(Received for publication, June 3, 1996, and in revised form, October 2, 1996)
From the Physiology & Pharmacology Department and Centre for
Molecular & Cellular Biology and the Drug Design Centre,
University of Queensland, St. Lucia, Brisbane,
Queensland 4072, Australia
While there is a considerable amount of evidence that signal transduction by the growth hormone (GH) receptor requires receptor homodimerization, there has been no systematic study of the role of receptor dimerization domain residues in this process. In conjunction with the distances derived from the crystal structure of the hGH-hGH receptor (extracellular domain) complex, we have used a luciferase-based c-fos promoter reporter assay in transiently transfected Chinese hamster ovary (CHO) cells, and stable receptor expressing CHO cell populations to define the dimerization domain residues needed for effective signaling. In addition to alanine substitution, we have used both aspartate and lysine substitutions to allow us to provide evidence for proximity relations through charge complementation. Introduced cysteine substitutions were also used, but unlike the erythropoietin receptor, these were unable to generate constitutively active receptor. We conclude that serine 145, histidine 150, aspartate 152, tyrosine 200, and serine 201, but not leucine 146 or threonine 147 are required for effective signal transduction through the dimerization domain. This information may be valuable in designing small molecule antagonists of GH and other cytokines that block dimerization by binding to the dimerization domain.
Dimerization of receptors induced by ligand binding is a common
means of triggering signal transduction in mammalian cells (1). In a
number of members of the tyrosine kinase receptor family
(e.g. platelet-derived growth factor and epidermal growth factor), two ligand molecules bind to two receptors to form a L2R2 complex, which is followed by activation
of the receptor tyrosine kinase. By contrast, in the cytokine receptor
superfamily, one ligand molecule promotes dimerization of heterologous
receptor subunits or dimerization of two identical subunits (2, 3). Currently, the best characterized example of ligand-induced receptor dimerization in the cytokine receptor superfamily is the GH receptor (GHR).1 The crystal structure of the
ligand-bound complex (4) shows that hGH binds to two identical receptor
(hGHBP) subunits to induce receptor dimerization, which is aided by a
dimerization domain in the C-terminal sandwich (domain 2). Eight
residues were identified as being involved in this dimerization domain,
and the relationship of these is summarized in Table I. The crystal
structure verified previous studies based on several techniques, which
demonstrated a 1:2 stoichiometry of hormone to receptor (5). GHR
dimerization occurs sequentially, i.e. hGH binds to the
first hGHR through "site 1" of hGH and subsequently binds to the
second hGHR through "site 2" of hGH (5). Cunningham et
al. (5) proposed that receptor dimerization results in signal
transduction, and this proposal was verified by studies from the same
group with GH mutants and GH receptor monoclonal antibodies using a
proliferation assay for FDC-P1 cells expressing a chimeric hGH-mouse
granulocyte colony-stimulating factor receptor (6). A study of
GH-induced tyrosine phosphorylation of cellular proteins also showed
that tyrosine phosphorylation was inhibited by treating IM-9 cells with
a GH mutant that prevents receptor dimerization (7). Furthermore, a
mathematical model for the quantitative evaluation of the bell-shaped
dose-response curves associated with sequential receptor dimerization
and the use of the nondimerizing hGH mutant
(G120R)2 indicated that GHR dimerization is
essential for GH stimulation of both lipogenesis in primary rat
adipocytes and receptor down-regulation in cultured human IM-9
lymphocytes (8). In support of these studies, a single amino acid
substitution (D152H) in the dimerization region of the human GHR
identified in a Laron dwarf abolished receptor dimerization (9). GHR
dimerization is thought to activate Jak2 kinase, which then
phosphorylates both the receptor and downstream signaling components
such as the Stats, IRS-1 and Shc (10). Currently, no systematic study
of the importance of receptor dimerization domain residues in signal
transduction has been carried out by biological function assay. In
addition, it is not proven that those residues involved in dimerization
in the crystal structure indeed represent residues interacting in
situ. Accordingly, eight residues in the putative dimerization
region of RGHR (based on the crystal structure of hGHBP; Ref. 4) were
converted to alanine, to cysteine, to lysine, and to aspartate
individually or in combination, and the importance of these residues in
signal transduction was examined using a c-fos promoter
luciferase assay (11) which has been useful in defining
structural components of the receptor needed for signaling (12).
