| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ARTICLES |
Pro Extracellular Domain Mutation of the Fatty Rat1
Department of Medicine, Division of Endocrinology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Jeffrey S. Flier, M.D., Division of Endocrinology, Center, RN, 99 Brookline Avenue, Boston, Massachusetts 02215. E-mail: jflier{at}bidmc.harvard.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
pro) in the
extracellular domain of the leptin receptor. We have characterized the
effects of this mutation on the two major isoforms of the leptin
receptor, Ob-Rb and Ob-Ra, by studying cell-surface expression, leptin
binding affinity, signaling capacity, and receptor-mediated
internalization and degradation of leptin in transfected mammalian cell
lines. Both Ob-Rb269 glnpro and
Ob-Ra269 glnpro have decreased cell-surface expression
and decreased leptin binding affinity. Ob-Rb269 glnpro
was shown to have defective signaling to the JAK-STAT pathway and
markedly diminished ability to activate transcription of the
egr-1 promoter. Constitutive ligand-independent
activation of Ob-Rb269 glnpro was observed for
activation of egr-1-luc but only under conditions when
JAK2 was coexpressed with Ob-Rb269 glnpro. Finally,
Ob-Ra269 glnpro has an increased ability to internalize
leptin but is less efficient at degrading leptin, as compared with
Ob-Ra. In conclusion, both Ob-Ra269 glnpro and
Ob-Rb269 glnpro have multiple functional defects. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Obese rodents have served as valuable models for the study of obesity, diabetes, and hypertension. The Zucker fatty rat (fa/fa) was first described in 1961 as a spontaneous mutation of Merck Stock M and Sherman rats (22, 23). These rats develop progressive obesity with hyperphagia noted post weaning (24). Metabolically, they have severe insulin resistance, with hyperinsulinemia, hyperglycemia, hyperlipidemia, and hypercortisolemia (25). Further, fa/fa rats have elevated serum leptin levels, compared with lean rats (25), suggesting that they are leptin resistant.
The cause of leptin resistance in fa/fa rats became clarified when the fa gene was mapped to a region syntenic to the db gene (26), which had already been shown to encode a mutant form of the leptin receptor (16, 27). Further studies identified the fa mutation as a single nucleotide substitution that resulted in an amino acid change from glutamine to proline at codon 269 in the extracellular domain of the leptin receptor (28, 29, 30). The Koletsky rat was also shown to have a mutation in the leptin receptor gene. In this strain of rats, a nonsense mutation truncates the receptor in the extracellular domain, creating what is predicted to be a null mutant for all leptin receptor isoforms (31).
Studies with the fa mutant long form of the receptor
(Ob-Rb269 glnpro) have begun to provide insights into
the behavior of this mutant receptor and have begun to explain the
mechanism for leptin resistance in Zucker rats. Both wild-type Ob-Rb
and Ob-Rb269 glnpro are reported to have similar levels
of mRNA expression in rat brain, as shown by RT-PCR analysis (32).
Specific functional leptin binding sites have been demonstrated and
found to be similar, using radiolabeled leptin and an alkaline
phosphatase leptin fusion protein, in choroid plexus of lean and obese
Zucker rat brain (33). Cell-surface expression and binding of the
mutant receptor have been studied using transient transfection in COS
cells and an alkaline phosphatase leptin fusion protein (30) or
iodinated leptin (34, 35) as ligand. These studies suggested that
cell-surface expression of Ob-Rb269 glnpro is reduced,
compared with wild-type, with estimates varying between 2- to 3-fold
and 6- to 10-fold (30, 34, 35). The leptin binding affinity of
Ob-Rb269 glnpro has been described as both normal (30, 34) or reduced (36). Cell-surface expression of
Ob-Ra269 glnpro has been reported to be approximately
6- to 8-fold lower than wild-type Ob-Ra (35); however, there is no
reported data on the binding affinity of
Ob-Ra269 glnpro.
Wild-type Ob-Rb has been shown to possess a number of signaling
capabilities. These include activation of the JAK-STAT (17, 37, 38, 39, 40, 41) and
MAPK pathways (42), stimulation of tyrosine phosphorylation of IRS-1
(42), and increased transcription of fos and jun
(42, 43). Wild-type Ob-Ra was initially thought to be completely
incapable of signaling (17, 40). Although lacking the ability to
activate STAT signaling, Ob-Ra has recently been shown to be capable of
increasing transcription of early response genes fos and
jun in Chinese hamster ovary (CHO) cells stably
expressing Ob-Ra (43), and activation of JAK kinases in transient
transfection models (42). In limited studies, the signaling capability
of Ob-Rb269 glnpro has been shown to be reduced,
when compared with Ob-Rb (34, 36). However, whether this difference is
entirely explained by decreased cell-surface expression is not
resolved. Recently, reduced signaling by
Ob-Rb269 glnpro was suggested to be caused, in part, by
a capacity of the fa receptor to mediate constitutive
ligand-independent activation of signaling (35).
