The Leptin Receptor Activates Janus Kinase 2 and Signals for Proliferation in a Factor-Dependent Cell Line

Nico Ghilardi and Radek C. Skoda

Biozentrum University of Basel 4056 Basel, Switzerland


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The antiobesity effects of leptin are mediated by the obese receptor (OB-R), a member of the cytokine receptor superfamily. Several isoforms of OB-R that differ in the length of the cytoplasmic domain have been described. An isoform with a long cytoplasmic domain of 302 amino acids, termed OB-Rb, contains the conserved box 1 and box 2 motifs and is likely to be responsible for leptin-induced signaling. A point mutation in the OB-R gene of diabetes (db) mice generates a new splice donor that interferes with the correct splicing of the OB-Rb mRNA and is predicted to cause absence of the OB-Rb protein in db/db mice. Here we examined the signaling potential of the long isoform, OB-Rb, and of a short isoform, OB-Ra, in BaF3 cells, a factor-dependent hematopoietic cell line. The long isoform was able to generate a proliferative signal and upon leptin binding, activated janus kinase 2 (Jak2). Consistently, antibodies directed against the extracellular domain of OB-R coprecipitated Jak2. The short isoform, OB-Ra, was inactive in both proliferation and Jak activation. These results provide further support for the long isoform, OB-Rb, being the principal mediator of the effects of leptin and help to explain why db/db mice are resistant to leptin, despite the presence of the short OB-R isoforms.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The central role of leptin and its receptor in the control of body weight was discovered through the study of the two mouse mutants obese (ob) and diabetes (db) (1). Mice homozygous for ob or db alleles display obesity, hyperglycemia, and insulin resistance resembling type II diabetes. Parabiosis experiments performed by Douglas L. Coleman in the early 1970s (1) suggested that the ob gene encodes a humoral satiety factor, whereas the db gene produces the receptor for this ligand. The first prediction was confirmed through positional cloning of the ob gene (2). The ob gene product, leptin, is a 16-kDa secreted protein produced in adipocytes that acts as a regulator of metabolism and feeding behavior. A mutation in the leptin gene caused absence of leptin protein in the circulation of ob/ob mice (2), and purified recombinant leptin injected into ob/ob mice corrected the metabolic abnormalities (3, 4, 5).

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. 1Go). 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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Figure 1. OB-R Isoforms and Alternative Splicing

Amino acid sequences of the intracellular domains are shown in one-letter code; asterisk, stop codon. Box 1 and box 2 motifs are underlined. Alternative splicing is symbolized for OB-Ra and OB-Rb (solid lines). The nucleotide sequence of the intron is shown to explain the OB-Rc isoform. The mechanism of generation of OB-Rd is unknown (dashed line). TM, transmembrane domain (solid box).

 
Mutations in OB-R were found in mice with two independent db alleles, C57BL/KSdb and dbPAS, providing strong evidence that Coleman’s second prediction was also correct (7, 8, 9). A point mutation in the OB-R gene of C57BL/KS db/db mice causes abnormal splicing of mRNA for one isoform, OB-Rb, and is predicted to cause the absence of OB-Rb protein (7, 8). Although the mutation of dbPAS has not yet been defined in molecular detail, it was shown that mice homozygous for dbPAS express at least 20 fold less OB-R mRNA (9). Furthermore, the fatty Zucker rat, a mutant phenotypically similar to db, maps very close to OB-R in the rat genome (9) and carries a missense mutation in an exon of the OB-R gene encoding a conserved part of the extracellular domain of the OB-R protein (11).

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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To assess the signaling potential of OB-R isoforms we transfected a factor-dependent hematopoietic cell line, BaF3 (18), with expression constructs encoding the long (OB-Rb) or short isoform (OB-Ra) of the leptin receptor (Fig. 1Go). 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. 2Go). 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. 3AGo). 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. 3BGo). The maximal stimulation with leptin resulted in approximately 25% of the maximal incorporation seen with IL-3.



