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Biozentrum University of Basel 4056 Basel, Switzerland
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Recently, the gene for the leptin receptor, OB-R, was cloned and shown
to bind leptin with high affinity (6). Four OB-R isoforms that differ
in the length of their cytoplasmic domains have been described in the
mouse (6, 7, 8) and were termed OB-Ra through d (Fig. 1
).
These proteins are identical in the sequence of their extracellular and
transmembrane domains and also share the first 29 amino acids of the
cytoplasmic domain. The predicted protein sequences diverge after K889
and result in four alternative C-terminal amino acid sequences. A large
intron of approximately 15 kb is located at the position corresponding
to K889 in the OB-R gene, and alternative splicing is the mechanism
that generates at least two of the four isoforms, OB-Ra and OB-Rb
(8, 9, 10).
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OB-R shows sequence homology to members of the cytokine receptor superfamily (6, 12). The cytoplasmic parts of these receptors lack enzymatic domains, instead, for signaling they associate with cytoplasmic tyrosine kinases of the janus kinase family (13). Ligand binding leads to activation of receptor-bound Jak kinases, which phosphorylate tyrosines in the cytoplasmic domain of the receptor as well as in other cytoplasmic target proteins. Several pathways can be activated by Jak kinases, including the signal transducers and activators of transcription (STAT), ras/mitogen-activated protein kinase, and phosphoinositide-3 kinase pathways (13, 14, 15).
OB-Rb is likely to be the active signaling chain, because it has a long cytoplasmic domain of 302 amino acids including the conserved box 1 and box 2 motifs that are thought to be essential for signaling. Furthermore, the long isoform is preferentially expressed in the hypothalamus, the primary site where leptin is thought to be acting. Moreover, the mutation in the C57BL/KS db allele selectively reduces transcripts encoding the long OB-Rb protein and, despite the presence of mRNA encoding the short isoforms, results in the db phenotype (6, 7), underlining the importance of the long OB-Rb isoform. The physiological role of the short OB-R isoforms remains to be determined.
Leptin levels in the blood correlate with the fat content of the body (16, 17). In obese individuals, leptin levels are often elevated, suggesting that leptin resistance is an important pathogenic mechanism. Leptin resistance might be caused by alterations in the receptor, as in db/db mice, and/or its downstream signaling pathway. Therefore, we sought to define the components of the leptin signaling pathway. We have shown that the long isoform, OB-Rb, can activate STAT3, STAT5, and STAT6 but not STAT1, STAT2, and STAT4 in transiently transfected COS cells (10). In contrast, the short isoform, OB-Ra, was inactive in STAT signaling (10). Here we have extended these studies and examined the potential of two OB-R isoforms to generate a proliferative signal and to activate Jak kinases in BaF3 cells, a factor-dependent hematopoietic cell line (18). Activation of Jak kinases is a prerequisite for the activation of all other known signaling cascades (13). Furthermore, we determined which Jak family member is activated by the long isoform, OB-Rb.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). These two isoforms are conserved between
mouse and human (6, 8, 19) and are therefore likely to be
physiologically relevant. Untransfected parental BaF3 cells did not
express OB-R mRNA, as verified by a RNase protection assay (not shown)
and are nonresponsive to leptin. We selected stable transfectants and
identified several clones that express the OB-R protein on the cell
surface by a [125I]leptin-binding assay (Fig. 2). To test the capacity of OB-R isoforms to provide a
proliferative signal, we placed the cells in media containing leptin in
the absence of interleukin-3 (IL-3). Proliferation was measured by
[3H]thymidine incorporation (Fig. 3A).
BaF3 cells transfected with the long isoform, OB-Rb, proliferated in
response to leptin in a dose-dependent fashion. The half-maximal
response was observed at leptin concentrations of 50 pM. In
contrast, BaF3 cells expressing the short isoform, OB-Ra, were unable
to grow in response to leptin. The same result was obtained with three
additional clones for the long and two clones for the short isoform
(not shown). The response to WEHI-3-conditioned medium as a source of
IL-3 is shown for comparison (Fig. 3B). The maximal stimulation with
leptin resulted in approximately 25% of the maximal incorporation seen
with IL-3.