Although the greatest attention has focused on the the long
leptin receptor isoform, Ob-Rb, the short form, Ob-Ra, may also play a
key role in leptin biology. Ob-Ra may transport leptin into the central
nervous system (CNS) via a saturable transport system located in the
choroid plexus (44) and/or in brain capillary microvessels. The
presence of acid-resistant binding of iodinated leptin to isolated
human brain capillary microvessels has been reported (45) and is
consistent with the possibility that one or more leptin receptor
isoforms are capable of internalizing leptin, but kinetic studies with
specific leptin receptor species have not yet been reported. Further,
the extent to which receptor-mediated transport of leptin may influence
levels of cerebrospinal fluid (CSF) leptin has not been determined.
Serum leptin levels are high in Zucker rats, compared with control
rats, but CSF levels in controls and mutant rats are not statistically
different (46). This implies that efficient brain uptake of leptin
requires one or more leptin receptor species but that alternative
transport mechanisms must also exist. The capacities of both the
wild-type and Ob-Ra269 glnpro to mediate leptin uptake
and transport into the brain therefore require investigation.
To further investigate the mechanism by which the fa
mutation causes leptin resistance of the Zucker rat, we carried out a
systematic comparison of wild-type leptin receptor and
Ob-Rb269 glnpro, with respect to receptor cell-surface
expression, affinity of binding, and signaling potential. Both
Ob-Rb269 glnpro and
Ob-Ra269 glnpro bind leptin with reduced affinity,
compared with wild-type receptors. There is also an approximately
4-fold reduction in cell-surface expression for Ob-Rb269
glnpro and a >2.5-fold reduction for
Ob-Ra269 glnpro, compared with their wild-type
counterparts. Under conditions of similar cell-surface expression,
Ob-Rb269 glnpro had reduced capacity to signal in the
JAK-STAT pathway and negligible capacity to activate
egr-1 gene transcription. The fa
mutation also affects the capacity of Ob-Ra to internalize and degrade
leptin.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cloning of leptin receptor cDNAs
The mouse leptin receptor short form (Ob-Ra) and long form
(Ob-Rb) cDNAs were generated by RT-PCR from mouse brain total RNA
(isolated from C57BL mice) and cloned into pcNA3.1Zeo(-) (Invitrogen,
Carlsbad, CA), as previously described by Bjørbæk et al.
(42). The fa mutant of both Ob-Rb and Ob-Ra (269 glnpro)
was generated using the site-directed mutagenesis kit from CLONTECH.
The region of the point mutation was sequenced using standard
double-stranded plasmid techniques.
Cell culture and transient transfection
CHO cells were grown in F-12 nutrient mixture HAM (F-12)
supplemented with 10% FCS, 100 U/ml penicillin, and 10 µg/ml
streptomycin at 37 C in 5% CO2. HEK 293 cells were grown
in DMEM (high glucose), and as CHO cells, except that the plates were
coated with 0.1% gelatin. COS-1 cells were grown in DMEM (low
glucose), and as CHO cells. For the leptin binding assays, cells were
grown in 24-well plates and transfected using 2 µl Lipofectamine and
DNA amounts as per the manufacturer’s protocol. For the luciferase and
ß-galactosidase assays, cells were grown in 6-well plates and
transfected using 10 µl Lipofectamine and 1.0 µg plasmid DNA. In
all experiments including JAK2 cDNA, the amounts of transfected JAK2
were 1/14 of the total amount of DNA transfected. For Western blotting,
cells were grown in 10-cm dishes and transfected using 80 µl
Lipofectamine. All cells were serum starved for 12–15 h before
stimulation with hormones. Cells were harvested 48 h post
transfection for the luciferase and ß-galactosidase assays and lysed
in 500 µl lysis buffer A [25 mM glycylgycine, 15
mM MgSO4, 4 mM EGTA with 1% Triton
X-100 and 2 mM dithiothreitol (DTT)]. For Western
blotting experiments, cells were harvested 48 h post transfection
by aspirating the medium, rinsing in ice cold PBS, and scraping into
1000 µl ice cold lysis buffer B (1% Nonidet P-40, 0.5% Triton
X-100, 10% glycerol, 150 mM NaCl, 2 mM
Na3VO4, 20 mM NaF, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml
aprotinin, 50 mM Tris-HCl, pH 7.4). The lysate was
clarified by centrifugation at 23,000 x g for 15 min,
and the supernatant was immunoprecipitated as described below.
Initial 125I-leptin binding studies
To obtain similar levels of expression of wild-type Ob-R and
mutant Ob-R, initial binding experiments were done in 24-well plates.
