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Molecular Endocrinology 16 (4): 859-872
Copyright © 2002 by The Endocrine Society

Identification of the Critical Sequence Elements in the Cytoplasmic Domain of Leptin Receptor Isoforms Required for Janus Kinase/Signal Transducer and Activator of Transcription Activation by Receptor Heterodimers

Gregor Bahrenberg, Iris Behrmann, Andreas Barthel, Paul Hekerman, Peter Claus Heinrich, Hans-Georg Joost and Walter Becker

Institut für Pharmakologie und Toxikologie (G.B., A.B., P.H., H.-G.J., W.B.) and Institut für Biochemie, (I.B., P.C.H.), Medizinische Fakultät der Rheinisch-Westfälische Technische Hochschule Aachen, Aachen D-52057, Germany

Address all correspondence and requests for reprints to: Dr. Walter Becker, Institut für Pharmakologie und Toxikologie, Medizinische Fakultät der Rheinisch-Westfälische Technische Hochschule Aachen, Wendlingweg 2, D-52057 Aachen, Germany. E-mail: walter.becker{at}post.rwth-aachen.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two predominant splice variants of the leptin receptor (LEPR) are coexpressed in leptin-responsive tissues: the long form, LEPRb, characterized as the signal-transducing receptor, and the signaling-defective short form, LEPRa. It is unknown whether heterodimers of these isoforms are capable of signal transduction via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. To address this question, chimeric receptors were constructed consisting of the transmembrane and intracellular parts of LEPRb and LEPRa fused with the extracellular domains of either the {alpha}- or ß-subunit of the IL-5 receptor. This strategy allows the directed heterodimerization of different LEPR cytoplasmic tails and excludes homodimerization. In COS-7 and HEPG2 cells, chimeric receptor heterodimers of LEPRa and LEPRb failed to activate the JAK/STAT pathway, whereas receptor dimers of LEPRb gave rise to the expected ligand-dependent activation of JAK2, phosphorylation of STAT3, and STAT3-dependent promoter activity. Markedly lower amounts of JAK2 were found to be associated with immunoprecipitated LEPRa chimeras than with LEPRb chimeras. Analysis of a series of deletion constructs indicated that a segment of 15 amino acids in addition to the 29 amino acids common to LEPRa and LEPRb was required for partial restoration of JAK/STAT activation. Site-directed mutagenesis of the critical sequence indicated that two hydrophobic residues (Leu896, Phe897) not present in LEPRa were indispensable for receptor signaling. These findings show that LEPRa/LEPRb heterodimers cannot activate STAT3 and identify sequence elements within the LEPR that are critical for the activation of JAK2 and STAT3.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ADIPOCYTE-DERIVED HORMONE leptin plays a key role in energy homeostasis and the control of body weight. In rodents and humans, inactivating mutations within the genes for leptin (1, 2) or the leptin receptor (LEPR) (3, 4, 5) cause syndromes of morbid obesity, hyperglycemia, hyperinsulinemia, and reduced fertility. Two predominant isoforms of the LEPR exist, which differ in their capacity of signal transduction, and it is a matter of considerable interest to understand the functions and interactions of these isoforms.

The LEPR belongs to the class I cytokine receptor family of which gp130, the common signal transducer of IL-6-type cytokines, is a prototype (6, 7, 8, 9, 10). LEPR isoforms represent alternative splice products and have identical extracellular domains, but differ in the length of their intracellular parts (4, 11, 12). The long form of the receptor (LEPRb) is primarily expressed in specific nuclei of the hypothalamus, which are known to regulate food intake and body weight (13, 14, 15, 16). LEPRb contains a 302-amino acid cytoplasmic domain with the consensus sequences necessary for signal transduction via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway (7). Two short, conserved motifs in the membrane-proximal region (box 1 and box 2) are thought to mediate binding of JAK kinases [JAK2 in the case of LEPR (17)]. After binding of leptin to the receptor, activated JAK2 rapidly phosphorylates tyrosine-1138 (box 3 motif) within LEPRb (18, 19), creating a binding site for STAT factors (signal transducers and activators of transcription). It has been shown that ectopically expressed LEPRb has the capacity to mediate leptin-induced activation of STAT3 and induction of target promoters (7, 19, 20, 21). LEPRa, the isoform predominantly expressed in most tissues, has a short cytoplasmic domain of 34 amino acids that includes a putative box 1 motif but contains no tyrosine residues. Multiple studies have shown that LEPRa is unable to activate STATs by itself (7, 22). In fact, injections of leptin in vivo result in a dose-dependent activation of STAT3 in the hypothalamus within 15 min in wild-type mice, but not in db/db mice, which selectively lack LEPRb (23). Furthermore, the near identity of the ob/ob (leptin defect) and db/db (LEPRb deficiency) phenotypes indicate that the presence of LEPRb is essential for most, if not all, physiological effects of leptin (8). Although there are some reports of signal transduction by LEPRa when overexpressed in mammalian cells (18, 24), it is generally assumed that LEPRa is rather involved in leptin transport through the blood-brain barrier or leptin clearance (25, 26).

It has previously been shown that homodimers of LEPRb mediate leptin-induced activation of STAT3 (9, 10, 20). However, ligand-induced heterodimerization of LEPRa and LEPRb has also been demonstrated by coimmunoprecipitation from transfected cells (27). Given the excess of the naturally occurring LEPRa in all known tissues, it is important to understand the functions of such heterodimers. Considering that a single STAT docking site (box 3 motif) in a receptor complex suffices to elicit STAT responses in other cytokine receptors [gp130 (28)], we decided to analyze in detail the capacity of LEPRb/LEPRa heterodimers to initiate signaling via the JAK/STAT pathway.

