Human Leptin Receptor

Abstract

The leptin receptor (OB-R) is a member of the class I cytokine receptor family and mediates the weight regulatory effects of its ligand through interaction with cytoplasmic kinases. The extracellular domain of this receptor is comprised of two immunoglobulin-like and cytokine-receptor homology domains each and type III fibronectin domains. The extracellular domain of human leptin receptor was expressed in and purified from Chinese hamster ovary cells and was found to contain extensive N-glycosylation (approximately 36% of the total protein). The purified protein had a molecular weight of approximately 145,000 and exhibited ligand binding ability as evidenced by formation of ligand-receptor complex, followed by chemical cross-linking. The determined disulfide motif of the soluble leptin receptor contained several distinct cystine knots as well as 10 free cysteines. The N-glycosylation analysis revealed that Asn624 of the WSXWS motif (residues 622–626) within the C-terminal cytokine receptor homology domain was glycosylated, indicating that this region is solvent-exposed. On the other hand, the N-terminal WSXWS motif was not glycosylated. The leptin receptor (OB-R) is a member of the class I cytokine receptor family and mediates the weight regulatory effects of its ligand through interaction with cytoplasmic kinases. The extracellular domain of this receptor is comprised of two immunoglobulin-like and cytokine-receptor homology domains each and type III fibronectin domains. The extracellular domain of human leptin receptor was expressed in and purified from Chinese hamster ovary cells and was found to contain extensive N-glycosylation (approximately 36% of the total protein). The purified protein had a molecular weight of approximately 145,000 and exhibited ligand binding ability as evidenced by formation of ligand-receptor complex, followed by chemical cross-linking. The determined disulfide motif of the soluble leptin receptor contained several distinct cystine knots as well as 10 free cysteines. The N-glycosylation analysis revealed that Asn624 of the WSXWS motif (residues 622–626) within the C-terminal cytokine receptor homology domain was glycosylated, indicating that this region is solvent-exposed. On the other hand, the N-terminal WSXWS motif was not glycosylated. receptor cytokine receptor homology bis-disuccinimidyl suberate Chinese hamster ovary dimethyl sulfoxide disuccinimidyl suberate granulocyte colony-stimulating factor receptor 4-α-hydroxycyano-cinnamic acid matrix-assisted laser desorption ionization leptin receptor N-ethylmaleimide phenylthiohydantoin polyacrylamide gel electrophoresis high pressure liquid chromatography phosphate-buffered saline. The ob gene product, leptin, is an important circulating signal for regulation of body weight (1Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-431Crossref PubMed Scopus (11940) Google Scholar, 2Gainford T. Wilson T. Metcalf D. Handman E. McFarlene C. Ng A. Nicola N.A. Alexander W.S. Hilton D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14564-14568Crossref PubMed Scopus (678) Google Scholar, 3White D.W. Tartaglia L.A. Cytokine Growth Factor Rev. 1996; 7: 303-309Crossref PubMed Scopus (89) Google Scholar, 4White B.D. Martin R.J. Proc. Soc. Exp. Biol. Med. 1997; 214: 222-232Crossref PubMed Scopus (89) Google Scholar). Weight reducing effects of recombinant leptin were observed in both normal mice and mice with diet-induced obesity (5Coleman D.L. Diabetologia. 1993; 9: 294-298Crossref Scopus (570) Google Scholar). The biological function of leptin is mediated through its membrane-associated receptor, leptin receptor (OB-R).1 Because of the biological and therapeutic importance of the leptin/OB-R system, numerous studies have been carried out to understand how this interaction is related to body weight regulation (6Tartaglia L.A. Dembski M. Weng X. Deng N Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3267) Google Scholar, 7Baumann H. Morella K.K. White D.W. Dembski M. Bailon P.S. Kim H. Lai C.-F. Tartaglia L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8374-8378Crossref PubMed Scopus (767) Google Scholar, 8Tartaglia L.A. J. Biol. Chem. 1997; 272: 6093-6096Abstract Full Text Full Text PDF PubMed Scopus (1204) Google Scholar, 9White D.W. Kuropatwinski K.K. Devos R. Baumann H. Tartaglia L.A. J. Biol. Chem. 1997; 272: 4065-4071Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Human OB-R is a membrane-spanning glycoprotein consisting of a signal sequence, two immunoglobulin domains, two cytokine receptor homology (CRH) domains each containing a WSXWS motif, fibronectin type III domains, a transmembrane region, and an intracellular domain. The predicted extracellular domain consists of 839 amino acid residues and shows significant similarity to members of the class I cytokine receptor family (6Tartaglia L.A. Dembski M. Weng X. Deng N Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3267) Google Scholar), in particular the gp-130 signal-transducing component of various cytokine receptors as well as the granulocyte-colony-stimulating factor receptor (G-CSFR) (10Fukunaga R. Ishizaka-Ikeda E. Pan C.