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Molecular Endocrinology, doi:10.1210/me.2003-0078
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Molecular Endocrinology 18 (1): 150-161
Copyright © 2004 by The Endocrine Society

Functional Analysis of Leptin Receptor Activation Using a Janus Kinase/Signal Transducer and Activator of Transcription Complementation Assay

Lennart Zabeau, Delphine Defeau, José Van der Heyden, Hannes Iserentant, Joël Vandekerckhove and Jan Tavernier

Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research (VIB09), Ghent University, Ghent, Belgium

Address all correspondence and requests for reprints to: Jan Tavernier Ph.D., Flanders Interuniversity Institute for Biotechnology, VIB09, Department of Medical Protein Research, Ghent University, Faculty of Medicine and Health Sciences, Baertsoenkaai 3, B-9000 Ghent, Belgium. E-mail: Jan.Tavernier{at}rug.ac.be.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The leptin receptor (LR), a member of the class I cytokine receptor family, is composed of a single subunit. Its extracellular domain consists of two so-called cytokine receptor homology domains, separated by an Ig-like domain, and two additional fibronectin type III modules. Requirements for LR activation were examined using a complementation strategy. Two LR mutants, LR-FFY-{Delta}box 1 and LR-F3, deficient in Janus kinase or signal transducer and activator of transcription (STAT) activation, respectively, were only able to generate a STAT3-dependent signal when coexpressed. Based on the requirements for Janus kinase/STAT signaling, and on the lack of complementation with similar receptor constructs, but containing the extracellular domain of the homodimeric erythropoietin receptor, this observation can be explained only by higher order LR clustering. Using a panel of deletion mutants we were able to define a role for the cytokine receptor homology 1 and Ig-like domains in leptin signaling. Moreover, we demonstrate a nonredundant function for the individual receptor chains within the homomeric LR complex. Based on these data, we propose a possible model for LR clustering.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LEPTIN, THE PRODUCT of the ob gene (1), is a key player in energy homeostasis and body weight control. It is a 16-kDa circulating protein with a structure resembling 4-{alpha}-helical bundle cytokines (2). It is secreted mainly by adipose cells, and the circulating level of this hormone strongly correlates with white adipose tissue mass. Leptin regulates energy expenditure and food intake by activating its receptor in certain nuclei of the hypothalamus (3, 4, 5). Loss-of-function mutations within the genes for leptin (1, 6) or for its receptor (7, 8, 9) cause complex syndromes characterized by morbid obesity, hyperglycemia, hyperinsulinemia, and reduced fertility. Numerous data suggest that leptin also has direct effects on tissues outside the brain, which may help explain its role in basal metabolism, reproduction, and hematopoiesis (10, 11, 12, 13).

The leptin receptor (LR) is composed of a single subunit, encoded by the db gene (7, 8, 14, 15), and is a member of the class I cytokine receptor family. It contains two so-called cytokine receptor homology (CRH) modules, which are formed by two barrel-like domains, each 100 amino acids (aa) in length, and which resemble the fibronectin type III (FN III) and Ig folds. Two conserved disulfide bridges are found in the N-terminal domain, whereas a WSXWS motif is characteristic for the C-terminal domain. Both LR CRH modules are separated by an Ig-like domain and are followed by two membrane-proximal FN III domains (Fig. 1Go). Using a panel of deletion and substitution mutants, Fong et al. (16) showed that the membrane-proximal CRH domain is necessary and sufficient for leptin binding, whereas the two FN III domains are not involved in ligand binding but are needed for receptor activation. Thus far, six isoforms of the LR generated by alternative mRNA splicing have been recognized and termed LRa through LRf. The LR long form (LRlo or LRb) has an intracellular chain length of 302 aa and is the only isoform capable of efficient signaling. It is this LRlo isoform that is primarily expressed in specific nuclei of the hypothalamus (17, 18, 19), but expression at lower levels in other cell types has also been observed (20, 21, 22). A second isoform, LRa, is a variant lacking most of the cytosolic domain. This LR short form (LRsh) is much more widely expressed, often at higher levels compared with LRlo, e.g. in the choroid plexus, kidney, lung, and liver (23).



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  		Fig. 1. Expression of Wild-Type LR and LR Mutants in HEK293T Cells

A, Schematic representation of the different LR constructs used. Wild-type LR, the two signaling-deficient mutants LR-F3 and LR-FFY-{Delta}box 1, and the deletion variants {Delta}CRH1, and {Delta}CRH1,{Delta}Ig are shown. B, LR Western blot analysis. Lysates of HEK293T cells transfected with 2 µg plasmids encoding 1) LR; 2) LR {Delta}CRH1; 3) LR {Delta}CRH1,{Delta}Ig; 4) LR-F3; 5) LR-F3 {Delta}CRH1; 6) LR-F3 {Delta}CRH1,{Delta}Ig; 7) LR-FFY-{Delta}box 1; 8) LR-FFY-{Delta}box 1 {Delta}CRH1; 9) LR-FFY-{Delta}box 1 {Delta}CRH1,{Delta}Ig; 10) empty vector, were separated on a 7.5% polyacrylamide gel, and blotted onto a nitrocellulose membrane. LR and derived mutants were visualized using a polyclonal antibody directed against residues 942–953 of the cytoplasmic domain of the receptor and a horseradish peroxidase-labeled secondary antibody. C, Analysis of LR cell surface expression using a SEAP binding assay. Transfected HEK293T cells (same cells as in B), were incubated 48 h after transfection with a leptin-SEAP chimeric protein for 90 min at room temperature with NaN3. As a control for aspecific binding, wild-type leptin was added to a final concentration of 2 µg/ml. After inactivation of endogenous phosphatase activity, bound SEAP activity was determined using the CSPD substrate. Bars shown represent mean values, and SD values of triplicate measurements. Results are representative for five independent experiments.