To induce heterodimerization of the different cytoplasmic tails of LEPRa and LEPRb, we used a system of chimeric receptors based on the extracellular domains of the human IL-5 receptor (IL-5R) {alpha}- and ß-chains (28, 29), each fused to the cytoplasmic parts of LEPRb and LEPRa, respectively. In this system, IL-5 binding induces the formation of receptor complexes containing both IL-5R{alpha} and IL-5Rß chimeric molecules. The present data show that heterodimers of LEPRa and LEPRb are unable to mediate ligand-induced activation of JAK2 and STAT3. However, replacing the five LEPRa-specific residues by a 15-amino acid segment of LEPRb restored the signaling potential of heterodimeric receptor complexes. These data define the minimal segment of the intracellular domain of LEPRb that is sufficient for binding and activation of a JAK kinase.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
COS 7 Cells Transfected with the Long or the Short Isoform of the LEPR Only Show STAT3 Activation When Expressing the Long Form
COS-7 cells were transiently transfected with expression plasmids encoding either the long (pSVL-LEPRb) or the short (pSVL-LEPRa) isoform of the LEPR. On Western blots, the expressed proteins were detected as bands with apparent molecular masses of 200 kDa and 130 kDa, respectively (Fig. 1AGo). After stimulation with leptin, STAT3 was activated in LEPRb-transfected cells, as revealed by Western blot analysis with an antibody against phosphorylated STAT3 and the appearance of the respective band in the EMSA (Fig. 1AGo). This protein-DNA complex could be identified as a STAT3 dimer when compared with the migration of retarded complexes from nuclear extracts from IL-6-stimulated human HepG2 hepatoma cells or from COS-7 cells with overexpressed STAT3 (data not shown). In LEPRa- or vector only-transfected cells, neither phosphorylation nor DNA binding of STAT3 was detectable (Fig. 1AGo).



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  		Figure 1. Activation of STAT3 by LEPR Isoforms

A, COS-7 cells were transfected with the expression plasmid pSVL encoding the long (LEPRb) or the short (LEPRa) isoform of the LEPR. Expression of LEPR isoforms was detected by Western blotting of total cellular lysates ({alpha}LEPR). To assess STAT3 activation, cells were stimulated with leptin (100 ng/ml) for 15 min or with vehicle alone before nuclear extracts were prepared. Western blots of nuclear extracts were developed with an antibody specific for phosphorylated STAT3 ({alpha}P-STAT3). Specific bands are indicated by an arrow; the asterisks marks an unspecific band that serves as a loading control. Aliquots of the nuclear extracts were used in EMSAs with the m67SIE probe. The band resulting from STAT3 homodimers is indicated. B, Reporter gene assay. HepG2 cells were transfected with the indicated expression plasmids (pCDNA3.1 or pSVL) along with an {alpha}2-macroglobulin promoter luciferase reporter gene construct. One day after transfection, cells were stimulated with IL-6 (200 U/ml), leptin (Lep; 100 ng/ml), or citrate buffer (vehicle) for 24 h. Luciferase activity of lysates was normalized to the activity of coexpressed ß-galactosidase. One representative experiment is shown (columns represent the average of two measurements and bars reflect the differences between the two). Observations were confirmed in two additional independent experiments.

 
For reporter gene assays of STAT3, HepG2 cells were used because effects were much stronger than in COS-7 cells (data not shown). Induction of a luciferase gene under the control of the {alpha}2-macroglobulin promoter was measured. After transient transfection of HepG2 cells with LEPRb or LEPRa inserted into two different vectors (pSVL; pCDNA3.1), a strong induction upon leptin stimulation was only detectable in LEPRb-transfected cells (Fig. 1BGo). A still stronger induction of the reporter gene was achieved by stimulation of the endogenous IL-6 receptor. This indicates that in HepG2 cells the JAK/STAT pathway was not maximally activated by the overexpressed LEPRb. No induction was observed in mock-transfected or LEPRa-transfected cells. These data show that LEPRa was not able to activate STAT3 in our system.

Heterodimers of the LEPRa and LEPRb Cytoplasmic Domains Are Incapable of STAT3 Activation
We generated hybrid molecules composed of the extracellular parts of the human IL-5R {alpha}- and ß-chains, respectively, and the transmembrane and intracellular regions of LEPRb or LEPRa. The chimeric leptin receptors were designated {alpha}CLRa, ßCLRa, {alpha}CLRb, and ßCLRb to indicate the contributing receptor domains ({alpha}, IL-5R{alpha}; ß, IL-5Rß; a, LEPRa, b, LEPRb). Our previous studies have shown that ligand induced activation of receptor complexes depends on the interaction of IL-5R{alpha} with ß-chimeric molecules (28). High-affinity binding of IL-5 to the extracellular domain of IL-5R{alpha} is known to be independent of the transmembrane and intracellular parts of the receptor (30), and IL-5 binding affinities of the IL-5R chimeras were found unchanged by deletion or exchange of the cytoplasmic region (28). Therefore, the system should be suitable to delineate the potential of the cytoplasmic region of LEPRa in the chimeric receptor (CLRa) to transduce signal within a heterodimer with LEPRb chimera (CLRb).

