From the
Growth hormone (GH) is believed to signal by dimerizing its
receptor through two binding sites on the hormone. Previous attempts to
increase the biopotency of GH by increasing its site 1 affinity have
been unsuccessful, which has led to a bias toward engineering site 2
interactions in the quest for creation of super agonists. Here we
report that increasing site 1 affinity can markedly increase
proliferative bioactivity in FDC-P1 cells expressing full-length GHR.
In contrast, we find three site 1 mutants with affinities for site one
similar to or greater than wild type GH, which have markedly decreased
bioactivity. Through crystal structure analysis of the receptor
interactive regions of these GH analogues, we are able to suggest why
previous mutagenesis on human GH failed to improve biopotency, and thus
provide a new avenue for GH and cytokine agonist design.
Growth hormone (GH)(
Despite these advances there have been no reports of GH analogues
that show enhanced bioactivity, although a human GH (hGH) that exhibits
a 30-fold increased site 1 affinity has been produced(5) . The
failure of this hGH analogue to show a commensurate increase in
efficacy disagrees with conventional pharmacological theory, which
predicts that increased receptor occupancy should translate to an
increased biological response (10, 11), a concept supported by computer
simulations of the biological response to GH binding(12) . This
disagreement has not been resolved, although it was suggested by Fuh et al.(5) that further analysis of the on and off
rates of hormone binding may elucidate the issue. Because this high
affinity analogue did not have improved biopotency, it was proposed
that the rate-limiting step in the biological response was site 2
binding and that in order to create analogues with higher efficacy,
modification of binding site 2 was required(5) .
In
addressing the above issues, we have based our studies on the myeloid
line used by Fuh et al.(5) but rather than their
mG-CSF/hGH chimeric receptor, we have stably transfected the cells with
a full-length GHR. This alleviates concerns about the ability of the
chimeric mG-CSF/hGHR to accurately represent the physiological
signaling mechanism.
Using this assay system, we provide support for
our previous findings regarding the importance of the C terminus of
helix one as a receptor interactive epitope. In addition, we show that
mutations in this region and at a site in the center of helix 4
substantially decrease the biopotency even though they do not adversely
affect the affinity of binding to site one. From inspection of the
crystal structure, we suggest that two regions of the receptor, the
hinge region linking domains 1 and 2 and the final
We also report that deletion of the 8 C-terminal
residues containing the small disulfide loop results in a pGH analogue
with increased site 1 affinity and biological potency. This indicates
that improvements to site 1 binding can result in substantial gains in
bioactivity, in contrast to previously accepted views(5) .
Human GH, pGH, and pGH analogues were expressed in Escherichia coli and purified as described in Refs. 9 and 13.
L127V(
FDC-P1 cells were grown to mid-confluence and
transfected by a method modified from Ref. 15. In brief, to a 0.4-cm
cuvette was added 5
GH-dependent
FDC-P1 cells (clone FDC-P1-RGHR3B, stably expressing the rabbit GHR)
were grown to mid-confluence in phenol red-free RPMI 1640 containing 5%
FCS, 40 ng/ml hGH, and 1 µg/ml gentamicin and washed by pelleting
cells at 500
Some of our pGH analogues (K30Q R34E pGH, K30E R34E pGH, and H170D
pGH; numbering system used is in accord with Ref. 19) have been shown
to be divalent metal ion (Me
Curve fitting was
performed by linear regression using the DeltaGraph package for
Macintosh desktop computers (Delta Point, Inc., Monterey, CA). The
ED
The number of surface-expressed rabbit
GHRs in the transfected line was determined by growing 500 ml of
confluent FDC-P1-RGHR3B cells in phenol red-free RPMI 1640 supplemented
with 5% FCS, 1 µg/ml gentamicin, and either 40 ng/ml hGH or 100
units/ml IL-3. Cells were pelleted and washed in the same manner as
described above. The pellet was finally resuspended in 4 ml of isotonic
glucose binding buffer with 20 mM MgCl
The
addition of 2 mM Mg
To facilitate the identification of non-primate GH agonists
and antagonists, we have developed a sensitive in vitro assay
system based on GH-dependent survival of the murine myeloid precursor
cell line, FDC-P1. This line normally requires IL-3 for proliferation
but has been shown by Fuh et al.(5) to convert to GH
dependence upon stable transfection with a hybrid receptor consisting
of the extracellular domain of the hGHR and the transmembrane and
cytosolic domains of the murine G-CSF receptor. Use of this assay
system (5) provided support for the proposal that
hormone-induced dimerization is required for signal transduction by the
GHR, in agreement with the demonstration that GH has two binding sites,
each binding a separate GHR molecule(4, 6) .
