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(Received for publication, July 11, 1996, and in revised form, November 15, 1996)
From ABSTRACT The leptin receptor (OB-R) mediates the weight
regulatory effects of the adipocyte secreted hormone leptin (OB).
Previously we have shown that the long form of OB-R, expressed
predominantly in the hypothalamus, can mediate ligand-induced
activation of signal transducer and activator of transcription factors
1, 3, and 5 and stimulate transcription via interleukin-6 and
hematopoietin receptor responsive gene elements. Here we report that
deletion and tyrosine substitution mutagenesis of OB-R identifies two
distinct regions of the intracellular domain important for signaling.
In addition, granulocyte-colony stimulatory factor receptor/OB-R and
OB-R/granulocyte-colony stimulatory factor receptor chimeras are
signaling competent and provide evidence that aggregation of two
OB-R intracellular domains is sufficient for ligand-induced receptor activation. However, signaling by full-length OB-R appears to
be relatively resistant to dominant negative repression by signaling-incompetent OB-R, suggesting that mechanisms exist to permit
signaling by the long form of OB-R even in the pretence of excess
naturally occurring short form of OB-R.
INTRODUCTION Leptin (OB) is an adipose tissue-derived secreted hormone that is
thought to suppress appetite by regulating activities of satiety
centers in the brain (1). The weight reducing effects of leptin appear
to be mediated by interaction with the leptin receptor
(OB-R)1 in the hypothalamus, a region of
the brain implicated in the control of body weight (2-4). In mice,
mutations in the genes encoding either OB-R (db) or leptin
(ob) result in profound early-onset obesity (5, 6). Multiple
splice variants of OB-R mRNAs encoding proteins with different
length intracellular domains have been detected (7, 8). The mutant
allele (db) of the OB-R gene was shown to encode a receptor
with a truncated cytoplasmic domain (7, 8), and more recent data
suggest this receptor is signaling inactive (9). Thus, mounting
evidence suggests the ability of leptin to regulate body weight is
facilitated by downstream signaling events initiated by ligand-induced
OB-R activation.
Sequence homology and more recent functional data suggest OB-R is a
member of the class I cytokine receptor superfamily (4, 10, 11).
Receptors of this class lack intrinsic tyrosine kinase activity and are
activated by ligand-induced receptor homo- or hetero-dimerization and
in many cases require activation of receptor-associated kinases of the
Janus family (JAKs) (12). JAKs associate with the membrane-proximal
domain of the intracellular part of the cytokine receptors and serve to
initiate signal transduction pathways following ligand-induced receptor
activation. Included among the downstream targets of the JAK proteins
are members of the STAT ( Previously, we have shown that ligand-induced activation of OB-R
appears independent of the signal transducing subunit of IL-6 type
cytokine receptor accessory chain gp130 and results in activation of
members of the STAT family. Specifically, OB-R was found to activate
the DNA binding activity of STAT1, STAT3, and STAT5B and to stimulate
transcription of IL-6- and hematopoietin receptor-responsive gene
elements in hepatoma cells (9). These studies also indicated that STAT3
activation, but not STAT5B, was highly dependent on the presence of the
box 3 motif (YXXQ) of OB-R (amino acids (aa) 1141-1144).
This finding is consistent with previous observations that cytokine
receptor-mediated activation of STAT3 requires a functional box 3 motif
in the receptor intracellular domain (13-15).
In the present study, we define two distinct intracellular regions of
OB-R important for induction of gene expression. In addition, we find
that G-CSFR/OB-R and OB-R/G-CSFR chimeras can stimulate transcription
following ligand-induced receptor activation. These results indicate
that 1) aggregation of two OB-R intracellular domains is sufficient to
trigger downstream signaling events, and 2) leptin can homo-dimerize
OB-R extracellular domains. These combined data suggest that OB-R
signaling does not require participation of an accessory receptor
subunit.
MATERIALS AND METHODS COS-1, COS-7, and H-35 cells were cultured as
described previously (16). Cells were mock stimulated in medium
containing 0.5% fetal calf serum and 1 µM dexamethasone
or treated in the same medium supplemented with 100 ng/ml human leptin
(Roche), IL-6 (Genetics Institute), or G-CSF (Immunex Corp.).