In transfected COS-7 cells, {alpha}CLRa/ßCLRb and ßCLRa/{alpha}CLRb heterodimeric receptor complexes elicited no detectable phosphorylation or induction of DNA binding activity of STAT3 after IL-5 stimulation (Fig. 2AGo, lanes 1, 2, 5, and 6). In striking contrast, IL-5 stimulation of LEPRb homodimers (lane 3) and gp130 homodimers (used as an assay control; lane 7) induced strong STAT3 responses. STAT3 activation was 3–5 times stronger by chimeric gp130 complexes than by the corresponding CLRb dimers (Fig. 2AGo). Dimeric receptor products of CLRa or CLRb with only the transmembrane region of gp130 did not activate STAT3 at all. For expression control, lysates were probed in Western blot experiments with antibodies against STAT3 and human IL-5R {alpha}- or ß-chains (Fig. 2AGo). Western blots with IL-5R{alpha}- or IL-5Rß-specific antibodies confirmed that different STAT3 responses cannot be explained by different expression levels of the chimeric receptors. The experiment outlined in Fig. 2AGo was reproduced three times in COS-7 cells without overexpression of STAT3 with qualitatively comparable results (data not shown).



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  		Figure 2. Heterodimerization of the Cytoplasmic Parts of LEPRb and LEPRa Does Not Induce STAT3 Activation

A, COS-7 cells were transfected with expression plasmids encoding the IL-5R {alpha} and ß chimeras of LEPRb, LEPRa, gp130, and gp130 without cytoplasmic part (gp130{Delta}cyt), as indicated. Untransfected cells were supplemented with transfection reagent alone. Cells were cotransfected with 0.3 µg of STAT3 expression vector per 6-cm dish. Two days after transfection, cells were stimulated with IL-5 (80 ng/ml) for 30 min before nuclear extracts were prepared. Nuclear extracts were analyzed by Western blotting with an antibody against phosphorylated STAT3 or a gel shift experiment with the m67SIE probe. For expression control, postnuclear supernatants were analyzed by Western blot experiments with antibodies against STAT3, human (h)IL-5R {alpha}, and hIL-5Rß. Calculated molecular masses of the chimeric receptors are given on the top of the respective lanes. B, Reporter gene assays. HepG2 cells were transfected with the indicated expression plasmids, an {alpha}2-macroglobulin promoter luciferase reporter gene vector (pGL3), and a ß-galactosidase construct. One day after transfection, cells were treated with IL-5 (80 ng/ml) or water in serum-free medium (vehicle) for 24 h. Luciferase activity of lysates was normalized to the activity of ß-galactosidase. Data are given as fold induction relative to pSVL-transfected and stimulated cells. Columns represent the average values of two independent experiments (each based on two independent sets of transfections), and the bars reflect the differences between these average values.

 
Cell surface expression of the chimeric receptors was analyzed by flow cytometry using antibodies directed against IL-5R{alpha} and IL-5Rß. Consistently, surface expression of CLRa was considerably higher than of each CLRb molecule. CLRb exhibited lower surface expression than chimeric gp130 receptors (12% of the cells vs. 54% of the cells), providing a plausible explanation for the differences in STAT3 signaling by these receptors (data not shown).

The same combinations of chimeric receptor constructs as shown in Fig. 2AGo were transfected into HepG2 cells together with an {alpha}2-macroglobulin promoter luciferase reporter gene construct. In agreement with the above data, no promoter induction by heterodimers of CLRb with CLRa was observed (Fig. 2BGo). Induction by homodimers CLRb/CLRb and CRgp130/CRgp130 was 12.5- and 60-fold, respectively, in comparison to values of vector-transfected and stimulated cells (Fig. 2BGo).

The Chimeric LEPRa Has a Reduced Capacity of JAK2 Binding
To answer the question why heterochimeric LEPRs (CLRa/CLRb) do not induce STAT3 activation, we compared the ability of CLRb, CLRa, and CRgp130 to bind JAK2. COS-7 cells were cotransfected with expression vectors encoding ßCRgp130, ßCLRb, ßCLRa, or ßCRgp130{Delta}box1 (negative control), respectively, and JAK2. After lysis under mild conditions, binding of JAK2 to ßCLRb was at least as good as to ßCRgp130 (Fig. 3Go, upper panel). Binding to ßCLRa was reduced, but consistently detectable in four independent experiments (Fig. 3Go, lane 2). The reduced binding of JAK2 to ßCLRa may be due to the lack of a box 2 motif in LEPRa, whereas the residual binding of the kinase may be mediated by box 1. JAK2 bound only weakly to a gp130 construct with a deletion in the membrane-proximal region including the box 1 motif (Fig. 3Go). Western blots of cellular lysates demonstrate comparable expression levels of JAK2 and the chimeric receptors.



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  		Figure 3. JAK2 Association with gp130, LEPRa, and LEPRb

COS-7 cells were cotransfected with expression constructs encoding the indicated receptor chimeras (ß/gp130, ß/LEPRa, ß/LEPRb, or ß/gp130{Delta}box1) and JAK2. After mild lysis of the cells, receptor complexes were immunoprecipitated with the anti-IL-5Rß antibody S-16. Immunocomplexes or cellular lysates were immunoblotted with antibodies against JAK2 or IL-5Rß as indicated. Observations were confirmed in four independent experiments.