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). Transfected cell
lines were incubated in the presence or absence of leptin, and cell
lysates were prepared. For immunoprecipitation of Jak family members,
we used the pan-reactive antibody R80 (20). This antibody is directed
against the conserved C-terminal kinase domain of Jak kinases and is
able to immunoprecipitate all known Jak/Tyk family members (20). A
phosphorylated band of approximately 120 kDa was detected in leptin-
stimulated cell expressing the long isoform of OB-R, but not in cells
transfected with the short isoform or in unstimulated cells (Fig. 4A).
The absence of tyrosine-phosphorylated Jak/Tyk proteins in these lanes
was not due to inefficient immunoprecipitation as shown by stripping
and sequential reprobing of the membrane with antibodies directed
against Jak1, Jak2, Jak3, or Tyk2. The band visible with
anti-phosphotyrosine antibodies was superimposable with the band
reprobed with either Jak1 or Jak2, which have a very similar
electrophoretic mobility on SDS-PAGE, but not with Jak3 or Tyk2, which
run at different positions on SDS-PAGE. Thus, stimulation of the long
isoform of OB-R resulted in phosphorylation of a Jak/Tyk family member,
comigrating with Jak1 or Jak2, whereas the short isoform of OB-R was
inactive. The same result was obtained with two independent clones (not
shown).
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). In contrast,
this effect was not observed in BaF3 cells expressing the short
isoform, OB-Ra. Neither the long nor short OB-R isoform was able to
phosphorylate Jak1 or Tyk2 upon leptin binding, although all members of
the Jak family are present and can be activated in BaF3 cells (21, 22, 23).
The weak band visible in the Jak1 immunoprecipitates did not colocalize
to the Jak1 band seen after reprobing of the membranes with anti-Jak1
and therefore likely represents a cross-reacting phosphoprotein. A weak
phosphotyrosine band was seen in anti-Jak3 immunoprecipitates. The
intensity of this band did not change in the presence or absence of
leptin, and the same band was also observed in untransfected parental
BaF3 cells (not shown). The band was superimposable with the Jak3 band
after reprobing with anti-Jak3 antibodies. It is not clear whether this
band represents a colocalizing cross-reacting phosphoprotein or whether
our BaF3 cells have low levels of constitutively phosphorylated Jak3.
We favor the first possibility, since this band was not detectable in
R80 immunoprecipitations (Fig. 4A), where, due to the faster mobility
of Jak3, it should be visible below the phosphorylated Jak2 band.
Because we observe no increase in the intensity of this band upon
leptin stimulation (Fig. 4B) and because no phosphorylated band was
visible in the 116-kDa region with the R80 immunoprecipitates despite
presence of a 116- kDa band after reprobing with anti-Jak3 antibodies
(Fig. 4A), we conclude that Jak3 is not phosphorylated in response to
leptin stimulation in BaF3 cells. Thus, upon leptin binding, the long
isoform, OB-Rb, activated Jak2, whereas the short isoform, OB-Ra, was
unable to activate Jak kinases.
To demonstrate that Jak2 physically interacts with the leptin receptor,
we performed immunoprecipitations with antibodies raised against the
extracellular domain of the OB-R protein under conditions that allow
coprecipitation of associated molecules. Probing of the Western blot
with a specific anti-Jak2 antibody shows that Jak2 was coprecipitated
with the long form of OB-R (Fig. 5, lower
panel). This association is constitutive and occurs in the absence
of the ligand. Stripping and reprobing of the filter with the
pan-reactive antibody R80 resulted in a single band that was
superimposable with the Jak2 band, whereas no signal was detectable
upon reprobing with antibodies against Jak1, Jak3, or Tyk2 (not shown).
