“ functional fragment ” as used herein is a peptide or polypeptide , optionally carrying one or more modifications , which , when integrated in a suitable receptor molecule , functions as a binding site for one or more signaling molecules . this binding may be either constitutive or ligand induced . “ modification ” as used herein may be any modification of an amino acid known by the person of ordinary skill in the art , such as , as a nonlimiting example , phosphorylation , glycosylation or ubiquitinilation . “ essentially consisting ” as used herein means that the cited seq id no : is embedded in a total functional fragment of only 50 amino acids , preferably only 30 amino acids , even more preferably about 20 amino acids . “ compound ” as used herein means any chemical or biological compound , including simple or complex organic or inorganic molecules , peptides , peptido - mimetics , proteins , antibodies , carbohydrates , nucleic acids or derivatives thereof . “ bind ( ing )” as used herein means any interaction , be it direct or indirect . a direct interaction implies a contact between the binding partners . an indirect interaction means any interaction wherein the interaction partners interact in a complex of more than two compounds . the interaction can be completely indirect , with the help of one or more bridging molecules , or partly indirect , where there is still a direct contact between the partners , which is stabilized by the additional interaction of one or more compounds . “ signaling molecule ” as used herein means any molecule that is involved in the transfer or the inhibition of the transfer of an activated receptor to a reporter gene . in that respect , a molecule that is not directly involved in signaling itself , but that , by binding on the receptor can inhibit another molecule from binding and inducing the signaling pathway is also considered as a signaling molecule . “ reporter gene ” is any gene that leads to a detectable signal and can be , as a nonlimiting example , an antibiotic resistance gene , a toxin gene resulting in cell death , a gene encoding a fluorescent protein such as gfp , or a gene encoding an enzyme activity such as β - galactosidase . the coding sequence is placed under control of a suitable promoter , i . e ., a promoter that is induced by binding of a ligand to the receptor and consequent induction of the reporter pathway . monoclonal anti - flag antibody m2 was obtained from sigma . mouse recombinant leptin was purchased from r & amp ; d systems . peptides were synthesized using standard fmoc - amino - acid solid phase chemistry on an applied biosystems model 431a peptide synthesizer . biotin was manually esterified following similar procedures as on the synthesizer and added to the reaction mixture . incorporation of phosphotyrosine was realized by using a protected fmoc - tyrosine - phosphobenzylester ( novabiochem ). after purification , the mass of the peptides was confirmed by mass spectrometry . sequences of the peptides were biotin - qrqpsvk ( p ) y 985 atlvsndk ( seq id no : 7 ) and biotin - nhreksvc ( p ) y 1077 lgvtsvnr ( seq id no : 8 ). generation of the leptin receptor mutants , the lepr f3 deletion and mlr f3 point mutants . generation of the leptin receptor mutants was described by eyckerman et al . ( 1999 ). the mutant leptin receptor ( long isoform ) is indicated as mlr . mlr f3 is the mutant where all the cytoplasmic residues are mutated to phenylalanine . through site - directed mutagenesis ( quikchange ™, stratagene ), three deletion and one point mutant of the mlr f3 , cloned in the pmet7 expression vector , were constructed . the deletion mutants lr f3 de11 ( aa : 1 - 1103 ), lr f3 de12 ( aa : 1 - 1050 ) and lr f3 de13 ( aa : 1 - 952 ) were created by mutating ala on position 1104 ( primer combinations mbu - o - 993 and mbu - o - 994 ), ser on position 1051 ( primer combination mbu - o - 885 and mbu - o - 886 ), and cys on position 953 ( primer combination mbu - o - 887 and mbu - o - 888 ), to a stop - codon , respectively . in each case , an extra ecorv restriction site was simultaneously built in to distinguish mutated from parental dna . rat socs3 cdna was amplified using 5 ′- gaagatctgtgcgccatggtc acccacagcaagtt ( seq id no : 9 ) and 5 ′- gctctagattttgctccttaaagtg gagcatcata ( seq id no : 10 ) as forward and reverse primers , respectively , and using mrna from leptin - stimulated pc12 cells as template . cdna was prepared using a standard rt procedure with superscript reverse transcriptase ( lifetechnologies ). amplification was realized using pfu polymerase ( stratagene ). the socs3 fragment was re - amplified using forward primer 5 ′- gcgagatctcagaattcgtcacccacagcaagtttcc ( seq id no : 11 ) and the reverse primer described above , which allows bglii - xbai - based cloning in a pmet7 variant containing an n - terminal flag tag sequence ( mdykddddk ) ( seq id no : 12 ), resulting in pmet7fsocs3 . cis constructs were generated in the pmet7 vector under control of the srα promoter . using site - directed mutagenesis ( quikchange ™, stratagene ), a unique apai restriction site was generated between the srα promoter and the ecori site in the pmet7mcs construct , a pmet7 - derivative containing a multiple cloning site . the 158 amino acid c - terminal part of human gp130 was amplified using primers 5 ′- gacgggcccgccaccatggattacaa ggatgacgacgataagatctcgaccgtggtacacagtggc ( seq id no : 13 ) and 5 ′- gcgaattccgaaccgccctgaggcatgtagccgcc ( seq id no : 14 ). the forward primer also encodes the flag - tag sequence ( mdykddddk ) ( seq id no : 12 ) and contains apai and bglii sites . the reverse primer contains a ggs hinge sequence and an ecori restriction site allowing apai - ecori - based cloning in the pmet7 construct , resulting in the pmg1 basic vector . a fragment of the sv40 - t antigen ( amino acid 261 - 708 ) was amplified using primers 5 ′- gcgaattcgaagcagaggaaactaaacaagtg ( seq id no : 15 ) and 5 ′- cgtctagagcggccgcagatctcgagtcgcgattatgtttcaggttcagggggag ( seq id no : 16 ). the n - terminal part of sv40 - t containing the nuclear localization signal was deleted to prevent nuclear shuttling . the forward primer contains an ecori site and the reverse primer contains a stop codon and nrui , xhoi , bglih , noti and xbai restriction sites . ligating this fragment in the pmg1 vector results in the pmg1 - svt vector . murine cis was amplified with forward primer 5 ′- gcggaattcgtcctctgcg tacagggatc ( seq id no : 17 ) and with reverse primer 5 ′- gcctctagatcagagttggaa ggggtactg ( seq id no : 18 ). ecori - xbai based cloning in the pmg1 - svt construct resulted in the pmg1 - cis plasmid . a fragment of human cis2 ( aa 34 - 198 ) containing the sh2 domain and the socs box sequence was amplified via rt - pcr using primers mbu - o - 1046 and mbu - o - 1047 as forward and reverse primers , respectively . the forward primer contains an ecori site while the reverse primer contains the stop codon and a noti restriction site . rna from hek293 c116 cells was used as input in a one - step rt - pcr procedure ( qiagen onestep rt - pcr kit ). ecori - noti - based cloning in pmg1 - svt resulted in pmg1 - cis2 . a fragment of human vav1 ( vavs : aa 259 - 789 ) was amplified using pfu polymerase from mrna of the human tf1 cell line by standard rt - pcr techniques . primers were mbu - o - 737 and mbu - o - 738 as forward and reverse primers , respectively . mbu - o - 737 contains an extra ecori allowing in - frame fusion to gp130 , and mbu - o - 738 contains a stop codon and a xhoi recognition site . the amplified fragment was subcloned in de pcr ®- blunt vector and ligated in the pmg1 vector through an ecori - xhoi - based exchange , resulting in pmg1 - vavs . the put651 construct expressing β - galactosidase was obtained from eurogentec . generation of the pgl3 - rpap1 - 1uci construct was described by eyckerman et al . ( 1999 ). the full - length rpap1 promoter fragment was excised using partial digestion with kpni and xhoi and ligated into the kpni - xhoi - digested pxp2d2 vector ( gift from prof . s . nordeen ), resulting in the leptin - responsive pxp2d2 - rpap1 - 1uci reporter construct . the pxp2d2 vector is a derivative of pxp2 that lacks potential cryptic activator protein 1 sites ( grimm and nordeen , 1999 ). mbu - o - 737 hvavs primer f 5 ′- gcggaattcaagctggaggaatgttctca ( seq id no : 19 ) mbu - o - 738 hvavs primer r 5 ′- gcctcgagttacacgtagttggcagggaacc ( seq id no : 20 ) mbu - o - 993 mlr f3 del1 f 5 ′- tcctgtgcacattcccatgaccatggctgttcagtgacatca ( seq id no : 21 ) mutagenesis mbu - o - 994 mlr f3 del1 r 5 ′- tgatgtcactgaacagccatggtcatgggaatgtgcacagga ( seq id no : 22 ) mutagenesis mbu - o - 885 mlr f3 del2 f 5 ′- gatttcaccacaactttgatatccggggttggatgagc ( seq id no : 23 ) mutagenesis mbu - o - 886 mlr f3 del2 r 5 ′- gctcatccaaccccggatatcaaagttgtggtgaaatc ( seq id no : 24 ) mutagenesis mbu - o - 887 mlr f3 del3 f 5 ′- gaaagcagttctatttgatatcgtgaccagtgtaacag ( seq id no : 25 ) mutagenesis mbu - o - 888 mlr f3 del3 r 5 ′- ctgttacactggtcacgatatcaaatagaactgctttc ( seq id no : 26 ) mutagenesis mbu - o - 924 mlr fall f 5 ′- cacctccgtcaacagagcggctagcggtgtg cttttga ctggtg ( seq id no : 27 ) r225a / e226a mutagenesis mbu - o - 925 mlr fall r 5 ′- caccagtcaaaagcacaccgctagccgctctgttgacg gaggtg ( seq id no : 28 ) r225a / e226a mutagenesis mbu - o - 1045 cis2 primer f 5 ′- gcagaattcaccctgcggtgcctggagcc ( seq id no : 29 ) mbu - o - 1046 cis2 primer r 5 ′- gctgcggccgcttatacctggaatttatattcttcc ( seq id no : 30 ) culture conditions and transient transfection procedures for pc12 and generation of the pc12lr8 cell line were as previously described . ( eyckerman et al ., 1999 ; waelput et al ., 2000 .) hek293t cells were maintained in a 10 % co 2 humidified atmosphere at 37 ° c . and grown using dmem with 4500 mg / l glucose , 10 % fetal bovine serum and 50 μg / ml gentamycin ( all from lifetechnologies ). typically , 4 × 10 5 cells were seeded the day before transfection in a 6 - well plate and transfected overnight with approximately 2 μg plasmid dna using a standard calcium phosphate precipitation procedure . one day after transfection , cells were re - suspended with cell dissociation agent ( lifetechnologies ), seeded in a black well plate ( costar ) and stimulated overnight with 100 mg / ml leptin or left unstimulated . luciferase assays , binding assays using leptin - seap and northern blot hybridizations with socs3 and actin probes were described previously by eyckerman et al . ( 1999 ). β - galactosidase activity was measured using the galacto - star ™ chemiluminescent detection kit ( tropix ) and a topcount chemiluminescence counter ( packard ). approximately 10 6 hek293t cells were lysed in 150 μl 2 × loading buffer . after sonication , 30 μl was loaded on a 10 % polyacrylamide gel . after overnight blotting , flag - tagged socs3 was revealed using a 1 / 2500 dilution of anti - flag antibody . blotting efficiency was checked using ponceaus staining ( sigma ). for phosphopeptide affinity chromatography , approximately 3 × 10 7 hek293t cells transiently transfected as indicated were lysed in lysis buffer ( 20 mm hepes ph 7 ; 1 mm mgcl 2 ; 10 mm kcl ; 0 . 5 mm dtt ; 150 mm nacl ; 0 . 5 % np40 ; 1 mm navo 4 ; 5 mm naf ; 20 % glycerol ; complete ™ protease inhibitor cocktail ( roche )). precipitated material was cleared by 5 minutes centrifugation at 10 , 000 g . to eliminate a - specific interactions , supernatants were brought on a pre - column containing sepharose 4b beads prior to phosphopeptide affinity chromatography on streptavidin - coupled agarose beads ( sigma ). peptide concentrations were determined by a colorimetric assay using alkaline hydrolysis and a ninhydrine reagent . fifty μl of streptavidin - agarose slurry was incubated with 5 nmoles of peptide for each reaction . cleared lysate was incubated for two hours at 4 ° c . under slow stirring . after incubation , beads were washed four times with lysis buffer and re - suspended in 2 × loading buffer . the observation that most obese human patients show elevated levels of leptin suggests the existence of a so - called leptin resistance . ( maffei et al ., 1995 .) socs3 , a potential mediator of this resistance ( bjorbaek et al ., 1998 ), is a member of a family of sh2 domain - containing proteins mostly involved in negative regulation of signal transduction pathways . ( hilton et al ., 1998 .) through representational difference analysis , a pcr - based differential expression screening in pc12 cells expressing the mouse leptin receptor , several leptin - induced transcripts ( including socs3 ) were identified . in order to check the inhibitory role of socs3 on leptin signaling in the cell systems used , an expression vector containing rat socs3 was generated . transient expression of socs3 in pc12 lr8 cells , a pc12 clone that stably expresses the leptin receptor , together with the pgl3 - rpap11uci construct leads to marked inhibition of leptin - mediated reporter induction ( fig1 ). the rpap1 promoter is derived from the rat pancreatitis associated protein1 gene and shows strong induction in pc12 cells upon treatment with leptin plus forskolin . ( waelput et al ., 2000 .) similar results were obtained in hek293t cells transiently transfected with the leptin receptor , socs3 and the pxp2d2 - rpap11uci constructs . the pxp2d2 - rpap11uci construct showed in hek293t cells a four times increased signal / background ratio and a higher reproducibility in comparison to the pgl3 - based reporter construct . taken together , these findings confirm the inhibitory activity of socs3 in our cell - based reporter systems . the murine leptin receptor contains three tyrosine residues within its cytoplasmic domain ( fig2 a ). y1138 is situated within a box3 or stat3 recruitment motif and is critical for leptin - mediated gene induction . previously , a critical role for y985 and , to a lesser extent , also for y1077 in the murine leptin receptor in a negative feedback signal was shown . ( eyckerman et al ., 1999 .) to assess the effect of