 
Activation of STAT3 by Heterodimers of CLRb with Deletion Mutants of CLRb
We analyzed how many amino acids of LEPRb C-terminal of the splice site in a chimeric receptor construct were required to elicit the STAT3 response by dimers with the full-length receptor (CLRb). The chimeric receptor constructs of CLRa, CLRb, and deletion mutants of CLRb are shown in Fig. 4Go. The first cytoplasmic 29 amino acids proximal to the transmembrane region are identical in all constructs and contain the presumed box 1 signature of the JAK-binding motif according to the criteria established for class 1 cytokine receptors (31, 32). CLRa and CLRb possess 5 and 273 unique amino acids at the C terminus, respectively (Fig. 4BGo). Deletion mutants were designated by the number of C-terminal amino acids distal to the 29 common amino acids in CLRa and CLRb. None of the deletion constructs encodes an intracellular tyrosine residue.



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  		Figure 4. Structure of the Chimeric Receptor Constructs Used in This Study

A, Intracellular amino acid sequences of CLRa and CLRb. Presumed box 1 and box 2 motifs (17 35 36 ) are underlined (closed lines); the unique amino acids in the C terminus of CLRa are marked by a dashed line. IL-5R extracellular parts are illustrated as gray boxes; extracellular and transmembrane (TM) regions of the leptin receptor are indicated as white boxes. The positions where stop codons were inserted into CLRb to generate the indicated deletion mutants are illustrated by vertical lines. K889 of LEPR is the last amino acid of CLR-0. B, Schematic representation of deletion mutants of CLRb (in comparison to CLRa and CLRb). The splice site (after amino acid K889) is illustrated by a dashed line. The box 1–3 motifs are represented by black boxes. The membrane proximal 29 amino acids of the cytoplasmic part of the LEPR are identical in LEPRa and LEPRb and include a box 1 motif. Each construct is designated by the number of unique C-terminal amino acids. The amino acids unique in CLRa are illustrated by a hatched box. The deletion constructs contain no intracellular tyrosine residue. Lengths of receptor constructs are not drawn to scale.

 
In transiently transfected COS-7 cells, we investigated the STAT3 response of CLRb in combination with the deletion mutants CLR-95, CLR-36, CLR-19, CLR-6, or CLR-0. The signaling capacity of a dimer of CLRb with CLR-95 was similar to that of CLRb homodimers as assessed by Western blot analysis with an antibody against phosphorylated STAT3 (Fig. 5AGo). Tyrosine phosphorylation of STAT3 was also induced by chimeric complexes of CLRb with CLR-36 or CLR-19. However, ligand-induced phosphorylation of STAT3 was weaker in cells expressing CLR-19/CLRb heterodimers, regardless of their fusion to the {alpha}- or ß-subunit of IL-5R. This difference was also observed when dimers of CLRb with CLR-44 and CLRb with CLR-19 were compared (data not shown). Deletion mutants further shortened by 13 or 19 amino acids (CLR-6 or CLR-0) have totally lost their capacity of STAT3 phosphorylation (Fig. 5BGo). The ability of the deletion constructs to elicit phosphorylation of STAT3 correlates well with their capability to mediate ligand-induced activation of the {alpha}2-macroglobulin promoter in HepG2 cells (Fig. 5CGo), as CLR-19 was active, but CLR-6 was not. It should be noted that CLR-19 and CLR-15 do not include the box 2 motif of LEPR as proposed by Ghilardi and Skoda (17 ; Fig. 4Go). Thus, these data suggest that the amino acids 896–904 of LEPRb (i.e. the last 9 amino acids of CLR-15) are critically involved in STAT3 activation.



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  		Figure 5. Signaling of Deletion Mutants of CLRb in Heterodimeric Complexes with CLRb

A and B, COS-7 cells were cotransfected with expression plasmids encoding the indicated chimeric receptor constructs and a STAT3 expression vector. Thirty-two hours after transfection, cells were starved for 16 h and then treated with 80 ng/ml IL-5 or vehicle only for 30 min before nuclear extracts were prepared. Extracts were used for Western blot analysis with a phospho-specific STAT3 antibody. For expression control, lysates were probed with antibodies against STAT3, human (h)IL-5R{alpha}, and hIL-5Rß. C, HepG2 cells were transfected with expression plasmids encoding ß/CLRb and the indicated {alpha} chimera along with an {alpha}2-macroglobulin promoter luciferase reporter gene construct. One day after transfection, cells were stimulated with IL-5 (80 ng/ml) or vehicle only for 24 h. Luciferase activity of lysates was normalized to the activity of coexpressed ß-galactosidase. One representative experiment is shown. Columns represent the average of two independent transfections and bars reflect the differences between the two. The same differences between the average values of the expression constructs were observed in three additional experiments.

 
The STAT3 Signaling Capability of Heterodimerized LEPR Cytoplasmic Parts Parallels Their Capability to Induce JAK2 Phosphorylation
We hypothesized that the differences in STAT3 activation by the LEPR deletion constructs are due to their different capability to activate JAK2. Phosphorylation of JAK2 was analyzed in COS-7 cells transiently transfected with JAK2, ßCLRb, and {alpha}CLR constructs encoding receptors with intracellular domains of variable length. Phosphorylated JAK2 was detected by immunoprecipitation, followed by Western blot analysis with phosphotyrosine-specific antibodies. Alternatively, we used an antibody specific for phosphorylated JAK2 to detect activated JAK2 in total cellular lysates. Figure 6AGo shows that ligand-induced phosphorylation of JAK2 was mediated by complexes of ßCLRb with {alpha}CLRb, {alpha}CLR-95, and {alpha}CLR-19. In contrast, we did not obtain an increase of tyrosine phosphorylation of JAK2 after heterodimerization of CLRb with CLR-0 or CLRa. Thus, activation of JAK2 by the different chimeric receptor complexes parallels their capability to mediate STAT3 activation.