-X. Seto Y. Nagata S. EMBO J. 1991; 10: 2855-2865Crossref PubMed Scopus (273) Google Scholar). Although overall sequence identity between OB-R and gp-130 is low (approximately 24%), several key regions are conserved, particularly the second cytokine homology domain (CRH)-2 (6Tartaglia L.A. Dembski M. Weng X. Deng N Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3267) Google Scholar). In this paper, we report structural information including the disulfide motif and N-glycosylation map of the extracellular domain of OB-R. Pepsin was obtained from Sigma. Trypsin and thermolysin were purchased from Boehringer Mannheim. Cross-linkers BS and DSS were obtained from Pierce and 4-HCCA from Sigma. TheN- and O-glycanases were purchased from Genzyme (Cambridge, MA), and sialidase was from Boehringer Mannheim. Recombinant human leptin was prepared as described previously (11Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3914) Google Scholar). All chemicals used were of reagent grade or analytical grade. New Zealand White rabbits were injected subcutaneously on day 1 with 0.2 mg of soluble OB-R and an equal volume of Freund's complete adjuvant. Further boosters (days 7, 21, 35, and 56) were given with the substitution of Freund's incomplete adjuvant. Antibody titers were monitored by enzyme-linked immunoassay. After the third booster, 20 ml of blood was obtained from each animal. The antisera for leptin was affinity purified on a leptin-Sepharose column and then conjugated to horseradish peroxidase using a Freezyme Kit (Pierce). Samples were electrophoresed under reducing conditions on 4–20% SDS-PAGE gels (Novex, San Diego) and then transferred to BA83 nitrocellulose (Schleicher & Schuell) overnight at 10 V in 25 mm Tris, 192 mmglycine, 20% v/v methanol. After transfer, blots were treated for 1 h in 1× PBS, Tween 20 (Bio-Rad) containing 5% w/v non-fat dry milk before adding antisera (1/2000 dilution) or antibody (0.1 μg/ml) for an additional hour. The blots were then washed with PBS/Tween 20 four times, 10 min each. For detection of receptor, a secondary anti-rabbit Ig (Amersham Pharmacia Biotech.) was diluted 1/6000 in PBS/Tween 20 and reacted with the blot for 30 min, afterward repeating the washes. Blots were then treated with enhanced chemiluminescence detection reagents (Amersham Corp.) and exposed to Kodak AR film. DNA encoding the extracellular domain and signal sequence of the human leptin receptor (residues 1–839) (6Tartaglia L.A. Dembski M. Weng X. Deng N Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3267) Google Scholar) was cloned into the mammalian expression vector pDSRα (12DeClerck Y.A. Yean T.-D. Lu H.S. Ting J. Langley L. J. Biol. Chem. 1991; 266: 3893-3899Abstract Full Text PDF PubMed Google Scholar) and then transfected into CHO D− cells (13Wigler M. Silverstein S. Lee L.S. Pellicer A. Chang Y.C. Axel R. Cell. 1977; 11: 223-232Abstract Full Text PDF PubMed Scopus (907) Google Scholar). Individual cell colonies were selected based upon expression of a dihydrofolate reductase gene in the vector. The highest expressing cells were adapted to 30 mmmethotrexate to stimulate amplification of soluble OB-R expression. After expansion in spinner flasks, roller bottles were inoculated with 2 × 107 cells in 200 ml of Dulbecco's modified Eagle's medium:Ham's F12 supplemented with nonessential amino acids (Life Technologies, Inc.) and 5% fetal bovine serum (HyClone, Logan, UT). After the cells reached confluency (3–4 days), the medium was replaced with 200 ml of growth medium with no serum. Conditioned media were harvested after 7 days and immediately chilled on ice. CHO cell-conditioned media containing soluble OB-R were concentrated 10-fold with a Pellicon tangential flow ultrafiltration device (Amicon, Danvers, MA) fitted with a 100,000 molecular weight cut-off filter (Filtron, Bearfoot, MA). The concentrate was diafiltered with 4 volumes of 25 mm Tris-HCl, pH 7.8, at 4 °C and then loaded onto a Q-Sepharose column (80 × 140 mm) equilibrated with 25 mm Tris-HCl, 30 mm NaCl, pH 7.8. After eluting the column with a linear NaCl gradient from 30 to 350 mm in 20 column volumes, aliquots of column fractions were applied to 4–20% polyacrylamide gels (Novex, San Diego) (14Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (214196) Google Scholar) and run under reducing SDS-PAGE conditions. Protein bands were visualized by silver staining and a pool made of fractions containing soluble OB-R. This pool was reacted with hydroxylapatite for 4 h at 4 °C (Calbiochem) which bound contaminating proteins but very little soluble receptor. The hydroxylapatite unbound material was concentrated and applied to a Superose 12 column (35 × 600 mm) (Amersham Pharmacia Biotech) equilibrated with 10 mm sodium phosphate, 200 mm NaCl, pH 7.6. Column fractions were analyzed by SDS-PAGE as described above, except using Coomassie Blue to stain, and a pool consisting of more than 95% pure receptor was