Unfortunately the assay system of Fuh et al.(5) did
not utilize the full-length GHR, which raises doubts about the validity
of derived biopotency data since this construct will not express the
physiological signaling molecule (i.e. does not include the
GHR cytoplasmic domain). Moreover, this assay system is restricted to
use with primate GHs as a result of the inability of non-primate GHs to
bind to primate GHRs. By developing a bioassay system based on the
full-length rabbit GHR, we have overcome these limitations. Our stably
transfected FDC-P1-RGHR cells have 186 ± 12 receptors/cell when
grown in GH and 2128 ± 230 receptors/cell when grown in IL-3.
The latter number is approximately 2 times greater than the number of
expressed hybrid receptors reported by Fuh et al.(5) .
Affinity cross-linking of the rabbit GHR expressed in this cell line
revealed 65- and 116-kDa bands (Fig. 1A), a result
consistent with the presence of cleaved GH-binding protein and
full-length GHR(24) . Scatchard analysis (Fig. 1B) showed the affinity of hGH for these receptors
to be 7.6 ± 1.6
We have
used the MTT cell proliferation assay, which allows rapid and
convenient spectrophotometric quantification of cell growth and
viability(17) , to compare the biopotency of our pGH analogues
with wild type pGH (). Contrary to the view of Fuh et
al. (5), a marked gain in biopotency can be achieved through
higher affinity site 1 interaction, as evidenced by the C181S del
184-191 pGH analogue. It was observed in the alanine-scanning
mutagenesis study of Cunningham and Wells (8) that alanine
substitution at Glu-186 and Ser-188 increased affinity, while alanine
substitution at Gly-187 decreased it. These results indicate that
smaller side chains at this location are more advantageous for receptor
binding, and that larger residues cause steric hindrance. Accordingly,
Seely et al. (25) reported that loss of the disulfide bond
between Cys-181 and Cys-189 (C181N C189S pGH and a reduced form of pGH)
resulted not only in an analogue that was less likely to aggregate, but
one that displayed increased affinity for GHRs. We have made a pGH
analogue devoid of the 8 C-terminal residues, and also find increased
affinity for binding to rabbit liver GHR (4.8-fold, ).
However, our C181S del 184-191 pGH analogue shows a 5.2-fold
increased bioactivity compared to wild type pGH in the cell
proliferation assay ( and Fig. 3). On the basis of CD
spectra, the loss of the C terminus has not caused significant
conformational changes in the GH structure (results not shown), while
energy minimization calculations on the mutated analogue reveal little
predicted change (less than 1Å) in the overall backbone
structure, so no conformational change appears to have been induced at
binding site 2. In the crystal structure the C terminus is too distant
from site 2 to interact directly, so we conclude that increased site 1
affinity increases biopotency, in accord with the theoretical
simulations of Ilondo et al.(12) .
In an attempt to
create further pGH analogues with improved biopotency, we targeted P6
because bGH had a 3-fold higher biopotency compared to pGH and this was
one of the few side chains that differed from bGH in the N terminus.
This mutation resulted in a 2-fold loss in biopotency without adverse
affects on site 1 affinity (), indicating this residue is
not responsible for the improved potency of bGH. Additionally, our
finding is consistent with the observation of Cunningham and Wells (6) that this side chain is a site 2 determinant.
Two of our
analogues are seen to have site 1 affinities and biopotencies similar
to pGH. The K139E change is in the floppy loop region between helices 3
and 4, while the K180A mutation is at the end of helix 4. Neither of
these side chains are in close contact with either GHR1 or GHR2;
therefore, these pGH analogues act as convenient controls.