125I-leptin binding (2200 Ci/mmol) to cells transfected
with 200 ng/well Ob-Rb269 glnpro plasmid DNA was
compared with the binding of 125 I-leptin to cells
transfected with varying amounts of wild-type Ob-Rb DNA. The Ob-Rb cDNA
amounts were compensated with appropriate amounts of empty vector
[pcDNA3.1/Zeo(-)], so that the total amount of transfected DNA was
the same in all wells. Subsequently, all experiments were done, using
the appropriately lowered Ob-Rb amounts (compensated with vector DNA),
which gave similar binding to Ob-Rb269 glnpro, as
determined by these initial binding assays. In CHO cells, the binding
of 125I-leptin to cells expressing
Ob-Ra269 glnpro (200 ng DNA/well in 24-well plates) was
compared with cells expressing varying cDNA amounts of wild-type Ob-Ra.
Ob-Ra cDNA amounts were compensated with appropriate amounts of empty
vector DNA. Binding of 125I-leptin was carried out as
described below. Subsequently, all experiments were done with the
lowered amounts of Ob-Ra cDNA (compensated with vector DNA), which
produced similar cell-surface binding as
Ob-Ra269 glnpro.
125I-leptin binding assay
Transfections were carried out in triplicates in 24-well tissue
culture plates, as described above. Forty-eight hours post
transfection, including 15 h of serum deprivation, cells were
incubated in 200 µl binding buffer [DMEM (293 or COS-1 cells) or
F-12 (CHO cells), 0.1% BSA, with or without 200 nM
unlabeled leptin] with 105 cpm of human
125I-leptin and incubated at 4 C or 22 C for 4 h.
Kinetic assays showed that maximal specific binding occurred after 4–6
h of incubation at 20 C (data not shown). The relative specific binding
between mutant and wild-type receptor counterparts was not
significantly different when done for 6 h at 4 C or at 20 C (data
not shown). Cells were then washed 3 times in 2 ml binding buffer,
lysed in 500 µl lysis buffer C (1% NP-40, 0.5% Triton X-100, 1 N
NaOH), and bound 125I-leptin was determined in a 
counter.
Determination of binding affinity
For determination of binding affinity (kilodaltons),
transfections were carried out, as above, with 200 ng/well Ob-Rb,
Ob-Rb269 glnpro, or
Ob-Ra269 glnpro cDNAs or 100 ng/well of Ob-Ra
expression vectors. Cells were incubated for 6 h at 20 C with
105 cpm 125I-leptin and varying concentrations
(0, 10-10–10-6 M) unlabeled
human leptin in binding buffer. Cells were then washed, and bound
radioactivity was determined by counting the cell pellet in a
counter. Scatchard analysis was then performed (49).
Immunoprecipitation and immunoblotting
Immunoprecipitations were performed as described earlier (42).
For immunoblotting, proteins were boiled for 5 min and subjected to
SDS-PAGE, followed by transfer of the resolved polypeptides to
nitrocellulose membranes. Nitrocellulose membranes were blocked with
10% nonfat dried milk in Towbin buffer [20 mM Tris-HCl
(pH 7.4), 150 mM NaCl, 0.05% Tween 20] for 2 h at 22
C and then incubated with antibodies in 5% milk at 4 C for 12–15 h.
After removal of unbound antibodies by three washes in Towbin buffer,
membranes were incubated with horseradish peroxidase-conjugated
antirabbit or antimouse Ig in 2.5% milk for 1.5 h and finally
washed in Towbin buffer. The targeted proteins were detected using
enhanced chemiluminescence, as described by the manufacturer (Amersham
International, Buckinghamshire, United Kingdom). Stripping of
nitrocellulose membranes was done as described earlier (42).
Luciferase and ß-galactosidase assays
After lysis, 50-µl aliquots were used for the assay. Briefly,
150 µl of 0.75 mM luciferin (Molecular Probes, Eugene,
OR) and 150 µl assay buffer (lysis buffer A and 15 mM
K2HPO4, 6 mM ATP, 3 mM
DTT, pH 7.6) were injected simultaneously and measured for 20
sec by a Luminometer (LB 9501, EG&G Berthhold, Bad Wildbad, Germany).
ß-galactosidase activities were determined in 20-µl samples (from a
10-fold dilution of the lysate in lysis buffer A) using Galacton
(Tropix, Inc., Bedford, MA), as described by the manufacturer, and were
measured by a Luminometer (LB 9501).
Acid resistance study
Transfections were carried out in triplicate in 24-well plates,
with 200 ng/well of Ob-Ra269 glnpro cDNA and
appropriate levels of Ob-Ra cDNA, which produced similar cell-surface
expression (as described above). Forty-eight hours post transfection,
including 15 h of serum deprivation, cells were washed and
incubated with 200 µl binding buffer (F-12 HAM media, 0.1% BSA) and
105 cpm of human 125I-leptin, with or without
unlabeled murine leptin (100 nM), at either 37 C or 4 C.