y to f mutations within the leptin receptor on socs3 expression itself , northern blot analysis was performed using pc12 cells transiently transfected with leptin receptor mutants . forty - eight hours after transfection , cells were stimulated for 24 hours with leptin ( 100 ng / ml ) or were left untreated ( fig2 b ). previously obtained results indicate that socs3 transcription is rapidly induced in pc12 cells with an optimum around 30 minutes , but that weak socs3 expression persists until later time points . ( waelput et al ., 2000 .) results shown in fig2 b imply that during this late induction phase , mutation of y985 results in a strong up - regulation of socs3 transcription . this occurs only in conjunction with y1138 , a stat3 activation site , indicating that socs3 expression is stat - dependent as has been shown for signaling via the il - 6 and leukemia inhibitory factor receptors . ( auernhammer et al ., 1999 ; schmitz et al ., 2000 .) absence of the y1077 site does not lead to altered socs3 mrna expression , but the lack of both sites results in a further increased induction level above what is observed for the y985 mutant alone . these results are in line with the previous results for metallothionein ii mrna regulation . ( eyckerman et al ., 1999 .) a possible explanation for the elevated socs3 expression level could be the loss of negative feedback via either socs3 itself or , alternatively , via the sh2 - containing phosphatase shp - 2 . ( carpenter et al ., 1998 ; li and friedman , 1999 .) both socs3 and the cytokine inducible sh2 - containing protein ( cis ), another member of the socs family , were shown to bind activated receptors . socs3 binds to the gp130 signaling component of the il - 6 complex ( nicholson et al ., 2000 ; schmitz et al ., 2000 ), the erythropoietin receptor ( sasaki et al ., 2000 ) and the insulin receptor ( emanuelli et al ., 2000 ), while cis binds to the erythropoietin receptor , the β common chain ( yoshimura et al ., 1995 ) and to the leptin receptor ( see example 2 ). based on its functional similarity with the gp130 chain , the leptin receptor was tested for interaction with socs3 . leptin receptor variants containing y to f mutations at positions y985 , y1077 , or at both sites , were tested in a functional assay based on rpap1 - induction . signalling via the leptin receptor variants was analyzed upon transient transfection in hek293t cells with the pxp2d2 - rpap11uci and the flag - tagged pmet7 - fsocs3 expression construct . luciferase activity data were normalized by co - transfection with put651 and a β - galactosidase activity assay . expression of leptin receptor variants and socs3 were confirmed , respectively , by a binding assay using a leptin - seap fusion protein and western blot analysis using an anti - flag antibody ( fig3 ). lr expression levels were measured on transfected cells by incubation for 90 minutes with leptin - seap fusion protein with or without excess leptin ( 100 ×). the transfected cells were stimulated for 24 hours with leptin or left untreated . luciferase measurements were performed in triplicate and were normalized by co - transfection with the put651 β - galactosidase construct and a β - galactosidase activity assay . the results indicated a strong inhibitory activity of socs3 ( 80 - 90 % inhibition ) when the wild - type or the mutant y1077f leptin receptor was expressed , and a moderate inhibition ( 30 - 50 % inhibition ) upon expression of a y985f receptor variant . in the case of the double y985 / 1077f mutant , no inhibition was observed . the interaction sites of socs3 with the leptin receptor were confirmed using a biochemical approach . flag - tagged socs3 was expressed upon transient transfection of pmet7 - fsocs3 in hek293t cells and was analyzed for binding to ( phospho )- tyrosine - containing peptides matching the two motifs within the leptin receptor . lysates of approximately 3 × 10 7 transfected cells were incubated with biotinylated peptides encompassing residues y985 or y1077 in the leptin receptor . western blot analysis using an anti - flag antibody showed clear and specific binding of socs3 to the phosphorylated y985 peptide , while non - phosphorylated peptide did not bind any socs3 protein . socs3 also binds specifically to the phosphorylated y1077 peptide , but apparently with a much lower affinity confirming its accessory role in socs3 mediated inhibition ( fig4 ). taken together , these findings suggest that both tyrosines are involved in socs3 recruitment to the activated leptin receptor complex although binding to the y1077 position apparently occurs at significantly lower efficiency suggesting an accessory