 
Like all members of the class I cytokine receptor family, the LR has no intrinsic kinase activity, and uses cytoplasmic-associated Janus kinases (JAKs) for intracellular signaling. Two short conserved sequences in the membrane-proximal region (box 1 and a putative box 2) are thought to mediate binding and activation of the JAK kinases [i.e. JAK2 in the case of the LRlo (24)]. In a generally accepted model, leptin binding leads to formation of a receptor complex, allowing activation of JAK2 by cross-phosphorylation. Activated JAK2 then rapidly phosphorylates several tyrosine residues in the cytosolic domain of the receptor (i.e. in the case of the mouse LRlo, tyrosines at positions 985, 1077, and 1138). Phosphorylated tyrosine 1138 provides a binding site for STAT3 (signal transducer and activator of transcription 3) (25). STATs themselves are also substrates for JAKs and homo- or heterodimerize upon phosphorylation, translocate to the nucleus, and modulate transcription of target genes. The STAT molecule primarily involved in leptin signaling is STAT3. Leptin-mediated STAT3 activation has been confirmed by Vaisse et al. (26) in the hypothalamus of wild-type and ob/ob mice after injection of the leptin. In cell lines, however, leptin-mediated STAT1 and STAT5B activation was also shown (27). The other mouse LRlo cytoplasmic tyrosine residues (on position 985 and 1077) are involved in negative feedback of LR signaling, by recruitment of the SH2-containing tyrosine phosphatase-2 (25, 28), or suppressor of cytokine signaling-3 (29, 30, 31), or in coupling to other signaling molecules such as MAPK (32) and phosphoinositide 3 kinase (33).

The model in which the LR becomes activated upon mere ligand-induced dimerization, i.e. one leptin molecule clusters two receptor chains, has been questioned (34, 35). To address this issue, two signaling-deficient LR mutants were constructed. Based on complementation of these two mutants, we here provide direct evidence for leptin-induced higher order clustering of its receptor. Using a panel of deletion mutants, we further define the role of individual LR subunits and of LR extracellular subdomains in LR complex formation and functioning.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction and Expression of LR Mutants
To study the requirements for LR activation in more detail, we constructed two types of signaling-deficient LR mutants. In the LR-F3 mutant, all three cytoplasmic tyrosine residues were mutated to phenylalanine, thereby blocking recruitment of STAT3 molecules to the activated receptor. Signaling by the second mutant, LR-FFY-{Delta}box 1, was abolished by two proline to serine mutations at positions 876 and 878 in the box 1 motif. These mutations prevent binding and activation of the associated JAK kinases (36). To reduce negative feedback by regulators like SH2-containing tyrosine phosphatase-2 or suppressor of cytokine signaling-3, and activation of other signaling pathways such as activation of MAPK and phosphoinositide 3 kinase, we also introduced tyrosine to phenylalanine mutations at positions 985 and 1077. The only intact tyrosine, Y1138, in this mutant allows activation of and signaling via STAT3. This LR-FFY mutant shows an approximately 2- to 3-fold increased induction of STAT3-dependent reporter activity when compared with the wt LR (37). A schematic representation of both mutant receptors is given in Fig. 1AGo. Also shown is the expression of both mutants upon transfection in human embryonic kidney (HEK)293T cells, using Western blot analysis (Fig. 1BGo) and a binding assay with a leptin-secreted alkaline phosphatase (SEAP) chimera (Fig. 1CGo). Both data sets illustrate that mutations in the cytoplasmic portion of the receptor have no significant effect on the overall expression and cell surface anchoring of the mutant receptors.

To address the role of the membrane distal CRH (also referred to as CRH1) and Ig-like domains in LR signaling, a panel of deletion mutants was constructed. In the first set, we deleted this CRH1 module in the wild-type LR receptor (LR {Delta}CRH1) and in both signaling-deficient mutants (LR-F3 {Delta}CRH1 and LR-FFY-{Delta}box 1 {Delta}CRH1). Western blot analysis and leptin-SEAP binding data on transfected HEK293T cells illustrate that this deletion results in a minor, but significant, decrease in LR cell surface expression (see Fig. 1Go, B and C). In a similar way, receptors lacking both the CRH1 and Ig-like domains (LR {Delta}CRH1,{Delta}Ig; LR-F3 {Delta}CRH1,{Delta}Ig; and LR-FFY-{Delta}box 1 {Delta}CRH1,{Delta}Ig) were constructed. In contrast, additional deletion of the Ig-like domain may lead to a slightly enhanced LR expression (Fig. 1Go). Binding of leptin-SEAP was performed at room temperature, and NaN3 was added to prevent internalization of the ligand-bound receptors. Similar results were obtained when the binding experiment was done on ice (data not shown). It is of note that the expression patterns are in the same range for each set of extracellular deletion mutants, irrespective of the mutations in the cytosolic domains.