Cells were then washed with PBS and incubated with or without 500 µl
of 0.2 M acetic acid, 0.5 M NaCl (pH 2) for 6
min on ice. The acid wash was measured in a counter, as the acid
sensitive component of bound leptin. Radioactivity bound to the cells
after the two acid washes was taken as the acid resistant portion.
Total specific binding was determined in the parallel set of cells
without acid wash. Acid-resistant uptake was expressed as the
acid-resistant counts divided by the total specific counts bound
x 100.
Degradation study
Transfections were carried out in CHO cells in 6-well plates, as
described above. One µg/well Ob-Ra269 glnpro cDNA was
matched with an appropriate level of Ob-Ra cDNA, as determined by the
initial binding assay, to produce equivalent binding. Forty-eight hours
post transfection, including 15 h of serum deprivation, cells were
rinsed three times in PBS and incubated in binding buffer (F-12 HAM
media, 0.1% BSA) with 105 cpm 125I-leptin with
or without unlabeled (100 nM) leptin for 2 h at 4 C.
Cells were then washed three times in ice-cold PBS to remove unbound
leptin and were incubated in 500 µl F-12 HAM media at 37 C for
various times. The F-12 HAM media was then reserved in a separate tube
and 500 µl of a 10% trichloro acetic acid (TCA) in F-12 HAM media
was added. These samples were kept on ice for 1 h, after which
they were centrifuged at 10,000 x g for 5 min, and the
supernatant was removed and counted in a counter, as the TCA
soluble fraction. Cells were lysed in lysis buffer C and measured in a
counter, as the cell associated portion. Leptin degradation was
expressed as the (TCA-soluble counts divided by the total cell
associated radioactivity measured at time zero) x 100. To
study the effects of chloroquin or methylamine on degradation,
chloroquin or methylamine were added to the 500 µl F-12 HAM media
(final concentrations of 0.1 mM or 10 mM,
respectively) after the three washes in ice-cold PBS.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
pro, or Ob-Ra269 glnpro.
Scatchard analysis was performed to obtain dissociation constant
(Kd) values for each receptor. As shown in Table 1
, the Kd values were higher
for both mutant receptors, as compared with their wild-type
counterparts. The obtained Kd values for Ob-Rb and
Ob-Rb269 glnpro are similar to values shown by others
(36). The Kd value of the wild-type short leptin receptor
is also similar to that obtained by Tartaglia et al. (15).
The total number of 125I-leptin binding sites per well was
also lower for the short (>2.5-fold) and long (
4-fold) mutant
receptors, as compared with wild-type receptors (Table 1),
demonstrating that fewer mutant receptors are expressed on the cell
surface under conditions of transient expression in mammalian cell
lines.
|
pro is
capable of leptin-activated receptor phosphorylation via the JAK
kinases, Ob-Rb and Ob-Rb269 glnpro were expressed
transiently in COS-1 cells. Immunoblotting with antiphosphotyrosine
(pY) antiserum of Ob-R immunoprecipitates revealed ligand-dependent
specific tyrosine phosphorylation of both receptors, as shown in Fig. 1 (upper panel). Two major
tyrosine-phosphorylated protein bands with estimated molecular masses
of 190 and 230 kDa were identified. The identity of the 190-kDa protein
was verified by immunoblotting of the stripped nitrocellulose membrane
with antileptin receptor antibodies (Fig. 1, bottom panel).
The identity of the 230-kDa band is unclear, because the band was not
detected with the antireceptor antibody. It is possible, however, that
this band may correspond to a small fraction of leptin receptors that
is highly tyrosine phosphorylated but is below the detection limit by
the antireceptor antibody. Another possibility includes a yet
unidentified protein that contains leptin-dependent tyrosine
phosphorylation and is coimmunoprecipitated with the leptin receptor.
Under conditions of similar leptin-induced tyrosine phosphorylation of
wild-type and mutant receptors (Fig. 1, upper panel),
approximately 5-fold more Ob-Rb269 glnpro protein was
present in the immunoprecipitates, as determined by laser-scanning
densitometry of the autoradiogram (Fig. 1, lower panel).
This result is therefore consistent with the binding analysis from
above, also suggesting that the mutant receptor has a reduced ability
to be properly inserted or maintained at the cell surface.
|
propro, to obtain the same cell-surface
binding of tracer concentrations of 125I-leptin (data not
shown). Twenty-fold less cDNA was required for Ob-Ra vs.
Ob-Ra269 glnpro to obtain similar cell surface
expression (data not shown). Altogether, these results are also
consistent with the mutant receptors, having reduced cell-surface
expression, as compared with the wild-type counterparts.
