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Endocrinology Vol. 139, No. 9 3681-3690
Copyright © 1998 by The Endocrine Society

ARTICLES

Functional Properties of Leptin Receptor Isoforms Containing the Gln->Pro Extracellular Domain Mutation of the Fatty Rat1

Barbara A. da Silva2, Christian Bjørbæk2, Shigeo Uotani and Jeffrey S. Flier

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
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mutations of the leptin receptor have been found to cause obesity in rodents. The fa mutation that is responsible for obesity in Zucker rats is a missense mutation (269 gln->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 gln->pro and Ob-Ra269 gln->pro have decreased cell-surface expression and decreased leptin binding affinity. Ob-Rb269 gln->pro 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 gln->pro was observed for activation of egr-1-luc but only under conditions when JAK2 was coexpressed with Ob-Rb269 gln->pro. Finally, Ob-Ra269 gln->pro has an increased ability to internalize leptin but is less efficient at degrading leptin, as compared with Ob-Ra. In conclusion, both Ob-Ra269 gln->pro and Ob-Rb269 gln->pro have multiple functional defects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, the product of the ob gene, is an adipocyte-derived hormone (1) whose serum levels in the fed state correlate with total body fat mass (2). Leptin exerts a profound effect on energy homeostasis via effects on the hypothalamus (3, 4, 5, 6, 7, 8, 9, 10) and possibly on peripheral sites, as well (11, 12, 13, 14). Leptin acts via receptors that have strong sequence homology to the class 1 cytokine receptor family (15) and exist as multiple isoforms through alternative messenger RNA (mRNA) splicing (16). At least four of these isoforms contain identical extracellular and transmembrane domains, as well as unique intracellular domains of varying length (Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd). A soluble form of the receptor is also predicted to exist (Ob-Re) (16). The long form of the leptin receptor (Ob-Rb) is expressed most abundantly in the hypothalamus and may have more limited expression in one or more peripheral sites (16, 17). The dominant short form of the receptor (Ob-Ra) is expressed most highly in kidney, lung, and choroid plexus (15, 16, 17, 18, 19, 20, 21).

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 gln->pro) 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 gln->pro 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 gln->pro 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 gln->pro has been described as both normal (30, 34) or reduced (36). Cell-surface expression of Ob-Ra269 gln->pro 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 gln->pro.

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 gln->pro 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 gln->pro 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 gln->pro 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 gln->pro, with respect to receptor cell-surface expression, affinity of binding, and signaling potential. Both Ob-Rb269 gln->pro and Ob-Ra269 gln->pro 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 gln->pro and a >2.5-fold reduction for Ob-Ra269 gln->pro, compared with their wild-type counterparts. Under conditions of similar cell-surface expression, Ob-Rb269 gln->pro 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant mouse leptin was obtained from Eli Lilly (Indianapolis, IN). 125I-human leptin was obtained from New England Nuclear Life Science Products (Boston, MA). All reagents for cell culture and transfection were purchased from Life Technologies (Gaithersburg, MD). Monoclonal antibody 12 CA5 [antihemagglutinin peptide (HA)] was from Babco (Emeryville, CA). The JAK2- and phosphotyrosine-antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). The STAT3 antibody and the phospho-specific STAT3 antibody were purchased from New England Biolabs (Beverly, MA). The murine HA-STAT3 expression construct was provided by Dr. John Blenis (Harvard Medical School). JAK2 complementary DNA (cDNA) expression vector was provided by Dr. Rikiro Fukunaga (Osaka University, Osaka, Japan) and Dr. Linda Winston (Beth Israel Deaconess Medical Center). The leptin receptor antibody was generated as described by Bjørbæk et al. (47). The CMV-lacZ reporter construct was from CLONTECH (Palo Alto, CA). The egr-1-luc reporter plasmid (48) was provided by Dr. Gerd Walz (Beth Israel Deaconess Medical Center).