Complementation with Signaling-Deficient LR Mutants
We next analyzed leptin-induced signaling via the mutant LR constructs upon (co)-transfection in HEK293T cells. As a read out, the STAT3-responsive rPAP1 (rat pancreatitis-associated protein 1)-luciferase reporter construct was used as described previously (31). Using a substractive cloning strategy, rPAP1 was identified as a late target gene upon leptin stimulation (38). As expected, both LR-F3 and LR-FFY-{Delta}box 1 mutant receptors were inactive, because neither STAT3-dependent activation of a rPAP1-luciferase reporter construct nor STAT3 phosphorylation could be observed (Fig. 2AGo). Interestingly, when both receptor mutants were coexpressed, we observed functional complementation leading to a clear increase (<=70-fold) of the leptin-induced rPAP1-luciferase reporter activation (Fig. 2AGo) and a concomitant marked STAT3 phosphorylation (Fig. 2BGo). The complementation-dependent signal was less sensitive when compared with the LR-FFY, a control receptor also not subjected to negative feedback (31): approximately 10-fold higher leptin concentrations were required, and a 70% decrease in plateau value was also observed (Fig. 2CGo). This is not unexpected because inevitably, cotransfection leads to the formation of several inactive LR complexes (e.g. composed of only one mutant receptor). Importantly, the decrease in sensitivity is limited, and all experiments described below were performed using physiological leptin concentrations (10 ng/ml, or as indicated).



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  		Fig. 2. Complementation of JAK/STAT Signaling with LR Mutants

A, Signaling properties of LR mutants LR-F3 and LR-FFY-{Delta}box 1. Plasmids encoding the LR mutants LR-F3 and LR-FFY-{Delta}box 1 were transiently transfected, individually (2 µg) or in combination (1 µg each), in HEK293T cells. The pXP2d2-rPAP1-luci reporter construct was cotransfected to measure STAT3 activation. Transfected cells were either stimulated with leptin for 24 h, or were left unstimulated (NS). Luciferase measurements were performed in triplicate, and bars and error bars represent mean values and SD, respectively. Results are representative for at least four independent transfection experiments. B, JAK2 and STAT3 phosphorylation. Similar transfections were performed to check for phosphorylation of JAK2 and STAT3. To detect JAK2 phosphorylation and expression, 0.01 µg pRK5-JAK2 was cotransfected. Serum-starved cells were stimulated with 100 ng/ml leptin for 10 min, or were left unstimulated. Lysates were blotted onto a nitrocellulose membrane and analyzed using phospho-specific antibodies. Total amounts of JAK2 and STAT3 are also shown. C, Comparison of wild-type vs. complementation signaling. HEK293T cells were transfected with either the LR-FFY (2 µg, solid bars), or a combination of the LR-F3 and LR-FFY-{Delta}box 1 mutants (1 µg each, open bars), along with the rPAP1-luciferase reporter. Stimulations were with a serial dilution of leptin as indicated. Mean luciferase values and error bars of triplicate measurements are plotted. Results are representative for three independent transfection experiments.

 
Because two LR-F3 chains are needed for reciprocal activation of JAK kinases, and because at least one LR-FFY-{Delta}box 1 chain with a phosphorylated tyrosine residue is necessary for the recruitment of STATs, our complementation data may be explained by formation of a higher order LR complex.

No Complementation Observed with Erythropoietin Receptor (EpoR)-LR Chimeras
To test this hypothesis, we constructed EpoR-LR chimeras containing similar LR-F3 and LR-FFY-{Delta}box 1 mutations, but with the extracellular part of the LR replaced by that of the EpoR. It is generally accepted that the EpoR functions as a homodimer. As observed for the full-length LR mutants, cytoplasmic mutations in the EpoR chimeras did not significantly alter expression, as could be shown by immunoprecipitation with an antibody directed against the extracellular domain of the EpoR, and Western blot analysis with an anti-LR antibody (data not shown). As shown in Fig. 3Go, neither EpoR chimera separately, nor in combination, were able to generate a STAT3-dependent signal even at an erythropoietin (Epo) concentration of 50 ng/ml. As a positive control, EpoR-LR, with an intact box 1 motif and the critical tyrosine 1138, showed a clear Epo-mediated activation of the rPAP1-luciferase reporter, illustrating that simple dimerization of the LR cytoplasmic domains was sufficient for JAK2 and STAT3 activation. It is of note that stimulation of cells expressing the Epo-LR chimera with 50 ng/ml Epo, the concentration used to test complementation signaling, results in an optimal STAT3-dependent signal. Together these data show that receptor dimerization does not allow complementation and lends further support to the LR being a higher order complex. Furthermore, the behavior of the EpoR chimeras most likely illustrates that the formation of higher order LR complexes is mediated by the extracellular domain of the receptor.



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  		Fig. 3. Signaling Properties of EpoR-LR Chimeric Mutants

To determine whether complementation occurs when the extracellular part of the LR is replaced by that of the EpoR, plasmids (2 µg) encoding EpoR-LR (1 ), EpoR-LR-F3 (2 ), EpoR-LR-FFY-{Delta}box 1 (4 ), or a combination (1 µg plasmid for each) of the two plasmids encoding the signaling-deficient EpoR chimeric mutants (3 ), were cotransfected with the rPAP1-luciferase reporter in HEK293T cells. Stimulation was with 50 ng/ml Epo (solid bars) or cells were left unstimulated (open bars). Results presented are the mean values and SD for three independent experiments. Inset, To measure the biological activity of Epo on cells expressing the EpoR-LR chimera, the receptor was transfected along with the pXP2d2-rPAP1-luci reporter construct. Cells were stimulated with a serial dilution of Epo as indicated. Mean luciferase values and error bars of triplicate measurements are plotted. Results are representative for three independent experiments.

