Pleiotropy of leptin receptor signalling is defined
by distinct roles of the intracellular tyrosines
Paul Hekerman
1
, Julia Zeidler
1
, Simone Bamberg-Lemper
1
, Holger Knobelspies
1
, Delphine Lavens
2
,
Jan Tavernier
2
, Hans-Georg Joost
3
and Walter Becker
1
1 Institute of Pharmacology and Toxicology, Medical Faculty of the Aachen University, Germany
2 The Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research (VIB9), Ghent University, Belgium
3 German Institute of Human Nutrition (DIfE) Potsdam-Rehbru
¨
cke, Nuthetal, Germany
Leptin is an adipocyte-secreted hormone that informs
the brain about the status of the body’s energy stores.
It regulates energy homeostasis through effects on sati-
ety and energy expenditure and deficiencies of leptin or
the leptin receptor in humans or rodents result in
severe obesity, infertility, impaired growth and insulin
resistance [1]. In db ⁄ db mice that lack the signalling act-

STAT5 and ERK1 ⁄ 2 but failed to alter the phosphorylation of AMPK.
Each of the three intracellular tyrosine residues in LEPR exhibited different
signalling capacities: Tyr985 was necessary and sufficient for leptin-induced
activation of ERK1 ⁄ 2; Tyr1077 induced tyrosyl phosphorylation of
STAT5; and Tyr1138 was capable of activating STAT1, STAT3 and
STAT5. Consistent results were obtained in reporter gene assays with
STAT3 or STAT5-responsive promoter constructs, respectively. Further-
more, the sequence motifs surrounding the three tyrosine residues are
conserved in LEPR from mammals, birds and in a LEPR-like cyto-
kine receptor from pufferfish. Mutational analysis of the box3 motif
around Tyr1138 identified Met1139 and Gln1141 as important deter-
minants that define specificity towards the different STAT factors. These
data indicate that all three conserved tyrosines are involved in LEPR func-
tion and define the pleiotropy of signal transduction via STAT1 ⁄ 3, STAT5
or ERK kinases. Activation and inhibition of AMPK appears to require
additional components of the signalling pathways that are not present in
beta cells.
Abbreviations
AMPK, AMP-activated kinase; ERK, extracellular signal-regulated kinase; GH, growth hormone; JAK, janus kinase; LEPR, leptin receptor;
SH2, src-homology 2; SOCS3, suppressor of cytokine signalling 3; STAT, signal transducer and activator of transcription.
FEBS Journal 272 (2005) 109–119 ª 2004 FEBS 109
actions of leptin have also been described [3]. Two
effects have been particularly well studied: the stimula-
tion of proinflammatory immune responses by direct
action on T-lymphocytes [4,5], and the inhibition of
insulin secretion from pancreatic beta cells [6–9].
As a class I cytokine receptor, LEPRb activates the
janus kinase ⁄ signal transducer and activator of tran-
scription (JAK ⁄ STAT) signalling pathway [10,11]. Lig-
and binding to LEPRb results in the activation of

tyrosine residues in LEPRb, Tyr985 can recruit either
the tyrosine phosphatase SHP-2 or suppressor of cyto-
kine signalling 3 (SOCS3) [15,23–26]. Binding of
SOCS3 to Tyr-985 attenuates leptin signalling by inhi-
bition of the receptor-associated JAK kinase [25]. In
contrast, recruitment of SHP-2 does not alter JAK2
activity but results in GRB2 binding to SHP-2 and
activation of the RAS ⁄ RAF ⁄ ERK pathway [26,27]. In
contrast to Tyr985 and Tyr1138, the role of Tyr1077
in leptin signalling is not yet clear.
More recently, it has been shown that AMP-depend-
ent protein kinase (AMPK) appears to be a down-
stream mediator of leptin signalling. Leptin directly
stimulates phosphorylation and activation of the a2
catalytic subunit of AMPK in muscle [28]. In contrast,
leptin suppresses a2 AMPK activity in secondary
hypothalamic neurons indirectly via activation of
agouti-related protein (AGRP) neurons [29].
The aim of this study was to analyse the contribu-
tion of the intracellular tyrosine residues to LEPRb-
mediated effects on STAT factors, MAP kinase and
AMPK. These data show that LEPRb is capable of
activating a broader range of STAT factors than other
cytokines such as interleukin-6 (IL-6) and growth hor-
mone (GH). Analysis of point mutants revealed that
each of the individual tyrosine residues in the intracel-
lular part of LEPRb exhibits a different signalling
capacity. In particular, our data identify Tyr1077 as a
docking site for STAT5.
Results

