(Received for publication, October 23, 1995; and in revised form, December 11, 1995)
From the
The WSXWS motif in the extracellular domain defines members of the cytokine receptor family, yet its role in receptor structure and function remains unresolved. To address this question we have generated a panel of 100 mutants within the WSXWS motif of the erythropoietin receptor, which represents all single amino acid substitutions of these five amino acids. All mutants were synthesized at the same level; however, their passage from the endoplasmic reticulum to the Golgi apparatus differed. Because of this, expression of mutant receptors at the cell surface varied more than 300-fold. The tolerance of the tryptophan and serine residues to substitution was quite narrow; as a result, most of these mutants were retained in the endoplasmic reticulum and showed no cell surface expression or reduced cell surface expression. Although many mutants with substitutions at the middle residue of the motif reached the cell surface, it was notable that one mutant, A234E, was processed more efficiently than the wild type receptor and was expressed in elevated numbers at the cell surface. Despite this variation, all mutant receptors that reached the cell surface appeared able to bind erythropoietin and transduce a proliferative signal normally. These results are discussed in terms of a general model for WSXWS function in which the motif contributes to efficient receptor folding.
Erythropoietin (Epo) ()plays a central role in the
regulation of red blood cell formation. Epo exerts this effect by
binding to receptors expressed on the surface of erythroid progenitors
and stimulating these cells to proliferate and differentiate.
The
Epo receptor cDNA has been isolated(1) ; its sequence reveals
that the receptor is a member of the cytokine receptor
family(2, 3, 4, 5) . This group of
proteins includes the receptors for growth hormone, prolactin,
interleukins (ILs) 2, 3, 4, 5, 6, 7, 9, 11, and 13, granulocyte and
granulocyte-macrophage colony-stimulating factors, leukemia inhibitory
factor, oncostatin-M, ciliary neurotrophic factor, thrombopoietin, and
the p40 subunit of IL-12 and the IL-12
receptor(2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) .
At the primary sequence level, the most striking similarities between
these receptors are present in the extracellular domain and include
four cysteine residues, the spacing of which is conserved, a series of
aromatic residues, and the five-amino acid motif Trp-Ser-Xaa-Trp-Ser
(WSXWS). The receptors for interferon-/
,
interferon-
, and IL-10 are more distantly related to the cytokine
receptor family, as is the cell surface protein tissue
factor(3) . These molecules share one pair of conserved
cysteines with the cytokine receptor family but lack a definitive
WSXWS motif.
Using primary sequence alignments, Bazan (3) predicted that members of the cytokine receptor family
would contain two, and in some cases four, domains similar to those
present in members of the immunoglobulin superfamily. Each of the
domains contains seven -strands that form a barrel. Upon solution
of the structure of the growth hormone/growth hormone receptor
complex(32) , the general details of Bazan's prediction
were confirmed. The organization of
-strands in the growth hormone
receptor, however, more closely resembled the D2 domains of pap-D and
CD-4 than immunoglobulins.
The crystal structure of the growth
hormone receptor also revealed the position, and in certain cases
suggested a function, for residues conserved among cytokine receptors.
For example, the four cysteines and one conserved tryptophan are buried
within the interior of the first -barrel and presumably stabilize
this domain. In contrast, although the homolog of the WSXWS
motif in the growth hormone receptor, YGEFS, is close to a
solvent-accessible surface, it is neither part of the hormone-binding
nor the receptor dimerization surfaces(32) . Thus, few clues
could be found as to the function of the WSXWS motif.
In an
attempt to determine the function of this region of cytokine receptors,
several studies have been carried out in which residues of the
WSXWS motif of the granulocyte-macrophage colony-stimulating
factor receptor -chain, IL-2 receptor
-chain, Epo receptor,
growth hormone receptor, and prolactin receptor have been
mutated(33, 34, 35, 36, 37, 38, 39, 40) .
While the number of the mutations made in these studies were limited,
no consistent picture emerged concerning the function subserved by this
motif; rather, a wide spectrum of hypotheses were proposed, including
roles in ligand binding, receptor internalization, signal transduction,
intersubunit interactions, and protein
folding(33, 34, 35, 36, 37) .
Despite these suggestions, it seems unlikely that a motif such as
WSXWS, which is conserved among proteins with very little
overall sequence similarity, will perform a radically different
function in each receptor.