 
Role of the CRH1 and Ig-Like Modules in LR Activation and Clustering
We next investigated the role of specific subdomains of the LR extracellular part in LR binding, clustering, and activation. Deletion of the CRH1 module in the wild-type receptor (LR {Delta}CRH1) results in a limited, but clear, decrease in receptor cell surface expression and on leptin binding (both shown in Fig. 1Go). As with the full-length mutants, the {Delta}CRH1 deletion in LR-F3 and LR-FFY-{Delta}box 1 mutants did not affect the ability to signal in the complementation assay (Fig. 4AGo), excluding a strict requirement for this module in higher order clustering of the LR. However, a lower leptin-induced response was clearly observed when the CRH1 domain was deleted (Fig. 4BGo). Similar data were obtained with receptor constructs lacking cytosolic mutations (Fig. 4EGo).



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  		Fig. 4. Involvement of CRH1 and Ig-Like Domains in LR Signaling

A, Signaling via LR-F3 {Delta}CRH1 and LR-FFY-{Delta}box 1 {Delta}CRH1. Both mutant receptors were transfected alone (2 µg) or in combination (1 µg each), and transfected HEK293T cells were treated with leptin, or were left unstimulated (NS). Mean luciferase values and error bars of triplicate measurements are plotted. B, Deletion of CRH1 leads to a lower leptin signal. Combinations of LR-F3 + LR-FFY-{Delta}box 1 (solid bars) and LR-F3 {Delta}CRH1 + LR-FFY-{Delta}box 1 {Delta}CRH1 were transiently transfected in HEK293T cells. Stimulations were overnight with a serial dilution of leptin (as indicated). Mean luciferase values and error bars of triplicate measurements are plotted. C, Effect of the amount transfected DNA on LR expression. Different quantities of DNA encoding LR-F3 and LR-FFY-{Delta}box 1 or LR-F3 {Delta}CRH1 and LR-FFY-{Delta}box 1 {Delta}CRH1 were transfected in HEK293T as indicated. Leptin binding was measured as described in Fig. 1Go. Bars represent the mean values and SD values of triplicate chemiluminescence measurements. D, Complementation analysis of full-length vs. {Delta}CRH1 signaling-deficient mutants. HEK293T cells were transfected with 1 µg (solid circles), 0.75 (open circles), 0.5 µg (solid squares), 0.25 µg (open squares) DNA encoding full-length LR-F3 and LR-FFY-{Delta}box 1, or 1 µg (solid triangles) LR-F3 {Delta}CRH1 and LR-FFY-{Delta}box 1 {Delta}CRH1. Cells were stimulated overnight with a serial dilution of leptin, as indicated. E, Role for the LR Ig-like domain in leptin signaling. Signaling via the wild-type LR and two deletion variants thereof, LR {Delta}CRH1 and LR {Delta}CRH1,{Delta}Ig, were analyzed in HEK293T cells as described. Bars represent mean luciferase counts of triplicate measurements. F, JAK2 and STAT3 phosphorylation. Phosphorylated and total amounts of JAK2 and STAT3 proteins were determined as described in Fig. 2Go.

 
We next analyzed whether CRH1 deletion reduced signaling only as a consequence of lowered LR cell surface expression. In a titration experiment, different amounts of DNA encoding full-length LR-F3 and LR-FFY-{Delta}box 1 (1, 0.75, 0.5, and 0.25 µg each) were cotransfected in HEK293T cells. Leptin-dependent signaling in the transfected cells was measured using the rPAP1-luciferase reporter and compared with cells cotransfected with 1 µg LR-F3 {Delta}CRH1 and 1 µg LR-FFY-{Delta}box 1 {Delta}CRH1. As expected, decreasing the amount of transfected DNA caused reduced leptin binding (Fig. 4CGo) and signaling (Fig. 4DGo). Clearly, the combination LR-F3 and LR-FFY-{Delta}box 1 showed at least an equal (when 0.5 µg DNA was used) or a significantly higher response (0.75 µg) when compared with the LR-F3 {Delta}CRH1 and LR-FFY-{Delta}box 1 {Delta}CRH1 combination (1 µg transfected), even at lower LR cell surface expression levels (Fig. 4CGo). These results indicate that the membrane-distal CRH1 also contributes to optimal LR activation.

Interestingly, additional deletion of the Ig-like domain in the wild-type LR completely abolished STAT3-dependent activation of the rPAP1-luciferase reporter (Fig. 4EGo). Western blot analysis clearly illustrates that this is due to the inability of the LR {Delta}CRH1,{Delta}Ig variant to activate JAK2, and this in strong contrast to LR and LR {Delta}CRH1 (Fig. 4FGo). Thus far, the function of this Ig-like domain remained unexplored and was not yet related to LR clustering and/or signaling.

Different Roles for Receptor Chains in an Activated LR Complex
To further define structural requirements for LR activation, different combinations of the two signaling-deficient mutants, and deletion variants thereof, were tested. In Fig. 5Go, we compared the combined expression of LR-F3 and LR-FFY-{Delta}box 1 {Delta}CRH1,{Delta}Ig with the LR-F3 {Delta}CRH1,{Delta}Ig and LR-FFY-{Delta}box 1 combination. The data showed that leptin-dependent STAT3 activation occurred only when a full-length LR-F3 mutant was expressed (Fig. 5AGo). It is of note that the drop in luciferase activity at very high leptin concentrations (10 µg/ml) was not reproducible. Combined deletion of the CRH1 and Ig-like domains in LR-F3 completely abolished signaling, even at leptin concentrations of 10 µg/ml (Fig. 5BGo). As with the wild-type receptor lacking both CRH1 and Ig-like domain, the lack of rPAP1 promoter activation can be explained by the total loss of JAK2 phosphorylation and activation. Taken together, these data confirm that the presence of an intact Ig-like module is necessary for signaling and, more specifically, for activation of the JAK kinases. Moreover, it appears that Jak2 activation is only possible when the Ig domain and the box 1 region are on the same receptor.



