Pleiotropy of leptin receptor signalling is defined by distinct roles of the intracellular tyrosines Paul Hekerman1, Julia Zeidler1, Simone Bamberg-Lemper1, Holger Knobelspies1, Delphine Lavens2, Jan Tavernier2, Hans-Georg Joost3 and Walter Becker1 Institute of Pharmacology and Toxicology, Medical Faculty of the Aachen University, Germany The Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research (VIB9), Ghent University, Belgium German Institute of Human Nutrition (DIfE) Potsdam-Rehbrucke, Nuthetal, Germany ¨ Keywords leptin; leptin receptor; STAT; luciferase; insulinoma Correspondence W Becker, Institut fuer Pharmakologie und Toxikologie, Medizinische Fakultat der ă RWTH Aachen, Wendlingweg 2, 52074 Aachen, Germany Fax: +49 241 8082433 Tel: +49 241 8089136 E-mail: walter.becker@post.rwth-aachen.de (Received 14 July 2004, revised September 2004, accepted 13 September 2004) doi:10.1111/j.1432-1033.2004.04391.x The leptin receptor (LEPR) is a class I cytokine receptor signalling via both the janus kinase ⁄ signal transducer and activator of transcription (JAK ⁄ STAT) and the MAP kinase pathways In addition, leptin has been shown previously to activate AMP-activated kinase (AMPK) in skeletal muscle To enable a detailed analysis of leptin signalling in pancreatic beta cells, LEPR point mutants with single or combined exchanges of the three intracellular tyrosines were expressed in HIT-T15 insulinoma cells Western blots with activation state-specific antibodies recognizing specific signalling molecules revealed that the wild-type receptor activated STAT1, STAT3, STAT5 and ERK1 ⁄ but failed to alter the phosphorylation of AMPK Each of the three intracellular tyrosine residues in LEPR exhibited different signalling capacities: Tyr985 was necessary and sufficient for leptin-induced activation of ERK1 ⁄ 2; Tyr1077 induced tyrosyl phosphorylation of STAT5; and Tyr1138 was capable of activating STAT1, STAT3 and STAT5 Consistent results were obtained in reporter gene assays with STAT3 or STAT5-responsive promoter constructs, respectively Furthermore, the sequence motifs surrounding the three tyrosine residues are conserved in LEPR from mammals, birds and in a LEPR-like cytokine receptor from pufferfish Mutational analysis of the box3 motif around Tyr1138 identified Met1139 and Gln1141 as important determinants that define specificity towards the different STAT factors These data indicate that all three conserved tyrosines are involved in LEPR function and define the pleiotropy of signal transduction via STAT1 ⁄ 3, STAT5 or ERK kinases Activation and inhibition of AMPK appears to require additional components of the signalling pathways that are not present in beta cells Leptin is an adipocyte-secreted hormone that informs the brain about the status of the body’s energy stores It regulates energy homeostasis through effects on satiety and energy expenditure and deficiencies of leptin or the leptin receptor in humans or rodents result in severe obesity, infertility, impaired growth and insulin resistance [1] In db ⁄ db mice that lack the signalling active, long splice variant of the leptin receptor (LEPRb), this syndrome was largely corrected by neuron-specific transgenic complementation of LEPRb deficiency [2], supporting the notion that leptin acts predominantly on central pathways However, a number of peripheral Abbreviations AMPK, AMP-activated kinase; ERK, extracellular signal-regulated kinase; GH, growth hormone; JAK, janus kinase; LEPR, leptin receptor; SH2, src-homology 2; SOCS3, suppressor of cytokine signalling 3; STAT, signal transducer and activator of transcription FEBS Journal 272 (2005) 109–119 ª 2004 FEBS 109 Pleiotropic leptin receptor signalling actions of leptin have also been described [3] Two effects have been particularly well studied: the stimulation of proinflammatory immune responses by direct action on T-lymphocytes [4,5], and the inhibition of insulin secretion from pancreatic beta cells [6–9] As a class I cytokine receptor, LEPRb activates the janus kinase ⁄ signal transducer and activator of transcription (JAK ⁄ STAT) signalling pathway [10,11] Ligand binding to LEPRb results in the activation of JAK2 by transphosphorylation and subsequent phosphorylation of tyrosine residues in the cytoplasmic part of LEPRb [10,12,13] These phosphorylated tyrosines provide docking sites for signalling proteins with srchomology (SH2) domains A short splicing variant of the leptin receptor (LEPRa) is abundantly expressed in most tissues but lacks tyrosine residues and appears to be signalling-inactive [14] Murine LEPRb contains three intracellular tyrosine residues that are conserved in mammals and birds [15,16] Tyr1138 is located in a canonical box3 motif (Tyr-x-x-Gln) and recruits the transcription factor STAT3, which is subsequently phosphorylated by JAK2, dimerizes and translocates to the nucleus Here it binds to the promoter regions of target genes Leptin-induced phosphorylation and nuclear translocation of STAT3 has been demonstrated in vivo in the hypothalamus [17,18], in isolated T-lymphocytes [19] and in insulin secreting cells [20,21] Mice with a targeted mutation of Tyr1138 (leprS1138) are hyperphagic and obese, underscoring the essential role of STAT3 in energy