Here we examine in detail the structural restrictions placed on the murine Epo receptor WSXWS motif by the generation and analysis of all possible single amino acid substitutions of these five residues. The results are discussed in terms of a simple model in which the sequence WSXWS is a structurally important element of cytokine receptors and other proteins.
COS cells were transiently
transfected with 1-3 µg of DNA using the DEAE-dextran
method(48) . The transfected cells were incubated for 2 days at
37 °C in a fully humidified incubator containing 5% CO in air and were then used for binding assays or metabolic
labeling experiments. Stable transfection of hematopoietic cells was
achieved by electroporation as described(49) .
Immune Detection of Epo
Receptors-In order to metabolically label proteins for
immunoprecipitation, cells were washed with medium lacking methionine
and cysteine and then incubated in medium supplemented with 200
µCi/ml of [S]methionine and
[
S]cysteine for the indicated times. In some
experiments cells were chased by subsequent incubation, for various
lengths of time, in medium containing excess unlabeled methionine and
cysteine. Cells were washed with phosphate-buffered saline and lysed in
250 µl of 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1.0% (v/v) Triton X-100, and 2 mM EDTA. The nuclei
were removed by centrifugation in a microcentrifuge at 9,900
g for 1 min at 4 °C. Lysates were then mixed with an equal
volume of 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1.0% (v/v) Triton X-100, 1.0% (v/v) sodium deoxycholate,
0.2% (w/v) sodium dodecyl sulfate, and 2 mM EDTA and incubated
with antisera directed against the N terminus or C terminus of the
murine Epo receptor (49) for 1-2 h at 4 °C and then
with protein A-agarose beads for a further 1-2 h at 4 °C. The
beads were washed 3 times with 10 mM Tris-HCl, pH 7.4,
containing 150 mM NaCl, 1.0% (v/v) Triton X-100, 1.0% (w/v)
sodium deoxycholate, and 0.1% (w/v) SDS and once in phosphate-buffered
saline. The bound proteins were eluted with gel sample buffer
containing 2% (v/v)
-mercaptoethanol and resolved on 10%
SDS-polyacrylamide gels. The gels were fluorographed and dried, and the
radiolabeled proteins were visualized by autoradiography.
Figure 1:
Synthesis of Epo receptors with
substitutions in the WSXWS motif. A, 1
10
COS cells were transfected transiently with cDNAs
encoding every possible single amino acid substitution of the Epo
receptor at position 236. Cells were metabolically labeled for 60 min,
after which proteins were immunoprecipitated with an antiserum directed
against the N terminus of the mature Epo receptor and resolved by
SDS-polyacrylamide gel electrophoresis on a 10% gel. The gel was then
dried, fluorographed, and exposed to autoradiographic film. The
one-letter amino acid code above the autoradiograph identifies the mutants of the Epo receptor at Ser
transfected into COS cells. Am, amber stop codon;
-, untransfected COS cells; wt, COS cells transfected
with the wild type Epo receptor; tr, COS cells transfected
with a truncated Epo receptor containing amino acids 1-271. B, 5
10
Ba/F3 cells, stably transfected
with the designated Epo receptor mutants, were pulse-labeled for 15 min (P) or pulse-labeled for 15 min and then chased for 60 min (C). Proteins were then extracted and immunoprecipitated using
an antiserum directed against the N terminus of the mature Epo
receptor; half of each sample was treated with endo-H (+), while
the remainder was left untreated(-), prior to resolution by
SDS-polyacrylamide gel electrophoresis on a 10% gel and
autoradiography.