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 gln->pro) 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 gln->pro 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 gln->pro, as determined by these initial binding assays. In CHO cells, the binding of 125I-leptin to cells expressing Ob-Ra269 gln->pro (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 gln->pro.

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 {gamma} 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 gln->pro, or Ob-Ra269 gln->pro 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 {gamma} 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 gln->pro 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 {gamma} 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 gln->pro 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 {gamma} counter, as the TCA soluble fraction. Cells were lysed in lysis buffer C and measured in a {gamma} 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The fa mutation reduces binding affinity and cell-surface expression of both Ob-Ra and Ob-Rb isoforms
We carried out competitive binding studies with 125I-leptin and varying concentrations of unlabeled leptin in cells transiently expressing Ob-Rb, Ob-Ra, Ob-Rb269 gln->pro, or Ob-Ra269 gln->pro. Scatchard analysis was performed to obtain dissociation constant (Kd) values for each receptor. As shown in Table 1Go, 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 gln->pro 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 1Go), demonstrating that fewer mutant receptors are expressed on the cell surface under conditions of transient expression in mammalian cell lines.


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  		Table 1. Affinity and binding capacity of receptor variants

 
Leptin stimulates tyrosine phosphorylation of wild-type and mutant receptor
Members of the class 1 cytokine receptor family to which the leptin receptor belongs lack intrinsic kinase activity (50). Upon ligand binding, these receptors activate associated JAK kinases, which then phosphorylate tyrosine residues on the cytoplasmic tail of the receptor (51). To determine whether Ob-Rb269 gln->pro is capable of leptin-activated receptor phosphorylation via the JAK kinases, Ob-Rb and Ob-Rb269 gln->pro 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. 1Go (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. 1Go, 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. 1Go, upper panel), approximately 5-fold more Ob-Rb269 gln->pro protein was present in the immunoprecipitates, as determined by laser-scanning densitometry of the autoradiogram (Fig. 1Go, 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.



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  		Figure 1. Tyrosine phosphorylation of leptin receptors in COS-1 cells. The upper panel shows Western blots with antiphosphotyrosine antibodies of leptin-receptor immunoprecipitates. The lower panel shows blotting with antileptin receptor antibodies after stripping of the same membrane. Cells were transfected with empty vector DNA, Ob-Rb, or Ob-Rb269 gln->pro together with JAK2 cDNA. After 15 h of serum deprivation, cells were stimulated, or not, with 100 nM murine leptin for 10 min. Clarified lysates were immunoprecipitated with antileptin receptor antibodies and resolved by 7% SDS-PAGE.

 
Leptin-induced JAK2 phosphorylation is reduced by Ob-Rb269 gln->pro
To obtain comparable cell-surface expression of wild-type and mutant receptors, varying concentrations of cDNA encoding Ob-Rb were transiently transfected into the various cell lines (293, COS-1, or CHO cells), along with the cotransfected cDNAs, which were used in subsequent experiments (JAK2, STAT3, or egr-1). The total amount of transfected cDNA was maintained constant by addition of empty vector DNA. Six-fold less Ob-Rb cDNA was required, as compared with Ob-Rb269 gln->pro, 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 gln->pro 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 gln->pro receptor. To compare the extent of leptin-induced JAK2 phosphorylation produced by Ob-Rb vs. Ob-Rb269 gln->pro under conditions of similar cell-surface expression, 293 cells were cotransfected with JAK2 and either Ob-Rb or Ob-Rb269 gln->pro 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. 2Go, A and C). With Ob-Rb269 gln->pro, 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 gln->pro, but maximal response was much reduced, as compared with wild-type Ob-Rb (Fig. 2Go, 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.



