
M) which was close
to the mid-point of the plateau of the maximum response (Fig. 1),
we found that the addition of up to 114 n
M G120R did not
further increase this magnitude of response. However, higher doses of
G120R antagonized hGH in a concentration-dependent manner. The potency
of G120R as an antagonist of hGH was enhanced 4-fold by the addition of
10 µ
M Zn; a 50% reduction in the
response to IS 80/505 was observed with 90 n
M G120R. This
potentiating effect of Zn
has been described
previously
(11) .
The Effect of rhGHBP on Nb2 Cells Stimulated with
G120R
rhGHBP, which represents the extracellular domain of the
hGH receptor, interacted with G120R and inhibited stimulation of Nb2
cells by this mutant in a dose-dependent manner (Fig. 6). When
stimulated with a dose of G120R (1.2 n
M) which provokes a
near-maximal response (Fig. 1), a dose-dependent inhibition of this
response was first observed with a concentration of 0.23 n
M
rhGHBP, and concentrations of the binding protein in excess of 3.5
n
M abolished the response. The ICfor rhGHBP was
1.1 n
M; the molar ratios of rhGHBP:G120R were thus
approximately unity (0.9) at this midpoint in the antagonist
dose-response relationship.
.
This is consistent with normal binding of the analogue at its
unmodified binding site 1 to the first receptor and an inability to
induce receptor dimerization by binding to site 2. We have also
demonstrated that G120R interacts with the extracellular domain of the
hGH receptor at rhGHBP:G120R ratios of unity. However, these molar
ratios were much higher (100-300) when rhGHBP inhibited
stimulation by IS 80/505 and, also, its isohormone, recombinant
methionyl 20-kDa hGH, as reported in separate studies
(21) .
H]thymidine. Stimulation required high
concentrations of G120R and was unusually sensitive to added
Zn
. The inhibitory effect of Zn
probably reconciles the two separate investigations. Fuh et
al. (11) added 10 µ
M Zn
to
their medium when they tested for agonist properties, whereas we
performed our experiments with a medium which contained no special
addition of Zn
and has a basal concentration of only
2 µ
M Zn
(20) . In the MTT-ESTA
bioassay, the addition of 10 µ
M Zn
reduced the maximum agonist response to G120R by 85%, whereas it
had little effect on hGH agonist activity. It is also possible that the
lower serum concentration in the medium used by Fuh et al. (11) (0.5% horse serum as opposed to the 10% in the present
studies) exacerbated the inhibitory effect of Zn
, due
to a reduced concentration of zinc-binding protein in the medium, with
a corresponding increase in the concentration of free zinc.
. It is well recognized that this divalent
cation is required for site 1 association between hGH and the hPRL
receptor. It does not, however, influence the binding of hGH to the hGH
receptor. Scanning mutational analysis has identified three residues in
hGH (His
, His
, and Glu
) and one
residue on human prolactin-binding protein (hPRLBP), which represents
the extracellular domain of the hPRL (lactogenic) receptor, as the four
probable residues involved in Zn
binding
(13) . It would appear that Zn
may be used as
an intriguing discriminator between the different analogues of hGH,
since we find a wide spectrum of the effects of this ion, when
investigating lactogenic Nb2 cell receptors. These influences range
from marked inhibition of the agonist activity of G120R, potentiation
of its inhibitory effects, the slight enhancement of native hGH when
low doses of the latter are used, which changes to inhibition at higher
hormone concentrations, through to striking potentiation of the
bioactivity of the alternative splice variant of hGH, 20-kDa hGH
(20) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.