Transiently transfected COS cells provide an excellent system for rapidly assaying the expression of Epo receptor mutants, as well as for examining characteristics of Epo binding to cell surface receptors. However, a relatively small proportion of the receptor synthesized in COS cells reaches the cell surface. In contrast, Epo receptors expressed by the factor-dependent hematopoietic cell line Ba/F3 are processed more efficiently; these cells, therefore, allow comparisons to be made between the intracellular processing of various Epo receptor mutants. Fig. 1B shows that in stably transfected Ba/F3 cells, both wild type and mutant Epo receptors are initially synthesized as an endo-H-sensitive glycoprotein with an apparent molecular weight of 64,000. After an hour, approximately 25% of wild type Epo receptors had become endo-H-resistant, confirming previous findings (49, 52) that about 20-25% of newly made Epo receptors exit the endoplasmic reticulum to the Golgi. In contrast, endo-H-resistant forms of the mutants W232Y, S233Y, S233F, S233A, and A234W were undetectable, suggesting that all of these receptor molecules are retained in the endoplasmic reticulum. Endo-H-resistant forms of the mutant S233G were also difficult to detect at any time point of chase, even though this mutant was detected at the cell surface by iodinated Epo binding (Fig. 1B, Fig. 3, and data not shown). Remarkably, one mutant, A234E, appeared to be processed more efficiently than wild type, with 80-90% of the newly synthesized molecules showing resistance to endo-H resistance after 1 h. These differences were confirmed by more extensive pulse-chase experiments in which endo-H-resistant wild type and A234E Epo receptors were detectable after a 15-min chase and reached a plateau level of 25-30% and 70-80% of total pulse-labeled receptor after 30 min of chase, respectively(53) . The balance of newly made wild type and mutant receptors were degraded without exiting the endoplasmic reticulum(49, 52) .
Figure 3:
Binding
of Epo to receptors with substitutions at positions Ser and Ser
. Approximately 10
COS cells
were transiently transfected with DNA encoding Epo receptors harboring
the designated substitutions at position Ser
(upper
panel) and Ser
(lower panel). The salient
details are described in the legend to Fig. 2.
Figure 2:
Binding of Epo to receptors with
substitutions at positions Trp and Trp
.
Approximately 10
COS cells were transiently transfected
with DNA encoding Epo receptors harboring the designated substitutions
at position Trp
(upper panel) and Trp
(lower panel). Forty-eight hours after transfection,
cells were incubated with 400 pM
I-Epo on
replicate transfectants, in the presence or absence of an excess of
unlabeled Epo at 32 °C (open circles) and at 37 °C (closed circles). Cells were washed 3 times in cold DMEM
containing 10% (v/v) FCS and solubilized in 0.5 ml of 1 M NaOH. Specific binding was calculated for each point and was
expressed as a fraction of the specific binding observed at 37 °C
to COS cells expressing the wild type Epo receptor. The figure shows combined data from at least two independent experiments.
Untransfected COS cells and cells transfected with wild type Epo
receptor cDNA were included in each experiment. Binding to
untransfected COS cells in the presence or absence of unlabeled Epo
varied from 120 to 160 cpm. Specific binding to COS cells transfected
with the wild type Epo receptor was between 11,000 and 15,000 cpm. The
limit of detection was routinely 0.5-1.0% of binding seen to wild
type, that is 180-300 cpm with a background of 120-160
cpm.
Figure 4:
Binding of Epo to receptors with
substitutions at position Ala. Approximately 10
COS cells were transiently transfected with DNA encoding Epo
receptors harboring the designated substitutions at position
Ala
. The salient details are described in the legend to Fig. 2.
The number and type of substitutions tolerated differed at each
position of the WSXWS motif. The position most sensitive to
change was Trp (Fig. 2). COS cells expressing the
mutant W232F bound approximately 100-fold less
I-Epo than
cells expressing the wild type receptor, while the capacity of cells
expressing W232Y to bind Epo was below the limit of detection. COS
cells expressing Epo receptors with substitutions of Trp
to aliphatic hydrophobic residues, to polar residues, or to
charged residues bound no detectable
I-Epo. Mutants of
Trp
exhibited a similar pattern of
I-Epo
binding to those of Trp
(Fig. 2), although
substitution to other aromatic hydrophobic residues was slightly better
tolerated in the former. Binding of Epo to COS cells expressing the
mutants W235F and W235Y was 14- and 100-fold lower than to cells
expressing the wild type receptor. Again, expression of receptors
containing other substitutions at position Trp
did not
yield detectable Epo binding.