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  		Figure 2. Tyrosine phosphorylation of JAK2 by Ob-Rb and Ob-Rb269 gln->pro in 293 cells. The upper panels show Western blots of nitrocellulose membranes probed with anti-PY antibodies. The lower panels show blotting with anti-JAK2 antibodies after stripping of the same membrane. A, Time course. Cells were cotransfected with Ob-Rb269 gln->pro or Ob-Rb using cDNA amounts that produced similar cell-surface expression, together with JAK2 cDNA. After 15 h of serum starvation, cells were stimulated with 10 nM murine leptin for 0, 2, 10, 30, or 60 min. Clarified lysates were immunoprecipitated with anti-JAK2 antibodies and resolved on 7.5% SDS-PAGE. B, Dose response. Cells were cotransfected, as above, with Ob-Rb269 gln->pro or Ob-Rb cDNAs, together with JAK2 cDNA. After 15 h of serum starvation, cells were stimulated for 5 min with 0, 0.1, 0.3, 1, or 10 nM murine leptin. Clarified lysates were immunoprecipitated with anti-JAK2 antibodies and resolved on 7.5% SDS-PAGE. C and D, Laser-scanning densitometry of the autoradiograms shown in A and B, respectively. Shown are the results of phosphorylated JAK2 (upper panel) divided with JAK2 protein (lower panel), where 100 represents the highest value obtained in each figure. Squares, Ob-Rb; triangles, Ob-Rb269 gln->pro.

 
Ob-Rb269 gln->pro has decreased capacity to induce STAT3 activation in response to leptin
Activation of STAT3 by leptin signaling through Ob-Rb has been previously shown by us and others (17, 37, 38, 39, 40, 41, 42). To compare the ability of Ob-Rb269 gln->pro 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 gln->pro 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. 3Go, 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. 3Go, 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 gln->pro; 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 gln->pro has a signaling defect.



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  		Figure 3. Phosphorylation of tyrosine 705 of STAT3 by Ob-Rb and Ob-Rb269 gln->pro in COS-1 cells. The upper panels show Western blots of nitrocellulose membranes probed with anti-phospho-specific STAT3 antibodies. The lower panels show blotting with anti-STAT3 antibodies after stripping of the same membrane. A, Time course. Cells were cotransfected with Ob-Rb269 gln->pro or Ob-Rb at cDNA amounts that produced similar cell-surface expression, together with HA-STAT3 cDNA. After 15 h of serum starvation, cells were stimulated with 10 nM murine leptin for 0, 2, 10, 30, and 60 min. Clarified lysates were immunoprecipitated with anti-HA antibodies and resolved on 7.5% SDS-PAGE. B, Dose response. Cells were cotransfected, as above, with Ob-Rb269 gln->pro or Ob-Rb cDNAs, together with HA-STAT3 cDNA. After 15 h of serum starvation, cells were stimulated for 15 min with 0, 0.1, 0.3, 1, or 10 nM murine leptin. Clarified lysates were immunoprecipitated with anti-HA antibodies and resolved on 7.5% SDS-PAGE. C and D, Laser-scanning densitometry of the autoradiograms shown in A and B, respectively. Shown are the results of phosphorylated STAT3 (upper panel) divided with STAT3 protein (lower panel), where 100 represents the highest value obtained in each figure. Squares, Ob-Rb; triangles, Ob-Rb269 gln->pro.

 
Ob-Rb269 gln->pro is unable to activate transcription of an Egr-1 promoter luciferase construct
Egr-1 is a gene whose transcription is stimulated by many growth factors and cytokines (52). To test the ability of Ob- Rb269 gln->pro to activate transcription of egr-1, an egr-1 promoter-luciferase reporter construct was used (48). Ob-Rb, Ob-Rb269 gln->pro (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 gln->pro (Fig. 4Go). 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 gln->pro (Fig. 4Go). 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).



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  		Figure 4. Activation of egr-1 gene transcription in CHO cells by Ob-Rb and Ob-Rb269 gln->pro. Cells were cotransfected with Ob-Rb, or Ob-Rb269 gln->pro, at cDNA amounts that produced similar cell-surface expression, together with egr-1-luc cDNA. After 15 h of serum starvation, cells were stimulated with either 100 nM (shown in black) or 1 nM (shown in gray) murine leptin for 8 h. Cell extracts were prepared for luciferase assays, as described in Materials and Methods. Data shows fold stimulation (basal = 0.0), and each bar represents means (± SE) of three separate experiments, each done in triplicate.