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  		Figure 6. Stimulation of JAK2 Phosphorylation by LEPR Deletion Mutants

A, COS-7 cells were transiently transfected with the indicated expression plasmids and 0.7 µg pSVL-JAK2 per 10-cm petri dish. Two days after transfection (including 16 h serum deprivation), cells were stimulated with IL-5 (80 ng/ml), lysed in Triton buffer, and lysates were subjected to immunoprecipitation with an antibody against JAK2. Immunoprecipitates and aliquots of cellular lysates were analyzed by Western blotting. Detection was performed with a mixture of phosphotyrosine-specific antibodies (4G10/PY99) or with an antibody against phospho-JAK2 (pYpY1007/1008). As control, the same blots or identical blots with the same lysates were developed with an antibody against JAK2. The data are representative of three independent experiments with identical results. Western blot analysis of immunoprecipitates with an antibody against phosphorylated JAK2 consistently showed the same results as with antibodies against P-Tyr. B, COS-7 cells were transiently transfected with expression plasmids for LEPRb, LEPRa, and the indicated cytoplasmic deletion mutants of LEPRb together with 0.7 µg pSVL-JAK2 (10-cm petri dish, upper panel) or 0.37 µg pSVL-JAK2 (6-cm petri dish, lower panels). Western blots of total cellular lysates were developed with the indicated antibodies.

 
To exclude the possibility that the structure of the intracellular domain of the leptin receptor was not fully maintained in the chimeric constructs, we also tested homodimers of LEPRb, LEPRa, and intracellular deletion mutants of LEPRb for their capacities to stimulate phosphorylation of JAK2. As shown in Fig. 6BGo, leptin-induced tyrosine phosphorylation of JAK2 was detected in cells expressing LEPRb, LEPR-15, LEPR-19, and LEPR-36, respectively. The same result was obtained when JAK2 immunprecipitates were analyzed with a phosphotyrosine-specific antibody (data not shown). These data indicate that the capability of the LEPRb deletion mutants to activate JAK2 parallels the signaling capability of the corresponding chimeric receptor mutants. Surprisingly, a weak leptin-induced tyrosine phosphorylation of nuclear STAT3 was mediated by the truncated receptors CLR-15, CLR-19, and CLR-36, which have no intracellular tyrosine residue (bottom panel). It should be noted, however, that JAK2 was overexpressed in this experiment.

Function of LEPR Constructs Carrying Mutations in a 13-Amino Acid Region Crucial for STAT3 Activation
The present experiments show that heterodimers of CLRb with CLR-15 and CLR-19, but not with CLR-6, are capable of activating the JAK/STAT pathway, pointing to a crucial role of amino acids 896–908 of LEPRb. In this segment, 7 of 13 amino acids are conserved between mouse and chicken, the most distantly related species of which the LEPR sequence is known. To further define the residues within this region that are critical for induction of the JAK/STAT pathway, we constructed a series of point mutants of CLR-15 and CLR-19. These mutations target the seven residues conserved between mouse and chicken plus two glutamate residues (Glu891, Glu894) located further proximal (Fig. 7AGo). Of these mutations, the exchange of Leu896 and Phe897 abrogated the STAT3 response mediated by the heterodimeric receptors (Fig. 7BGo). In contrast, the other mutations did not alter STAT3 phosphorylation. In agreement with these data, CLRb/CLR-15 (LF->AA) heterodimers were completely inactive in reporter gene assays, whereas STAT3-responsive promoter activity was not affected by the KH->AA mutation (data not shown). Compared with CLRb/CLR-19 heterodimers, CLRb/CLR-15 receptors showed a reduced but clearly detectable STAT3 response (Fig. 7BGo) and also mediated ligand-induced activation of the {alpha}2-macroglobulin promoter (Fig. 5CGo and data not shown). Thus, a sequence of 44 intracellular amino acids (29 amino acids common to LEPRa and LEPRb plus 15 amino acids of CLR-15) is sufficient to induce signal transduction upon heterodimerization with CLRb.



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  		Figure 7. Mutational Analysis of a 13-Amino Acid Region Crucial for STAT3 Signaling by CLRb

A, Design of point mutants from CLR-15 and CLR-19. Sequences of the membrane proximal cytoplasmic segments of the LEPR of Mus musculus (EMBL database accession no. Y10298; upper sequence) and Gallus gallus (AF169827; lower sequence) are aligned. In CLR-15 and CLR-19, conserved amino acids were replaced by alanine (arrows). The box 1 motif (solid lines) and the proposed box 2 motif (dashed lines) (17 ) are boxed. The transmembrane region (TM) is indicated. B, COS-7 cells were transfected with expression plasmids encoding the IL-5R{alpha} and ß chimeras as indicated and a STAT3 expression vector. The experiment was performed as described in Fig. 5Go. Results for each construct were confirmed in three independent experiments.