We next studied the signaling potential of the
Ob-Rb269 glnpro receptor. To compare the extent of
leptin-induced JAK2 phosphorylation produced by Ob-Rb
vs. Ob-Rb269 glnpro under conditions of
similar cell-surface expression, 293 cells were cotransfected with JAK2
and either Ob-Rb or Ob-Rb269 glnpro cDNAs, with DNA
amounts based on the results of the above binding assays. After
stimulation with 10 nM murine leptin for varying times, the
cells were lysed and subjected to immunoprecipitation with anti-JAK2
antibodies. Western blots with anti-pY antibodies revealed a
time-dependent increase in JAK2 activation in cells expressing Ob-Rb,
with 40% of maximal activation occurring at 2 min (Fig. 2, A and C). With
Ob-Rb269 glnpro, a time-dependent JAK2 activation was
also observed; however, the maximal stimulation was much lower than the
intensity reached with the wild-type receptor. A leptin dose response
was examined at 5 min of stimulation. A dose-dependent JAK2
phosphorylation was found, with a maximal response at 10 nM
leptin, in cells expressing Ob-Rb. A dose-dependent JAK2
phosphorylation was also noted in cells expressing
Ob-Rb269 glnpro, but maximal response was much
reduced, as compared with wild-type Ob-Rb (Fig. 2, B and D). This
latter result suggests that, in addition to a reduced affinity for
leptin and a reduced expression on the cell surface, as shown above,
the mutant leptin receptor also has a defect in activation of
intracellular signaling. This signaling defect may, in fact, be even
more severe, because this experiment was carried out under conditions
of equal 125I-leptin-binding at sub-Kd
concentrations. Under such conditions, the mutant receptors are
probably expressed at slightly higher levels at the cell surface, as
compared with wild-type receptors, because of the lower affinity of the
mutant receptor.
|
pro has decreased capacity to induce
STAT3 activation in response to leptinpro to activate STAT3 with that of
wild-type receptor, COS-1 cells were cotransfected with HA-STAT3 cDNA
and either Ob-Rb or Ob-Rb269 glnpro cDNA, at amounts
that produce equal cell-surface 125I-leptin-binding. Cells
were stimulated with leptin for varying times, and lysates were
immunoprecipitated with anti-HA antibodies. Immunoblotting with
antiphosphospecific STAT3 antiserum showed a time-dependent STAT3
phosphorylation by both receptors. Near-maximal activation occurred as
early as approximately 10 min after addition of 10 nM
leptin in cells expressing Ob-Rb (Fig. 3, A and C). A similar time dependence was observed with
the mutant receptor, although the extent of STAT3 phosphorylation was
lower. A leptin dose response was examined at 15 min of stimulation. As
shown in Fig. 3, B and D, a dose-dependent STAT3 phosphorylation was
noted, with a maximal response at 10 nM, in cells
expressing Ob-Rb. A dose-dependent response was also noted in cells
expressing Ob-Rb269 glnpro; however, the maximal
response of STAT3 phosphorylation was reduced, compared with samples
from cells expressing Ob-Rb. This result is consistent with the JAK2
results from above, suggesting that Ob-Rb269 glnpro has
a signaling defect.
|
pro is unable to activate
transcription of an Egr-1 promoter luciferase constructpro to activate transcription of
egr-1, an egr-1 promoter-luciferase reporter
construct was used (48). Ob-Rb, Ob-Rb269 glnpro (using
cDNA amounts that would produce similar cell-surface expression), were
cotransfected with the egr-1-luc plasmid into CHO cells. A
time course for egr-1-luc transcription with 1
nM murine leptin revealed a maximal stimulation at 8 h
of stimulation (data not shown). Cells were then stimulated for 8
h with 1 nM murine leptin, and luciferase activities were
measured, as described in Materials and Methods. Leptin
induced a 2.8-fold activation of transcription over basal levels in
cells expressing Ob-Rb, but only a 0.4-fold activation in cells
expressing Ob-Rb269 glnpro (Fig. 4). Because the 1 nM leptin
concentration is near the receptor’s Kd value, stimulation
was also carried out at 100 nM leptin for 8 h, and
similar results were obtained: 2.0-fold activation in cells expressing
Ob-Rb, and a 0.4-fold activation in cells expressing
Ob-Rb269 glnpro (Fig. 4). A dose of 100 nM
leptin had no effect on a CMV-lacZ control construct (data
not shown) or on cells not expressing leptin receptors (data not
shown).
|
pro
using the Egr-1 promoter luciferase constructpro causes
constitutive ligand-independent activation of signaling (35). This was
shown in a variety of cell lines, including 293 cells stably expressing
Ob-Rb. Constitutive ligand-independent activation of signaling had not
been observed in our studies. Therefore, to investigate this further,
we cotransfected 293 cells with Ob-Rb or
Ob-Rb269 glnpro expression vectors at conditions that
produced similar levels of expression, together with
egr-1-luc and CMV-lacZ reporter constructs, with
or without JAK2 cDNA. When JAK2 cDNA was not cotransfected into the
cells, the basal luciferase activities were similar for cells
expressing both Ob-Rb and Ob-Rb269 glnpro, and leptin
had no effect or little effect on egr-1 promoter activities
in cells expressing either receptor (Fig. 5). When JAK2 was coexpressed together
with Ob-Rb269 glnpro, however, the basal values
increased 4-fold over the basal values for Ob-Rb. Upon stimulation with
leptin, transcriptional activation through Ob-Rb went up 3.2-fold,
whereas Ob-Rb269 glnpro only induced
egr-1-luc transcription 1.6-fold.