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Constructs
Receptor Dimerization Domain MutantsA rabbit GHR (RGHR)
expression plasmid (pECE-RGHR) was constructed by inserting rabbit
growth hormone receptor (RGHR) cDNA (13) into the SV40-based
mammalian expression vector pECE at KpnI and XbaI
sites. All RGHR mutants were inserted into the mammalian expression
vector pECE as for wild type pECE-RGHR. The following mutations were
made by oligonucleotide-mediated mutagenesis using the Altered Sites
in vitro mutagenesis system (Promega) with pSELECT and
approximately 30 bp of oligonucleotides incorporating 1-3 altered
bases, synthesized on a Milligen/Biosearch 7500 DNA synthesizer, and
purified by polyacrylamide gel electrophoresis. Multiple alanine substituted mutants were made with the following oligonucleotides: 5-G
ACT TTA CTG
T GTT
C
A
CC GGG ATT
T GCA G
T ATT CAA GTG
C-3
for A143/A145/A146/A147/A150/A152 (6 Ala); 5
-CT GTA TTG TCG ACA
TCA GTT CCT GTG
C
CG TTA AG-3
for
A200/A201; 5
-CCT GTA TTG TCG ACA TCA GTT CCT GTG TAC
CG
TTA-3
for S201A; 5
-GTT AGC TTA ACC GGG ATT
T GCA
T ATT CAA G-3
for A150/A152. Eight Ala, 4 Ala, and 3 Ala
mutants were made by combining these primers, to give the particular
Ala substitutions given below.
In this construct, 8 residues in the extracellular region identified in the crystal structure as interacting across the dimerization domain (asparagine 143, serine 145, leucine 146, threonine 147, histidine 150, aspartate 152, tyrosine 200, and serine 201) were converted to alanine.
pECE-RGHR-6AIn this construct, 6 residues (asparagine 143, serine 145, leucine 146, threonine 147, histidine 150, and aspartate 152) were converted to alanine.
pECE-RGHR-4AFor this construct, 4 residues (histidine 150, aspartate 152, tyrosine 200, and serine 201) were converted to alanine.
pECE-RGHR-3AHere, 3 residues (histidine 150, aspartate 152, and serine 201) were converted to alanine.
pECE-RGHR-Y200,S201A (RGHR-200,201A)In this construct, 2 residues (tyrosine 200 and serine 201) were converted to alanine.
Other ConstructsOther constructs created with the Altered Sites system were: pECE-RGHR-S201A, pECE-RGHR-S145C, pECE-RGHR-D152C, pECE-RGHR-Y200C, pECE-RGHR-S201C, pECE-RGHR-152,200C, pECE-RGHR-L146D, pECE-RGHR-H150D, pECE-RGHR-Y200D, pECE-RGHR-S201D, pECE-RGHR-N143K, pECE-RGHR-S145K, pECE-RGHR-L146K, pECE-RGHR-T147K, pECE-RGHR-H150K, pECE-RGHR-D152K, pECE-RGHR-S201K, pECE-RGHR-H150K, D152K(RGHR-150, 152K), and pECE-RGHR-Y200,S201K (RGHR-200,201K). All constructs were verified by double-strand dideoxy sequencing using the Pharmacia T7 kit.
c-fos Luciferase Reporter ConstructPlasmid pfos
396219-81 was constructed by inserting the fragment of the human
c-fos promoter from
396 to +42 into pGL.Basic after
deleting the 138 bp from
219 to
81 (11).