To verify that our affinity-purified antibodies immunoprecipitated both
the short and the long OB-R isoform, we stripped and reprobed the
filter with the anti OB-R antibody (Fig. 5, upper panel).
The short OB-Ra isoform migrates as a strong band of approximately 150
kDa and a weaker band of approximately 116 kDa. Two nonspecific faster
migrating proteins were detected in all lanes including the
nontransfected parental BaF3 control. Treatment of the
immunoprecipitate before electrophoresis with endoglycosidase F, an
enzyme that removes N-linked sugar molecules, yielded a single
immunoreactive band of approximately 100 kDa, which is in good
agreement with the predicted size of the unmodified OB-Ra protein (not
shown). The long isoform, OB-Rb, is also present as two bands that
migrate at approximately 230 kDa and 190 kDa and likewise can be
converted into a single band of approximately 170 kDa by
endoglycosidase F treatment (not shown). The predicted size of the
unmodified OB-Rb protein is 130 kDa. The basis of this discrepancy of
approximately 40 kDa is presently not clear. The correctness of the
transfected cDNA construct has been confirmed by sequencing and
restriction mapping. The long isoform may carry additional covalent
modifications that are responsible for the higher apparent mol wt.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) and are conserved between
mouse and humans (6, 8, 19). Upon stimulation with leptin, the long
isoform, OB-Rb, can provide a proliferative signal in BaF3 cells (Fig. 3A). It is not known whether this proliferative signal of OB-Rb is
relevant under physiological conditions in vivo. Although it
seems unlikely that a proliferative signal would be important in the
adult hypothalamus, this signal might play a role during embryogenesis
of the nervous system. A proliferative signal by OB-Rb might also be
required for the formation of the murine reproductive tract, because
mice homozygous for the db mutation display atrophy of the
uterus and ovaries (28), and OB-Rb mRNA is expressed in these organs
(10). In addition, a mitogenic signal might also play a role in
hematopoiesis, as OB-Rb is expressed in the lymph nodes (10) and on
hematopoietic stem cells (19, 29).
The long isoform, OB-Rb, activates Jak2 (Fig. 4, A and B). In general,
cytokine receptors that signal as homodimers all activate Jak2. This
might be linked to the ability of Jak2 to autophosphorylate efficiently
(13). Jak2 is associated with the receptor and can be
coimmunoprecipitated both in the presence or absence of the ligand
(Fig. 5). Preassociation of Jak2 with the receptor has been described
for other members of the cytokine receptor superfamily including the
erythropoietin receptor (30) and the PRL receptor (31), and
oligomerization of the receptor/Jak complex through ligand binding is
thought to be the crucial step in initiating the signaling cascade
(13). OB-R is structurally closely related to the gp130 subfamily (6).
Most of these receptors form multimeric signaling complexes with gp130
(32). OB-Rb can signal for proliferation without gp130, at least in the
artificial setting of transfected BaF3 cells, since BaF3 cell do not
express gp130 (33). In contrast, the receptor for leukemia-inhibitory
factor-Rß when transfected into BaF3 cells required cotransfection
with gp130 to signal for proliferation (34). Consistent with our
results, addition of blocking antibodies against gp130 did not alter
signaling by OB-R in HepG2 cells (35). However, we cannot exclude the
possibility that under physiological conditions OB-R signals in
association with gp130 or other as yet unidentified proteins. We have
shown that the long isoform, OB-Rb, can activate STAT-3, STAT-5, and
STAT-6 in transfected COS cells (10). Recently, it was shown that
STAT-3 can be activated by leptin in the hypothalamus (36). The
db phenotype is not prevented by the presence of the short
isoforms. Therefore, it appears that the long isoform, OB-Rb, is the
only functional leptin receptor isoform.