homeostasis [22] However, whereas db ⁄ db mice are infertile, short and diabetic, leprS1138 mice are fertile, longer and appear to be less hyperglycemic This result clearly indicates that STAT3-independent pathways play an important role in LEPRb signalling Of the two other intracellular tyrosine residues in LEPRb, Tyr985 can recruit either the tyrosine phosphatase SHP-2 or suppressor of cytokine signalling (SOCS3) [15,23–26] Binding of SOCS3 to Tyr-985 attenuates leptin signalling by inhibition of the receptor-associated JAK kinase [25] In contrast, recruitment of SHP-2 does not alter JAK2 activity but results in GRB2 binding to SHP-2 and activation of the RAS ⁄ RAF ⁄ ERK pathway [26,27] In contrast to Tyr985 and Tyr1138, the role of Tyr1077 in leptin signalling is not yet clear More recently, it has been shown that AMP-dependent protein kinase (AMPK) appears to be a downstream mediator of leptin signalling Leptin directly stimulates phosphorylation and activation of the a2 catalytic subunit of AMPK in muscle [28] In contrast, leptin suppresses a2 AMPK activity in secondary hypothalamic neurons indirectly via activation of agouti-related protein (AGRP) neurons [29] 110 P Hekerman et al The aim of this study was to analyse the contribution of the intracellular tyrosine residues to LEPRbmediated effects on STAT factors, MAP kinase and AMPK These data show that LEPRb is capable of activating a broader range of STAT factors than other cytokines such as interleukin-6 (IL-6) and growth hormone (GH) Analysis of point mutants revealed that each of the individual tyrosine residues in the intracellular part of LEPRb exhibits a different signalling capacity In particular, our data identify Tyr1077 as a docking site for STAT5 Results Leptin receptor signal transduction in HIT-T15 and RINm5F insulinoma cells We used HIT-T15 insulinoma cells as a model system to characterize leptin receptor signalling in pancreatic beta cells The cells were stimulated either with leptin, IL-6 or GH to compare the activation of downstream signalling pathways by the different cytokines Although HIT-T15 cells have previously been reported to be leptin responsive [30,31] we observed no leptininduced phosphorylation of STAT1, STAT3, STAT5 or ERK1 ⁄ ERK2 in untransfected cells (Fig 1A) IL-6 and GH elicited the expected responses, i.e tyrosine phosphorylation of STAT3 and STAT5, respectively In HIT-T15 cells transfected with cDNA of LEPRb, phosphorylation of STAT1, STAT3, STAT5 and the ERK kinases was induced by leptin The weak response of STAT5 to leptin compared to that elicited by GH can be partially explained by the rather ineffective transfection of the HIT-T15 cells (estimated at 10–20% of transfected cells) Cells expressing the short splice variant of the leptin receptor (LEPRa) showed no detectable response to leptin We used rat RINm5F cells as a second insulinoma cell line to confirm these results Contrary to published data [20], treatment of nontransfected cells with leptin failed to elicit a detectable reponse to leptin (data not shown) Therefore, we constructed a retroviral vector in which the MuMLV long-terminal repeat controlled the expression of murine LEPRb Infection of RINm5F cells with the recombinant retrovirus generated polyclonal pools of cells stably expressing LEPRb In these cells (Fig 1B), leptin and IL-6 stimulated tyrosine phosphorylation of STAT3 to a similar degree, but leptin again induced activation of a broader spectrum of STAT factors (STAT1, STAT3, STAT5, STAT6) Leptin has recently been reported to activate AMPK in muscle cells, thereby stimulating expression of enzymes involved in fatty acid oxidation [28] FEBS Journal 272 (2005) 109–119 ª 2004 FEBS P Hekerman et al A Pleiotropic leptin receptor signalling B C Fig Leptin signalling in insulinoma cell lines (A) HIT-T15 cells were transfected with expression plasmid encoding either the short (LEPRa) or the long splicing variant (LEPRb) of the leptin receptor or were not transfected Cells were serum-starved for 22 h and stimulated with the indicated cytokine (Lep, leptin; sIL-6R, soluble IL-6 receptor; GH, growth hormone) for 15 Total cellular lysates were used for Western blot analysis with phospho-specific antibodies against STAT1, STAT3, STAT5A ⁄ B, and ERK1 ⁄ The STAT5 antibody does not discriminate between STAT5A and STAT5B (B) RINm5F cells stably expressing LEPRb were stimulated with cytokines for 15 Nuclear extracts (left panels) or total cellular lysates (right panels) were subjected to Western blot analysis with the indicated phospho-specific antibodies The phospho-STAT6 antibody crossreacted with phospho-STAT5 (indicated by asterisks) (C) HIT-T15 cells (left panels) or RINm5F cells (right panels) ectopically expressing LEPRb were treated with leptin (Lep) or with vehicle alone (Ø) The activating phosphorylation of AMPK was detected by immunoblotting with a phosphospecific antibody As positive controls for AMPK activation, HIT-T15 cells were glucose deprived for 15 in phosphate buffered saline, and RINm5F cells were starved by overnight-incubation without change of the medium Tyrosyl phosphorylation of STAT3 is shown as a positive control for the leptin effect (lower panel) Previously, leptin has also been shown to prevent