Cell Culture
Transient Transfection of CHO K1 CellsThis was undertaken as for Chen et al. (11). Briefly, CHO K1 cells maintained in 2 ml of Ham's F-12 medium containing 5% FCS were dispensed into six-well plates at 2.2 × 105 cells/well and left for 20-24 h. After aspiration of the medium, 160 µl of transfection reagent-DNA mixture containing 1 µg of receptor mutant expression plasmid, 2 µg of reporter gene, and 20 µl of DOTAP in HBS (20 mM Hepes, 150 mM NaCl, pH 7.4) was added to each well and mixed gently for 30 s. Three ml of Ham's F-12 medium containing 0.5% FCS was then added to each well. After incubation for 40 h, transfected cells were treated with 200 ng/ml rhGH. Negative control cells were treated with the same volume of serum-free Ham's F-12 medium (medium control). After further incubation for 5.5 h, the cells were harvested for luciferase assay according to Promega's instructions, and normalized to protein content. Constructs were transfected in triplicate for each experiment, and all experiments were repeated a minimum of three times. Transfection controls were as described in Ref. 11, and always included wild type receptor.
Stable Transfection of CHO K1 CellsCHO K1 cells maintained in 2 ml of Ham's F-12 medium containing 5% FCS were dispensed into six-well plates at 2.2 × 105 cells/well and left for 20-24 h. After aspiration of the medium, 160 µl of transfection reagent-DNA mixture containing 2.7 µg of wild type or mutant RGHR expression plasmid, 0.3 µg of NeoR expression plasmid, and 20 µl of DOTAP in HBS (20 mM Hepes, 150 mM NaCl, pH 7.4) was added to each well and mixed gently for 30 s. Two ml of Ham's F-12 medium containing 0.5% FCS was then added to each well. After incubation for 16 h, the culture medium was changed to 3 ml of Ham's F-12 medium containing 10% FCS, and the cells were incubated for an additional 48-72 h. Stable transfectants were selected by including G418 in the cell culture medium at 300 ng/ml until the surviving cells reached clonal confluence. The surviving clones were then selected and cultured. An aliquot of each clone was stored frozen, while the reminder of the cells were used for analyses.
Hormone Binding Analysis
Hormone binding constants for cell surface receptor mutants were
determined by Scatchard assay using recombinant human GH (a gift from
Genentech Inc., South San Francisco, CA) with receptor mutants stably
expressed as populations in CHO-K1 cells, since it was not possible to
measure receptor transiently expressed, as in the luciferase assay. GH
was radiolabeled by the IODOGEN method (14) following by separation on
a Sephadex G100 column to give a specific activity range of 100-150
µCi/µg. For assays, 2 × 105 cpm iodinated hormone
(less than 1 week old) and eight concentrations (0, 3, 10, 30, 50, 100, 1000, and 10,000 ng/ml) of unlabeled competitor in triplicate were
added to cultured cells transfected with wild type or mutant GH
receptor. CHO K1 cells stably expressing wild type or mutant RGHR were
grown to confluence in six-well plates. Medium was then changed to 25 mM Tris-HCl (pH 7.5), 130 mM NaCl, 0.1% BSA,
and 20 mM MgCl, and the assay was carried out essentially as in Ref. 15. After incubation at 10 °C for 16 h, the CHO K1 cells were washed with ice-cold phosphate-buffered saline and harvested
by addition of 1 ml of 0.1 M NaOH and incubation for 15 min
at room temperature. At 10 °C, internalization is minimal, and the
cell membrane is in a highly ordered, gel phase (27). Cell
surface-associated counts were quantified using an LKB 1275 counter. The resulting data were analyzed and the binding constants calculated using the McPherson LIGAND program (Elsevier Biosoft). Scatchard plots are linear below 50 pM receptor
concentration for a dimerizing GH receptor (8), and all estimations
herein gave linear Scatchard plots and receptor concentrations below 30 pM.