clonal pools of cells stably expressing LEPRb. In these
cells (Fig. 1B), leptin and IL-6 stimulated tyrosine
phosphorylation of STAT3 to a similar degree, but lep-
tin again induced activation of a broader spectrum of
STAT factors (STAT1, STAT3, STAT5, STAT6).
Leptin has recently been reported to activate AMPK
in muscle cells, thereby stimulating expression
of enzymes involved in fatty acid oxidation [28].
Pleiotropic leptin receptor signalling P. Hekerman et al.
110 FEBS Journal 272 (2005) 109–119 ª 2004 FEBS
Previously, leptin has also been shown to prevent lipo-
toxicity in pancreatic islets by upregulating expression
of fatty acid oxidation-related enzymes (carnitine
palmitoyl transferase, acyl CoA oxidase) [32,33].
Therefore, we analysed the effect of leptin on AMPK
in the insulinoma cell lines. As shown in Fig. 1C, lep-
tin treatment failed to alter the activating phosphoryla-
tion of AMPK in HIT-T15 cells or in RINm5F cells.
As a positive control, AMPK phosphorylation was
readily stimulated in glucose-depleted cells, indicating
that essential components of the AMPK pathway were
present in these cell lines.
Role of the intracellular tyrosine residues
in LEPRb
To determine the role of the three intracellular tyrosine
residues (Tyr985, Tyr1077, Tyr1138) in LEPR-medi-
ated activation of downstream signalling events, con-
structs in which phenylalanine(s) replaced either one of
the three tyrosines or combinations of them were
expressed in HIT-T15 cells. The specific signalling

STAT response elements. Two different reporter con-
structs were used. In the first one (a
2
M), luciferase
expression is driven by the IL-6 responsive element of
the a
2
-macroglobulin promoter, which is controlled
by STAT3 [34]. The second reporter plasmid (spi2.1)
contains the GH-responsive element of the rat serine
protease inhibitor 2.1 (spi2.1) gene, whose expression
is controlled by STAT5 [35]. Assays with the different
promoter constructs were performed under identical
conditions to analyse the ability of the LEPR point
mutants to specifically activate STAT3- and STAT5-
driven promoter activity (Fig. 3). Consistent with the
detection of tyrosine phosphorylated STAT factors by
Western blot analysis, all LEPRb constructs contain-
ing the Tyr1138fiPhe mutation (YYF, FYF, YFF,
FFF) were severely reduced in their capacity to sti-
mulate a
2
M reporter gene activity. It is likely that
the residual activation by the mutants retaining
Tyr1077 (YYF, FYF) can be explained by the action
of STAT5. Spi2.1 promoter activity was stimulated
by all constructs containing either Tyr1077 or
Tyr1138, in full agreement with the presumed control
by STAT5. As expected, Tyr985 was not able to
induce reporter gene activity driven by STAT-depend-

binding of STAT1 by the IL-6 receptor gp130 [37] and
was exchanged for glycine. A consensus sequence for
binding of STAT5 has not yet been explicitly defined
but in most cases the phosphorylated tyrosine is fol-
lowed by an aliphatic hydrophobic residue such as leu-
cine, isoleucine, valine or methionine [38–41]. The
constructs were transiently expressed in HIT-T15 cells,
and leptin-induced activation of STAT factors was
monitored by Western blot analysis with phospho-spe-
cific antibodies and by reporter gene assays (Fig. 4).
Consistent with the established consensus sequence for
binding of STAT3, mutation of Gln1141 abolished lep-
tin-induced phosphorylation of STAT3 and decreased
the activation of the a
2
M-derived promoter but did
not affect STAT5 phosphorylation or induction of the
spi2.1 promoter. In contrast, exchange of Met1139 for
alanine or arginine eliminated phosphorylation of
STAT5 and reduced induction of the spi2.1 promoter
construct. This promoter is probably also responsive
to STAT1 and ⁄ or STAT3. Mutation of Pro1140
strongly reduced activation of all three STAT proteins.
Taken together, these results indicate that the combi-
nation of a hydrophobic residue in the P+1 position
and the glutamine in P+3 allows the binding of either
STAT1, STAT3 or STAT5 to pTyr-1138 in LEPRb.
Discussion
Class I cytokine receptors such as LEPRb transmit
extracellular signals by recruiting SH2 domain-contain-

)1
) for 18 h. The
luciferase activity was determined and normalized to coexpressed
b-galactosidase activity. Data are expressed as fold stimulation rel-
ative to unstimulated cells. Bars reflect means ± SEM of three
(a2M) or four to five independent experiments (spi2.1). The bottom
panel of Fig. 2 provides an expression control for the different
LEPR mutants as aliquots of the same DNA samples were trans-
fected in this experiment.
P. Hekerman et al. Pleiotropic leptin receptor signalling
FEBS Journal 272 (2005) 109–119 ª 2004 FEBS 113
expected to observe a reduced or increased phosphory-
lation of AMPK in response to leptin in the pancreatic
beta cell lines. However, concentrations of leptin that
maximally activated STAT3 failed to alter AMPK phos-
phorylation (Fig. 1C). Glucose deprivation induced the
anticipated activation of AMPK, indicating that the
upstream kinase is present in the cells. Consistent with
our results, Leclerc et al. [42] recently reported that
leptin did not change AMPK activity in murine MIN6
insulinoma cells and in isolated rat islets. Thus, we
conclude that pancreatic beta cells lack a component
required for leptin-induced activation of AMPK, pos-
sibly the c3 subunit of AMPK which appears to speci-
fically expressed in skeletal muscle [42].
Our conclusion that Tyr1077 in murine LEPRb
plays an important role in leptin signalling is
supported by the fact that the surrounding sequence is
strikingly conserved in mammals and birds [15],
although the intracellular domains of murine and