lipotoxicity in pancreatic islets by upregulating expression of fatty acid oxidation-related enzymes (carnitine palmitoyl transferase, acyl CoA oxidase) [32,33] Therefore, we analysed the effect of leptin on AMPK in the insulinoma cell lines As shown in Fig 1C, leptin treatment failed to alter the activating phosphorylation of AMPK in HIT-T15 cells or in RINm5F cells As a positive control, AMPK phosphorylation was readily stimulated in glucose-depleted cells, indicating that essential components of the AMPK pathway were present in these cell lines FEBS Journal 272 (2005) 109–119 ª 2004 FEBS Role of the intracellular tyrosine residues in LEPRb To determine the role of the three intracellular tyrosine residues (Tyr985, Tyr1077, Tyr1138) in LEPR-mediated activation of downstream signalling events, constructs in which phenylalanine(s) replaced either one of the three tyrosines or combinations of them were expressed in HIT-T15 cells The specific signalling capacities of each tyrosine residue in the intracellular domain of LEPRb can be deduced from the results presented in Fig Tyr985 is necessary and sufficient 111 Pleiotropic leptin receptor signalling P Hekerman et al Fig Signalling by leptin receptor mutants in HIT-T15 cells HIT-T15 cells were transfected with expression plasmids for the indicated LEPRb point mutants (left panels, single point mutants; right panels, double mutants) and for STAT5B Cells were treated with 100 ngỈmL)1 leptin or vehicle for 15 before nuclear extracts were prepared Leptin-induced phosphorylation of downstream signalling molecules was assayed by Western blotting and immunodetection with phospho-specific antibodies Postnuclear supernatants were probed with a LEPRspecific antibody to verify comparable expression of the LEPRb mutants One representative experiment out of three is shown for activation of ERK1 ⁄ ERK2, either Tyr1077 or Tyr1138 is required for leptin-induced tyrosyl phosphorylation of STAT5, and Tyr1138 is essential for activation of STAT1 and STAT3 Note that leptininduced STAT5 phosphorylation was weakly detectable in cells transfected with the triple mutant of LEPRb (FFF); this effect was variable in its magnitude and may be due the overexpression of STAT5B in this experiment We next studied the capacity of the LEPRb point mutants to induce reporter gene activity driven by STAT response elements Two different reporter constructs were used In the first one (a2M), luciferase expression is driven by the IL-6 responsive element of the a2-macroglobulin promoter, which is controlled by STAT3 [34] The second reporter plasmid (spi2.1) contains the GH-responsive element of the rat serine protease inhibitor 2.1 (spi2.1) gene, whose expression is controlled by STAT5 [35] Assays with the different promoter constructs were performed under identical conditions to analyse the ability of the LEPR point 112 mutants to specifically activate STAT3- and STAT5driven promoter activity (Fig 3) Consistent with the detection of tyrosine phosphorylated STAT factors by Western blot analysis, all LEPRb constructs containing the Tyr1138fiPhe mutation (YYF, FYF, YFF, FFF) were severely reduced in their capacity to stimulate a2M reporter gene activity It is likely that the residual activation by the mutants retaining Tyr1077 (YYF, FYF) can be explained by the action of STAT5 Spi2.1 promoter activity was stimulated by all constructs containing either Tyr1077 or Tyr1138, in full agreement with the presumed control by STAT5 As expected, Tyr985 was not able to induce reporter gene activity driven by STAT-dependent promoters Interestingly, the data point to an inhibitory function of Tyr1138 in these assays, as the mutation of Tyr1138 enhanced luciferase activity (YYF vs WT, P < 0.001, double-sided t-test) This result is consistent with the known requirement of Tyr1138 for induction of the feedback-inhibitor protein SOCS3 [27] FEBS Journal 272 (2005) 109–119 ª 2004 FEBS P Hekerman et al Fig Effects of leptin receptor mutants on STAT-responsive promoter elements HIT-T15 cells on six-well plates were transfected with luciferase reporter constructs driven by the IL-6-response element of the a2 macroglobulin promoter (a2M) or by the GHresponse element of serine protease inhibitor 2.1 promoter (spi2.1), b-galactosidase reporter control plasmid, and the indicated LEPRb expression plasmids Twenty-four hours after transfection, cells were treated or untreated with leptin (100 ngỈmL)1) for 18 h The luciferase activity was determined and normalized to coexpressed b-galactosidase activity Data are expressed as fold stimulation relative to unstimulated cells Bars reflect means ± SEM of three (a2M) or four to five independent experiments (spi2.1) The bottom panel of Fig provides an expression control for the different LEPR mutants as aliquots of the same DNA samples were transfected in this experiment Structural basis for the recruitment of different STAT factors by Tyr1138 The unusual capacity of the box3 motif in LEPRb to mediate activation of STAT1, STAT3 and STAT5 stimulated us to characterize the structural determinants required for binding of each of these proteins Four point mutations targeting the residues C-terminal of Tyr1138 were generated to test their potential role in binding of the