The
greatest decreases in biopotency were seen with the K30Q R34E (5-fold)
and K30E R34E pGHs (7.7-fold), even though their affinities for the
receptor were not reduced. Similarly, the H170D analogue has wild type
affinity but a 3-fold reduction in biopotency. These mutations involve
interactions with the GHR at site 1(9) . Since the binding assay
measures the affinity of binding site 1 only(6) , it follows
that the loss in bioactivity seen with these mutants must be a result
of unfavorable site 2 interactions, induced either by direct
interaction with GHR2 or indirectly through a conformational change in
the GH/GHR1 complex that is disadvantageous to receptor 2 binding (i.e. to receptor dimerization). We do not favor the former of
these proposals because analysis of the crystal structure (4) reveals that the closest contact between side chain head
groups in the C terminus of helix 1 of GH and GHR2 is 14.1 Å and
12.2 Å between Lys-30 and Arg-34 of pGH and Gln-166 of GHR2,
respectively, while His-170 sits in the middle of the site 1
interface(8) . These contacts are too distant to have a
significant effect on site 2 binding, and since all side chains are
solvent exposed, it is unlikely that mutations at these positions would
induce a conformational change in the hormone. Since energy
minimization of these analogues reveals no difference in their overall
structures compared to the wild type pGH, direct effects on the site 2
interaction are difficult to envisage, thus unfavorable interactions
are most likely to occur through site 1.
We have shown previously
that pGH residues Lys-30, Glu-34, and His-170 are in reasonably close
contact (between 4 and 7 Å) with Glu-126, Glu-127, and Glu-220 of
the GHR(9) . There is considerable evidence that these and
closely associated side chains may be important mediators of signaling
through the GHR. The Glu-126 and Glu-127 side chains are unique in that
they are within the 4-residue segment linking domain 1 and 2 of the
extracellular portion of the GHR (Fig. 4A). This segment
was proposed by De Vos et al.(4) to be involved in
orientating domain 1 and to be the primary reason why domain
positioning differs from that seen with the homologous immunoglobulin
domains. Indeed, the importance of this hinge region in domain
orientation was further highlighted upon the release of the
hGH/prolactin receptor crystal structure (26) as superimposing
the hGH/hGHR complex onto the hGH/human prolactin receptor complex
showed a significant difference in domain orientation of the homologous
PRL and GH receptors. A Glu
Conformational changes of this
type could explain why the high affinity hGH analogue (H21A R64K E174A
hGH) of Fuh et al.(5) did not have enhanced
biopotency, as two of the three mutations (H22A and E173A if using the
numbering system of Ref. 28) used to increase site 1 affinity are
juxtaposed to the conformation-sensitive region described here. His-22
and Glu-173 are both in close contact with the
In conclusion, our binding and biological activity data
show that care must be taken in the design of binding site 1 GH
analogues, as it is possible to adversely affect biological activity
without loss of site 1 binding affinity. Through analysis of the
crystal structure of the GH(GHR)
Scatchard
analysis was used to determine the affinity of each GH and was
performed as in Ref. 9. The biopotency of the GHs relative to pGH was
determined as described in Fig. 1. One plate was used to assay each
mutant, with the plate divided in half to allow a within plate
comparison with wild type pGH. Eight GH concentrations were used per
assay, allowing each GH concentration to be assessed in sextuplicate.