Alanine substitution (scanning) is minimally perturbing for secondary and tertiary structure and is generally regarded as the best means of selective removal of interactive residues involved in salt bridges and hydrogen bonds (16, 17). To confirm that alanine-substituted RGHRs were expressed correctly, and to define any impairment of ligand binding, Scatchard assays were performed with stable CHO K1 transfectants. These showed that pECE-RGHR-8A, pECE-RGHR-6A, and pECE-RGHR-S201A were expressed with wild type RGHR affinity and at comparable levels, although pECE-RGHR-8A was expressed at only 28% of wild type number (Table II). These data indicate that alanine mutation in the dimerization domain has no effect on binding affinity of RGHR, and that receptor was expressed at an acceptable level. Fig. 1A shows that in the transient cotransfection assay, induction of luciferase activity by GH with pECE-RGHR-8A, pECE-RGHR-6A, pECE-RGHR-4A, pECE-RGHR-3A, and pECE-RGHR-200,201A was less than 25% of wild type pECE-RGHR, and for pECE-RGHR-S201A was 67 ± 4% that of wild type. These results indicate that the residues in the dimerization region observed from crystal structure are indeed involved in signal transduction, particularly Tyr-200 and His-150/Asp-152. Moreover, although the alanine mutants pECE-RGHR-8A, pECE-RGHR-6A, pECE-RGHR-4A, and pECE-RGHR-3A exhibited reduced induction of luciferase activity by GH, they exhibited significantly higher (over 2-fold) basal activity than wild type RGHR (Fig. 1B). Therefore, it is possible that these alanine mutants are actually facilitating dimerization through hydrophobic interaction.
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Cysteine Substitutions
The homologous erythropoietin receptor
is able to exhibit constitutive activation through disulfide
bond-initiated homodimerization in the R129C erythroleukemia mutation
(18), and also in E132/133C putative dimerization domain mutants (19).
To investigate whether an analogous constitutive activation of the GH
receptor could be obtained, we substituted four dimerization domain
residues (Ser-145, Asp-152, Tyr-200, and Ser-201) with cysteine, and we also created a double cysteine mutant (pECE-RGHR-D152C,Y200C). As
indicated in Table I, the latter two residues appear to
be hydrogen-bonded in the crystal structure. These mutants were found to be expressed with essentially wild type affinity in CHO-K1 stable
populations, and at levels from 76 to 278% of wild type (Table
II). However, transient transfection assay showed that basal luciferase activities were similar to that of wild type RGHR with
all of these cysteine mutants, ranging from 79% (pECE-RGHRS145C) to
115% (pECE-RGHRS201C) of wild type (Fig.
2B). Cysteine substitution of Ser-145 almost
abolished GH induction of luciferase, exhibiting just 5.3 ± 2.9%
(mean ± S.E.) the activity of wild type RGHR (Fig. 4). GH
induction with pECE-RGHR-D152C, pECE-RGHR-Y200C, pECE-RGHR-S201C, and
pECE-RGHR-152,200C was 12 ± 3%, 42 ± 5%, 64 ± 10%,
and 10 ± 2.0% that of wild type RGHR, respectively (Fig.
2A). Accordingly, these substitutions further support the
important role of dimerization domain residues (particularly Ser-145
and Asp-152) in signal transduction, but provide no evidence of
constitutive activation of the receptor.
Aspartate Substitutions
Charge reversal is a useful tool for
defining interactive residues (20-22). Conversion of residues in the
dimerization region of RGHR to aspartic acid should abrogate
dimerization by repulsion unless divalent cations shield these negative
charges (21). Accordingly, four dimerization domain residues (leucine
146, histidine 150, tyrosine 200, and serine 201) were converted to
aspartic acid. Scatchard analysis of stable CHO K1 populations
expressing these mutant receptors showed that hormone affinity was not
significantly different from wild type RGHR, and cell surface receptor
expression ranged from 38-80% of wild type (Table II). Inductions of
luciferase activity by GH with pECE-RGHR-L146D, pECE-RGHR-H150D,
pECE-RGHR-Y200D, and pECE-RGHR-S201D were 90 ± 5%, 11 ± 3%, 27 ± 3.3%, and 13 ± 4% that of wild type RGHR,
respectively (Fig. 3A). Wild type RGHR and
the four aspartic acid mutants had similar basal luciferase activity
(Fig. 3B). These data support a role for His-150, Tyr-200, and Ser-201 in the dimerization process, but not Leu-146.
Lysine Conversions
As in the case of aspartic acid,
substitution of dimerization domain residues with lysine would be
expected to inhibit dimerization by charge repulsion. Accordingly, 8 residues (asparagine 143, serine 145, leucine 146, threonine 147, histidine 150, aspartate 152, tyrosine 200, and serine 201) were
converted to lysine. With the exception of Ser-145, hormone affinity
did not differ from wild type in stable transfectants (Table II). As in
the case of the cysteine mutant RGHR-S145C, the binding affinity of
RGHR-S145K was somewhat lower than that of wild type RGHR (39%), which
may indicate a minor contribution to hormone binding, presumably
indirectly through the sandwich. Receptor expression in these
stable populations ranged from 54 to 173% of wild type (Table II).