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  		Fig. 5. The Role of the Individual Receptor Chains in the LR Complex Is Not Redundant

HEK293T cells were transfected with the plasmid combinations (each 1 µg transfected) LR-F3 + LR-FFY-{Delta}box 1 {Delta}CRH1,{Delta}Ig (A) and LR-F3 {Delta}CRH1,{Delta}Ig + LR-FFY-{Delta}box 1 (B). The pXP2d2-rPAP1-luci reporter construct was cotransfected to measure STAT3 activation. Cells were stimulated overnight with a serial dilution of leptin as indicated. Luciferase measurements were as described earlier, and bars represent mean values. JAK2 (0.01 µg pRK5-JAK2 was cotransfected) and STAT3 phosphorylated and total amounts are shown as insets. Experimental procedures were as described in Fig. 2Go. A schematic representation of the mutant receptor combinations used is shown on the right. Data are representative for four independent transfection experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The receptor for leptin, an adipose tissue-derived cytokine involved in body weight homeostasis, was cloned in 1995 (14). In contrast to other well-studied cytokine receptors, i.e. the homodimeric erythropoietin and GH receptors, the granulocyte-colony stimulating factor receptor, or the more complex glycoprotein 130 (gp130)-based receptor family, mechanisms of LR clustering and activation remain largely unknown. In this study, we examined the structural requirements for LR activation using a novel complementation strategy based on two crucial, distinct steps in LR signaling, i.e. JAK2 activation and STAT3 recruitment. Two LR mutants, LR-F3, unable to bind and activate STAT factors, and LR-FFY-{Delta}box 1, deficient in JAK activation, only generate a STAT-dependent signal when coexpressed (Fig. 2Go). It could not be excluded a priori that the observed effect is due to mobility of the JAK kinases. This model would imply that upon stimulation of the LR-F3 mutant, the activated JAK kinases might dissociate from the receptor and phosphorylate in trans the tyrosine 1138 in the LR-FFY-{Delta}box 1 receptor, leading to subsequent STAT3 activation. To exclude this possibility, we analyzed the behavior of EpoR-LR mutants. Several lines of evidence argue for the active state of the EpoR being a dimer: 1) the constitutively active R129C EpoR mutant was found to exist as Epo-independent disulfide-linked dimers when expressed in cells (39); 2) biophysical studies identified two interaction sites on Epo for EpoR extracellular domain monomers (40); and 3) crystal structures of the EpoR extracellular domain in complex with Epo (41), with an Epo-mimetic (42), or with an Epo-antagonistic peptide (43), all showed a 1:2 stoichiometry. Substitution of the extracellular part of the mutant LRs by that of the EpoR led to a complete loss of complementation (Fig. 3Go). Taking into account the inherent requirements for JAK/STAT signaling, i.e. two JAK activating receptors and at least one STAT recruitment site, and the differential behavior of the EpoR-LR mutant chimeras, the observed complementation can be explained only by assuming a higher order LR clustering. It is noteworthy that the EpoR-LR chimera with intact JAK/STAT activation motifs generate a clear STAT3-dependent signal (Fig. 3Go), implying that Epo binding leads to correct positioning of JAKs, allowing their activation and subsequent STAT3 recruitment.

Leptin-dependent signaling by functional complementation of LR mutants is less efficient when compared with LR-FFY signaling (Fig. 2CGo). This is most likely due to the formation of noncomplementing and therefore inactive mutant LR complexes. Compared with the wild-type situation, here mimicked by the LR-FFY mutant, the decrease in sensitivity is rather moderate. Because physiological leptin concentrations can be used (leptin levels in healthy people vary around 5 ng/ml), data obtained from the complementation assays reflect normal leptin-LR interactions.

Higher order clustering of the LR has been suggested previously by White et al. (34). This may help explain why the LRlo is only moderately sensitive to the presence of excess LRsh. Despite the presence of a box 1 motif, this short isoform is unable to activate JAK2 at physiological intracellular kinase levels (44) and is hence signaling deficient. Two groups showed independently that, next to the box 1 motif, the intracellular residues 31–36 in the long form are critical for mediating JAK2 activation (44, 45). These residues are absent in the LR short form. LRsh is often coexpressed with LRlo, accounting for up to 95% of LR mRNA in many tissues (7) and may function in leptin clearance or transport through the blood-brain barrier (14). Our findings support oligomeric clustering (i.e. dimerization of dimers) of the LR and, given the presence of the additional N-terminal CRH1, even higher order clustering may occur. Obviously, in more complex receptor configuration, the presence of LRsh variants is less likely to completely block signaling. Alternatively, differential sorting of the LR short and long isoforms, perhaps due to their different ability to bind JAK2, could also explain this lack of interference (34).