Because some of the pGH analogues have been shown to be
Ca
We thank Ela Knapik for excellent technical assistance
in purification of human and porcine GHs and Christine Wells for her
tissue culture assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
has a wide variety
of applications both clinically and agriculturally, where it has been
found to produce substantial increases in growth rate and protein
accretion along with decreases in carcass fat
content(1, 2) . Improvement in the efficacy of GHs is
dependent on an understanding of the molecular basis of GHR (GHR)
binding, and the most significant studies delineating this interaction
have come from Wells et al.(3) . In agreement with
those studies, the crystal structure of the GH(GHR)
complex
shows the hormone to contain two separate binding sites for the
GHR(4) . For the propagation of a biological response there is a
requirement for sequential binding of the first receptor subunit to
site 1 of GH, followed by binding of the second receptor subunit to
site 2 to form the GH(GHR)
complex(5) . Because of
its role in promoting formation of the biologically effective receptor
dimer, the site 2 interaction is thought to be critical in regulation
of the biological response(5) . Hormone binding site 2 involves
residues in the N terminus of GH as well as side chains in helix 3,
while site 1 has been proposed to consist of four discontinuous
segments, namely the central part of helix 1, residues 38-47 and
54-74, and the C terminus of helix 4(4, 6) . The C
terminus of helix 1 has consistently been overlooked as an interactive
epitope in mutagenesis studies (7, 8) despite its
proximity to receptor 1 in the crystal structure. We have recently
shown this region to be an important site 1 binding domain and to be
largely responsible for the contrasting Ca
dependence
of binding of non-primate and primate GHs to the rabbit GHR(9) .
-turn before
the membrane, may be important regions that mediate transmission of the
biological signal.
)
bovine GH was a gift from Monsanto
(Chesterfield Village, MO), while oPRL-16 was from the National Hormone
and Pituitary Program (Baltimore, MD). Far-UV CD spectra were performed
on all pGHs, and no measurable change in
-helical content was seen
between wild type pGH and pGH analogues. CD spectra are not shown but
were identical to those previously reported(13) . In addition to
DNA sequencing, all analogue pGHs underwent mass spectrometry to verify
the incorporation of the desired mutational changes. Protein
concentration of mutants was obtained by laser densitometric scan of
silver-stained gels and by amino acid analysis.
Establishment of Cell Lines Expressing the Rabbit
GHR
FDC-P1 cells are an IL-3-dependent murine myeloid cell
line(14) , and along with IL-3 were a gift from Andrew Hapel
(John Curtin School of Medical Research, Australian Capital Territory,
Australia). Cells were routinely passaged in 5% CO, 95%
O
at 37 °C, RPMI 1640 medium supplemented with
gentamicin at 1 µg/ml, 5% fetal calf serum (FCS), (from Life
Technologies, Inc., Glen Waverly, Victoria, Australia) containing IL-3
at 50 units/ml.
10
FDC-P1 cells in 200 µl
of growth media and 0, 5, 10, 15, or 20 µg of pCIS2.RGHR1 DNA (16) in 50 µl of PBS. Cells were electroporated at 960
microfarads and 300 V with a Bio-Rad Gene Pulser apparatus connected to
a capacitance extender (Bio-Rad Laboratories Pty. Ltd., North Ryde,
Australia). After electroporation the cells were placed in a
25-cm
flasks containing 10 ml of fresh growth media
supplemented with 50 units of IL-3/ml. Cells were left for 36-48
h (to allow GHR expression), after which they were transferred to
growth medium devoid of IL-3 but containing 40 ng/ml hGH. GH selection
was continued for 2 weeks with medium changes at 48-72-h
intervals. Stably transfected lines were then cloned by repeated
limiting dilution in the presence of 40 ng/ml hGH.
MTT Assay
This cell proliferation assay was
originally developed for the spectrophotometric quantification of cell
growth and viability (17) and so provides a rapid and convenient
means by which the proliferation of GH-dependent cell lines can be
assessed in response to the addition of mutant GHs.
g for 5 min, aspirating the medium, and
resuspending in the same volume of sterile PBS. This step was repeated
twice to ensure the removal of all free hGH. The cells were then
resuspended in phenol red-free RPMI 1640, 5% FCS with 1 µg/ml
gentamicin, diluted to a final concentration of 8
10
cells/ml, and 50 µl of this suspension was dispensed into
each well of a 96-well plate. This was followed by 100 µl of
appropriately diluted hormone made up in the same medium. One plate was
used to assay each mutant, with the plate divided in half to allow a
within plate comparison with wild type pGH. Eight GH concentrations
were used per assay, allowing each GH concentration to be assessed in
sextuplicate. Plates were placed without lids (to maintain uniform gas
exchange) in a humidified chamber for 20-24 h at 37 °C in 5%
CO
, after which 50 µl of 4 mg/ml MTT was added and the
plates left for another 3-4 h(18) . Assays were terminated
by lysing the cells in 120 µl of isopropanol with trituration, and
plates were stored in the dark at 22 °C for 10 min before reading
the absorbance at 595 nm in a microplate reader (model 450, Bio-Rad).