|
pro has altered capacity for leptin
internalizationpro to internalize leptin, we used the acid
resistance method (54). CHO cells were transiently transfected with
either Ob-Ra or Ob-Ra269 glnpro cDNA at levels that
produce similar cell-surface expression. The percent acid resistant
leptin binding for both Ob-Ra and Ob-Ra269 glnpro at 37
C and 4 C, is shown in Fig. 6.
Ob-Ra269 glnpro has a markedly increased ability to
mediate leptin internalization in CHO cells, as indicated by a greater
percent of acid resistant 125I-leptin uptake, when compared
with Ob-Ra, at 15 and 60 min. At 4 degrees, internalization is minimal,
and accordingly, there is no time-dependent increase in the percent of
acid resistance. Because this experiment was performed with continuous
presence of 125I-leptin in the medium, the increased
capability of the mutant short-form leptin receptor to internalize
leptin may, in part, result from faster recycling of this receptor on
the cell surface and/or changes in rates of degradation and de
novo synthesis. Further studies with pulse-chase
35S-methionine labeling, in combination with
internalization studies without continuous presence of
125I-leptin in the medium, will be required to specifically
address these questions.
|
pro degrades internalized leptin less
efficiently than wild-type Ob-Rapro cDNA were transiently transfected into
CHO cells, in amounts producing equal cell-surface binding at 4 C. The
appearance of TCA-soluble 125I-leptin in the medium, with
time, is shown in Fig. 7A. The TCA
soluble fraction of 125I-leptin represents the amount of
initially bound ligand that has been degraded inside the cells and is
subsequently extruded into the medium (55). Both Ob-Ra and
Ob-Ra269 glnpro degrade leptin at similar rates, with
approximately 20% of cell associated 125I-leptin being
degraded at 60 min. However, because Ob-Ra269 glnpro
internalizes more leptin over the same time course (Fig. 6
), the mutant
receptor must degrade leptin less efficiently than the wild-type
receptor. Receptor-mediated ligand degradation typically takes place in
lysosomes, and lysosomal inhibitors have been used to verify that
degradation occurs in this organelle (55). We used the lysosomal
inhibitors, chloroquin and methylamine. As shown in Fig. 7B, in cells
expressing either Ob-Ra or Ob-Ra269 glnpro, each of the
lysosomal inhibitors markedly reduced the amount of TCA-soluble
125I-leptin present in the medium (15-fold reduction
after 120 min).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
pro) in the
extracellular domain that is common to all receptor isoforms (28, 29, 30).
Although leptin-receptor mutations causing severe obesity have also
been found in humans (57), comprehensive studies in large cohorts of
humans with different racial origins have shown that mutations in the
leptin-receptor gene are not a common cause of human obesity
(58, 59, 60). However, given the enormous literature on the
pathophysiology of these rats, a detailed characterization of the
functional consequences of this mutation is necessary. In this study,
we have examined cell-surface expression, binding affinity, and
signaling capacity of Ob-Rb269 glnpro; and cell-surface
expression, binding affinity, and capacity for ligand internalization
and degradation of Ob-Ra269 glnpro.
Reduced cell-surface expression has been previously reported in
Ob-Rb269 glnpro (30, 34, 35) and
Ob-Ra269 glnpro (35), leading to the conclusion, which
our results support, that reduced cell-surface expression is a major
mechanism for reduced signaling and function by these receptors
in vivo. A second abnormality in these mutant receptors is a
reduced affinity of leptin binding, which has been observed in some
(but not all) prior studies with the Ob-Rb isoform (30, 34, 36). Here,
we provide the first data on the ligand binding affinity of both mutant
Ob-Ra and Ob-Rb isoforms, and our observation of reduced affinity of
Ob-Rb269 glnpro clearly defines this as a second defect
responsible for some of the impaired signaling by the receptor.