The
expression vectors for the long form of human OB-R (4), full-length
G-CSFR or truncated G-CSFR( COS-1 and H-35 cells were
transfected by the DEAE-dextran method (20) and COS-7 cells by the
lipofectamine method (4). For analysis of STAT protein activation, COS
cells were maintained for 16 h in serum-free medium, followed by
treatment of cells with 100 ng/ml leptin or G-CSF for 15 min.
For CAT assays, transfected cell cultures were subdivided and treated
with ligands for 24 h. CAT reporter activities were determined and
are expressed relative to values obtained for untreated control
cultures for each experimental series. All experiments were performed a
minimum of three times. Mean ± S.D. values are shown in Figs.
1B, 3B, 4C, and 5,
A-E.
DNA binding by STAT proteins was analyzed by electromobility shift
assay (EMSA) using whole cell extracts as described previously (9).
Radiolabeled double-stranded oligonucleotides SIEm67 (for STAT1 and
STAT3) and TB-2 (for STAT5B) served as binding substrates in the EMSA.
Receptor expression in COS cells was analyzed by quantitative cell
surface binding of AP-OB fusion protein as described previously
(21).
All immunoblotting was done as described
previously (9), and immunoreactive proteins were visualized by enhanced
chemiluminescence detection as described by the manufacturer (Amersham
Corp.). Rabbit polyclonal antiserum specific for STAT5B was from Santa
Cruz Biotechnology. Goat polyclonal antiserum against bacterially
expressed extracellular domain of human G-CSFR was prepared at Roswell
Park Cancer Institute Springville Laboratories.
RESULTS To define
regions of the OB-R intracellular domain required for signaling, a
series of C-terminal deletion mutants were constructed (Fig.
1A). These constructs were transiently
co-transfected into H-35 cells with either IL-6RE- or HRRE-CAT reporter
constructs and assayed for their ability to stimulate transcription
(Fig. 1B). C-terminal truncations that remove the consensus
box 3 motif (aa 1141-1144) of OB-R abolish transcriptional activation
via IL-6-RE (Fig. 1B, upper panel). This result is
consistent with previous observations that a Tyr to Phe mutation in the
single box 3 motif of OB-R completely disrupts signaling in H-35 cells via IL-6RE (9). In contrast, OB-R signaling through HRRE was minimally
reduced by removal of extreme C-terminal sequences and was not
completely disrupted until removal of aa between 868 and 965 (Fig.
1B, lower panel).
To ensure that the expression vectors for the various OB-R mutants
directed the synthesis of surface localized receptor proteins, COS
cells transfected with each construct were assayed for receptor expression by AP-OB binding studies. C-terminal truncations of OB-R
generate proteins that are expressed at the surface and bind ligand
(Fig. 2A). Moreover, we observed that the
expression level increased with progressive truncation of the
intracellular domain.
We have shown previously that OB-R gene induction via IL-6RE correlates
with activation of STAT1 and STAT3, whereas OB-R gene induction via
HRRE was found to correlate with activation of STAT5B (9). To further
evaluate the correlation between HRRE stimulation and STAT5B
activation, COS cells were co-transfected with expression plasmids for
STAT5B and the OB-R deletion mutants. Immunoblotting was performed on
extracts prepared from these cells to ensure that STAT5B was expressed
at relatively equal amounts in each of the transfected cultures (Fig.
2B). Cells were treated with leptin, and EMSA analysis was
performed. Progressive C-terminal truncations of OB-R result in a
reduced ability to activate STAT5B (Fig. 2B), and detectable
STAT5B activation was lost only with removal of the membrane proximal
OB-R segment (construct pOBR To define the relative contribution of the conserved intracellular
domain tyrosine residues and of the membrane proximal box 1 motif to
signaling by OB-R via HRRE, we generated mutants OB-RY1141F, OB-RY986F,
OB-RY1079F, and OB-R(box1mt) (Fig. 3A)
(previously we have demonstrated that OB-R tyrosine 1141 of the box 3 element minimally contributed to signaling by OB-R via HRRE (9)). When analyzed in COS cells, AP-OB binding studies demonstrate that these
mutants are expressed at the cell surface approximately as well as
wild-type OB-R (data not shown). When transfected into H-35 cells,
OB-RY986F and OB-RY1079F were unchanged in their ability to regulate
HRRE (Fig. 3B). In contrast, mutation of the OB-R box 1 motif results in a complete loss of regulation of gene induction through this element. Thus, the box 1 motif of OB-R appears to be an
important determining factor for the ability of OB-R to activate
pathways that can modulate gene induction via HRRE.