role for this site . this observation could explain why , at late time points post - stimulation , the single y1077 mutant does not lead to detectable differences on induction of metallothionein ii and socs3 genes when compared to the wild - type receptor , whereas a more pronounced induction is observed when the double y985 / 1077f mutant is compared to the y985f receptor variant ( fig2 ). in line with these findings , recruitment of socs3 at the single phosphorylated y1077 motif shows a moderate inhibition of leptin signaling . perhaps this accessory role of the y1077 motif is only functional when expression levels of socs3 are highly elevated , suggesting different threshold levels for leptin receptor signaling may exist . although several groups were not able to show leptin - dependent phosphorylation of the y1077 site in the leptin receptor using anti - phosphotyrosine antibodies ( li and friedman , 1999 ; banks et al ., 2000 ), this disclosure indicates a functional phosphorylation - dependent role for this site in leptin signaling . it is of note that a stretch of ten amino acids downstream of the y1077 motif is very conserved throughout evolution . this pronounced conservation of the y1077 motif also underscores its functional importance in leptin signaling . cis binds to the highly conserved y985 and y1077 motifs in the leptin receptor cis expression is rapidly induced by leptin , but recruitment to the leptin receptor was never shown . using a functional assay , binding of cis to both phosphorylated y985 and y1077 motifs have been demonstrated . leptin receptor mutants that contained the y1138f mutation were used and resulted in lack of stat ( baumann et al ., 1996 ) and rpap1 promoter ( eyckerman et al ., 1999 ) activation . y1138f receptor mutants containing additional y to f substitutions at positions y985 , y1077 , or at both , were transiently co - transfected with the gp130 - cis construct and the pxp2d2 - rpap1 - 1uci and put651 reporter vectors . normalized luciferase data are shown in fig5 and indicate comparable functional recruitment of cis to both single y985 and y1077 tyrosine motifs within the leptin receptor . in line with the use of both sites , a stronger signal is obtained for the single y1138f mutant receptor . no luciferase activity is induced in case of cells transfected with pmet7 - lr - f3 or with empty vector . no difference in expression levels of the mutant receptors was observed . mutual comparison and cross - species relationship of the amino acid contexts of the y985 and y1077 residues support the functional data on cis recruitment on both sites . vav is recruited by the conserved y1077 region , even without phosphorylation of the tyrosine residue the functionality of the mlr f3 deletions were tested by the following transfections in hek293t cells : transfection was performed as follows : a 300 μl precipitation mixture was prepared containing 3 . 05 μg dna ( 0 . 05 μg of put651 ; 1 μg of each other vector ). two hundred μl of this mixture was added to 4 × 10 5 cells for 18 hours . after one wash with pbs - a , cells were further incubated in 3 ml dmem medium for 24 hours . cells were re - suspended with 200 μl cell dissociation agent and dmem medium was added to a total volume of 2 ml . fifty μl of this cell suspension was transferred into a 96 - well plate for each transfection and stimulation with leptin ( final concentration 100 ng / ml ) was performed in triplicate . twenty - four hours after stimulation , luciferase and β - galactosidase measurements were performed as described herein . as shown in fig6 , the results illustrate that from the three deletion mutants , only the mlr f3 de11 ( transfection b ) shows a significant induction of the rpap1 promoter . transfections c and d , compared to the mlr f3 ( transfection a ), lack induction of luciferase activity with leptin . cis2 binds to both the y985 and y1077 motifs in the leptin receptor a similar functional assay in hek293t cells as in example 2 was performed . variant leptin receptor mutants were co - transfected with the pmg1 - cis2 construct and both the pxp2d2rpap11uci and put651 reporters . fig7 shows normalized luciferase data . no difference in expression levels of the mutant receptors was observed . data shown is the mean of triplicate measurements . cis2 binds predominantly to the y1077 site , while binding to the y985 site is less pronounced . no significant luciferase induction is observed when the lr - f3 mutant was transfected . attoub , s ., noe , v ., pirola , l ., bruyneel , e ., chastre , e ., mareel , m ., wymann , m . p . and gespach , c . 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