Fig. 4A shows that pECE-RGHR-L146K and
pECE-RGHR-T147K gave essentially wild type induction of luciferase
activity by GH and pECE-RGHR-N143K showed minor loss of inducibility,
while pECE-RGHR-S145K, pECE-RGHR-H150K, pECE-RGHR-D152K,
pECE-RGHR-S201K, pECE-RGHR-150,152K, and pECE-RGHR-200,201K showed
clearly reduced induction, particularly the last three mutants. This
indicates that substitution of asparagine 143, leucine 146, and
threonine 147 with lysine has little effect on signal transduction to
c-fos, whereas again Ser-145, His-150, Asp-152, and
Tyr-200/Ser-201 are needed for effective signaling. Interestingly, pECE-RGHRN143K displayed a significantly higher basal luciferase activity (264 ± 4% of wild type RGHR), although basal expression of other lysine mutants was normal (Fig. 4B).
Taken together, these alanine, cysteine, aspartic acid, and lysine mutations indicate that particular dimerization domain residues are involved in signal transduction by the RGHR. However, the decrease in induction of luciferase activity by GH with these mutants could also result from a less specific perturbation in conformation in the C-terminal domain of the RGHR extracellular region, which affects signal transduction but not binding affinity. Therefore, to verify whether these residues are directly involved in dimerization, and to gain information about proximities of important dimerization domain residues, charge complementation studies (20) were undertaken. Plasmids encoding negatively charged mutants (pECE-RGHR-H150D, pECE-RGHR-Y200D, and pECE-RGHR-S201D) and positively charged mutants (pECE-RGHR-S145K, pECE-RGHR-L146K, pECE-RGHR-H150K, pECE-RGHR-D152K, pECE-RGHR-S201K, pECE-RGHR-150,152K, and pECE-RGHR-200,201K) were either transfected alone or co-transfected into CHO K1 cells with the c-fos reporter plasmid. The total amount of receptor expression plasmid was held constant, so that in the absence of complementation, a value between the individual values for single charge conversions would be predicted. Complementation of induction through electrostatic pairing would suggest that the decreased signaling observed when each mutant is expressed alone is specifically a result of disrupted dimerization.
Results with negatively charged mutants alone, and positively charged mutants alone or with both charge mutant combinations are summarized in Table III. These results showed that signal transduction was partially restored (p < 0.05 to p < 0.01) by charge complementarity between the 150/152 couple (p < 0.05), the 201/201 couple (p < 0.01), and the 200/150-152 couple (p < 0.05). Weak complementation probably also occurs between the 143/150 couple and the 200/201 couple, but not among the remaining charge complementations. Since these conclusions are based on the assumption that co-expressed plasmids are expressed as the sum of each separately, we tested this by co-transfecting three positive complementations and measuring total cell surface expression by Scatchard analysis. Table II shows that for the 150/152 couple and the 201/201 couple, receptor expression resulting from co-transfection is not significantly different from the sum of separate expressions. However, expression of cotransfected pECE-RGHR-Y200D and pECE-RGHR-150,152K is somewhat less than either construct alone; the effect of this will be to minimize the observed complementation.
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It has been demonstrated that GH binding leads to dimerization and
receptor signaling (6), but with the exception of a case of Laron
dwarfism involving Asp-152 (9), the importance of individual residues
within the dimerization domain has not been demonstrated. Indeed, to
our knowledge, a mutagenic analysis of known receptor dimerization
domain residues needed for signaling has not been carried out with any
cytokine receptor, although a study on four putative dimerization
domain residues has been reported for the erythropoietin receptor (19).