Like the tryptophan residues,
relatively few mutants containing substitutions at the serine residues
of the WSXWS motif were able to bind Epo when expressed
transiently in COS cells (Fig. 3). Cells expressing the mutants
S233T and S233G bound 2- and 50-fold less I-Epo than
those expressing the wild type receptor. Mutation of Ser
to other residues resulted in receptors that displayed no ability
to bind Epo. Substitutions at position Ser
were less
deleterious than the corresponding mutants of Ser
(Fig. 3). For example, COS cells expressing Epo receptors
with the changes S236T and S236G bound
I-Epo at levels
comparable with wild type, while those expressing S236C and S236A were
also functional, at 25 and 12% of wild type, respectively. Cells
synthesizing S236Y also bound
I-Epo, although this
binding was approximately 30- to at least 100-fold lower than wild
type. In common with mutations at Ser
, no
I-Epo binding could be detected to COS cells expressing
mutants containing substitutions of Ser
to charged or
hydrophobic residues.
The middle residue of the WSXWS motif
of the Epo receptor is alanine. COS cells expressing all mutants at
this position, except A234W, bound I-Epo. Not all mutants
of Ala
, however, were equivalent (Fig. 4). As we
recently described, cells expressing A234E reproducibly bound
3-5-fold more
I-Epo than cells expressing the wild
type Epo receptor(53) . A234E was, indeed, the only mutant of
the 100 examined that exhibited such a phenotype. Cells expressing
receptors carrying substitutions of Ala
to other polar
and charged residues bound
I-Epo at levels similar to
wild type, and in general, hydrophobic residues were less well
tolerated at position 234 than hydrophilic residues. An increase in the
hydrophobicity of the side chain at position 234 was correlated with a
decrease in the amount of
I-Epo bound. For example, cells
expressing the mutants A234W, A234F, and A234Y bound, respectively,
less than 0.5%, 0.6-0.9%, and 2-3% of the
I-Epo bound by cells expressing the wild type Epo
receptor, while for the mutants A234I, A234L, and A234V the levels were
2-3%, 6-8%, and 20%, respectively.
To determine whether
mutant receptors that failed to bind Epo were expressed at the cell
surface, we attempted to detect Epo receptors using a polyclonal
antiserum raised against a peptide from the N terminus of the mature
Epo receptor. Initial experiments using immunofluorescence microscopy
on transiently transfected COS cells suggested that the mutant
receptors that were unable to bind Epo were not expressed at the cell
surface. Such data, however, could not be quantitated (data not shown).
To overcome this problem, we purified, using protein A, the IgG
fraction of antisera directed to the N-terminal and C-terminal peptides
of the Epo receptor. The IgG fraction was then radioiodinated and used
in a binding assay. No specific binding of the N-terminal or C-terminal
antibodies could be detected to untransfected COS cells. Binding of the
labeled anti-N-terminal IgG but not the anti-C-terminal IgG was
observed to COS cells transiently expressing the wild type Epo
receptor. This binding was specific, since it was inhibited by
unlabeled IgG from the N-terminal antiserum but not by IgG purified
from antisera to the C-terminal peptide or an irrelevant antigen (data
not shown). Using this technique we examined the cell surface
expression of a number of WSXWS mutant Epo receptors. Although
each mutant was synthesized at an equivalent level (Fig. 1A and data not shown), the amount of receptor detected at the cell
surface varied at least 10-fold. The mutants A234T, S233T, S236T, and
S236G were, for example, expressed on the surface at similar levels to
the wild type receptor, while A234V, A234L, S236A, S236C, and W235F
were 3-20-fold lower and A234F, A234W, S233G, S233A, S233C,
W232Y, and W232F were undetectable ( Fig. 5and data not shown).
The mutant A234E again proved exceptional in that it was expressed on
the surface at 1.5-3-fold higher levels than wild type (see also (53) ). Although immunodetection proved to be about
10-20-fold less sensitive than I-Epo binding in
detecting cell surface receptors, there was an excellent correlation
between the two methods (Fig. 5). In summary, the mutant A234E
was expressed at higher levels on the cell surface than the wild type
Epo receptor and mutants such as S233T and S236T, while cell surface
expression of other mutants was reduced or undetectable. These results
further support the notion that the primary effect of mutating the
WSXWS motif is to affect receptor folding and, as a
consequence, transport of the receptor from the endoplasmic reticulum
and its expression at the cell surface.
Figure 5:
Immunodetection of Epo receptors. COS
cells were transiently transfected with various Epo receptor mutants.