We have defined Ob-Rb269 glnpro as being capable
of leptin-stimulated, JAK-dependent tyrosine phosphorylation of
the receptor, thereby providing the first analysis of the earliest
steps in leptin signaling by Ob-Rb269 glnpro. Given the
reduced capacity for membrane expression and reduced ligand binding
affinity of this mutant, we performed further studies to determine the
efficiency with which membrane associated
Ob-Rb269 glnpro can engage downstream signaling
pathways. To do this, we established conditions in which, by varying
the amount of transfected cDNA, similar expression of the wild-type and
mutant receptors were obtained. With this approach, we clearly
determined that membrane-associated Ob-Rb269 glnpro is
also defective at the level of signal transduction, with a reduced
ability to stimulate tyrosine phosphorylation of JAK2, and STAT3, as
well as a markedly reduced ability to stimulate transcription of an
egr-1 promoter luciferase construct. Furthermore, by
studying signaling at high concentrations of leptin, we have
demonstrated that the reduced capability of
Ob-Rb269 glnpro to activate intracellular signal
transduction is independent of the reduced leptin-binding affinity of
Ob-Rb269 glnpro. One possibility for this signaling
defect could be an impaired ability of the receptor to dimerize upon
ligand binding, although this remains to be shown. Thus, this mutant
receptor has defective signal transduction capabilities, in addition to
the defects in plasma membrane expression and binding affinity. These
studies extend the reports of White et al. (35).
An in vivo study has shown that
intracerebroventricular leptin administration, at doses which reduced
food intake and body weight of lean rats, produced no effect on
fa/fa rats (61). Another group showed that fa/fa
rats given 10 x the intracerebroventricular dose of leptin had a
reduction in weight gain and a reduction in neuropeptide Y (NPY)
content of the arcuate and paraventricular nuclei (62). These findings
suggest that, with extremely large doses of leptin,
Ob-Rb269 glnpro can signal. We observed very little
effect on transcriptional activation of the egr-1 promoter
luciferase construct, even when higher doses (100 nM) of
leptin were used. However, we did detect some activation of JAK2 and
STAT3 phosphorylation using high doses of leptin, which may be
sufficient for the physiological changes observed in vivo
when administrating large doses of leptin to fa/fa rats
(62).
We also report here the first functional studies of the
Ob-Ra269 glnpro isoform. The Ob-Ra short receptor
isoform was the first form of the leptin receptor to be cloned (15). It
was cloned from RNA derived from choroid plexus, where it is heavily
expressed and presumed to play a role in receptor-mediated transport
into the CNS. This receptor isoform is also expressed in a number of
other peripheral sites, including kidney, where it may play a role in
clearance and degradation of leptin (15, 16, 17, 18, 19, 21, 63). In addition, it
has been shown that human brain microvessels can mediate leptin
internalization, raising the possibility that leptin receptors at the
blood-brain-barrier could play a role in transport of leptin into the
brain (45). We have here, for the first time, characterized the
capacity of the short form of the leptin receptor to internalize and
degrade leptin, using a transient transfection model, and have compared
it to Ob-Ra269 glnpro. Two functional
consequences of Ob-Ra269 glnpro were noted. First,
as assessed by the acquisition of acid-resistant cell-associated
leptin, Ob-Ra269glnpro mediates a faster rate of leptin
internalization than does Ob-Ra. Despite this increased rate of
internalization of bound leptin, the apparent rate of leptin
degradation by a lysosomal pathway is reduced.
Other members of the class 1 cytokine receptor family to which Ob-R
belongs have been shown to mediate ligand uptake, and evidence for the
PRL receptor suggests that amino acid motifs in the cytoplasmic tail of
the receptor are important for this function (53, 64). These motifs are
thought to induce a ß turn conformation in the cytoplasmic tail of
the PRL receptor (53). This formation may be recognized by plasma
membrane adaptor proteins, which then promote aggregation of the PRL
receptor into clathrin-coated pits (53), after which internalization
occurs. Whether a similar mechanism exists for the leptin receptor
internalization is not yet clear. How the missense mutation in the
extracellular domain alters the internalization capability of the
leptin receptor remains unresolved. The fa mutation does
occur in a well-conserved region of the Ob-R (28, 32), and a mutation
in this area may alter the conformation of the receptor in a manner
that promotes the ability of Ob-Ra269 glnpro to
internalize.
Because Zucker rats have a low CSF/plasma leptin ratio (46), it may be
speculated that these animals have a reduced leptin receptor-mediated
transport of leptin into the CNS. This might caused by a low
cell-surface expression and/or a low leptin-binding affinity of
Ob-Ra269 glnpro in the choroid plexus and/or the brain
capillary microvessels. Another possibility is that
Ob-Ra269 glnpro has a specific defect in transport of
intact leptin across the blood/CSF and/or the blood-brain barriers. The
extent to which leptin receptor-mediated transport of leptin may
influence levels of leptin in the CSF, however, has not been
determined. Altogether, our findings could have important implications
for our understanding of the mechanisms and pathways of leptin uptake,
transport, and clearance.