The role of the short isoforms remains to be defined. The short
isoform, OB-Ra, was unable to bind or activate Jak kinases (Figs. 4 and 5) and was also inactive in the proliferation assay (Fig. 3A). The
intracellular domain of the short isoform, OB-Ra, consists of 33 amino
acids and comprises the conserved box 1 motif but lacks a box 2 motif.
For the PRL receptor and for the GH receptor, isoforms or mutants
missing box 2 have been reported to be partially functional in
providing a proliferative signal (37, 38). However, in all other
receptors studied, both box 1 and box 2 were required for binding and
activation of Jak kinases (13). Consistent with the defect in Jak
activation, the short isoform, OB-Ra, was also unable to activate STATs
(10). Since OB-Ra was found in both humans and mice (19) and shows high
degree of sequence homology (the C-terminal amino acid sequence RTDTL
in mice is almost identical to RTDIL in humans), it is tempting to
speculate that OB-Ra might have a function other than signaling. This
isoform is abundant, accounting for up to 95% of OB-R mRNA in many
tissues (7, 10) and might therefore act as a binding protein for
leptin, regulating the free leptin concentration, or function as a
transport protein.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Proliferation Assay
Cells were washed free of IL-3-containing medium and plated in
96-well plates in triplicates at 5 x 103 cells per
well in 100 µl of RPMI medium with 10% bovine calf serum. Purified
recombinant leptin (gift of Michele Chiesi, Ciba-Geigy, Basel) was
added to a final concentration of 10 nM, and a 1:4 dilution
series was established. After 48 h, 1 µCi
[3H]thymidine was added to each well, and incorporation
of [3H]thymidine was measured after 6 h in a
ß-counter.
Production of Antibodies against OB-R
A histidine-tagged peptide corresponding to amino acids 39–189
of the OB-R protein was expressed in Escherichia coli,
purified by nickel-nitrilotriacetic acid chromatography (40) and by
preparative SDS-PAGE, and used for immunization of rabbits. Serum was
affinity purified against the antigen coupled to cyanogen
bromide-activated Sepharose beads (Pharmacia, Uppsala, Sweden).
Stimulation and Immunoprecipitation
Cells were washed three times in PBS and starved in RPMI
medium without serum and without cytokines for 4 h. Purified
recombinant leptin was added to a final concentration of 50
nM and incubated for 15 min at 37 C. The cells were then
placed on ice, washed once in cold PBS, and lysed in 1 ml lysis buffer
containing 50 mM Tris, pH 8, 150 mM NaCl, 10
mM Na pyrophosphate, 10 mM NaF, 10
mM EDTA, 1 mM orthovanadate, 0.5 mM
dithiothreitol, 1% (wt/vol) Brij 96, and proteinase inhibitors (0.2
mM phenylmethylsulfonylfluoride, 2 ng/ml aprotinin, 1 ng/ml
leupeptin, 1 ng/ml pepstatin). After 20 min on ice, the lysates were
centrifuged at 20,000 x g at 4 C, and the supernatants
were used for immunoprecipitations. The following polyclonal rabbit
antibodies were used for immunoprecipitations: an anti Jak/Tyk antibody
R80 (gift from Dwayne L. Barber) (20) and an anti-Jak1 antibody (41)
(gift from Andrew Ziemiecki). Anti-Jak2 and anti-Jak3 antibodies were
purchased from Upstate Biotechnology Inc. (Lake Placid, NY), and the
anti-Tyk2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
For immunoprecipitations 5 µl of antibody were incubated with cell
lysates, and immune complexes were precipitated with protein A
Sepharose (Pharmacia). After washing three times with Tris-buffered
saline (TBS)/0.1% Triton-X100, the immunoprecipitated proteins were
separated by SDS-PAGE and transferred to nitrocellulose by Western
blot. The membranes were blocked with 2% BSA in TBS/0.05% Tween 20
and incubated with a 1:1 mixture of mouse monoclonal
anti-phosphotyrosine antibodies 4G10 (Upstate Biotechnology Inc.) and
PY20 (Transduction Laboratories, Lexington, KY) at a 1:1000 dilution.