Here we have shown that Ser-145, His-150, Asp-152, Tyr-200, and Ser-201
constitute the important residues required for effective signal
transduction to the c-fos promoter, which integrates two of
the major signaling pathways originating from the activated receptor:
STAT (ignal
ransducer and
ctivator of
ranscription) and
mitogen-activated protein kinases (11). We have also used a charge
complementation approach derived from Barnard (20) to demonstrate that
the loss in signaling seen with mutated residues is not a result of
nonspecific conformational perturbation, and to demonstrate proximity
of certain of the dimerization domain residues involved in signal
transduction. Charge reversals have been used successfully in the study
of a number of protein-protein interactions (e.g. Refs. 21
and 22).
Since the sequences of the rabbit and human GH receptors are totally homologous within the dimerization domain (13), in the ensuing discussion of individual interactions we shall refer to the distances determined for the human (GHBP)2 hGH complex (4).
In the crystal structure, the distance between the oxygen atom of
serine 201 (receptor 1 or bp 1) and the
oxygen atom of tyrosine 200 (bp 2) is 3.3 Å, and the distance between the
oxygen atom of
serine 201 (bp 2) and the nitrogen atom of leucine 146 (bp 1) is 3.1 Å (Table I), indicating hydrogen bonding. When serine 201 was converted
to alanine, cysteine, aspartic acid, or lysine, induction of luciferase
activity by GH with these mutants was 67%, 64%, 17%, and 11% that
of wild type RGHR, respectively, and induction of luciferase activity
by GH with pECE-RGHR-S201D and pECE-RGHR-S201K combined was 33% that
of wild type RGHR. These results show that induction of luciferase
activity by GH is greater when there is no repulsion between two
position 201 residues, which means that proximity of both 201 residues
is required for effective signal transduction by the RGHR, and indeed,
these two residues are within salt bridge distance in the crystal
structure. Charge complementation did not fully restore signaling,
possibly because of steric hindrance in the dimerization region or
because not all transiently co-transfected cells expressed an equal
amount of both constructs. The fact that signaling with S201C was not constitutive may mean that an additional hormone-induced conformational change is required, or that the disulfide bond did not form. Western blots of cysteine-substituted stable CHO K1 populations obtained from
non-reduced, N-ethylmaleimide-extracted cells did not show receptor dimer, but did show a large amount of immunoreactive material
at the stacking gel interface.3
In the crystal structure of the complex the oxygen atom of tyrosine
200 (bp 2) is 2.7 Å from the
2 oxygen atom of aspartic acid 152 (bp
1) and 3.3 Å from the
oxygen atom of serine 201 (bp 1), and it is
believed that tyrosine 200 forms an important hydrogen bond stabilizing
the receptor dimer (Table I and De Vos et al. (4)). When
tyrosine 200 was converted to cysteine and aspartic acid, the induction
of luciferase activity by GH were 42% and 24% that of wild type RGHR,
respectively, without significant change in hormone binding affinity.
Furthermore, induction of luciferase activity by GH with
pECE-RGHR-S201A and pECE-RGHR-200,201A was 67% and 21% that of wild
type RGHR, respectively. These results verify that tyrosine 200 is
important for signal transduction by the RGHR but has no significant
effect on hormone binding affinity, as expected. In the case of Y200C,
where the phenolic ring of tyrosine was replaced by a thiol group,
conformational perturbation or weakening of the hydrogen bond is
expected, with minor loss in signaling. However, in the case of Y200D,
the mutant resulted in repulsion between two aspartic acids, so
induction of luciferase activity by GH with pECE-RGHR-Y200D was lower
than that of pECE-RGHR-Y200C, even accounting for decreased expression
of the aspartate mutant (Table II). Further evidence for a role of
tyrosine 200 in dimerization leading to signaling came from the charge
reversals. Combinations of pECE-RGHR-150,152K with pECE-RGHR-Y200D and
pECE-RGHR-D152K with pECE-RGHR-Y200D led to a greater induction of
luciferase activity by GH than with RGHR-150,152K, pECE-RGHR-Y200D, and
pECE-RGHR-D152K alone (Table III). Interestingly, pECE-RGHR-200,201K
with pECE-RGHR-Y200D and pECE-RGHR-S201K with pECE-RGHR-Y200D did not
give a greater induction of luciferase activity by GH than
pECE-RGHR-200,201K, pECE-RGHR-Y200D, and pECE-RGHR-S201K alone,
respectively (Fig. 5). These results suggested that
tyrosine 200 interacts with aspartic acid 152 rather than serine 201. This suggestion was also supported by the minimal GH induction seen
with the non-repulsive mutant pECE-RGHR-152,200C (Fig. 2). It may be
that the inability to obtain constitutive activation with the
pECE-RGHR152,200C mutant is a result of an excessive distance between
thiol groups where disulfide bonds cannot be formed when the rigid
sheet structures are aligned, or a result of formation of non-signaling
higher order polymers, as indicated above.