The primary structure of OB-R suggests that it is
closely related to the signaling subunits of the class I cytokine
receptors. Members of this group can be activated by either
heterodimerization or homodimerization (10, 11). Included among the
former are the receptors for IL-6, leukemia inhibitory factor,
oncostatin M, IL-11, and ciliary neurotrophic factor, all of which
share the common signal transducer, gp130 (10, 22). However, previously we have found that OB-R appears to signal independently of gp130 (9).
Therefore, OB-R may function in the presence of another accessory chain
such as the common signaling subunit utilized by receptors for either
IL-3, granulocyte macrophage-colony stimulating factor (GM-CSF), and
IL-5 (IL-3R To analyze whether the G-CSFR/OB-R chimeric receptor could propagate a
ligand-induced signal comparable with that for wild-type OB-R, the
chimera was tested for STAT activation (Fig. 4B) and for
transcriptional stimulation (Fig. 4C). Co-transfection of G-CSFR/OB-R with STAT proteins yielded a G-CSF-induced activation of
STAT1, STAT3, and STAT5B. This result is similar to the STAT protein
activation induced by OB in OB-R transfected cells (9). Expression of
the chimeric receptor was confirmed by immunoblot analysis of cultures
transfected with G-CSFR/OB-R (Fig. 4B). These results
suggest that G-CSF-mediated dimerization of OB-R cytoplasmic domains
can generate an OB-R type activation of STAT proteins. We also
determined whether the G-CSFR/OB-R chimera could stimulate transcription as detected by measurement of gene induction in H-35
cells following receptor co-transfection with the IL-6RE and HRRE
reporter constructs (Fig. 4C). We found that the chimera was
able to stimulate transcription via these response elements and that
the response elicited was similar to an induction of the reporter gene
constructs by either OB-R or endogenous IL-6R.
The above results indicate that homodimerization of two OB-R
cytoplasmic domains can initiate signaling by OB-R, similar to the
mechanism mediating signaling by wild-type G-CSFR. However, the
G-CSFR/OB-R chimera could not definitively prove that leptin has the
capability to dimerize OB-R extracellular domains. Consequently, we
analyzed signaling activity by the reciprocal chimera containing the
OB-R extracellular domain joined to the G-CSFR intracellular domain
(Fig. 4A). Indeed, the OB-R/G-CSFR chimera could mediate gene induction comparable with that by wild-type OB-R, G-CSFR/OB-R, and
wild-type G-CSFR (Fig. 4C). Thus, taken together, these
results suggest that OB-R does not require an accessory chain for
signaling and that aggregation of two OB-R intracellular domains
appears sufficient for receptor activation.
The results
presented in the preceding section demonstrate that aggregation of two
OB-R intracellular domains is sufficient to generate a signal following
ligand-induced activation and suggests that OB-R may function by
receptor homodimerization. Consequently, we predicted that signaling by
OB-R could be "poisoned" by overexpression of a homodimerizing
partner that is signaling deficient, similar to what has been shown for
members of the receptor tyrosine kinase family (25-28). As described
above, OB-R containing only the membrane proximal 6 aa of the
cytoplasmic domain is signaling defective (Fig. 1B).