After 48 h cells were incubated for 4 h, at 4 °C, with either I-Epo or
I-antibody in the presence or
absence of unlabeled competitor. Subsequently the radiolabeled protein
was removed, the cells were washed 3 times with cold DMEM containing
10%(v/v) FCS, and the cells with receptor-bound label were solubilized
in 0.5 ml of 1 M NaOH. The mean specific binding of three
replicate transfections is shown for
I-Epo and
I-antibody.
Figure 6:
Equilibrium and steady-state analyses of
Epo binding to COS cells expressing various WSXWS mutant Epo
receptors. A, replicate wells containing 10 COS
cells were transiently transfected with the cDNAs encoding the
designated mutant Epo receptors. After 48 h cells were incubated for 16
h at 4 °C with various concentrations of
I-Epo (30
pM to 4 nM) in the presence or absence of a 300-fold
excess of unlabeled Epo. Subsequently the amount of Epo that was
specifically bound to cells and the amount free in solution were
determined. The data is graphed according to the method of Scatchard (59) . B, replicate wells containing 10
COS cells were transiently transfected with the cDNAs encoding
the wild type Epo receptor and the designated mutants. COS cells were
incubated with 400 pM
I-Epo at 37 °C for 6 h
in the presence or absence of an excess of unlabeled Epo. Free
I-Epo was separated from bound
I-Epo, and
the cells were washed twice in ice-cold DMEM containing 10% (v/v) FCS.
The cells were rinsed with 1 ml of ice-cold phosphate-buffered saline
containing 4% (v/v) acetic acid to remove surface-bound
I-Epo, washed with phosphate-buffered saline, and
solubilized with 1 M NaOH to allow quantitation of
internalized
I-Epo. The ratio of the amount of
I-Epo specifically bound to surface receptors to the
amount that had been internalized is shown for replicate
transfectants.
A similar relationship was
observed in Ba/F3 and 32-D cells, although the numbers of receptors
expressed by these cell lines were far lower than for COS cells. In
both factor-dependent hematopoietic cell lines the affinity of wild
type and mutant receptors for Epo varied from 450 to 1,100 pM;
however, while there were 1000 binding sites at the cell surface
of Ba/F3 cells expressing the wild type Epo receptor, there were
3,000-7,000 sites on cells expressing the A234E mutant but only
100-200 on cells expressing the S233G mutant (data not shown).
The characteristics of Epo binding by WSXWS mutant
receptors expressed by COS cells were explored further by analyzing the
kinetics of Epo association and dissociation as well as the ability of
each receptor to internalize Epo. The kinetic dissociation constant for
the complex between Epo and each of the mutant receptors was between
0.02 and 0.04 min at 4 °C, the same as for the
wild type receptor (data not shown). The curves depicting the progress
of Epo binding to equilibrium were also similar, suggesting that the
kinetic association rate constant governing the formation of
Epo
Epo receptor complexes at 4 °C was also the same for each
of the mutant receptors (data not shown). Finally, for each mutant
receptor examined, the ratio of
I-Epo specifically
internalized to that bound at the cell surface was 2.5-3.5 at 37
°C under steady state conditions (Fig. 6B),
indicating that receptor endocytosis and degradation of bound ligand
was not altered for these mutant receptors.
Figure 7:
Signal transduction by WSXWS
mutant Epo receptors. 300 Ba/F3 cells were cultured in agar for 7 days
in the absence of factor or in the presence of the specified
concentration of human erythropoietin. Cultures in which interleukin-3
was used as a stimulus were prepared as a positive control. For each
cell type, interleukin-3 stimulated the growth of 180-235
colonies. A, parental Ba/F3 cells () and Ba/F3 cells
expressing the constitutive mutant R129C (
). B, Ba/F3
cells expressing the wild type Epo receptor (
), the mutant A234E
(
), or S233G (
). C, Ba/F3 cells expressing the
Epo receptor mutants S233F (
) and W232F
(
)
Ba/F3 cells expressing various WSXWS mutants responded differently to Epo. Like parental Ba/F3 cells (Fig. 7A), those lines expressing mutant Epo receptors that did not reach the cell surface (i.e. W232F, W232Y, S233Y, S233F, and A234W) did not proliferate in response to Epo at concentrations from 0.005 to 10 units/ml (Fig. 7C and data not shown). These cells were also unable to survive with Epo as the sole growth factor (data not shown). Ba/F3 cells expressing the mutants A234E and S233G exhibited 3000-7000 and 100-200 receptors per cell, compared with cells expressing wild type Epo receptors that exhibited 1500-2000 receptors/cell. Despite differences in the expression of cell surface receptors, each cell line exhibited a similar dose-dependent ability to proliferate in response to Epo (Fig. 7B).