 
In conclusion, our results define three regions critical for STAT3 activation via dimers of CLRb with its deletion mutants: 1) The reduced STAT3 response elicited by CLR-19 vs. CLR-36 (Fig. 5Go) indicates that the 17-amino acid region from 909–925 of LEPR is required for maximal activation; 2) The four additional amino acids of CLR-19 vs. CLR-15 (905–908) prove to be involved in STAT3 signaling as STAT3 response is weaker in CLRb/CLR-15 dimers; and 3) The failure of CLR-6 to induce STAT3 phosphorylation reveals that the segment of nine amino acids (896–904) that distinguishes CLR-6 and CLR-15 is essential for CLRb.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Among members of the class I cytokine receptor family, it is a distinctive feature of the LEPR system that the signaling-competent receptor isoform, LEPRb, is coexpressed with an excess of a truncated splice variant, LEPRa, that appears incapable of signal transduction. The present studies were performed to address the question of whether LEPRa may contribute to leptin signaling by forming heterodimers with LEPRb. Taking advantage of the IL-5R chimeric receptor system, we showed that heterodimeric receptors containing the different cytoplasmic tails of LEPRa and LEPRb are not able to mediate ligand-induced phosphorylation of JAK2, phosphorylation of STAT3, and reporter gene induction driven by a STAT3-responsive promoter. Replacing the five LEPRa-specific amino acids by a 15-amino acid segment of LEPRb reconstituted the signaling capacity of LEPRa in heterodimers with LEPRb. It is concluded that the failure of LEPRa/LEPRb heterodimers to induce JAK/STAT signaling is due to LEPRa’s lack of a complete JAK binding sequence, which must be present in two copies in a receptor complex to allow the mutual activation of two JAKs by transphosphorylation.

In addition to demonstrating the signaling incompetence of heterodimers of LEPRb and LEPRa, we have employed the chimeric receptor system to map the amino acids in LEPR critical for association and activation of JAK2. In class I cytokine receptors, two main structural motifs essential for JAK kinase activation have been identified in the membrane-proximal region of the cytoplasmic subdomain (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46). The box 1 motif is characterized by two essentially invariant prolines and has previously been identified in LEPRb by mutagenesis (18, 20); this motif is also present in LEPRa. In addition, a box 2 motif has been defined in certain receptors by mutation analysis (32, 40, 41, 47). Deletion of box 2 has been shown to abrogate JAK kinase activation by gp130 (48). A putative box 2 motif has been identified in LEPRb by sequence comparisons (17, 20), but no data exist to demonstrate its role in signal transduction. This motif is absent in LEPRa (see Fig. 4Go). In the present study, sequence elements necessary for JAK2 activation were defined in the LEPR by studying the signaling capacity of heterodimers of LEPRb with a series of deletion constructs and point mutants.

The present findings clearly indicate that the putative box 2 motif as proposed by Ghilardi and Skoda (17) is not essential for activation of JAK2 and STAT3. However, a segment of 15 amino acids immediately carboxy terminal of the intracellular 29 amino acids common to LEPRa and LEPRb was found to be indispensable for signal transduction by heterodimeric receptor complexes. The ability of this sequence element to support signal transduction by the LEPR in the absence of the box 2 motif was unexpected and caused us to analyze this region in more detail. Within this segment, we identified a pair of hydrophobic amino acids (Leu896/Phe897) that were crucial for signaling. Sequence comparisons of all known LEPR sequences revealed that these residues were fully conserved in seven species (mouse, human, macaque, sheep, pig, chicken, turkey), as were the basic residues Lys899 and His900. However, mutation of Lys899 and His900 and of five other amino acids between 890 and 908 did not affect STAT3 activation (Fig. 7Go). A crucial role of hydrophobic residues has previously been reported for several experimentally defined JAK binding motifs, whereas the role of charged amino acids appears to vary in different receptors (32, 46, 47, 49). A glutamate residue is highly conserved in the box 2 consensus sequence and was found essential for JAK activation by the EPO receptor (47) and IFN{alpha} receptor (49). In contrast, our data indicate that any of the three glutamates distal of the box 1 motif in the LEPR is dispensable for signaling by CLR-15. In support of this conclusion, two of the glutamates (Glu894, Glu902) are not conserved in sheep and humans, respectively.

It should be noted that although the membrane-proximal 44 amino acids of LEPRb were sufficient for activation of STAT3, maximal activation of STAT3 required the presence of 21 additional amino acids of the cytoplasmic domain. This region comprises the sequence motif previously proposed as box 2, a segment of eight amino acids that is conserved between mammals and chicken (Fig. 7Go). This segment comprises a LLEP-motif that is also present in the extended box 2 of the erythropoietin receptor (50).

The use of the chimeric receptor system enabled the targeted dimerization of the cytoplasmic tail of LEPRb with mutated versions of the receptor and allowed us to assess not only phosphorylation of JAK2 but also activation of STAT3. In these experiments (Figs. 5–7GoGoGo), a single STAT-binding module within a heterodimeric receptor complex was sufficient for phosphorylation and activation of STAT3. This finding is fully consistent with previous results obtained with chimeric cytokine receptors (28) and the IL-3 receptor (IL-3R) complex, which lacks an intracellular tyrosine residue in the IL-3R{alpha} chain (51). STAT3 has been identified as one of the physiological mediators of leptin action because STAT3 is activated by leptin in a dose-dependent fashion in vivo (23). In our experiments, tyrosine phosphorylation of STAT3 and reporter gene activity could be determined without the need of overexpressing JAK2. Therefore, we think that our experimental setup reliably reflects physiological conditions. However, JAK2 expression was so low that kinase expression plasmids had to be transfected for assays of JAK binding (Fig. 3Go) or phosphorylation (Fig. 6Go). As high levels of JAK may favor nonphysiological interactions (43), it was important to carefully control the level of expression of JAK2 to reduce ligand-independent phosphorylation to an acceptable level (Fig. 6Go).