Consequently, experiments were performed to determine whether
expression of a truncated, signaling deficient OB-R could disrupt
signaling by full-length OB-R. Cells were co-transfected with
increasing amounts of truncated receptor OB-R Our interpretation of the above experiments assumes that the amount of
transfected input DNA correlates with the amount of cell surface
receptor expression. However, we had previously observed that OB-R cell
surface expression levels increased with progressive intracellular
domain truncation (Fig. 2). Consequently, an experiment was performed
to assess cell surface expression levels of full-length OB-R and
OB-R One potential explanation for the weak dominant negative repression of
OB-R is that interaction of two OB-R molecules may require functional
domains residing in the intracellular region of the receptor. To
address this possibility, we assessed the dominant negative repression
of OB-R by a mutant receptor rendered signaling defective by a single
aa substitution (Y1141F) in the OB-R box 3 motif. As described
previously, this mutation completely abolished the ability of OB-R to
modulate gene induction via IL-6RE in H-35 cells (9). Consequently, we
analyzed OB-R(Y1141F) for its ability to inhibit wild-type OB-R
signaling via this enhancer element. Similar to our observations when
experiments were performed with OB-R DISCUSSION In this report we have mutationally separated two distinct
signaling activities of the OB-R intracellular domain. Previously we
have shown that OB-R can induce gene induction in hepatoma cells
through IL-6RE and HRRE. Here we find that gene induction by OB-R
through IL-6RE requires sequences near the extreme C terminus of OB-R
(Fig. 1B). In contrast, OB-R gene induction through HRRE does not appear to require these C-terminal sequences. Moreover, gene
induction via this element is only minimally affected by removal of
OB-R intracellular domain sequences between amino acids 965 and 1165 but is dependent upon membrane proximal sequences between amino acids
868 and 965. Consequently, the proposed box 2 motif of OB-R (8) (human
OB-R aa 1066-1075) does not appear to contribute to gene induction
through HRRE. EMSA analysis suggests gene induction of HRRE correlates
with the ability of OB-R to activate STAT5B. Interestingly, OB-R Previously, we have reported that OB-R can signal in hepatoma cells in
the presence of neutralizing antibodies to the gp130 signal transducing
component of the IL-6-type cytokine receptors (9). Moreover, these
hepatoma cells do not express the other characterized cytokine receptor
accessory chains IL-2R As would be predicted for receptors that are activated by simple
homodimerization, signaling by full-length G-CSFR and the G-CSFR/OB-R
chimera can be greatly diminished by co-expression of a signaling
deficient homodimerizing partner (Fig. 5). However, OB-R We have previously speculated that the short forms of OB-R serve a
transport or clearance function in the body (4, 41). However, our
observations that the short forms of OB-R can modulate the long form of
OB-R raises the intriguing possibility that in vivo the
short form of OB-R can regulate activities of the long form.
Interestingly, we have found that the major naturally occurring non-signaling short form of OB-R in the mouse (containing a 34-aa intracellular domain), which also corresponds to the mutant OB-R found
in the db/db mouse, can similarly modulate long
form receptor signaling (data not shown). Experiments are
presently underway to identify tissues in which the long and
short forms of OB-R are co-expressed.
In conclusion, we have further defined the mechanism of
ligand-induced OB-R triggering and regions of the OB-R
cytoplasmic domain required for activation of STAT signal
transduction pathways. We believe a detailed knowledge of the pathways
regulated by OB-R will prove invaluable for understanding homeostatic
mechanisms governing normal body weight regulation. Identifying the
important target genes whose transcription is differentially regulated
by these pathways is the exciting challenge ahead.
FOOTNOTES Acknowledgments We thank Immunex Research Corp. for G-CSF and
the expression vector for G-CSFR; Genetics Institute for IL-6; Drs. J. Ripperger and G. H. Fey (Friedrich Alexander University, Erlangen,
Germany) for STAT1, -3, and -5B cDNA; and Dr. D. W. Tweardy
(Pittsburgh Cancer Institute) for the bacterially expressed human
G-CSFR extracellular domain.
REFERENCES
Volume 272, Number 7,
Issue of February 14, 1997
pp. 4065-4071
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
CYTOPLASMIC DOMAIN MUTATIONAL ANALYSIS AND EVIDENCE FOR
RECEPTOR HOMO-OLIGOMERIZATION*
,
and
Millennium Pharmaceuticals,
Cambridge, Massachusetts 02215-2406, § Roswell Park Cancer
Institute, Department of Molecular and Cellular Biology,
Buffalo, New York 14263, and ¶ Roche Research Gent,
Jozef Plateaustraat 22, B-9000 Gent, Belgium
ignal 
ransducers and
ctivators of ranscription) family of transcription factors (12). The STATs are DNA binding transcription factors that contain Src homology (SH2) domains that interact with
receptor molecules through phosphorylated tyrosine residues. STAT
proteins are activated by tyrosine phosphorylation, form hetero- or
homodimers, translocate to the nucleus, and modulate transcription of
target genes.
cyto) (17), and rat STAT1, STAT3, and
STAT5B have been described previously (13, 18). pOB-R1115,
pOB-R1065, and pOB-R965, encoding carboxyl-terminal truncated
human OB-R, were generated by PCR. Briefly, oligonucleotides spanning
the intracellular domain of human OB-R were used to generate in-frame
stop codons 3
to the specified amino acids. The PCR fragments were
digested with EcoRV and XbaI and subcloned into human OB-R that had been digested with EcoRV and
XbaI. A similar strategy was used to generate pOB-R868
but with primers generating an MscI-XbaI fragment
that replaced endogenous human OB-R sequences. pOB-RY1141F, encoding
human OB-R with a mutated box 3 sequence, has been described previously
(9). OB-R mutants pOB-R(box1mt), containing PNP to SNS changes in the
OB-R box 1 motif (aa 876 and 878), and mutants pOB-RY986F and
pOB-RY1079F were generated by overlap extension PCR using synthetic
oligonucleotides encoding the specified aa substitutions (19). The
G-CSFR/OB-R chimeric receptor was generated by PCR and encodes the
extracellular domain of human G-CSFR (aa 1-598) joined to the
transmembrane and intracellular domain of human OB-R (aa 829-1165).