 
Coexpression of JAK2 causes constitutive ligand-independent activation of Ob-Rb269 gln->pro using the Egr-1 promoter luciferase construct
It has been suggested that Ob-Rb269 gln->pro 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 gln->pro 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 gln->pro, and leptin had no effect or little effect on egr-1 promoter activities in cells expressing either receptor (Fig. 5Go). When JAK2 was coexpressed together with Ob-Rb269 gln->pro, 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 gln->pro only induced egr-1-luc transcription 1.6-fold.



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  		Figure 5. Egr-1 gene transcription in 293 cells expressing Ob-Rb or Ob-Rb269 gln->pro with and without cotransfection of JAK2 cDNA. HEK 293 cells were cotransfected with Ob-Rb or Ob-Rb269 gln->pro at cDNA amounts that produce similar cell-surface expression, together with egr-1-luc and CMV-lacZ reporter constructs, with or without JAK2 cDNA. After 15 h of serum deprivation, cells were not stimulated (black bars) or stimulated with 1 nM leptin (gray bars) for 8 h. Cell extracts were prepared for luciferase and ß-galactosidase assays, as described in Materials and Methods. Data are depicted as relative to results obtained from the nonstimulated cells expressing Ob-Rb without cotransfected JAK2 cDNA (= 1.0 relative luciferase units). Each value is corrected for differences in transfection efficiency by normalizing the luciferase activities with the ß-galactosidase activities from the same sample. Each bar represents data from three separate experiments, each done in triplicate.

 
Ob-Ra269 gln->pro has altered capacity for leptin internalization
Members of the class 1 cytokine receptor family are capable of undergoing receptor/ligand internalization, degradation, and down-regulation (53). Ob-Ra was initially cloned from the choroid plexus (15) and has been hypothesized to transport leptin across the blood-brain barrier and/or the blood-CSF barrier. An early step in the process of receptor-mediated transport is the internalization of ligand. To test the ability of both Ob-Ra and Ob-Ra269 gln->pro to internalize leptin, we used the acid resistance method (54). CHO cells were transiently transfected with either Ob-Ra or Ob-Ra269 gln->pro cDNA at levels that produce similar cell-surface expression. The percent acid resistant leptin binding for both Ob-Ra and Ob-Ra269 gln->pro at 37 C and 4 C, is shown in Fig. 6Go. Ob-Ra269 gln->pro 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.



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  		Figure 6. Receptor-mediated leptin internalization by Ob-Ra and Ob-Ra269 gln->pro in CHO cells. Cells were transfected with Ob-Ra or Ob-Ra269 gln->pro at cDNA amounts that produced similar cell-surface expression. After 15 h of serum starvation, cells were incubated with 125I-leptin, with and without 100 nM unlabeled leptin, for 15, 60, or 120 min at either 37 C or 4 C and were washed three times with PBS. Half of the samples were then washed with acid buffer (pH 2), and the acid buffer was counted in a {gamma} counter (acid-sensitive portion), and cell-associated radioactivity was taken to be the acid resistant portion. The other half of the samples were lysed without acid wash and counted as total cell binding. Shown is the percent acid resistant radioactivity over time for Ob-Ra269 gln->pro (crosses) and Ob-Ra (squares) at 37 C, and Ob-Ra269 gln->pro (triangles) and Ob-Ra (circles) at 4 C. The data represents the mean (± SE) of at least three separate experiments, each done in triplicate.