The present data clearly demonstrate that LEPRa lacks critical sequence elements required for activation of JAK2 and is incapable of activating the JAK/STAT pathway, even in heterodimers with LEPRb. Therefore, the ability of LEPRa to form heterodimers with LEPRb in the presence of leptin (27) would be expected to markedly suppress LEPRb signaling because LEPRa and LEPRb are coexpressed in many tissues, including the hypothalamus. Similarly, the signaling-competent isoform of the PRL receptor (PRLR-L) is widely coexpressed with a short splice variant (PRLR-S) that contains a box 1 motif but lacks a box 2 sequence (52). Heterodimers of PRLR-L and PRLR-S have been found to be unable to induce ligand-dependent signaling, and it has been suggested that PRLR-S has a modulatory function as a dominant-negative isoform (53). This possibility has already been ruled out for the LEPR system: Signaling by LEPRb was only marginally susceptible to dominant negative repression by LEPRa in coexpression experiments (20, 27). This lack of interaction may by explained by the fact that LEPRb and LEPRa exist in preformed homomeric complexes in the absence of leptin, whereas lower levels of heterodimers of LEPRa and LEPRb are only detectable after leptin treatment (27). In conclusion, LEPRa appears neither to contribute to leptin-induced signal transduction nor to inhibit leptin signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
Restriction enzymes, Klenow enzyme, and T4-DNA ligase were obtained from Promega Corp. (Madison, WI), Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK), MBI-Fermentas (Vilnius, Lithuania), or Roche Diagnostics (Mannheim, Germany). Oligonucleotides were from MWG Biotech (Ebersberg, Germany). DMEM and DMEM/F12 were purchased from Life Technologies, Inc. (Eggenstein, Germany) and FCS from Seromed (Berlin, Germany). Human IL-5 was obtained from Cell Concepts (Umkirch, Germany) and recombinant murine leptin from PeproTech (London, UK). Recombinant human IL-6 was kindly provided by A. Küster (Aachen, Germany). Diethylaminoethyl-dextran, chloroquine, and protease inhibitors were purchased from Sigma (Deisenhofen, Germany). The following primary antibodies were used: anti-phospho-STAT3 (Tyr705) and anti-STAT3 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), antimouse LEPR goat antibody from R&D Systems (Wiesbaden, Germany), antihuman IL-5Rß (N-20) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antihuman IL-5R{alpha} goat antibody from R&D Systems, anti-pTyr antibodies PY99 from Santa Cruz Biotechnology, Inc., 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and PY20 from Transduction Laboratories, Inc. (Lexington, KY). Antirabbit horseradish peroxidase-labeled IgG (IgG-POD) was obtained from Pierce Chemical Co. (Rockford, IL), antimouse IgG-POD from Amersham Pharmacia Biotech, and antigoat IgG-POD from Dianova (Hamburg, Germany). For flow cytometry, the monoclonal antibody 16–4 specific for the human IL-5R {alpha}-chain (kindly provided by J. Tavernier, Ghent, Belgium) and the monoclonal antibody S-16 specific for the human IL-5Rß-chain (Santa Cruz Biotechnology, Inc.) were used. PE-labeled goat antimouse IgG-F(ab')2 was obtained from Dianova. Antiserum against JAK2 was a kind gift from A. Ziemiecki (Bern, Switzerland). The antibody against phosphorylated JAK2 (pYpY1007/1008) was purchased from BioSource Technologies, Inc. (Camarillo, CA).

Plasmid Construction
First-strand cDNA was synthesized from mouse liver and hypothalamus RNA samples with oligo(dT) as primer (first strand cDNA synthesis kit, Amersham Pharmacia Biotech). cDNAs for LEPRa and LEPRb were PCR amplified using the Expand High Fidelity PCR system with an enzyme mix of Taq DNA and Pwo DNA polymerases (Roche Diagnostics). For expression studies, the LEPR-cDNA was inserted into vector pSVL (Amersham Pharmacia Biotech), using restriction sites (XhoI, SacI) introduced by the PCR primers as described previously (54). For some reporter gene assays in HepG2 cells, the complete LEPR-cDNA was cloned into the vector pcDNA 3.1(-) (Invitrogen, Leek, The Netherlands). The expression plasmids for the chimeric receptors IL-5R/gp130, IL-5Rß/{Delta}cyt and IL-5Rß/gp130{Delta}box1 have been described previously (28, 55). The chimeras IL-5R/LEPR were cloned from the expression plasmid pSVL-IL5R/gp130 by exchanging the EcoRI/BamHI-fragment encoding the transmembrane and cytoplasmic part of gp130 against the EcoRI/BglII-fragment encoding the corresponding fragment of LEPRb (amino acids 826–1,162). Mutants of the cytoplasmic part of chimeric LEPRb were created by amplification of the corresponding EcoRI/BglII-fragment with mutated PCR primers. Deletion mutants of LEPRb were generated by replacing the cDNA fragment encoding the cytoplasmic part of LEPRb in the pSVL-LEPRb construct with appropriate PCR products coding for the deleted cytoplasmic tails via an internal DraIII site in the LEPRb cDNA and a SacI site in pSVL. The integrity of all ligation products was verified by DNA sequencing. The expression plasmid pSVL-JAK2 was constructed from pRK5-JAK2 kindly provided by I. Kerr (London, UK). The STAT3 expression vector has been described previously (56).