)-dependent in binding to
the rabbit GHR(9, 20) , and since phenol red-free RPMI
1640 medium was calculated to contain 125 mM Me
(120 mM Na
and 5 mM K
) and 0.8 mM Me
(0.4
mM Ca
and 0.4 mM Mg
), it was possible that this was insufficient
to achieve maximal binding and therefore a maximal mitogenic response.
For this reason each of the Me
-dependent pGH
analogues and pGH were assayed in the presence or absence of an
additional 2 mM MgCl
.
was calculated after subtracting the base line from a
maximal GH dose, this being 900 ng/ml for pGH assays and 450 ng/ml for
bGH, hGH, and C181S del 184-191 pGH assays (Fig. 3).
Figure 3:
Transformed data from MTT assay showing
difference in biopotency between pGH (), C181S del 184-191
pGH (
), and K30E R34E pGH (
) analogues. A protein
concentration correction based on laser densitometry of silver-stained
gels and amino acid analysis (9) was applied to these results for
calculation of biopotencies. Curve fitting was performed by linear
regression using the DeltaGraph package for Macintosh desktop computers
(Delta Point, Inc.). The ED
was calculated after
subtracting the base line from a maximal GH dose, this being 900 ng/ml
for pGH assays and 450 ng/ml for bGH, hGH, and C181S del 184-191
pGH assays. This result was typical of three preparations of the C181S
del 184-191 pGH analogue and two preparations of wild type
pGH.
[
Cell proliferation assays using
[H]Thymidine Incorporation
Assays
H]thymidine were set up in an identical fashion
to the MTT assays. The cells were incubated for 14-16 h in the
presence of hormone prior to pulsing with 0.5 mCi of
[
H]thymidine in 50 µl of RPMI 1640, 5% FCS, 1
µg/ml gentamicin for 5-6 h before harvesting on a Titer Tek
cell harvester (Flow Laboratories, ICN Biomedicals Pty. Ltd., Seven
Hills, New South Wales, Australia). Glass filters were counted on a
Packard 1900 CA
spectrometer with quench correction.
Determination of Affinity Constants for GHs and
Characterization of Rabbit GHRs Expressed in FDC-P1
Cells
Affinity values for bGH and all pGH analogues were
assessed using rabbit liver microsomes as described in Ref. 9. For
determination of hGH affinity for receptors expressed in the FDC-P1
cell line, I-labeled hGH was displaced by increasing
dilutions of unlabeled hGH.
(IGBBM)(22) . To 12
75-mm glass tubes was then
added 200 µl of IGBBM, 100 µl of
I-hGH
(approximately 200,000 cpm), 100 µl of unlabeled hGH at increasing
dilutions, and finally 100 µl (2
10
and 2
10
cells/ml for those grown in GH and IL-3,
respectively) of cells. Assays were shaken gently at 12 °C (to
prevent receptor internalization; Ref. 20) for 14 h and terminated by
adding 2 ml of ice-cold IGBBM to each tube, followed by centrifugation
for 25 min at 4 °C at 1600
g. Pellets were counted
on an LKB 1274
spectrometer. Data analysis and curve fitting were
performed as described previously(9, 20) . The identical
assay procedure as described above was used to bind
I-hGH
to FDC-P1-RGHR3B cells prior to cross-linking, except that Tris was
replaced by Hepes in the IGBBM assay buffer and 1.5
10
cells were incubated with 1
10
dpm of
I-hGH in a final volume of 2 ml. Cross-linking was
performed in the manner described by Barnard and Waters(23) .
Crystal Structure Analysis
The complete
co-ordinates for the hGH(hGHR extracellular) complex were
kindly made available to us by A. De Vos and A. Kossiakoff. These were
converted to the pGH(pGHR extracellular)
complex by use of
the homology package, and energy minimized using the Discover program
(Biosym Technologies, San Diego, CA) as described in Ref. 9.