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Figure 2. Binding of [125I]Leptin to BaF3 Clones Transfected with OB-R Isoforms

L, Long isoform OB-Rb; S, short isoform OB-Ra; wt, untransfected parental BaF3 cells. Bars indicate the median ± SD of triplicates. The P values were calculated by ANOVA: *, P < 0.01; **, P < 0.005; ***, P < 0.001. Cells were incubated with 1 nM [125I]leptin in the absence (solid bars) or presence (open bars) of 100 nM of unlabeled leptin.

 


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Figure 3. Proliferation Assay with BaF3 Cells Expressing OB-R Isoforms

[3H]thymidine incorporation in response to leptin (A) or WEHI-3-conditioned media as a source of IL-3 (B). Solid circles, BaF3 clone expressing the long isoform OB-Rb (L46); open triangles, BaF3 clone expressing the short isoform, OB-Ra (S44), of OB-R.

 
To study activation of Jak kinases by OB-R isoforms, we stimulated the transfected BaF3 clones with leptin and analyzed phosphorylation of immunoprecipitated Jak proteins by Western blots using an anti-phosphotyrosine antibody (Fig. 4Go). 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. 4AGo). 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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Figure 4. Activation of Jak Kinase Members in Transfected BaF3 Cells in Response to Leptin.

BaF3 clones transfected with the long isoform, OB-Rb (L), or short isoform, OB-Ra (S), were incubated for 15 min in the presence or absence of leptin. A, Immunoprecipitation (IP) was carried out with the R80 antibody followed by immunodetection of Western blots with anti-phosphotyrosine ({alpha}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 ({alpha}PY) antibodies. Shown below are the membranes after stripping and reprobing with the same Jak-specific antibodies as used for the immunoprecipitations.

 
To identify which member of the Jak/Tyk kinase family was activated by the leptin receptor, we performed immunoprecipitations with monospecific antibodies against individual Jaks in the place of R80. We observed phosphorylation of Jak2 when BaF3 cells carrying the long isoform, OB-Rb, were stimulated with leptin (Fig. 4BGo). 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. 4AGo), 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. 4BGo) 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. 4AGo), 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. 5Go, 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. 5Go, 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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Figure 5. Association of Jak2 with OB-R Isoforms

Untransfected parental BaF3 cells (-) and BaF3 clones transfected with the short isoform, OB-Ra (S), or long isoform, OB-Rb (L), were incubated for 10 min in presence (+) or absence (-) of leptin. Immunoprecipitation (IP) was carried out with an affinity-purified antibody against OB-R ({alpha}OB-R) followed by immunoblotting with {alpha}OB-R (upper panel) or an antibody-specific for Jak2 (lower panel). The position of molecular mass markers is indicated in kilodaltons.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Alternative splice variants resulting in proteins with truncated cytoplasmic domains are commonly found among members of the cytokine receptor superfamily (24, 25, 26, 27). The physiological role of these variants is poorly understood. For the OB-R, a long isoform and three truncated isoforms with alternative C termini have been described in the mouse (6, 7, 8). We examined the signaling potential of the long isoform, OB-Rb, and of one short isoform, OB-Ra. These two isoforms are generated by alternative splicing (Fig. 1Go) 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. 3AGo). 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. 4Go, 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. 5Go). 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. 4Go and 5Go) and was also inactive in the proliferation assay (Fig. 3AGo). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Transfections
The cDNAs for the long and short isoform of OB-R (10) were subcloned into the expression vector pGD (39) and transfected into BaF3 cells (18) by electroporation. G418-resistant clones were selected and screened for cell surface expression of OB-R by their capacity to bind [125I]leptin (gift of Michele Chiesi, Ciba-Geigy, Basel) as previously described (10). For competition, 0.1 µM unlabeled leptin was added in the control samples. Clones expressing the long OB-Rb isoform (L44, L46) or the short OB-Ra isoform (S29, S44) were used for further analysis.

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.


    ACKNOWLEDGMENTS
 
We wish to thank Michele Chiesi, (Ciba-Geigy, Basel) for mouse recombinant leptin, Dwayne Barber for R80 antibody, Andrew Ziemiecki for anti-Jak1 antibody, and Jeannette Holenstein for help with tissue culture.


    FOOTNOTES
 
Address requests for reprints to: Dr. Radek C. Skoda, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland.

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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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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