The OB-R/G-CSFR chimeric receptor was generated by PCR and encodes the
mouse OB-R extracellular domain and transmembrane sequences (aa 1-860)
joined to the intracellular domain of the human G-CSFR (aa 631-813).
The CAT reporter gene constructs, pHRRE-CAT and pIL-6-CAT, have been
described previously (13, 15).
Fig. 1.
Regulation of gene induction by C-terminal
truncated OB-R proteins in H-35 cells. A, structures of
C-terminally truncated OB-R proteins. The names and predicted length of
the OB-R mutants are shown at the top. The OB-R
extracellular domain is shown as shaded, the transmembrane
region as black, and the cytoplasmic domain as
white. The locations of tyrosine residues in the cytoplasmic domain of OB-R are indicated; those conserved between the mouse and
human receptors are in boldface. The length of the
cytoplasmic domains (in aa) are listed at the bottom of the
structures. B, regulation of IL-6RE-CAT and HRRE-CAT by OB-R
mutants. H-35 cells were transfected with cDNAs encoding the
indicated OB-R forms and either Il-6RE-(upper panel) or
HRRE-CAT (lower panel). Subcultures of cells were treated
for 24 h with serum-free medium alone (
) or containing mouse
leptin (+). CAT activity was determined and is expressed relative to
values obtained for untreated control cultures. Autoradiograms show the
thin layer patterns of the CAT assays from one experimental series. The
bar graphs summarize the data of three separate transfection
experiments (an exception is OB-R.1065 that has been analyzed only
once).
[View Larger Version of this Image (26K GIF file)]
Fig. 3.
Box 1 is required for gene induction through
HRRE. A, structures of OB-R mutants OB-RY1079F, OB-RY986F,
and OB-R(box1mt). Locations of tyrosine residues 986 and 1079 and the
box 1 sequence are indicated. B, H-35 cells were
co-transfected with HRRE-CAT and expression plasmids for either
OB-RY986F, OB-RY1079F, or OB-R(box1mt). Subcultures of cells were
treated for 24 h with serum-free medium containing human leptin.
CAT activity was determined and is expressed relative to values
obtained for untreated control cultures.
[View Larger Version of this Image (25K GIF file)]
Fig. 4.
Structure and signaling activities of
receptor chimeras. A, structures of wild-type OB-R
(open) and G-CSFR (closed) or OB-R/G-CSFR and
G-CSFR/OB-R chimeras. Identities of the receptor molecules are shown at
the top. The extracellular and intracellular domains and the
major structural elements are compared. The location of the predicted
Box 1, Box 2, and Box 3 motifs in the cytoplasmic domains of OB-R and G-CSFR are shown. B, STAT protein
activation by the G-CSFR/OB-R chimera. COS-1 cells were co-transfected
with expression plasmids for the G-CSFR/OB-R chimera and either STAT1, STAT3, or STAT5B, and 24 h later cells were incubated for 16 h in serum-free media followed by stimulation with G-CSF for 15 min.
STAT protein DNA binding activity was determined by EMSA (left
panel) and expression of the G-CSFR/OB-R chimera was quantitated by immunoblotting (right panel). C, regulation of
gene induction by receptor chimeras. H-35 cells were co-transfected
with expression plasmids for the indicated receptors (IL-6R refers to
endogenous receptor) and the IL-6RE-(left panel) or HRRE-CAT
(right panel) reporter constructs. Cells were stimulated
with the appropriate ligands, and CAT activity was determined as
described in Fig. 1. All values are expressed relative to untreated
control cultures.
[View Larger Version of this Image (29K GIF file)]
Fig. 5.