different STAT factors (Fig 4) A glutamine found three amino acids after the phosphorylated tyrosine (position P+3, where P is the phosphorylation site), fitting the consensus STAT3-binding FEBS Journal 272 (2005) 109–119 ª 2004 FEBS Pleiotropic leptin receptor signalling motif (YXXQ) [36], was exchanged for valine, which is found in this position in the Tyr1077 motif A proline residue at P+2 has been proposed to be important for binding of STAT1 by the IL-6 receptor gp130 [37] and was exchanged for glycine A consensus sequence for binding of STAT5 has not yet been explicitly defined but in most cases the phosphorylated tyrosine is followed by an aliphatic hydrophobic residue such as leucine, isoleucine, valine or methionine [38–41] The constructs were transiently expressed in HIT-T15 cells, and leptin-induced activation of STAT factors was monitored by Western blot analysis with phospho-specific antibodies and by reporter gene assays (Fig 4) Consistent with the established consensus sequence for binding of STAT3, mutation of Gln1141 abolished leptin-induced phosphorylation of STAT3 and decreased the activation of the a2M-derived promoter but did not affect STAT5 phosphorylation or induction of the spi2.1 promoter In contrast, exchange of Met1139 for alanine or arginine eliminated phosphorylation of STAT5 and reduced induction of the spi2.1 promoter construct This promoter is probably also responsive to STAT1 and ⁄ or STAT3 Mutation of Pro1140 strongly reduced activation of all three STAT proteins Taken together, these results indicate that the combination of a hydrophobic residue in the P+1 position and the glutamine in P+3 allows the binding of either STAT1, STAT3 or STAT5 to pTyr-1138 in LEPRb Discussion Class I cytokine receptors such as LEPRb transmit extracellular signals by recruiting SH2 domain-containing proteins to phosphorylated tyrosine residues Until now, only two of the three conserved tyrosines in LEPRb (Tyr985 and Tyr1138) have been demonstrated to play a role in leptin signalling [22–27] Our analysis of LEPRb point mutants in insulinoma cell lines indicates that the presence of Tyr1077 as the only intracellular tyrosine residue was sufficient to induce tyrosine phosphorylation of STAT5 (in HIT-T15 and RINm5F cells), and to stimulate STAT5-driven reporter gene activity (in HIT-T15 cells) These results establish that all of the three intracellular tyrosines in murine LEPRb participate in leptin signal transduction and have different capacities to activate downstream signalling pathways Tyr985 is required for the activation of the RAS ⁄ RAF ⁄ ERK pathway, Tyr1077 mediates the activation of STAT5, and Tyr1138 can stimulate tyrosine phosphorylation of STAT1, STAT3 or STAT5 Leptin has previously been reported to stimulate AMPK in muscle and, more recently, to inhibit the kinase in hypothalamic nuclei [28,29] We therefore 113 Pleiotropic leptin receptor signalling P Hekerman et al A B Fig Mutational analysis of the box3 motif and sequence conservation of the intracellular tyrosine motifs in LEPRb (A) Mutations of the amino acids following Tyr1138 were introduced into LEPRb-FFY HIT-T15 cells were transfected with the LEPRb constructs indicated by the sequence of the wild type (YMPQ, identical with FFY in Figs and 3) or mutated box3 motif (YRPQ, YAPQ, YMGQ, YMPV, and FMPQ, which is identical with FFF) Western blot analysis of STAT phosphorylation in nuclear extracts (left panels) and reporter gene assays (right diagrams) were performed as described in the legends to Figs and Expression levels of the overexpressed proteins were assessed by Western blot analysis of postnuclear supernatants (STAT5, LEPR) Luciferase activities are represented as percent of the YMPQ construct Bars (open bars, no leptin; filled bars, 100 ngỈmL)1 leptin) reflect means ± SD of three to five independent experiments (except n ¼ for YAPQ) (B) Sequences from murine, human, chicken and pufferfish (Tetraodon nigroviridans) LEPR are shown as representatives for mammals, birds and fish Consensus sequences are given in the bottom line (/, hydrophic residue) The localization of the tyrosines in murine LEPRb is illustrated (985, 1077, 1138) Proteins recruited to the phosphotyrosine motifs are indicated below the alignments NCBI protein database accession numbers for the LEPR sequences are P48356 (mouse); NP_002294 (human), AAF31355 (chicken) and AAR25693 (Tetraodon) expected to observe a reduced or increased phosphorylation of AMPK in response to leptin in the pancreatic beta cell lines However, concentrations of leptin that maximally activated STAT3 failed to alter AMPK phosphorylation (Fig 1C) Glucose deprivation induced the anticipated activation of AMPK, indicating that the upstream kinase is present in the cells Consistent with our results, Leclerc et al [42] recently reported that leptin did not change AMPK activity in murine MIN6 insulinoma cells and in isolated rat islets Thus, we conclude that pancreatic beta cells lack a component 114 required for leptin-induced activation of AMPK, possibly the c3 subunit of AMPK which appears to specifically expressed in skeletal muscle [42] Our conclusion that Tyr1077 in murine LEPRb plays an important role in leptin signalling is supported by the fact that the