from pufferfish contains 30% of identical amino acids
with murine LEPRb and 27% with murine gp130, the
signal transducing subunit of the IL-6 receptor (gaps
> 50 amino acids were not penalized). The intracellu-
lar domain shows no significant similarity with any
known mammalian protein except for LEPRb.
Tyr-1077 has previously been shown to play a role
in down-regulation of LEPRb signalling, presumably
by serving as a docking site for SOCS3 [15]. The con-
servation of the aliphatic hydrophobic residue in the
P+1 position after Tyr-1077 is also compatible with
the known requirements of STAT5-binding as deter-
mined in different receptors [38,40,41]. Leptin-induced
activation of STAT5 has already been described in the
first papers reporting STAT signalling by the LEPRb
[10,43]. Later, in vivo studies suggested that only
STAT3 is activated upon leptin administration in the
hypothalamus of mice and rats [17,44]. However,
leptin-induced tyrosine phosphorylation has been
observed in various cell types, e.g. hypothalamic GT1-
7 cells [45], intestinal L-cells [46], enterocyte-like
CaCo-2 cell line [47], and H-35 hepatoma cells [48].
Our results are also consistent with earlier reports that
mutant constructs of the human LEPRb either with a
substitution of Tyr1141 for phenylalanine or with a
deletion of the C-terminus including Tyr1141 were still
able to induce DNA binding of overexpressed
STAT5B in electrophoretic mobility shift assays
[10,49]. It should be noted that leptin-induced activa-
tion of STAT5 in the insulinoma cell lines was detect-

same sequence motifs in gp130 [TyrLeuProGln(905–
908)] and [TyrMetProGln(915–918)] do not activate
STAT5, indicating that the hydrophobic residue in
P +1 is not the only residue required for binding of
STAT5.
Experimental procedures
Reagents
Recombinant murine leptin was obtained from PeproTec
(London, UK) and GH from Bachem (Bubendorf, Switzer-
land). Recombinant human IL-6 and soluble IL-6 receptor
were kindly provided by Gerhard Mu
¨
ller-Newen (Depart-
ment of Biochemistry, Aachen University). The following
primary antibodies were used: polyclonal rabbit antibodies
against p(Y701)-STAT1, p(Y705)-STAT3, p(Y694)-STAT5,
p(Y641)-STAT6, STAT1, STAT3, p(T172)-AMPK, anti-
AMPK, anti-pTyr Ig PY100, and phospho p42 ⁄ 44 MAP
kinase from Cell Signalling Technology (Beverly, MA),
anti-STAT5A ⁄ B from Upstate (Charlottesville, VA, USA),
goat anti-(mouse LEPR) Ig from R&D Systems (Wiesba-
den, Germany), antibody against phosphorylated JAK2
(pYpY1007 ⁄ 1008) from BioSource Technologies (Cama-
rillo, CA) and anti-pTyr Igs PY20 from Transduction
Laboratories, Inc. (San Diego, CA). Horseradish peroxi-
dase-labeled anti-(rabbit IgG) (IgG-POD) was obtained
from Pierce Chemical Co. (Rockford, IL), anti-(mouse
IgG-POD) from Amersham (Buckinghamshire, UK), and
anti-(goat IgG-POD) from Dianova (Hamburg, Germany).
Cell culture, transient transfection, and retroviral

TM
Site-Directed Mutagenesis
Kit (Stratagene).
For stable expression in RINm5F cells, LEPRb was sub-
cloned from pSVL-LEPRb into the retroviral vector
pWZL-Neo. Viruses were produced in 293T cells, and con-
fluent RINm5F cells were infected with pWZL-neo-LEPR
viruses [58]. Selection was performed for 14 days in
1mgÆmL
)1
of G418.
Western blot analysis and EMSA
Cells were incubated in serum-free medium for 18–22 h
before leptin (100 ngÆmL
)1
), IL-6 (200 U mL
)1
), or GH
(500 ngÆmL
)1
) was added for 15 min. All assays of transi-
ently transfected HIT-T15 cells were performed 48 h after
transfection. Nuclear extracts were prepared by hypotonic
lysis [34]. To prepare total cellular lysates, cells were
washed with phosphate buffered saline and lysed in 1%
(w ⁄ v) SDS, 20 mm Tris ⁄ HCl pH 7.4 in a boiling water bath
for 5 min. For Western blot analysis, protein samples were
separated by SDS ⁄ PAGE (8% gels), blotted on to nitrocel-
lulose, and specific proteins were detected by chemilumines-
cence using the primary antibodies mentioned above and

data were normalized to b-galactosidase activities.
Acknowledgements
We thank Drs Lars-Arne Haldosen, Gerhard Mu
¨
ller-
Newen, Peter C. Heinrich, Annette Schu
¨
rmann, Dag-
mar Meyer zu Heringdorf for generous donations of
reagents and cell lines. This work was supported by
the Deutsche Forschungsgemeinschaft (SFB 542).
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