 
Ob-Ra269 gln->pro degrades internalized leptin less efficiently than wild-type Ob-Ra
To evaluate receptor-mediated degradation of leptin, Ob-Ra or Ob-Ra269 gln->pro 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. 7AGo. 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 gln->pro degrade leptin at similar rates, with approximately 20% of cell associated 125I-leptin being degraded at 60 min. However, because Ob-Ra269 gln->pro internalizes more leptin over the same time course (Fig. 6Go), 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. 7BGo, in cells expressing either Ob-Ra or Ob-Ra269 gln->pro, each of the lysosomal inhibitors markedly reduced the amount of TCA-soluble 125I-leptin present in the medium (~15-fold reduction after 120 min).



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  		Figure 7. Leptin degradation mediated by Ob-Ra and Ob-Ra269 gln->pro. CHO cells were transfected with Ob-Ra or Ob-Ra269 gln->pro at cDNA amounts which produced similar cell-surface expression. After 15 h of serum starvation, cells were incubated with 125I-leptin with and without 100 nM unlabeled leptin, at 4 C for 2 h. After washing, the cells were further incubated at 37 C for 0, 30, 60 or 120 min The medium was then incubated with TCA to determine leptin degradation, as described in the methods. Total cell associated radioactivity was also determined. Represented in A is the percent TCA soluble over time for Ob-Ra (squares) and Ob-Ra269 gln->pro (squares). Each bar represents the mean (± SE) of three separate experiments, each done in triplicate. In B, the percent TCA soluble for Ob-Ra and Ob-Ra269 gln->pro at 120 min without inhibitors (dotted bars), or treated with 0.1 mM chloroquin for 120 min (black bars), or treated with 10 mM methylamine (striped bars) for 120 min, as described in Materials and Methods. Each bar represents the mean (± SE) of three separate experiments, each done in triplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Leptin, the product of the ob gene (1), exerts important effects on energy homeostasis (3, 4, 5, 6, 7, 8, 9, 10), and lack of leptin causes severe obesity in both rodents and humans. With the cloning of the leptin receptor gene, it became evident that mutations at this locus were responsible for severe obesity, as well (56). In db/db mice, a mutation within the leptin receptor gene causes truncation of the intracellular domain of the long form of the leptin receptor (Ob-Rb), replacing it with the short form, Ob-Ra (16, 27), clearly demonstrating that the Ob-Rb isoform is obligatory for normal energy homeostasis. Koletsky rats, also leptin resistant and obese, have a nonsense mutation that truncates the receptor in the extracellular domain (31), creating a rat that is apparently null for all receptor forms. Zucker rats have a missense mutation, causing a single amino acid substitution (ObR269 gln->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 gln->pro; and cell-surface expression, binding affinity, and capacity for ligand internalization and degradation of Ob-Ra269 gln->pro.

Reduced cell-surface expression has been previously reported in Ob-Rb269 gln->pro (30, 34, 35) and Ob-Ra269 gln->pro (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 gln->pro clearly defines this as a second defect responsible for some of the impaired signaling by the receptor.

We have defined Ob-Rb269 gln->pro 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 gln->pro. 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 gln->pro 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 gln->pro 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 gln->pro to activate intracellular signal transduction is independent of the reduced leptin-binding affinity of Ob-Rb269 gln->pro. 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 gln->pro 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 gln->pro 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 gln->pro. Two functional consequences of Ob-Ra269 gln->pro were noted. First, as assessed by the acquisition of acid-resistant cell-associated leptin, Ob-Ra269gln->pro 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 gln->pro 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 gln->pro in the choroid plexus and/or the brain capillary microvessels. Another possibility is that Ob-Ra269 gln->pro 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 gln->pro 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 gln->pro, with respect to receptor phosphorylation, or phosphorylation of JAK2 or STAT3. We did, however, observe constitutive ligand-independent activity for Ob-Rb269 gln->pro 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 gln->pro 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 gln->pro and Ob-Rb269 gln->pro 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
 
1 This work was supported by NIH Grant-DK-R37 28082 and a grant from Eli Lilly (both to J.S.F.). Back

2 These two authors have contributed equally. Back

Received January 2, 1998.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
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