surrounding sequence is strikingly conserved in mammals and birds [15], although the intracellular domains of murine and chicken LEPRb show little overall sequence similarity (25% of identical amino acids distal of the JAK kinase binding motif) Moreover, our database searches for FEBS Journal 272 (2005) 109–119 ª 2004 FEBS P Hekerman et al LEPR homologues in more distantly related vertebrates identified LEPR-related sequences from green pufferfish (Tetraodon nigroviridans; NCBI protein accession AAR25693) and zebrafish (Danio rerio; ENSEMBL predicted protein ENSDARP00000011908) containing the three tyrosine phosphorylation motifs (Fig 4B) Sequence comparisons indicated that these proteins are more closely related with LEPR than with any other mammalian cytokine receptor: the sequence from pufferfish contains 30% of identical amino acids with murine LEPRb and 27% with murine gp130, the signal transducing subunit of the IL-6 receptor (gaps > 50 amino acids were not penalized) The intracellular domain shows no significant similarity with any known mammalian protein except for LEPRb Tyr-1077 has previously been shown to play a role in down-regulation of LEPRb signalling, presumably by serving as a docking site for SOCS3 [15] The conservation of the aliphatic hydrophobic residue in the P+1 position after Tyr-1077 is also compatible with the known requirements of STAT5-binding as determined in different receptors [38,40,41] Leptin-induced activation of STAT5 has already been described in the first papers reporting STAT signalling by the LEPRb [10,43] Later, in vivo studies suggested that only STAT3 is activated upon leptin administration in the hypothalamus of mice and rats [17,44] However, leptin-induced tyrosine phosphorylation has been observed in various cell types, e.g hypothalamic GT17 cells [45], intestinal L-cells [46], enterocyte-like CaCo-2 cell line [47], and H-35 hepatoma cells [48] Our results are also consistent with earlier reports that mutant constructs of the human LEPRb either with a substitution of Tyr1141 for phenylalanine or with a deletion of the C-terminus including Tyr1141 were still able to induce DNA binding of overexpressed STAT5B in electrophoretic mobility shift assays [10,49] It should be noted that leptin-induced activation of STAT5 in the insulinoma cell lines was detectable with endogenous levels of STAT factors (Figs 1, and 4) Changing the ratio of STAT factors in a cell can strikingly alter the downstream effects of a receptor, particularly when different STATs compete for binding to the same tyrosine residue [50] In pancreatic b-cells, prolactin and growth hormone are physiological inducers of STAT5 [51] Both hormones stimulate insulin production and b-cell proliferation via STAT5-dependent pathways [52] and may contribute to islet hyperplasia in pregnancy [51] Interestingly, leptin has also been reported to stimulate proliferation and suppress apoptosis of islet cells [53–55] It is conceivable that this effect of leptin is mediated by activation of STAT5 FEBS Journal 272 (2005) 109–119 ª 2004 FEBS Pleiotropic leptin receptor signalling An unexpected finding was the capability of Tyr1138, which is located within a canonical box3 consensus motif, to mediate activation of STAT5 The results of our mutational analysis are consistent with findings obtained with other cytokine receptors: the position after the phosphotyrosine (P +1) is critical for binding of STAT5 but not of STAT1 or STAT3, whereas the glutamine in position P +3 is required for binding of STAT1 and STAT3, but is not relevant for STAT5 This result is consistent with the fact that residues C-terminal of the phosphotyrosine are important for binding of SH2 domains [55a] Equivalent results have been obtained by mutational analysis of the STAT binding motif of the IL-9 receptor TyrLeuProGln(367–370), which is also capable to activate STAT1, STAT3, and STAT5 [39,56] It should be noted, however, that the same sequence motifs in gp130 [TyrLeuProGln(905– 908)] and [TyrMetProGln(915–918)] not activate STAT5, indicating that the hydrophobic residue in P +1 is not the only residue required for binding of STAT5 Experimental procedures Reagents Recombinant murine leptin was obtained from PeproTec (London, UK) and GH from Bachem (Bubendorf, Switzerland) Recombinant human IL-6 and soluble IL-6 receptor were kindly provided by Gerhard Muller-Newen (Departă ment of Biochemistry, Aachen University) The following primary antibodies were used: polyclonal rabbit antibodies against p(Y701)-STAT1, p(Y705)-STAT3, p(Y694)-STAT5, p(Y641)-STAT6, STAT1, STAT3, p(T172)-AMPK, antiAMPK, anti-pTyr Ig PY100, and phospho p42 ⁄ 44 MAP kinase from Cell Signalling Technology (Beverly, MA), anti-STAT5A ⁄ B from Upstate (Charlottesville, VA, USA), goat anti-(mouse LEPR) Ig from R&D Systems (Wiesbaden, Germany), antibody against phosphorylated JAK2 (pYpY1007 ⁄ 1008) from BioSource Technologies (Camarillo, CA) and anti-pTyr Igs PY20 from Transduction Laboratories, Inc (San Diego, CA) Horseradish peroxidase-labeled anti-(rabbit IgG) (IgG-POD) was obtained from Pierce Chemical Co (Rockford, IL), anti-(mouse IgG-POD) from Amersham (Buckinghamshire, UK), and anti-(goat IgG-POD) from Dianova (Hamburg, Germany) Cell culture, transient