WSXWS mutants differ in their ability to exit the endoplasmic reticulum. The results described in this paper represent the first systematic study of the relationship between the structure of a cytokine receptor WSXWS motif and its function. A series of 100 point mutations representing all possible single amino acid substitutions in this region were created in the WSXWS motif of the murine Epo receptor. The salient property of the mutants generated in this study was that they differed in their ability to exit the endoplasmic reticulum and as a consequence to be expressed at the cell surface. This is most likely due to a difference in the ability of the mutants to fold into a normal conformation since endo-H-sensitive forms of the wild type receptor are unable to bind Epo, while endo-H-resistant forms are able to bind(37) . Thus the endoplasmic reticulum-localized forms of the wild type receptor are in various stages of folding and appear to acquire the native conformation just prior to or coincident with transport through the medial Golgi, where resistance of oligosaccarides to endo H is acquired. While the majority of mutants showed a defect in transport through the secretory pathway and presumably in folding, one, A234E, was transported and folded more efficiently than wild type. Importantly, the mutants that were transported to the cell surface had properties indistinguishable from the wild type receptor: each bound Epo with normal affinity, internalized Epo at the normal rate and, where tested, were capable of transducing a normal proliferative signal.
We examined the fate of WSXWS mutants of the Epo receptor expressed transiently in COS cells and stably in the factor-dependent hematopoietic cell line, Ba/F3. Although newly synthesized wild type receptor is processed to very different extents in the two cell types, the effect of mutations in the WSXWS motif appears analogous. In COS cells, the Epo receptor is transiently expressed at very high levels. The vast majority of Epo receptor synthesized, however, never exits the endoplasmic reticulum and is degraded. Despite inefficient processing, extremely high levels of Epo receptor synthesis ensure that a large number of receptors are expressed at the surface of the transfected COS cells. Differences in receptor processing in COS cells are therefore best assessed indirectly by measuring Epo receptors at the cell surface. In COS cells each mutant was synthesized at a similar level to the wild type receptor; however, cell surface expression levels varied over 300-fold, with the mutant A234E being expressed at 3-5-fold higher levels than wild type receptor and others being expressed at levels similar to or lower than the wild type.
We have shown
previously that the endogenous Epo receptor expressed in murine fetal
liver cells is inefficiently processed and transported from the
endoplasmic reticulum. In addition, Epo receptors ectopically expressed
in a variety of hematopoietic cell lines, including Ba/F3 cells, are
poorly processed, independent of the level of receptor
expression(53) . The extent of Epo receptor processing and
transport through the endoplasmic reticulum is, however, more efficient
in hematopoietic cells than in COS cells, although the overall levels
of receptor expression are lower (data not shown). In Ba/F3 cells
25% of newly synthesized Epo receptors exit the endoplasmic
reticulum and acquire endo-H-resistant carbohydrates (Fig. 1, B and C, and (49) and (52) ). In
Ba/F3 cells it is therefore possible to monitor the effect of mutations
directly on intracellular receptor processing. Strikingly, the mutant
(A234E) that was expressed at higher levels on the surface of COS cells
was also more efficiently processed in Ba/F3 cells, with 80-90%
of newly synthesized receptor leaving the endoplasmic reticulum within
an hour and 2-3-fold more receptors being present at the cell
surface(53) . Likewise we showed that there was an excellent
correlation between reduced cell surface expression of mutants such as
S233G and W232F in COS cells and a reduction in the efficiency of
receptor processing, measured by endo-H sensitivity, and cell surface
expression in Ba/F3 cells. The cell type-independent nature of the
phenotype of WSXWS mutants suggests that this motif plays an
important role in generating a correctly folded protein.
The motif is located on a
distorted region at the top of strand G in the second barrel.
This region of the receptor is not directly involved in binding growth
hormone, nor is it implicated in the formation of receptor homodimers (Fig. 8A; (32) ). Importantly, this observation
argues against theoretical predictions that have suggested that the
WSXWS motif lies on the ``floor of the binding crevice'' (3) and that have been previously used to assess the function
of the WSXWS motif in cytokine
receptors(33, 34, 35) .