This mixture gave a stronger signal than each of the individual
antibodies alone. After six washes with TBS, the membranes were
incubated with 1:5000 diluted horseradish peroxidase-coupled sheep
anti-mouse antibodies (Amersham, Buckinghamshire, England) and washed.
Enhanced chemiluminescence (ECL) was detected following the
instructions of the manufacturer (Amersham). Before reprobing, the
membranes were stripped for 30 min at 50 C in 62.5 mM
Tris-HCl (pH 6.8), 2% SDS, and 100 mM ß-mercaptoethanol,
blocked in 2% BSA, and incubated with specific anti-Jak or anti-Tyk2
antibody.
Coimmunoprecipitation
BaF3 cells were starved as described above. Recombinant
leptin was added to a final concentration of 50 nM, and
5 x 108 cells per condition were incubated for 10 min
at 37 C. The cells were then placed on ice, washed once in cold PBS and
lysed in 4 ml lysis buffer (see above). Lysis was carried out for
1 h at 4 C on an end-over-end shaker. The lysates were then
centrifuged at 12,300 x g to remove nuclei and debris.
The supernatants were preadsorbed to Protein-A-Sepharose beads
(Pharmacia) for 1 h and incubated overnight with affinity-purified
anti-OB-R antibodies and protein-A-Sepharose beads. The immune
complexes were washed five times in lysis buffer, subjected to
SDS-PAGE, and analyzed by western blotting with antibodies against
Jak1, Jak2, Jak3, and Tyk2 at a 1:1000 dilution, or the
affinity-purified anti-OB-R at a 1:500 dilution.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by grants from the Swiss National Science Foundation to R.C.S.
Received for publication June 13, 1996. Revision received January 15, 1997. Accepted for publication January 16, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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M. Tena-Sempere, L. Pinilla, F.-P. Zhang, L. C. González, I. Huhtaniemi, F. F. Casanueva, C. Dieguez, and E. Aguilar Developmental and Hormonal Regulation of Leptin Receptor (Ob-R) Messenger Ribonucleic Acid Expression in Rat Testis Biol Reprod, February 1, 2001; 64(2): 634 - 643. [Abstract] [Full Text]
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 S. ATTOUB, V. NOE, L. PIROLA, E. BRUYNEEL, E. CHASTRE, M. MAREEL, M. P. WYMANN, and C. GESPACH Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase-, Rho-, and Rac-dependent signaling pathways FASEB J, November 1, 2000; 14(14): 2329 - 2338. [Abstract] [Full Text]
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 M. W. Nickola, L. E. Wold, P. B. Colligan, G.-J. Wang, W. K. Samson, and J. Ren Leptin Attenuates Cardiac Contraction in Rat Ventricular Myocytes : Role of NO Hypertension, October 1, 2000; 36(4): 501 - 505. [Abstract] [Full Text] [PDF]
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Y.-B. Kim, S. Uotani, D. D. Pierroz, J. S. Flier, and B. B. Kahn In Vivo Administration of Leptin Activates Signal Transduction Directly in Insulin-Sensitive Tissues: Overlapping but Distinct Pathways from Insulin Endocrinology, July 1, 2000; 141(7): 2328 - 2339. [Abstract] [Full Text] [PDF]
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S. Dridi, N. Raver, E. E. Gussakovsky, M. Derouet, M. Picard, A. Gertler, and M. Taouis Biological activities of recombinant chicken leptin C4S analog compared with unmodified leptins Am J Physiol Endocrinol Metab, July 1, 2000; 279(1): E116 - E123. [Abstract] [Full Text] [PDF]
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A. S. Banks, S. M. Davis, S. H. Bates, and M. G. Myers Jr. Activation of Downstream Signals by the Long Form of the Leptin Receptor J. Biol. Chem., May 5, 2000; 275(19): 14563 - 14572. [Abstract] [Full Text] [PDF]