When aspartic acid 152 was converted to cysteine and to lysine,
induction of luciferase activity by GH was 12% and 23% that of wild
type RGHR, respectively, although hormone binding affinity and
expression were similar to wild type RGHR (Table II). These results
indicate that aspartic acid 152 is involved in signal transduction of
RGHR and are consistent with a recent report that Laron dwarfism occurs
when aspartic acid 152 is replaced by histidine (9). Combination of
pECE-RGHR-150,152K with pECE-RGHR-H150D and of pECE-RGHR-D152K with
pECE-RGHR-H150D resulted in complementation of luciferase induction by
GH relative to pECE-RGHR-150,152K, pECE-RGHR-D152K, and pECE-RGHR-H150D
alone, which indicates that interaction between residues 150 and 152 is
required for signal transduction. Indeed, in the wild type RGHR, the
1 nitrogen atom of histidine 150 (bp 1) is 3.8 Å from the
1
oxygen atom of aspartic acid 152 (bp 2), forming a weak salt
bridge.
In the crystal structure, the distance between the 2 nitrogen atom
of histidine 150 (bp 1) and the
oxygen atom of asparagine 143 (bp
2) is 2.9 Å, which suggests a hydrogen bond between them (4).
Surprisingly, when histidine 150 was converted to either aspartic acid
or lysine, signaling was reduced to less than 20% of wild type RGHR
(Figs. 3 and 4), indicating the undesirability of a full charge at this
location. Examination of the crystal structure shows that this is
probably a result of the close proximity of Asp-152 on the one hand,
and steric hindrance to the introduction of lysine on the other.
Interestingly, when nearby asparagine 143 was converted to lysine,
induction of c-fos promoter by GH was 67% that of wild type
RGHR, while the basal luciferase activity was 265% that of wild type
RGHR. Indeed, the small decrease of inducibility by GH seen with this
mutant may be a result of higher basal luciferase activity rather than
decreased dimerization of RGHR. Thus, constitutive signaling by RGHR
appears to occur with this mutant. Coexpression of pECE-RGHRH150D and
pECE-RGHRN143K showed lower GH induction of luciferase activity than
pECE-RGHRN143K alone (despite similar receptor number), indicating that
a salt bridge between aspartic acid 150 and lysine 143 is not forming due to repulsion by Asp-152. Examination of the crystal structure shows
that lysine at 143 (bp 2) is within optimum H bond distance (3 Å) from
Thr-147 (bp 1), whereas with asparagine at 143, the distance to
threonine 147 is too great (6.4 Å). Thus, in the Lys-143 mutant, a new
strong hydrogen bond forms, which may account for the apparent
constitutive activity.
In the model of DeVos et al. (4), the distance between the
oxygen atom of threonine 147 (bp 1) and the
1 oxygen atom of
aspartic acid 152 (bp 2) is 2.7 Å, and the distance between the
nitrogen atom of leucine 146 (bp 1) and the
oxygen atom of serine
201 (bp 2) is 3.1 Å. When threonine 147 was converted to lysine,
induction of luciferase activity by GH with this mutant was 80% that
of wild type RGHR and the binding affinity of this lysine mutant was
83% that of wild type RGHR, i.e. no significant effect was
observed. Similarly, when leucine 146 was converted to lysine and to
aspartic acid, induction of luciferase activity by GH with these
mutants were 88% and 89% that of wild type RGHR, and binding affinity
of these mutants were 87% and 95% that of RGHR, respectively. In
addition, the combination of pECE-RGHRS201D with pECE-RGHRL146K
exhibited a lower induction of luciferase activity by GH than
pECE-RGHRL146K alone, suggesting little interaction (Table III). These
results indicate that leucine 146 and threonine 147 are not involved in
signal transduction by RGHR.