Dominant negative repression of OB-R
signaling. H-35 cells were co-transfected HRRE-CAT (A)
or IL-6RE-CAT (B-E) reporter constructs and the
listed amounts of OB-R and OB-R868 (A), OB-R/G-CSFR and
OB-R868 (B), G-CSFR and G-CSFR(cyto) (C),
G-CSFR/OB-R and G-CSFR(cyto) (D), and OB-R and OB-RY1141F
(E). Cells were stimulated with the appropriate ligands, and
CAT activity was determined as described in Fig. 1. All values are
expressed relative to untreated cultures in each experimental series.
F, ligand binding activities of co-expressed OB-R and
OB-R868. COS-7 cells were transfected or co-transfected with the
indicated quantities of OB-R constructs, and 48 h later cells were
incubated with 1 nM mouse AP-OB fusion protein. Columns
show the average of two binding determinations, and bars
reflect the differences between the two.
[View Larger Version of this Image (30K GIF file)]
Fig. 2.
Quantitation of receptor expression level and
STAT protein activation by OB-R mutants. A, ligand binding
activity of mutant OB-R proteins in COS cells. COS-7 cells were
transfected with the indicated OB-R constructs, and 48 h later
cells were incubated with 1 nM mouse AP-OB fusion protein.
Columns show the average of two binding determinations and
bars reflect the difference between the two. B,
STAT5B activation by OB-R mutants. COS-1 cells were transfected with
cDNAs encoding the indicated OB-R mutants and expression plasmid
for STAT5B. 24 h post-transfection, cells were incubated for
16 h in serum-free medium, treated for 15 min with leptin, and
extracts were prepared. DNA binding activity of the STAT proteins was
analyzed by EMSA (upper panel), and STAT protein levels were
quantitated by Western blotting (lower panel).
[View Larger Version of this Image (42K GIF file)]
868). Thus, there appears to be a
correlation between loss of OB-R STAT5B activation and gene induction
via HRRE.
), or IL-2, IL-4, IL-7, and IL-9 (IL-2R
). However,
OB-R signals in hepatoma cells, which do not express either IL-3R or
IL-2R (14, 15). Alternatively, OB-R may be activated by
homodimerization as is found for the granulocyte-colony stimulating
factor receptor (G-CSFR) (23, 24). Therefore, to determine whether OB-R
has the ability to dimerize and signal as a homodimer, we constructed
chimeric receptors containing the extracellular domain of G-CSFR joined
to the intracellular domain of OB-R or the reciprocal receptor having
the extracellular domain of OB-R joined to the intracellular domain of
G-CSFR (Fig. 4A).
868 relative to
full-length OB-R, and the ability of these complexes to stimulate expression of a reporter gene construct was assayed. Co-transfection of
increasing amounts of truncated OB-R does result in decreased signaling
by wild-type receptor (Fig. 5A). However,
even at a large excess of truncated to full-length receptor, the
signaling repression observed did not approach the degree of reduction
observed for repression of G-CSFR signaling by overexpressed and
signaling-deficient truncated G-CSFR(cyto) (Fig. 5, compare
A and C). Moreover, we find that the differing
sensitivity to dominant negative repression observed for OB-R and
G-CSFR was a property of their extracellular domains as shown by
dominant negative studies with the receptor chimeras (Fig. 5,
B and D).
868 in these co-transfection experiments. Briefly, COS cells
were co-transfected with cDNAs encoding full-length OB-R and
OB-R868 at ratios identical to that described for Fig. 5A. Transfected cells were then analyzed for cell surface
leptin binding activity by standard AP-OB binding analysis. Cells
transfected with DNA encoding only full-length OB-R exhibit a small
increase in binding activity relative to mock transfected cells (Fig.
5F). However, co-transfection of the same amount of pOB-R
and an equal amount of pOB-R868 results in greatly increased binding
activity and suggests that OB-R868 is expressed at the cell surface
approximately 6-7-fold more efficiently than full-length OB-R.
Moreover, increased pOB-R868 input further enhances AP-OB binding
activity of the transfected cells. These data are consistent with our
prediction that high level expression of signaling defective OB-R
results in only moderate dominant negative repression of wild-type
OB-R.
868, increasing the ratio of
transfected mutant OB-RY1141F to wild-type receptor did not strongly
repress signaling (Fig. 5E). Thus, the OB-R box 3 mutant and
OB-R868 behave similarly in their ability to trans-repress signaling
by wild-type OB-R. Interestingly, low level expression of either
truncated or box 3 mutant OB-R receptor generates a slight enhancement
of signaling by wild-type OB-R. Moreover, a similar pattern was also
observed for OB-R/G-CSFR signaling in the presence of increasing
amounts of truncated OB-R868 (Fig. 5, A, B, and
E).