Affinity of GH Analogues
The relative affinities
of hGH and bGH were 2.4- and 3.5-fold greater, respectively, than pGH (). Of the pGH analogues tested, the K30E R34E pGH, E33K
pGH, and C181S del 184-191 pGH analogues showed a greater than
2-fold increase in affinity for the rabbit GHR relative to wild type
pGH (2.4-, 2.1-, and 4.8-fold, respectively). The increased affinity of
the K30E R34E pGH analogue relative to pGH was still evident when
assayed under physiological conditions (1.8-fold increase), but was
less than the increase in affinity obtained when binding was performed
in 20 mM Mg. All other pGH analogues
exhibited an affinity similar to wild type pGH ().
Establishment of Stably Transfected Cells
Several
clones of FDC-P1-RGHR cells were obtained, with each of these being
able to specifically bind GH and showing a dose-response curve almost
identical to that of selected FDC-P1-RGHR3B line (data not shown).
Affinity cross-linking to the FDC-P1-RGHR3B cells with I-hGH revealed bands at around 65 and 116 kDa after
subtraction of the hormone component (Fig. 1A).
Scatchard analysis from 3 independent assays on this clone revealed 186
± 12 GHRs/cell with an affinity of 7.6 ± 1.6
10
M
when cells were supported
with GH (Fig. 1B). When cells were supported with IL-3,
the affinity of hGH for the GHR was similar, 9.69 ± 2.1
10
M
, however, the receptor
number was increased to 2128 ± 230 receptors/cell (n = 3).
Figure 1:
A, autoradiograph of I-hGH cross-linked to FDC-P1-RGHR3B cells.
I-hGH was bound to 1.8
10
cells alone
or displaced by 2 µg/ml hGH or 1 µg/ml ovine PRL. Cross-linking
of GH to the GHR was performed according to ``Materials and
Methods.'' Reduced cross-linked cell homogenates were loaded (arrow indicates top of resolving gel) and run on a 10%
SDS-polyacrylamide gel, with molecular size standards from Bio-Rad or
Pharmacia (Amrad Pharmacia, Cannon Hill, Queensland, Australia). The
dried gel was exposed to x-ray film for 48 h prior to development of
the autoradiograph. B, Scatchard analysis of
I-hGH binding to FDC-P1-RGHR3B cells. Cells were grown to
confluence in RPMI 1640 supplemented with 5% FCS and 40 ng/ml hGH.
After three washes with PBS to remove hGH, the assay was set up in the
manner described under ``Materials and Methods,'' with 1.8
10
cells added per assay tube. Each point in the
above assay represents the mean of a triplicate determination. The
number of receptors per cell was 186 ±
12.
Biopotency of GH Analogues by MTT
Assay
Application of the MTT assay to the FDC-P1-RGHR3B cell
line provided a fast, sensitive, and non-radioactive method for
determining the bioactivity of GH analogues. In all cases the base-line
(0 ng/ml) and maximal GH response to pGH and the test analogue were
virtually identical, enabling an accurate assessment of the
ED. A value of 900 ng/ml was used to obtain the maximal GH
response for pGH and helix 1 and 4 mutants, whereas 450 ng/ml was used
for bGH, hGH, and the C181S del 184-191 pGH analogue, these
values being in the center of the plateau in the bell-shaped curve (Fig. 2). Because each analogue was assayed against wild type pGH
in the same plate, interassay variation was not a concern.
Figure 2:
A, bell-shaped dose-response
curve for hGH () and pGH (
) with FDC-P1-RGHR3B cells
using the [
H]thymidine assay. The assay protocol
is described under ``Materials and Methods.'' Each point is
the mean of a triplicate determination with S.E. indicated. The above
curve represents one of three similar assays. B, bell-shaped
dose-response curve for hGH (
) and pGH (
) with
FDC-P1-RGHR3B cells using the MTT assay. The assay was performed as
described under ``Materials and Methods.'' Each point in the
above curve is the mean of a sextuplicate determination with S.E.
indicated. Two additional experiments performed in the same manner gave
nearly identical dose-response curves.