Given the structural complexity of the extracellular domain of the LR, very few data related to the function of individual structural modules are available to date. A rather unique feature of the LR within the family of cytokine receptors is the presence of two distinct CRH modules. Other receptors containing two CRH modules are the ßC chain, shared in the receptors for IL-3, IL-5, and granulocyte-macrophage colony stimulating factor, the leukemia inhibitory factor receptor (LIFR), and thrombopoietin receptor. Crystallographic structure analysis of the ßC extracellular domain, consisting of two adjacent CRH modules, provides evidence that the receptor is expressed at the cell surface as an interlocked homodimer, even in the absence of ligand (46). Here, the G strand of the N-terminal domain of the membrane-distal CRH module interacts with the N-terminal domain of the membrane-proximal CRH of the other partner in the dimer and vice versa. The role for the CRH modules in the LIFR, now separated by an Ig-like domain, is less well defined. Both CRH modules are involved in ligand binding, while the membrane-proximal CRH is also involved in receptor dimerization (47). He et al. (48) showed that membrane-distal CRH of LIFR associates in vitro with the soluble ciliary neurotrophic factor receptor, even in the absence of ciliary neurotrophic factor. Here we show that the membrane distal CRH1 domain of the LR is not strictly required for wild-type LR signaling or for higher order clustering of the receptor, because LR-F3 and LR-FFY-{Delta}box 1 mutants lacking this domain are still able to generate a STAT3-dependent signal in a complementation setup (Fig. 4AGo). However, a clearly reduced signal and a marked decrease in JAK2 and STAT3 phosphorylation are observed upon deletion of CRH1 when compared with the full-length receptor (Fig. 4Go, B, E, and F). This effect can be explained by both lowered cell surface expression (Fig. 1Go) and reduced signaling capacity (Fig. 4Go, C and D). Two possible roles for the CRH1 can be put forward: 1) CRH1 provides further clustering of activated core LR complexes (see below), thereby enhancing the local concentrations of signaling components. We recently showed that antibody-induced formation of higher order clustering of the IL-5R potentiates signaling (49). 2) Alternatively, the CRH1 domain may also play a role in sorting or anchoring the LR to distinct membrane regions, such as lipid rafts or caveolae, thereby enhancing leptin responsiveness. So far, however, no evidence for expression of the LR in specialized membrane subdomains has been obtained. Therefore, additional studies are required to establish the precise role of the CRH1 module.

Combined deletion of both the CRH1 and the Ig-like domains in the wild-type LR led to a complete loss of JAK2 phosphorylation and concomitant STAT3- dependent signaling (Fig. 4EGo). Expression and membrane anchoring of the resulting LR mutant is comparable, however, to that of the full-length receptor (Fig. 1Go). A role for the Ig-like domain related to ligand binding and receptor activation is well established in the granulocyte colony stimulating factor receptor (50, 51), the LIFR (52, 53), and gp130 (54, 55), but was not yet shown for the LR.

Based on 1) the observed complementation data, 2) the postulated involvement of the Ig-like domain, and 3) the lack of a strict requirement for CRH1 in signaling, we propose a model wherein leptin functions as a trivalent ligand (Fig. 6BGo). Two leptin binding sites (sites I and II) interact with two membrane-proximal CRH2 modules of two distinct LR subunits, and residues belonging to a third site (site III) interact with the Ig-like domain of a third LR chain (Fig. 6BGo).



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  		Fig. 6. Model for the LR Complex

A schematic representation of the IL-6 receptor (A) and of the LR complexes (B) is shown. Panels C and D show receptor complexes composed of the LR-F3 + LR-FFY-{Delta}box 1 {Delta}CRH1,{Delta}Ig (C) and LR-F3 {Delta}CRH1,{Delta}Ig + LR-FFY-{Delta}box 1 (D) receptor combination, illustrating the functional asymmetry within the LR complex (see arrow). IL-6R{alpha}, IL-6 receptor {alpha}-chain; Ig, Ig-like domain; I, II, and III, putative interaction sites on the ligand.

 
This model strongly resembles that of receptor complexes in the gp130-binding cytokine family. Mutagenesis and binding studies have indicated that these cytokines interact via three distinct binding epitopes with their receptor subunits (56, 57, 58, 59, 60, 61). This model was recently supported by data from the crystal structure of Kaposi’s sarcoma-associated Herpes virus IL-6 in a complex with the extracellular part of gp130 (54). Two vIL-6 molecules bind to two gp130 CRH2 modules via their site I and to two gp130 Ig-like domains via their site III, leaving the sites II available for interaction with the specific IL-6R{alpha} chains (Fig. 6AGo).

At this moment, we cannot exclude the possibility that additional ligand-independent receptor-receptor interactions can contribute to higher order clustering and that one of the binding sites (I, II, or III) may not be present. For example, homotypic CRH2-CRH2 interactions could functionally replace interaction sites I or II in the complex. To date, very few data concerning the structure-function correlation of the LR are available. However, this model can help explain the unexpected finding that signaling strongly differs when the combinations LR-F3 + LR-FFY-{Delta}box 1 {Delta}CRH1,{Delta}Ig and LR-F3 {Delta}CRH1,{Delta}Ig + LR-FFY-{Delta}box 1 are compared (Fig. 5Go). Leptin-induced complex formation in both combinations is most likely identical because the same extracellular receptor parts (full length + {Delta}CRH1,{Delta}Ig) and similar cytosolic domains are used. According to the model, simultaneous binding of leptin to one LR membrane-proximal CRH2 and to the Ig-like domain of a second LR chain leads to clustering of both receptors in such a spatial configuration that the JAKs become properly orientated allowing their cross-phosphorylation and activation (illustrated by the arrow in Fig. 6CGo). Much in contrast, recruitment of a JAK-associated receptor (in this case, LR-F3 or LR-F3 {Delta}CRH1,{Delta}Ig) by the putative site II, does not lead to JAK activation (Fig. 6DGo). This underscores the critical role for the Ig-like domain in JAK activation and explains why a LR lacking both CRH1 and Ig-like modules is not able to generate a STAT3-dependent signal (Fig. 4BGo). This strict requirement for JAK orientation is in marked contrast to STAT activation, because deletion of the CRH1 and Ig-like domains in LR-FFY-{Delta}box 1 has no major effect on signaling. That precise JAK positioning is a prerequisite for efficient signaling is in line with recent reports showing that introduction of one to four alanine residues in the {alpha}-helical transmembrane domain or juxtamembrane intracellular region dramatically affects JAK activation (62, 63). The nonredundant use of individual receptor subunits within a homomeric receptor complex, however, is a novel and unexpected finding and could only be revealed thus far using this complementation strategy. It will be of interest to apply this approach to other receptor systems, including heteromeric cytokine receptors.