965,
which has been deleted of all intracellular domain tyrosine residues
and therefore all potential SH2 docking sites, is still capable of low
level STAT5B activation and transcriptional stimulation through HRRE.
Only when membrane proximal sequences of OB-R are removed (OB-R868) are both HRRE gene induction and STAT5B activation completely abolished. Consistent with this, OB-R(box1mt), containing a mutated box
1 motif, is similarly unable to induce gene induction through HRRE and
would be predicted to be unable to activate STAT5B.
or IL-3R (14, 15). Consequently, it is
possible that OB-R may function by a mechanism involving receptor
homodimerization. Among members of the class I cytokine receptor
family, signaling by the G-CSFR is predicted to be initiated by
ligand-induced receptor homodimerization (23, 24). Since chimeric
receptor complexes have proven quite productive for the analysis of the
mechanism of cytokine receptor activation (15, 29, 30), we constructed
OB-R/G-CSFR and G-CSFR/OB-R chimeras as a means to analyze the
mechanism of OB-R signaling. We find that the G-CSFR/OB-R chimera can
strongly activate transcription of both the IL-6RE- and HRRE-CAT
reporter constructs (Fig. 4C). Since G-CSFR is thought to
form a homodimer when G-CSF is bound, our result implies that the
aggregation of two intracellular OB-R domains is sufficient to initiate
receptor signaling. In a similar manner, the OB-R/G-CSFR chimera also
mediates transcriptional activation through IL-6RE and HRRE (Fig.
4C). These results show that leptin binding can dimerize two
OB-R extracellular chains thus inducing the association of at least two
intracellular G-CSFR domains and activation of the receptor complex.
Moreover, our results using the receptor chimeras suggest that it may
be possible to generate small molecules or antibodies that act as
OB-R agonists through simple cross-linking of two OB-R chains.
868 was
unable to as efficiently repress signaling by full-length OB-R or the
OB-R/G-CSFR chimera. It is therefore possible that leptin binding to
cell surface receptors can result in higher order oligomerization
(receptor number>2/complex) as has been shown for IL-10 receptor
complexes (31) and for members of the activin/transforming growth
factor-R family (32-36). According to this model, ligand binding by
full-length OB-R or OB-R/G-CSFR chimera can lead to aggregation of more
than two receptor chains, yet juxtaposition of only two intracellular
domains is sufficient for signal generation. Such complexes would be
predicted to be highly resistant to dominant negative repression. The
strong repression of signaling by G-CSFR(cyto) in complexes
containing the G-CSFR/OB-R chimera demonstrates that OB-R intracellular
domain can be efficiently repressed when placed in the context of a
simple homodimer structure (Fig. 5). Although it is possible that
OB-R868 localizes to a different region of the membrane than
wild-type OB-R, we believe it is unlikely that mutation of a single
tyrosine residue of the OB-R intracellular domain (Y1114F) would result
in altered receptor membrane localization. Thus, our observation of
similar repression effects mediated by either OB-R868 or OB-RY1141F
suggests our results are not due to altered membrane localization.
However, it is still possible that post-translational mechanisms exist within the cell that do not permit efficient association of wild-type and mutant OB-R chains. In contrast, G-CSFR, which appears highly sensitive to dominant negative repression, is apparently not subject to
this mechanism. As previously noted, low expression levels of either
OB-R868 and OB-RY1141F generate a small enhancement of signaling for
full-length OB-R and the OB-R/G-CSFR chimera. We speculate that this
effect is attributable to either ligand presentation (37-39) or ligand
passing as has previously been observed for the tumor necrosis factor
receptor (40).
To whom correspondence should be addressed: Roswell Park
Cancer Institute, Dept. of Molecular and Cellular Biology, Elm and Carlton Sts., Buffalo, NY 14263. Tel.: 1-716-845-4587; Fax:
1-716-845-8389.
1
The abbreviations used are: -R, receptor; JAK,
Janus kinase; STAT, signal transducer and activator of transcription;
SH-2, Src homology domain; IL-, interleukin; HRRE, hematopoietin
receptor response element; G-CSF, granulocyte-colony stimulatory
factor; PCR, polymerase chain reaction; CAT, chloramphenicol
acetyltransferase; aa, amino acid; EMSA, electrophoretic mobility shift
assay.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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