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T. J. Kieffer and J. F. Habener The adipoinsular axis: effects of leptin on pancreatic beta -cells Am J Physiol Endocrinol Metab, January 1, 2000; 278(1): E1 - E14. [Abstract] [Full Text] [PDF]
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M. Caprio, A. M. Isidori, A. R. Carta, C. Moretti, M. L. Dufau, and A. Fabbri Expression of Functional Leptin Receptors in Rodent Leydig Cells Endocrinology, November 1, 1999; 140(11): 4939 - 4947. [Abstract] [Full Text]
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G. Paolisso, M. R. Tagliamonte, M. Galderisi, G. A. Zito, A. Petrocelli, C. Carella, O. de Divitiis, and M. Varricchio Plasma Leptin Level Is Associated With Myocardial Wall Thickness in Hypertensive Insulin-Resistant Men Hypertension, November 1, 1999; 34(5): 1047 - 1052. [Abstract] [Full Text] [PDF]
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C. Bjorbak, K. El-Haschimi, J. D. Frantz, and J. S. Flier The Role of SOCS-3 in Leptin Signaling and Leptin Resistance J. Biol. Chem., October 15, 1999; 274(42): 30059 - 30065. [Abstract] [Full Text] [PDF]
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 C. Li and J. M. Friedman Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction PNAS, August 17, 1999; 96(17): 9677 - 9682. [Abstract] [Full Text] [PDF]
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V. A. Barr, K. Lane, and S. I. Taylor Subcellular Localization and Internalization of the Four Human Leptin Receptor Isoforms J. Biol. Chem., July 23, 1999; 274(30): 21416 - 21424. [Abstract] [Full Text] [PDF]
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C. Bjørbæk, J. K. Elmquist, K. El-Haschimi, J. Kelly, R. S. Ahima, S. Hileman, and J. S. Flier Activation of SOCS-3 Messenger Ribonucleic Acid in the Hypothalamus by Ciliary Neurotrophic Factor Endocrinology, May 1, 1999; 140(5): 2035 - 2043. [Abstract] [Full Text]
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P. Magni, R. Vettor, C. Pagano, A. Calcagno, E. Beretta, E. Messi, M. Zanisi, L. Martini, and M. Motta Expression of a Leptin Receptor in Immortalized Gonadotropin-Releasing Hormone-Secreting Neurons Endocrinology, April 1, 1999; 140(4): 1581 - 1585. [Abstract] [Full Text]
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T. Thomas, F. Gori, S. Khosla, M. D. Jensen, B. Burguera, and B. L. Riggs Leptin Acts on Human Marrow Stromal Cells to Enhance Differentiation to Osteoblasts and to Inhibit Differentiation to Adipocytes Endocrinology, April 1, 1999; 140(4): 1630 - 1638. [Abstract] [Full Text]
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 D. G. Baskin, M. W. Schwartz, R. J. Seeley, S. C. Woods, D. Porte , Jr., J. F. Breininger, Z. Jonak, J. Schaefer, M. Krouse, C. Burghardt, et al. Leptin Receptor Long-form Splice-variant Protein Expression in Neuron Cell Bodies of the Brain and Co-localization with Neuropeptide Y mRNA in the Arcuate Nucleus J. Histochem. Cytochem., March 1, 1999; 47(3): 353 - 362. [Abstract] [Full Text]
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 F. J. King, E. Hu, D. F. Harris, P. Sarraf, B. M. Spiegelman, and T. M. Roberts DEF-1, a Novel Src SH3 Binding Protein That Promotes Adipogenesis in Fibroblastic Cell Lines Mol. Cell. Biol., March 1, 1999; 19(3): 2330 - 2337. [Abstract] [Full Text] [PDF]
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T. L. Horvath, S. Diano, and A. N. van den Pol Synaptic Interaction between Hypocretin (Orexin) and Neuropeptide Y Cells in the Rodent and Primate Hypothalamus: A Novel Circuit Implicated in Metabolic and Endocrine Regulations J. Neurosci., February 1, 1999; 19(3): 1072 - 1087. [Abstract] [Full Text] [PDF]