There may also be an interaction between serine 145 (bp 1) and aspartic acid 152 (bp 2), since the distance between them is 3 Å, according to the crystal structure (Table I). Accordingly, when serine 145 was converted to cysteine or lysine, induction of luciferase activity by GH with these mutants was 5% and 32%, respectively, and the binding affinity of these mutants were 48% and 39% of wild type, respectively. These results indicate that serine 145 is involved in dimerization and also has a minor influence on affinity for hormone, possibly through perturbation of loop 5 and 6 interactions with the hormone (4).
In summary, our results suggest that signal transduction by RGHR, presumably as a result of dimerization, requires interaction between residues asparagine 143 and histidine 150, between histidine 150 and aspartate 152, between aspartate 152 and tyrosine 200, and between serine 201 and serine 201. The distribution of these residues is shown in Fig. 5, where it can be seen the important residues form two small pockets, of area less than 60 Å2. Despite the predominance of apolar residues in the dimerization domain (4), it can be seen from this figure that there is a cluster of hydrogen bonding/charge residues at the functional center of this domain.
Finally, while the results of the initial alanine scan support the individual charge and cysteine substitutions discussed above, pECE-RGHR-8A, pECE-RGHR-6A, pECE-RGHR-4A, and pECE-RGHR-3A exhibited a higher basal level of luciferase activity than wild type RGHR. Indeed, it is difficult to determine if the decrease in induction of c-fos promoter by GH seen with these mutants results from high basal activity as a result of enhanced dimerization or from elimination of dimerization. It is certainly possible that multiple alanine substitutions create a hydrophobic surface facilitating hydrophobic interactions through the methyl side chains, somewhat analogous to a leucine zipper. Consideration of Fig. 5 indicates that such a hydrophobic pocket is likely to occur on alanine substitution, and calculation (23, 24) of the binding free energy for a 100-Å2 buried hydrophobic area (well within the available surface area shown in Fig. 5) yields a value of 10-11 kJ/mol, close to the value for the hormone-(GHBP)2 complex (25). We attempted to determine if the increased basal luciferase was a result of constitutive dimerization by performing the post transfection and hormone treatment in the presence of 12.5 nM monoclonal antibody 5, which is known to block receptor dimerization (5). While this was effective in blocking signaling through the wild type receptor, it was without effect on either the basal activity of pECE-RGHR-8A or its residual GH induction, presumably because the epitope for monoclonal antibody 5 was destroyed, as demonstrated for the D152H Laron mutation (9).4
In relation to the creation of constitutively active receptors, it should be noted that this occurs both spontaneously with the erythropoietin receptor R129C erythroleukemia mutation (18) and can be induced by substituting cysteines at positions 132 and 132 within the putative dimerization domain of this homodimerizing cytokine receptor (19). However, in the latter case, the proportion of disulfide-bonded dimers was quite small, and no indication of the extent of higher polymers was given. As indicated, these prevented us from definitely concluding we had created disulfide-bonded GH receptor dimers. It is interesting that the same residues are able to form disulfide dimers in the erythropoietin receptor (e.g. residues 129), whereas our complementation data suggest that different residues interact across the dimerization domain, except for Ser-201. Also, alanine substitution of four putative dimerization domain residues with the erythropoietin receptor did not affect proliferative signaling (19), whereas in our case, blockade of GH induction was observed. Clearly there are major differences between these homologous receptors. These differences emphasize the lack of homology that exists between potential dimerization domain sequences (26). This would be an advantage in the design of specific small molecule antagonists capable of inhibiting dimerization, hence signaling. The studies presented here, which demonstrate that signaling can be inhibited by modifying 5 dimerization domain residues (Ser-145, His-150, Asp-152, Tyr-200, and Ser-201) in a tight cluster (Fig. 5), provide a target for rational design of such antagonists. In principle, this approach could find application in the creation of other cytokine receptor antagonists for use in the treatment of a variety of cancers.
We thank Thea Monks for expert assistance in tissue culture, and Ross Barnard and Scott Rowlinson for helpful discussions.