The most
significant reductions in biopotency of the pGH analogues were seen
with K30Q R34E pGH, K30E R34E pGH, H170D pGH, and P6S pGH, which had 5,
7.7-, 3-, and 2-fold reductions respectively in potency compared with
wild type pGH. In contrast, bGH, hGH, and C181S del 184-191 pGH
showed increased biopotency compared with wild type pGH ().
All other pGH analogues had similar biopotency to wild type pGH.
to the growth medium
elevated nonspecific growth and thus the assay base line at 0 ng/ml;
however, the ED
for all of the hormones tested under this
condition was not significantly different from that obtained in the
control assay performed in the absence of additional Mg
().
Cell Proliferation Determined by
[
Over a
GH range from 0 to 3 H]Thymidine Incorporation
10
ng/ml, a bell-shaped
dose-response curve was seen with the
[
H]thymidine assay (Fig. 2A) as
reported by Fuh et al.(5) , similar to that seen with
the MTT assay (Fig. 2B). This assay also shows the
higher potency of hGH compared to pGH, and the fact that inhibition of
cell proliferation at the highest hormone concentrations was more
pronounced with hGH than with pGH.
10
M
and 9.6 ± 2.1
10
M
when the cells were grown in GH and
IL-3, respectively, similar to the values of Gobius et al.(22) and Leung et al.(16) , who transiently
expressed the identical rabbit GHR construct in COS-7 cells.
Tyr change in the hinge region at
position 127 was assigned as essentially responsible for the domain
shift.
Figure 4:
Crystal
structure (4) of the pGH(pGHR) complex detailing. A, pGH (in orange) with the C terminus of helix 1 and
His-170 highlighted in yellow. This region is juxtaposed to
the hinge region linking domains 1 and 2 (Glu-126 and Glu-127), and the
last
-loop in domain 2 (Glu-220) (all highlighted in yellow) of the pGHR1 (in green). pGHR2 is in white. B, examination of the crystal structure of the
GH/GHR1 complex reveals two distinct but adjacent clusters binding in
the complex, with pGH and pGHR1 side chains having blueand yellow Van der Waals radii, respectively.
The Lys-30-Glu-33-Arg-34-Arg-42/Glu-126-Glu-127-Glu-220 cluster
(distances within this cluster are summarized in Ref. 9) is separated
from the His-22-His-170-Glu-173/Asn-218 cluster (distances between
Asn-218 and His-22 and between Asn-218 and Glu-173 are 4.3 and 3.2
Å, respectively) by a distance of approximately 10 Å. These
two clusters are connected through residues Asn-218 and Glu-220, both
being found on or near the F/G
-bend in domain 2 of pGHR1.
Therefore, there is a strong likelihood that mutations affecting
intermolecular interactions in one cluster would have an influence on
interactions within the other.
Glu-220 is positioned directly after the -bend
connecting strands F and G in domain 2 (4) (Fig. 4A). This
-bend protrudes upward,
away from the cell surface and toward the hormone, forming the bottom
of a ``cupped hands'' configuration that holds the GH
molecule in the GH(GHR)
complex. Significantly, there is a
difference in
-loop structure that is observed when GHR1 and GHR2
are superimposed (Fig. 5). The
-loop in GHR1 appears to have
collapsed as a result of the interaction with GH side chains,
principally His-21 and Glu-173, suggesting a hormone-induced
conformational change (this is analogous to the conformational change
observed in the loop comprising residues 163-168 of the
hGHR(4) ). Since this
-bend is the last turn before the
extracellular domain of the GHR inserts into the plasma membrane, it is
plausible that this conformational change is involved in initiation of
the biological response.