transfection, and retroviral infection HIT-T15 hamster insulinoma cells (gift of A Schurmann, ă Potsdam) and RINm5F rat insulinoma cells (gift of D Meyer zu Heringdorf, Essen) were cultivated in RPMI 1640 115 Pleiotropic leptin receptor signalling medium with l-glutamine, 10% (v ⁄ v) fetal bovine serum, 100 unitsỈmL)1 penicillin, and 100 mgỈmL)1 streptomycin The medium was further supplemented with 5% (v ⁄ v) horse serum for culture of HIT-T15 cells For analysis of total cell lysates (Fig 1), · 105 HIT-T15 cells on six-well plates plates were transfected with 1.0 lg of pSVL-LEPR plasmids [14] For preparation of nuclear extracts (Fig 2), · 106 cells on 6-cm plates were transfected with 1.5 lg of pMET7-mLRlo constructs [57] using the JET-PEI transfection reagent (Polyplus-transfection, Illkirch, France) If indicated (Fig 2), 1.0 lg of pECE-STAT5B expression plasmid was cotransfected Point mutants targeting the residues C-terminal of Tyr1138 in LEPRb were generated with the help of the QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene) For stable expression in RINm5F cells, LEPRb was subcloned from pSVL-LEPRb into the retroviral vector pWZL-Neo Viruses were produced in 293T cells, and confluent RINm5F cells were infected with pWZL-neo-LEPR viruses [58] Selection was performed for 14 days in mgỈmL)1 of G418 Western blot analysis and EMSA Cells were incubated in serum-free medium for 18–22 h before leptin (100 ngỈmL)1), IL-6 (200 U mL)1), or GH (500 ngỈmL)1) was added for 15 All assays of transiently transfected HIT-T15 cells were performed 48 h after transfection Nuclear extracts were prepared by hypotonic lysis [34] To prepare total cellular lysates, cells were washed with phosphate buffered saline and lysed in 1% (w ⁄ v) SDS, 20 mm Tris ⁄ HCl pH 7.4 in a boiling water bath for For Western blot analysis, protein samples were separated by SDS ⁄ PAGE (8% gels), blotted on to nitrocellulose, and specific proteins were detected by chemiluminescence using the primary antibodies mentioned above and horseradish peroxidase-labeled secondary antibody Blots were re-used after stripping the primary antibody by incubation in 2% (w ⁄ v) SDS, 50 mm Tris ⁄ HCl, 150 mm NaCl, pH 7.4 in the presence of 100 mm 2-mercaptoethanol Reporter gene assays Luciferase reporter constructs contained the promoter region )215 to +8 of the rat a2-macroglobulin gene (pGL3a2 m-215Luc; kindly provided by P C Heinrich, Department of Biochemistry, Aachen, Germany) for assays of STAT3-driven promoter activity [59] or six copies of the GH-responsive GAS-like element (GLE) from the rat spi2.1 gene (pSpi-GLE-Luc, gift of L.-A Haldosen, Karolinska Institutet, Huddinge, Sweden) for assays of STAT5-dependent promoter activity [35] HIT-T15 cells on six-well plates (3.5 · 105 cells per well) were transfected with 0.3 lg of pMET7-LEPRb expression plasmids along with 0.75 lg each of the luciferase reporter construct and 116 P Hekerman et al the b-galactosidase reporter control plasmid pSVb-gal (Promega) Transcription of the lacZ gene in this control vector is driven by the SV40 early promoter and enhancer Twenty-four hours after transfection, the cells were stimulated with 100 ngỈmL)1 leptin for 22 h in serum-free medium Luciferase activities were determined from duplicate wells with the help of a commercial kit (Promega), and data were normalized to b-galactosidase activities Acknowledgements We thank Drs Lars-Arne Haldosen, Gerhard Mulleră Newen, Peter C Heinrich, Annette Schurmann, Dagă mar Meyer zu Heringdorf for generous donations of reagents and cell lines This work was supported by the Deutsche Forschungsgemeinschaft (SFB 542) References Friedman JM & Halaas JL (1998) Leptin and the regulation of body weight in mammals Nature 395, 763–770 Kowalski TJ, Liu SM, Leibel RL & Chua SC Jr (2001) Transgenic complementation of leptin-receptor deficiency I Rescue of the obesity ⁄ diabetes phenotype of LEPR-null mice expressing a LEPR-B transgene Diabetes 50, 425–435 Margetic S, Gazzola C, Pegg GG & Hill RA (2002) Leptin: a review of its peripheral actions and interactions Int J Obes Relat Metab Disord 26, 1407–1433 Lord GM, Matarese G, Howard JK, Baker RJ, Bloom SR & Lechler RI (1998) Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression Nature 394, 897–901 Lord GM, Matarese G, Howard JK, Bloom SR & Lechler RI (2002) Leptin inhibits the anti-CD3-driven proliferation of peripheral blood T cells but enhances the production of proinflammatory cytokines J Leukoc Biol 72, 330–338 Kulkarni RN, Wang ZL, Wang RM, Hurley JD, Smith DM, Ghatei MA, Withers DJ, Gardiner JV, Bailey CJ & Bloom SR (1997) Leptin rapidly suppresses insulin release from insulinoma cells, rat and human islets and, in vivo, in mice J Clin Invest 100, 2729–2736 Ookuma M, Ookuma K & York DA (1998) Effects of leptin on insulin secretion from isolated rat pancreatic islets Diabetes 47, 219–223 Kieffer TJ & Habener JF (2000) The adipoinsular axis: effects of leptin on pancreatic beta-cells Am J Physiol Endocrinol Metab 278, E1–E14 Seufert J, Kieffer TJ & Habener JF (1999) Leptin inhibits insulin gene transcription and reverses hyperinsulinemia in leptin-deficient ob ⁄ ob mice Proc Natl Acad Sci USA 96, 674–679 FEBS Journal 272 (2005) 109–119 ª 2004 FEBS P Hekerman