In summary, we have demonstrated the use of a novel JAK/STAT complementation assay in unraveling structural requirements for LR complex formation and activation. We have provided evidence for 1) higher order LR complex formation, 2) a role for the CRH1 in optimal leptin receptor activation, 3) a critical role for the Ig-like domain in leptin binding and JAK activation, and 4) a functional nonredundant role for individual LR chains within the homomeric LR complex.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Growth Factors and Antibodies
Recombinant mouse leptin and human erythropoietin (Epo) were purchased from R&D Systems (Minneapolis, MN). Typical stimulations were performed with 100 ng/ml leptin (= 6.25 nM), or 50 ng/ml Epo (= 2.5 nM), unless indicated otherwise.

A peptide was synthesized according to the primary sequence of the mouse LR at position aa 942–953. The KLH-coupled peptides were emulsified in TiterMax Gold Adjuvant (cytRx Corp., Los Angeles, CA) and injected intradermally into New Zealand rabbits. Animals were boosted several times sc and im. The final antisera were passed over Protein-A Sepharose (Amersham Pharmacia Biotech, Arlington Heights, IL) and eluted with 100 mM glycine, 50 mM NaCl, pH 3.0. The IgG fraction was further affinity purified by binding to the corresponding peptide coupled to EAH-Sepharose (Amersham Pharmacia Biotech). Final elution was performed with 50 mM diethylamine, 10% glycerol, 50 mM NaCl (pH 11.7), and neutralization was achieved by addition of 1.5 M Tris-HCl (pH 6.8).

Vectors
The pMET7-mouse LR (National Center for Biotechnology Information nucleotide accession no. U46135) long form (pMET7-mLR) was kindly provided by L. Tartaglia. The mutant leptin receptor LR-F3 was generated as described previously (31). In brief, the three cytoplasmic tyrosine residues (Y985, Y1077, and Y1138) were mutated to phenylalanines using the QuikChange site-directed mutagenesis procedure (Stratagene, La Jolla, CA). In the second LR variant, LR-FFY-{Delta}box 1, the tyrosines on positions 985 and 1077 were mutated to phenylalanines to reduce the negative feedback of the signal to a similar level as for the LR-F3. To eliminate JAK activation, two proline residues on positions 876 and 878 in the box 1 motif, were mutated to serines using the mutagenic oligonucleotides 5'-GTTGTTTTGGGACGATGTTTCAAACTCTAGAAATTGTTCCTGGGCACAAGG-3', and 5'-CCTTGTGCCCAGGAACAATTTCTAGAGTTTGAAACATCGTCCCAAAACAAC-3'. The LR-FFY variant was constructed as described previously (31).

Generation of the EpoR-mLR chimera, containing the extracellular part of EpoR, and the transmembrane and intracellular parts of the mLR, as well as generation of the derived EpoR-LR-F3, has been described previously (64). The EpoR-LR-{Delta}box 1 was constructed in a way similar to the LR-FFY-{Delta}box 1 mentioned above.

LR variants lacking the membrane distal CRH module (aa 1–308) were generated using a site-directed mutagenesis strategy. Using the mutagenic oligonucleotides, 5'-GGCATATCCAATCTCTCCCCCTCGAGATTTAAGTTGTTTTGTGG-3', and 5'-CCACAAAACAACTTAAATCTCGAGGGGGAGAGATTGGATATGCC-3', a XhoI site was introduced at position 90, just after the signal peptide-encoding sequence. A second XhoI site was generated at position 966 (nucleotides 5'-GACTGGAGTTCACCTCGAGTCTTTACCACACAAG-3' and 5'-CTTGTGTGGTA-AAGACTCGAGGTGAACTCCAGTC-3'). The resulting construct was digested with XhoI, and the smallest fragment (228 bp) was ligated back into the opened vector. Deletion of the membrane-distal CRH and the neighboring Ig-like domain (aa 309–447) was performed along a similar strategy. A third XhoI restriction site was introduced at position 1275 (nucleotides 5'-GCTGAATTATACGTGCTCGAGCCGTCAATATCAATATATC-3' and 5'-GATATATTGATATTGACGGCTCGAGCACGTATAATTCAGC-3'), and the vector was circularized after digestion with XhoI. All constructs were verified by DNA sequence analysis.

Generation of the pXP2d2-rPAP1 (rat pancreatitis associated protein 1)-luciferase reporter was described previously (31). Activation of this reporter is dependent on STAT3, because overexpression of dominant-negative STAT3, but not of dominant-negative STAT1, completely blocks transcriptional activation (65).

Cell Lines and Transfection Procedures
HEK293T cells were cultured in 10% CO2 humidified atmosphere at 37 C, and grown using DMEM with 4500 mg/liter glucose, 10% fetal bovine serum, and 50 µg/ml gentamicin (all from Invitrogen, San Diego, CA).

For transfection experiments, HEK293T cells were freshly seeded and cultured overnight. Cells were transfected overnight with approximately 2 µg (unless stated otherwise) plasmid DNA using a standard calcium phosphate precipitation procedure. One day after transfection, cells were washed with PBS-A (PBS without calcium, magnesium and sodium bicarbonate), and cultured until further use.