Figure 5:
Crystal structure of the -loop
linking strands F and G in domain 2 of hGHR 2 (in red; side
chains in green) superimposed onto hGHR 1 (in light
blue; Ser-219 and Asn-221 side chains in yellow). The
distances between
-carbonyl O of residues 217-224 taken from
the superimposed hGHR1 and hGHR2 complex are as follows: Arg-217, 0.36
Å; Asn-218, 0.40 Å; Ser-219, 2.35 Å; Gly-220, 3.24
Å; Asn-221, 2.78 Å; Tyr-222, 0.64 Å; Gly-223, 0.39
Å; Glu-224, 0.51 Å. As highlighted previously (4), the loop
connecting strands F and G in domain 2 is one of the few regions of the
hormone binding interface that is not shared between hGHR 1 and 2.
Since this is the case, any difference in the structure within this
region is presumably a direct result of GH interaction with GHR1. Thus
we predict that the displacement of residues 219-221 and the
180° rotation of Asn-221 is due to contact of hGH side chains
His-22 and Glu-173 with Asn-218 of the
-loop.
We suggest that the loss of bioactivity
associated with the K30Q R34E, K30E R34E pGH, and H170D pGH analogues
is due to unfavorable interactions within either or both of the
locations discussed above. Since the GH molecule interacts with GHR1 at
the hinge region (Glu-126/Glu-127) and the F/G -bend (Asn-218 and
Glu-220) in the second domain of pGHR1, it seems likely that the
measured angle between domains 1 and 2 of GHR1 in the GH/GHR complex is
modulated by these interactions (Fig. 4B). The angles
between the two domains in hGHR1 and hGHR2 differ considerably, and
this is a reflection on the different modes of binding of these
subunits with the GH molecule. The mutated side chains that interact
with the hinge region and
-bend could induce a small movement that
manifests a large change in the orientation of receptor domain 2, in
much the same way as Tyr-127 is responsible for the difference in
domain orientation between the hGH and PRL receptors. A change in the
orientation of domain 2 relative to domain 1 in the GHR could affect
its ability to dimerize with the identical domain in GHR2. Similarly,
an unfavorable displacement of the
-bend could also effect
dimerization analogously, or as suggested by crystal structure evidence (Fig. 5), induce an unfavorable conformational change that is
transmitted to the submembrane domain, which thereby influences
signaling. In support of this proposal, we have recently shown that
mutation of Tyr-222 (at the base of the
-bend) to Ala results in
complete loss in signal transduction despite only a 5-fold decrease in
affinity for hormone(27) .
-bend in domain 2
of the receptor (Fig. 3B). The conversion of these side
chains to Ala would decrease the chances of inducing the above
mentioned conformational change in the
-bend. Close contact with
the
-bend is also an important in the interaction between hGH and
the homologous prolactin receptor, as the histidines in helix 1 and
Glu-173 in helix 4 of hGH and histidine 218 in the PRL receptor are
involved in zinc chelation(26, 29) . Accordingly, we
propose the mutations used by Fuh et al.(5) to make
their 30-fold higher affinity hGH analogue were inappropriate for
demonstrating the relationship between higher site 1 affinity and
biopotency.
complex we suggest that
there are two regions on the receptor (the hinge region between domains
1 and 2, and the
-bend) that are conformationally sensitive and
important for signal transduction. Confirmation of this hypothesis is
required through further mutagenic analysis of the hGH/hGHR
interactions or through crystallization of a non-primate GH/GHR
complex. Importantly, however, this study shows that it is possible to
make analogues of GH that have increased site 1 affinity with a
commensurate increase in biopotency. This is consistent with
pharmacologic theory and indicates that conventional theory does apply
to situations where receptor dimerization is necessary for signal
transduction. This realization calls for a re-examination of approaches
used in the design of more potent GHs and of members of the
structurally homologous cytokine family(28) .
Table: Combined affinity (K) and
biopotency values for hGH, bGH, pGH, and pGH analogues
-dependent (9), additional Mg
was
supplemented to the growth medium to ensure a maximal mitogenic
response. Each analogue underwent Scatchard and biopotency analysis a
minimum of three times; S.E. is indicated. The values in parentheses
are the decreases in bioactivity relative to wild type pGH. *, p < 0.05;**, p < 0.01 indicate significant change
relative to pGH.
, monovalent cation; Me
, divalent
cation; pGH, porcine growth hormone.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.