Figure 8:
Alignment of members of the cytokine
receptor and thrombospondin families. The predicted amino acid sequence
of representative members of the cytokine and interferon receptor
family (A) and the thrombospondin family (B) were
aligned by eye. Aromatic and charged residues that form a hydrophobic
sheath are shown in boldface type, and their interactions are
shown by the lines above the alignment. The serine
residues in the WSXWS and the residues with which they
hydrogen bond are underlined and connected by lines below the alignment. The positions of the -strands of the
growth hormone receptor are also shown. The abbreviations are the
standard single-letter amino acid code, and sequences were obtained
from the GenBank
data base.
In addition to suggesting that the WSXWS motif does not participate directly in ligand binding or receptor homodimerization, the structure of the growth hormone receptor permits the residues that interact with the YGEFS sequence to be identified. This process is illuminating since it raises the possibility that the WSXWS motif might primarily act in a structural role.
In addition to the presumptive stabilizing role of the WSXWS motif, the hydrophobic interactions involving the WSXWS motif of the growth hormone receptor provide a scaffold for a series of solvent-accessible polar and charged residues. A striking pattern is observed in the growth hormone receptor, in which one face of this surface contains exposed oxygen atoms and the other exposed nitrogens. The position of these residues is conserved within the cytokine receptor family; however, their identity varies (Fig. 8A). This pattern of solvent-exposed charged and polar residues, supported by the hydrophobic scaffold provided by the WSXWS motif, represents an ideal surface for intermolecular interaction. As this surface is apparently uninvolved in either cytokine binding or homodimerization, it is attractive to speculate that it might be important in the formation of ternary receptor complexes. Substitution of these polar residues with residues of different charge (e.g. E173Q or E173K, K215E or K215Q in the growth hormone receptor) may be illuminating in determining the function of these residues and the veracity of this hypothesis.
It is interesting to note that there is a second extensive family of proteins that contain a WSXWS motif - those that contain a thrombospondin type I repeat (Fig. 8B). This family includes thrombospondin, F-spondin, the terminal components of complement C6, C7, C8, and C9, properdin, and the thrombospondin-related surface proteins of various parasites(4, 55, 56, 57, 58) . Like the WSXWS motif in the cytokine receptor family, the residues in the thrombospondin motif are not absolutely conserved, with phenylalanine and tyrosine substituting for tryptophan and with threonine, glycine, or alanine replacing serine. As discussed, members of the cytokine receptor family contain a series of alternating hydrophobic and positively charged residues upstream of the WSXWS motif. This pattern is not observed upstream of the WSXWS motifs found in members of the thrombospondin family: rather, a similar set of residues is present the same distance toward the C terminus and may act in an analogous manner to stabilize this molecule and produce a surface for intermolecular interaction (compare Fig. 8A with Fig. 8B).
The importance of a hydrogen bond between
the serine hydroxyl group and the adjacent main chain amine is
highlighted by the substitutions at positions 233 and 236 described in
this study. Serine and threonine are well tolerated at both positions,
while mutant receptors containing cysteine or tyrosine at position
Ser, which are also be expected to form hydrogen bonds,
also exit the endoplasmic reticulum and are expressed at the cell
surface, although at far reduced numbers compared with the wild type
receptor. Additionally, the tolerance, albeit less well, of small
residues such as glycine and, in the case of Ser
,
alanine, may reflect their ability to be accommodated in this region of
the receptor without markedly distorting local structure.
The results presented in this study support the notion that the conserved WSXWS motif plays a pivotal role in the generation of a properly folded cytokine receptor. The details of this role in the overall folding process might be further addressed in vivo by comparing the interactions of resident endoplasmic reticulum proteins such as BiP and protein disulfide isomerase with wild type and mutant Epo receptors and by examining the structure of receptors retained in the endoplasmic reticulum using monoclonal antibodies to conformational epitopes. In addition the folding of the Epo receptor might also be followed in vitro. Finally, if the structural model of the WSXWS motif proposed in this paper proves generally applicable, then the interactions made by the motif in each cytokine receptor should closely resemble the interactions made by the sequence YGEFS in the growth hormone receptor. The solution of the structures of other members of the cytokine receptor family will enable such comparisons to be made.