It has recently been suggested that Ob-Rb269 glnpro
mediates constitutive ligand-independent transcriptional activity, and
it has been speculated that this activity may, by an as-yet unknown
mechanism, promote leptin resistance (35). In our investigation, we did
not observe elevated basal activity of Ob-Rb269 glnpro,
with respect to receptor phosphorylation, or phosphorylation of JAK2 or
STAT3. We did, however, observe constitutive ligand-independent
activity for Ob-Rb269 glnpro when studying
stimulation of egr-1 promoter luciferase activity. However,
this occurred only when JAK2 was cotransfected with receptor and
egr-1-luc cDNA. Thus, increased ligand-independent activity
of Ob-Rb269 glnpro is seen in some mammalian cell lines
under conditions of high levels of JAK2. Although JAK2 is thought to be
ubiquitously expressed (50), it remains unresolved whether this
ligand-independent activity occurs in vivo and whether it
contributes to leptin resistance.
In conclusion, we have studied the expression and functional properties
of Ob-Ra269 glnpro and Ob-Rb269 glnpro
and have defined multiple factors underlying the leptin resistance
brought about by this mutation. In addition to reduced cell-surface
expression and reduced affinity of binding, this mutation causes a
decreased ability of the expressed receptor to signal. Finally,
receptor-mediated internalization and degradation by Ob-Ra are also
altered by the fa mutation. Further investigation will be
required to determine the consequences of these changes for leptin
resistance in the fa/fa rat.
|
Footnotes |
|---|
2 These two authors have contributed equally.
Received January 2, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
pro extracellular domain
fatty mutation. Proc Natl Acad Sci USA 94:10657–10662This article has been cited by other articles:
 |
|
 |
|
  M. Matheny, Y. Zhang, A. Shapiro, N. Tumer, and P. J. Scarpace Central overexpression of leptin antagonist reduces wheel running and underscores importance of endogenous leptin receptor activity in energy homeostasis Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2009; 297(5): R1254 - R1261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E W Livingstone, S. L Grassick, G. L Currie, B. R Walker, and R. Andrew Dysregulation of glucocorticoid metabolism in murine obesity: comparable effects of leptin resistance and deficiency J. Endocrinol., May 1, 2009; 201(2): 211 - 218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. W. Williams, A. Zsombok, and B. N. Smith Rapid Inhibition of Neurons in the Dorsal Motor Nucleus of the Vagus by Leptin Endocrinology, April 1, 2007; 148(4): 1868 - 1881. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. W. Williams and B. N. Smith Rapid inhibition of neural excitability in the nucleus tractus solitarii by leptin: implications for ingestive behaviour J. Physiol., June 1, 2006; 573(2): 395 - 412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. de Assis, M. Wang, S. Goel, A. Foxworth, W. Helferich, and L. Hilakivi-Clarke Excessive Weight Gain during Pregnancy Increases Carcinogen-Induced Mammary Tumorigenesis in Sprague-Dawley and Lean and Obese Zucker Rats J. Nutr., April 1, 2006; 136(4): 998 - 1004. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bjorbaek and B. B. Kahn Leptin Signaling in the Central Nervous System and the Periphery Recent Prog. Horm. Res., January 1, 2004; 59(1): 305 - 331. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. L. Dobbins, L. S. Szczepaniak, W. Zhang, and J. D. McGarry Chemical sympathectomy alters regulation of body weight during prolonged ICV leptin infusion Am J Physiol Endocrinol Metab, April 1, 2003; 284(4): E778 - E787. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Hwa, L. Ghibaudi, J. Gao, and E. M. Parker Central melanocortin system modulates energy intake and expenditure of obese and lean Zucker rats Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2001; 281(2): R444 - R451. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Overton, T. D. Williams, J. B. Chambers, and M. E. Rashotte Cardiovascular and metabolic responses to fasting and thermoneutrality are conserved in obese Zucker rats Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2001; 280(4): R1007 - R1015. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. F. Wildman, S. Chua Jr., R. L. Leibel, and G. P. Smith Effects of leptin and cholecystokinin in rats with a null mutation of the leptin receptor Leprfak Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2000; 278(6): R1518 - R1523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. V. Zlokovic, S. Jovanovic, W. Miao, S. Samara, S. Verma, and C. L. Farrell Differential Regulation of Leptin Transport by the Choroid Plexus and Blood-Brain Barrier and High Affinity Transport Systems for Entry into Hypothalamus and Across the Blood-Cerebrospinal Fluid Barrier Endocrinology, April 1, 2000; 141(4): 1434 - 1441. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Cittadini, C. S. Mantzoros, T. G. Hampton, K. E. Travers, S. E. Katz, J. P. Morgan, J. S. Flier, and P. S. Douglas Cardiovascular Abnormalities in Transgenic Mice With Reduced Brown Fat : An Animal Model of Human Obesity Circulation, November 23, 1999; 100(21): 2177 - 2183. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Chen and A. Garg Monogenic disorders of obesity and body fat distribution J. Lipid Res., October 1, 1999; 40(10): 1735 - 1746. [Abstract] [Full Text]
|
|
|
|
|
|
Editorial: Further Insights into Leptin Action Endocrinology, September 1, 1998; 139(9): 3679 - 3680. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