Cell Culture, Transient Transfections, and Stimulations
Simian monkey kidney cells (COS-7) were maintained in DMEM, human hepatoma cells (HepG2) in DMEM/F12 medium supplemented with 10% FCS, 100 mg/liter streptomycin, and 60 mg/liter penicillin. Approximately 1.5 x 107 COS-7 cells on 10-cm petri dishes were transiently transfected with 10–20 µg plasmid DNA using the diethylaminoethyl-dextran method, as described elsewhere (29). Alternatively, 0.8–2.0 x 105 HepG2- or COS-7 cells on 6-cm plates were transfected with 2.8 µg plasmid DNA using the FuGENE 6 transfection reagent (Roche Diagnostics) according to the manufacturer’s instructions. Cells grown to 80% confluence were stimulated with 100 ng/ml leptin for 15 min, as described previously (54). Cells with overexpressed IL-5 chimeras were starved for 16 h and stimulated with 80 ng/ml IL-5 for 30 min. All assays were performed 48 h after transfection. Protein concentrations were determined with a BCA protein assay kit from Pierce Chemical Co.

Immunoprecipitation
COS-7 cells were washed twice with PBS, scraped off the dish, and lysed in Brij97-lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1% BRIJ97 (Sigma, Deisenhofen, Germany), 1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin] for 30 min on ice. Cell lysates were centrifuged at 14,000 rpm for 10 min. The supernatants were used for immunoprecipitation of the receptor chimeras using the anti-IL-5Rß antibody S-16. After overnight incubation at 4 C, immune complexes were collected on protein A-Sepharose (Amersham Pharmacia Biotech) during a 60-min incubation, washed twice with 1% Brij97-lysis buffer, and boiled for 5 min in Laemmli buffer at 95 C. The proteins were separated by 8.0% SDS-PAGE, followed by electroblotting onto a nitrocellulose membrane (Schleicher \|[amp ]\| Schuell, Inc., Dassel, Germany). Sizes of the expressed proteins were estimated with the help of molecular mass standards (molecular mass range, 30,000–200,000 kDa) from Sigma. Western blot analysis was conducted with the indicated antibodies and the BM chemiluminescence Western blotting kit (Roche Diagnostics) or the SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical Co.) according to the supplier’s instructions. For stripping of blots, the membranes were incubated in 1x TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 100 mM 2-mercaptoethanol and 2% SDS with gentle shaking for 30 min at 50 C.

For immunoprecipitations of JAK2, amounts of cDNA for JAK2 (0.7 µg pSVL/JAK2 on a 10-cm petri dish) had to be kept low to reduce nonspecific phosphorylation in the absence of ligand. Immunoprecipitations using the anti-JAK2 antibody or antiphosphotyrosine antibody (PY20) were conducted as described above, except that 1% Triton buffer was used [1% (vol/vol) Triton X-100 (Serva, Heidelberg, Germany), 50 mM HEPES, pH 7.6, 150 mM NaCl, 1 mM EDTA, 10% (vol/vol) glycerol, 1 mM dithiothreitol, 1 mM benzamidine, 0.5 mM phenylmethylsulfonylfluoride, 10 mM NaF, 1 mM Na3VO4, 30 mM Na4P2O7].

EMSA
EMSAs were performed as described previously (57) using a double-stranded 32P-labeled mutated m67SIE oligonucleotide from the c-fos promotor (m67SIE, 5'-GATCCGGGAGGGATTTACGGGAAATGCTG-3') (55). The protein DNA-complexes were separated on a 4.5% polyacrylamide gel containing 7.5% glycerol in 0.25-fold TBE (20 mM Tris, 20 mM boric acid, 0.5 mM EDTA) at 20 V/cm for 4 h. Gels were fixed in 10% methanol, 10% acetic acid, and 80% water for 30 min, dried, and autoradiographed.

Flow Cytometry
Fluorescence-activated cell sorting analysis was conducted as described earlier (29) using the monoclonal anti-IL-5R{alpha} antibody (16–4) or anti-IL-5Rß antibody (S-16) as primary antibodies. Cells were analyzed in a FACScalibur (Becton Dickinson and Co., Mountain View, CA) equipped with a 488-nm argon laser.

Reporter Gene Assays
pGL3{alpha}2M-215Luc contains the promotor region -215 to +8 of the rat {alpha}2-macroglobulin gene upstream of the luciferase-encoding sequence (58). For reporter gene assays, HepG2 cells on six-well plates were transfected with 0.6 µg of luciferase reporter construct, ß-galactosidase control plasmid pSVß-gal (Promega Corp.) and each receptor expression vector, respectively. Twenty-four hours after transfection, the cells were stimulated with 80 ng/ml IL-5 for another 24 h. Luciferase assays were performed using the Promega Corp. luciferase assay system. All values were normalized to ß-galactosidase activity.


   ACKNOWLEDGMENTS
 
We wish to thank Simone Bamberg-Lemper and Hanna Czajkowska for excellent technical assistance. We thank Jan Tavernier for providing antibodies, Michael Igel for murine LEPR cDNAs, Ian Kerr for the pRK5-JAK2 vector, Andrea Küster for IL-6, and Andrew Ziemiecki for the gift of JAK2 antibody.


   FOOTNOTES
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 542) and the Fonds der Chemischen Industrie (Frankfurt/Main, Germany).

Abbreviations: CLR, Chimeric leptin receptor; IgG-POD, antirabbit horseradish peroxidase-labeled IgG; IL-3R, IL-5R, IL-3 and IL-5 receptors, respectively; JAK, Janus kinase; LEPR, leptin receptor; PRLR-L, signaling competent form of the PRL receptor; PRLR-S, short splice variant of PRL receptor; STAT, signal transducer and activator of transcription.

Received for publication April 16, 2001. Accepted for publication December 3, 2001.


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