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 P. M. Smith, V. Mollaret, and A. V. Ferguson Leptin acts in the rat hypothalamic paraventricular nucleus to induce gastric mucosal damage Am J Physiol Regulatory Integrative Comp Physiol, December 1, 1998; 275(6): R2081 - R2084. [Abstract] [Full Text] [PDF]
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K. C. McCowen, J. C. Chow, and R. J. Smith Leptin Signaling in the Hypothalamus of Normal Rats in Vivo Endocrinology, November 1, 1998; 139(11): 4442 - 4447. [Abstract] [Full Text] [PDF]
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N. M. Morton, V. Emilsson, Y.-L. Liu, and M. A. Cawthorne Leptin Action in Intestinal Cells J. Biol. Chem., October 2, 1998; 273(40): 26194 - 26201. [Abstract] [Full Text] [PDF]
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 J Harvey and M L J Ashford Insulin occludes leptin activation of ATP-sensitive K+ channels in rat CRI-G1 insulin secreting cells J. Physiol., September 15, 1998; 511(3): 695 - 706. [Abstract] [Full Text] [PDF]
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B. A. da Silva, C. Bjorbak, S. Uotani, and J. S. Flier Functional Properties of Leptin Receptor Isoforms Containing the Gln->Pro Extracellular Domain Mutation of the Fatty Rat Endocrinology, September 1, 1998; 139(9): 3681 - 3690. [Abstract] [Full Text] [PDF]
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J Harvey and M L J Ashford Role of tyrosine phosphorylation in leptin activation of ATP-sensitive K+ channels in the rat insulinoma cell line CRI-G1 J. Physiol., July 1, 1998; 510(1): 47 - 61. [Abstract] [Full Text] [PDF]
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M.-L. Hakansson, H. Brown, N. Ghilardi, R. C. Skoda, and B. Meister Leptin Receptor Immunoreactivity in Chemically Defined Target Neurons of the Hypothalamus J. Neurosci., January 1, 1998; 18(1): 559 - 572. [Abstract] [Full Text] [PDF]
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C. Bjorbak, S. Uotani, B. da Silva, and J. S. Flier Divergent Signaling Capacities of the Long and Short Isoforms of the Leptin Receptor J. Biol. Chem., December 19, 1997; 272(51): 32686 - 32695. [Abstract] [Full Text] [PDF]
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C. Bjorbak, R. M. Buchholz, S. M. Davis, S. H. Bates, D. D. Pierroz, H. Gu, B. G. Neel, M. G. Myers Jr., and J. S. Flier Divergent Roles of SHP-2 in ERK Activation by Leptin Receptors J. Biol. Chem., February 9, 2001; 276(7): 4747 - 4755. [Abstract] [Full Text] [PDF]
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C. Bjorbak, H. J. Lavery, S. H. Bates, R. K. Olson, S. M. Davis, J. S. Flier, and M. G. Myers Jr. SOCS3 Mediates Feedback Inhibition of the Leptin Receptor via Tyr985 J. Biol. Chem., December 15, 2000; 275(51): 40649 - 40657. [Abstract] [Full Text] [PDF]
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I. Szanto and C. R. Kahn Selective interaction between leptin and insulin signaling pathways in a hepatic cell line PNAS, February 29, 2000; 97(5): 2355 - 2360. [Abstract] [Full Text] [PDF]
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PY) antibodies. Membranes were stripped and
sequentially reprobed with antibodies specific for individual Jak
kinase members as indicated. B, Identification of specific Jak kinase
members activated by leptin. Immunoprecipitation (IP) was carried out
with antibodies against individual Jak kinase members as indicated.
Western blots were immunodetected with anti-phosphotyrosine (