et al 10 Baumann H, Morella KK, White DW, Dembski M, Bailon PS, Kim H, Lai CF & Tartaglia LA (1996) The full-length leptin receptor has signalling capabilities of interleukin 6-type cytokine receptors Proc Natl Acad Sci USA 93, 8374–8378 11 Tartaglia LA (1997) The leptin receptor J Biol Chem 272, 6093–6096 12 Bjørbaek C, Uotani S, da Silva B & Flier JS (1997) Divergent signaling capacities of the long and short isoforms of the leptin receptor J Biol Chem 272, 32686–32695 13 Ghilardi N & Skoda RC (1997) The leptin receptor activates janus kinase and signals for proliferation in a factor-dependent cell line Mol Endocrinol 11, 393–369 14 Bahrenberg G, Behrmann I, Barthel A, Hekerman P, Heinrich PC, Joost HG & Becker W (2002) 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 Mol Endocrinol 16, 859–872 15 Eyckerman S, Broekaert D, Verhee A, Vandekerckhove J & Tavernier J (2000) Identification of the Y985 and Y1077 motifs as SOCS3 recruitment sites in the murine leptin receptor FEBS Lett 486, 33–37 16 Horev G, Einat P, Aharoni T, Eshdat Y & FriedmanEinat M (2000) Molecular cloning and properties of the chicken leptin-receptor (CLEPR) gene Mol Cell Endocrinol 162, 95–106 17 Vaisse C, Halaas JL, Horvath CM, Darnell JE Jr, Stoffel M & Friedman JM (1996) Leptin activation of Stat3 in the hypothalamus of wild-type and ob ⁄ ob mice but not db ⁄ db mice Nat Genet 14, 95–97 18 Hubschle T, Thom E, Watson A, Roth J, Klaus S & ă Meyerhof W (2001) Leptin-induced nuclear translocation of STAT3 immunoreactivity in hypothalamic nuclei involved in body weight regulation J Neurosci 21, 2413–2424 19 Maccarrone M, Di Rienzo M, Finazzi-Agro A & Rossi A (2003) Leptin activates the anandamide hydrolase promoter in human T lymphocytes through STAT3 J Biol Chem 278, 13318–13324 20 Morton NM, Emilsson V, de Groot P, Pallett AL & Cawthorne MA (1999) Leptin signalling in pancreatic islets and clonal insulin-secreting cells J Mol Endocrinol 22, 173–184 21 Briscoe CP, Hanif S, Arch JR & Tadayyon M (2001) Fatty acids inhibit leptin signalling in BRIN-BD11 insulinoma cells J Mol Endocrinol 26, 145–154 22 Bates SH, Stearns WH, Dundon TA, Schubert M, Tso AW, Wang Y, Banks AS, Lavery HJ, Haq AK, Maratos-Flier E, Neel BG, Schwartz MW & Myers MG Jr (2003) STAT3 signalling is required for leptin regulation of energy balance but not reproduction Nature 421, 856–859 FEBS Journal 272 (2005) 109–119 ª 2004 FEBS Pleiotropic leptin receptor signalling 23 Carpenter LR, Farruggella TJ, Symes A, Karow ML, Yancopoulos GD & Stahl N (1998) Enhancing leptin response by preventing SH2–containing phosphatase interaction with Ob receptor Proc Natl Acad Sci USA 95, 6061–6066 24 Li C & Friedman JM (1999) Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase modulates Ob receptor signal transduction Proc Natl Acad Sci USA 96, 9677–9682 25 Bjørbaek C, Lavery HJ, Bates SH, Olson RK, Davis SM, Flier JS & Myers MG Jr (2000) SOCS3 mediates feedback inhibition of the leptin receptor via Tyr985 J Biol Chem 275, 40649–40657 26 Bjørbaek C, Buchholz RM, Davis SM, Bates SH, Pierroz DD, Gu H, Neel BG, Myers MG Jr & Flier JS (2001) Divergent roles of SHP-2 in ERK activation by leptin receptors J Biol Chem 276, 4747–4755 27 Banks AS, Davis SM, Bates SH & Myers MG Jr (2000) Activation of downstream signals by the long form of the leptin receptor J Biol Chem 275, 14563– 14572 28 Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D & Kahn BB (2002) Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase Nature 415, 339–343 29 Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferre P, Birnbaum MJ, Stuck BJ & Kahn BB (2004) AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus Nature 428, 569–574 30 Shimizu H, Ohtani K, Tsuchiya T, Takahashi H, Uehara Y, Sato N & Mori M (1997) Leptin stimulates insulin secretion and synthesis in HIT-T 15 cells Peptides 18, 1263–1266 31 Tsiotra PC, Tsigos C & Raptis SA (2001) TNFalpha and leptin inhibit basal and glucose-stimulated insulin secretion and gene transcription in the HIT-T15 pancreatic cells Int J Obes Relat Metab Disord 25, 1018– 1026 32 Zhou YT, Shimabukuro M, Koyama K, Lee Y, Wang MY, Trieu F, Newgard CB & Unger RH (1997) Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation Proc Natl Acad Sci USA 94, 6386–6390 33 Zhou YT, Shimabukuro M, Wang MY, Lee Y, Higa M, Milburn JL, Newgard CB & Unger RH (1998) Role of peroxisome proliferator-activated receptor alpha in disease of pancreatic beta cells Proc Natl Acad Sci USA 95, 8898–8903 34 Wegenka UM, Buschmann J, Lutticken C, Heinrich ă PC & Horn F (1993) Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level Mol Cell Biol 13, 276–288 117 Pleiotropic leptin receptor signalling 35 Wood TJ, Sliva D, Lobie PE, Goullieux F, Mui AL, Groner B, Norstedt G & Haldosen LA (1997) Specificity of transcription enhancement via the STAT responsive element in the serine protease inhibitor 2.1 promoter Mol Cell Endocrinol 130, 69–81 36 Stahl N, Farruggella TJ, Boulton TG, Zhong Z, Darnell JE Jr & Yancopoulos GD (1995) Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors Science 267, 1349–1353 37 Gerhartz C, Heesel B, Sasse J, Hemmann U, Landgraf C, Schneider-Mergener J, Horn F, Heinrich PC & Graeve L (1996) Differential activation of acute phase response factor ⁄ STAT3 and STAT1 via the cytoplasmic domain of the interleukin signal transducer gp130 I Definition of a novel phosphotyrosine motif mediating STAT1 activation J Biol Chem 271, 12991– 11298 38 May P, Gerhartz C, Heesel B, Welte T, Doppler W, Graeve L, Horn F & Heinrich PC (1996) Comparative study on the phosphotyrosine motifs of different cytokine receptors involved in STAT5 activation FEBS Lett 394, 221–226 39 Demoulin JB, Uyttenhove C, Van Roost E, DeLestre B, Donckers D, Van Snick J & Renauld JC (1996) A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9 Mol Cell Biol 16, 4710–4716 40 Pezet A, Ferrag F, Kelly PA & Edery M (1997) Tyrosine docking sites of the rat prolactin receptor required for association and activation of STAT5 J Biol Chem 272, 25043–25050 41 Mayr S, Welte T, Windegger M, Lechner J, May P, Heinrich PC, Horn F & Doppler W (1998) Selective coupling of STAT factors to the mouse prolactin receptor Eur J Biochem 258, 784–793 42 Leclerc I, Woltersdorf WW, Da Silva Xavier G, Rowe RL, Cross SE, Korbutt GS, Rajotte RV, Smith R & Rutter GA (2004) Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion Am J Physiol Endocrinol Metab 286, E1023–E1031 43 Ghilardi N, Ziegler S, Wiestner A, Stoffel R., Heim MH & Skoda RC (1996) Defective STAT signaling by the leptin receptor in diabetic mice Proc Natl Acad Sci USA 93, 6231–6235 44 McCowen KC, Chow JC & Smith RJ (1998) Leptin signaling in the hypothalamus of normal rats in vivo Endocrinology 139, 4442–4447 45 Kaszubska W, Falls HD, Schaefer VG, Haasch D, Frost L, Hessler P, Kroeger PE, White DW, Jirousek MR & Trevillyan JM (2002) Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line Mol Cell Endocrinol 195, 109– 118 118 P Hekerman et al 46 Anini Y & Brubaker PL (2003) Role of leptin in the regulation of glucagon-like peptide-1 secretion Diabetes 52, 252–259 47 Morton NM, Emilsson V, Liu YL & Cawthorne MA (1998) Leptin action in intestinal cells J Biol Chem 273, 26194–26201 48 Wang Y, Kuropatwinski KK, White DW, Hawley TS, Hawley RG, Tartaglia LA & Baumann H (1997) Leptin receptor action in hepatic cells J Biol Chem 272, 16216–16223 49 White DW, Kuropatwinski KK, Devos R., Baumann H & Tartaglia LA (1997) Leptin receptor (OB-R.) signaling Cytoplasmic domain mutational analysis and evidence for receptor homo-oligomerization J Biol Chem 272, 4065–4071 50 Costa-Pereira AP, Tininini S, Strobl B, Alonzi T, Schlaak JF, Is’harc H, Gesualdo I, Newman SJ, Kerr IM & Poli V (2002) Mutational switch of an IL-6 response to an interferon-gamma-like response Proc Natl Acad Sci USA 99, 8043–8047 51 Nielsen JH, Svensson C, Galsgaard ED, Moldrup A & Billestrup N (1999) Beta cell proliferation and growth factors J Mol Med 77, 62–66 52 Friedrichsen BN, Richter HE, Hansen JA, Rhodes CJ, Nielsen JH, Billestrup N & Moldrup A (2003) Signal transducer and activator of transcription activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta-cells Mol Endocrinol 17, 945–958 53 Shimabukuro M, Wang MY, Zhou YT, Newgard CB & Unger RH (1998) Protection against lipoapoptosis of beta cells through leptin-dependent maintenance of Bcl2 expression Proc Natl Acad Sci USA 95, 9558–9561 54 Islam MS, Sjoholm A & Emilsson V (2000) Fetal pancreatic islets express functional leptin receptors and leptin stimulates proliferation of fetal islet cells Int J Obes Relat Metab Disord 24, 1246–1253 55 Okuya S, Tanabe K, Tanizawa Y & Oka Y (2001) Leptin increases the viability of isolated rat pancreatic islets by suppressing apoptosis Endocrinology 142, 4827–4830 55a Waksman G, Shoelson SE, Pant N, Cowburn D & Kuriyan J (1993) Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms Cell 72, 779–790 56 Demoulin JB, Van Roost E, Stevens M, Groner B & Renauld JC (1999) Distinct roles for STAT1, STAT3, and STAT5 in differentiation gene induction and apoptosis inhibition by interleukin-9 J Biol Chem 274, 25855–25861 57 Eyckerman S, Waelput W, Verhee A, Broekaert D, Vandekerckhove J & Tavernier J (1999) Analysis of Tyr to Phe and fa ⁄ fa leptin receptor mutations in the PC12 cell line Eur Cytokine Netw 10, 549–556 FEBS Journal 272 (2005) 109–119 ª 2004 FEBS P Hekerman et al 58 Kohn AD, Barthel A, Kovacina KS, Boge A, Wallach B, Summers SA, Birnbaum MJ, Scott PH, Lawrence JC Jr & Roth RA (1998) Construction and characterization of a conditionally active version of the serine ⁄ threonine kinase Akt J Biol Chem 273, 11937– 11943 FEBS Journal 272 (2005) 109–119 ª 2004 FEBS Pleiotropic leptin receptor signalling 59 Schaper F, Gendo C, Eck M, Schmitz J, Grimm C, Anhuf D, Kerr IM & Heinrich PC (1998) Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression Biochem J 335, 557–565 119 ... of LEPRb, phosphorylation of STAT1, STAT3, STAT5 and the ERK kinases was induced by leptin The weak response of STAT5 to leptin compared to that elicited by GH can be partially explained by the. .. activation of STAT5 The results of our mutational analysis are consistent with findings obtained with other cytokine receptors: the position after the phosphotyrosine (P +1) is critical for binding of. .. (1997) Divergent signaling capacities of the long and short isoforms of the leptin receptor J Biol Chem 272, 32686–32695 13 Ghilardi N & Skoda RC (1997) The leptin receptor activates janus kinase and