Reporter Assay, Leptin Binding Assay, Western Blot Analysis, and Immunoprecipitation
Cells were resuspended 48 h after transfection with cell dissociation agent (Invitrogen) and seeded in a black 96-well plate (Costar). Cells were stimulated overnight with the appropriate cytokine, and luciferase activity was measured by chemiluminescence. Lysates were prepared (lysis buffer: 25 mM Tris, pH 7.8; 2 mM EDTA; 2 mM dithiothreitol; 10% glycerol; 1% Triton X-100), and 35 µl luciferase substrate buffer [20 mM Tricine; 1.07 mM (MgCO3)4Mg(OH)2·5 H2O; 2.67 mM MgSO4·7 H2O; 0.1 mM EDTA; 33.3 mM dithiothreitol; 270 µM coenzyme A; 470 µM Luciferin; 530 µM ATP; final pH 7.8] was added per 50 µl lysate. Light emission was measured for 5 sec in a TopCount Chemiluminescence Counter (Packard Instruments, Meriden, CT).

Cell surface expression of wild-type LR or LR mutants was measured using a binding assay with a mouse leptin-SEAP chimeric protein. Generation of this leptin-SEAP chimera has been described previously (14). Cells were washed (wash buffer: DMEM, 0.1% NaN3, 20 mM HEPES, pH 7.0, 0.01% Tween 20) 2 d after transfection and incubated for 90 min at room temperature with a 1:50 dilution of a Cos-conditioned medium containing the leptin-SEAP chimera (final concentration, 10 ng/ml). After three successive washing steps, cells were lysed (lysis buffer: Triton X-100, 10 mM Tris-HCl, pH 7.4). Endogenous phosphatases in the lysates were inactivated (65 C, 30 min), and secreted alkaline phosphatase activity was measured using the 1% chemiluminescent AP substrate disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)-phenylphosphate (CSPD) substrate method (PhosphaLight, Tropix, Inc., Bedford, MA) in a TopCount Chemiluminescence Counter (Packard Instruments).

Expression of wild-type LR or LR mutants was also demonstrated using Western blot analysis. Briefly, 4 x 105 HEK293T cells in a six-well were transfected with plasmids encoding the LR variants. Cells were directly lysed 60 h post transfection in 300 µl loading buffer. After sonication, 40 µl of the lysates were loaded on a 7.5% polyacrylamide gel. After overnight blotting onto nitrocellulose sheets, LR constructs were revealed using an in-house-made polyclonal antibody directed against the intracellular part of the receptor (see above), and a donkey-antirabbit horseradish peroxidase-coupled antibody.

For STAT3 and JAK2 phosphorylation, HEK293T cells were transfected with the appropriate LR variants. Cells were starved 65 h later in serum-free medium for 5 h and were left untreated or stimulated with 100 ng/ml leptin for 15 min. Gel electrophoresis and blotting were as described above. STAT3 phosphorylation was checked using the phospho-STAT3-Tyr705 antibody (Cell Signaling), according to the manufacturer’s guidelines. STAT3 expression levels were verified using an anti-STAT3 antibody (Transduction Laboratories, Inc., Lexington, KY). To detect JAK2 phosphorylation and expression, 0.01 µg pRK5-JAK2 was cotransfected and JAK2 was revealed using an antiphospho-JAK2 (Y1007, Y1008) antibody (Upstate Biotechnology, Inc., Lake Placid, NY), or an anti-JAK2 antibody (Upstate Biotechnology, Inc.).


   ACKNOWLEDGMENTS
 
We thank Dr. D. Broekaert and Dr. F. Peelman for critical reading the manuscript, and M. Goethals for synthesis and coupling of the peptides.


   FOOTNOTES
 
This work was supported by grants from the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie (to L.Z.) and The Fonds voor Wetenschappelijk Onderzoek (Grant 1.5.446.98).

Abbreviations: aa, Amino acids; CRH, cytokine receptor homology; Epo, erythropoietin; EpoR, Epo receptor; FN-III, fibronectin type III; gp130, glycoprotein 130; HEK, human embryonic kidney; JAK, Janus kinase; LIFR, leukemia inhibitory factor receptor; LR, leptin receptor; LRlo, LR long form; LRsh, LR short form; rPAP1, rat pancreatitis-associated protein 1; SEAP, secreted alkaline phosphatase; STAT, signal transducer and activator of transcription.

Received for publication March 7, 2003. Accepted for publication September 26, 2003.


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F. Peelman, H. Iserentant, A.-S. De Smet, J. Vandekerckhove, L. Zabeau, and J. Tavernier
Mapping of Binding Site III in the Leptin Receptor and Modeling of a Hexameric Leptin{middle dot}Leptin Receptor Complex
J. Biol. Chem., June 2, 2006; 281(22): 15496 - 15504.
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L. Zabeau, D. Defeau, H. Iserentant, J. Vandekerckhove, F. Peelman, and J. Tavernier
Leptin Receptor Activation Depends on Critical Cysteine Residues in Its Fibronectin Type III Subdomains
J. Biol. Chem., June 17, 2005; 280(24): 22632 - 22640.
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H. Iserentant, F. Peelman, D. Defeau, J. Vandekerckhove, L. Zabeau, and J. Tavernier
Mapping of the interface between leptin and the leptin receptor CRH2 domain
J. Cell Sci., June 1, 2005; 118(11): 2519 - 2527.
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F. Peelman, K. Van Beneden, L. Zabeau, H. Iserentant, P. Ulrichts, D. Defeau, A. Verhee, D. Catteeuw, D. Elewaut, and J. Tavernier
Mapping of the Leptin Binding Sites and Design of a Leptin Antagonist
J. Biol. Chem., September 24, 2004; 279(39): 41038 - 41046.
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