made. A stable complex was made by incubating soluble OB-R (100 μg) with a 2-fold molar excess of recombinant human leptin at 4 °C for 24 h. The ligand-receptor complex was purified by Superose 12 (10 × 300 mm) equilibrated with 1× PBS, pH 7.8. Cross-linking was performed by treating the complex with 1 mm DSS (dissolved in Me2SO) or BS (dissolved in water) for 1 h at room temperature. The reaction was terminated by addition of 0.1m ammonium acetate. The reactions were analyzed by SDS-PAGE under reducing conditions. Cross-linked products were detected by both silver staining and Western blotting using an affinity purified, horseradish peroxidase-conjugated, polyclonal antibody raised against human leptin. In order to obtain Cys-containing peptides, soluble OB-R (100 μg) was digested with pepsin (2 μg) in 0.02 n HCl, pH 2, for 20 h at 37 °C. Under acidic conditions, disulfide rearrangements can be avoided. The digestion was terminated by direct injection into an HPLC column. Tryptic digestion of soluble OB-R was performed in the presence of 5 mm iodoacetate in 0.1 m Tris-HCl, pH 7.5. The digestion was carried out at 37 °C for 24 h with an enzyme:substrate ratio of 1:50 (w/w). Thermolytic or of were performed in 0.1 pH at 37 °C for 20 h with an enzyme:substrate ratio of approximately (w/w). of OB-R (100 μg) was performed with 2 mg of in of The reaction was to for 24 h at 25 °C in the and was by direct HPLC The were treated in 0.1 m pH and then digested with followed by in the with an enzyme:substrate ratio of 1:50 (w/w). OB-R was and by adding to and to mm and then at °C for 5 was then to before the and receptor with an equal volume of of were then digested with sialidase or at 37 °C for h. A reaction containing no was treated to the digested of which were then analyzed by SDS-PAGE on polyacrylamide gels (Novex, San Diego) under reducing conditions. Protein bands were visualized by silver staining and Western of was performed by with in 0.1m pH at 37 °C for 24 h. The digested material was purified by HPLC for were by HPLC using a column × mm) with two linear from 2 to 30 min and from to 10 min at a flow of ml were on a column × mm) using a linear gradient from 2 to 30 The flow was the as N-terminal sequence of and proteins were performed on a or from For analysis of an was used with a liquid analysis was performed with the analysis for protein The were loaded onto for The that contained disulfide was with 10 at 25 °C for 5 min in The was with 20 mm N-ethylmaleimide for 30 min at room after to pH with 1 m as described previously H. in Scholar). The modified were after by HPLC using a column × mm) as described laser desorption ionization of the was performed using a or Samples were in and then on the with a 4-HCCA Cys-containing were analyzed using a with an using a The was with acid at 5 The was with a of The and were using Human soluble OB-R was expressed in CHO cells and purified from medium by and gel The purified soluble OB-R exhibited a on to approximately 145,000 of the purified soluble OB-R the molecular to approximately indicating extensive N-glycosylation with sialidase and its that is but not The N-terminal sequence of the purified protein was to be (residues the C-terminal sequence of (residues was from a The observed and C-terminal were to predicted from the sequence by Tartaglia (6Tartaglia L.A. Dembski M. Weng X. Deng N Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3267) Google Scholar), which is in the soluble OB-R purified contained amino acid residues residues to with a molecular weight of indicating that the OB-R contained approximately 36% of its total acid sequence of soluble OB-R. The sequence was from sequence (6Tartaglia L.A. Dembski M. Weng X. Deng N Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3267) Google Scholar). The sequence shows a signal The complex was prepared by the receptor with a 2-fold molar excess of leptin. After at 4 °C for 24 the was to chromatography using a Superose 12 column (10 × 300 mm) equilibrated with PBS, pH 7.8. were to complex and free leptin. the in Superose 12 is that the complex contained of OB-R. The complex was to HPLC using a × which that contained a ratio of leptin and OB-R In order to ligand binding of the purified complex was with 1 mm BS and followed by SDS-PAGE gel was and other to nitrocellulose and detected with an affinity purified, polyclonal antibody raised against recombinant human leptin. In the gel 4 a protein that to free receptor is 4 shows the Western blot of this gel using an the presence of leptin to the in this The soluble OB-R was by in numerous by HPLC sequence analysis was performed on and that more than Cys-containing were I shows the of the sequence of the Cys-containing including both and free cysteines. contained two peptides, (residues and (residues in which was to be a to the that and were not through a disulfide but as free and Although the two are in from the HPLC column under the conditions Cys-containing from N-glycosylation the determined after N N-glycosylation the determined after in a contained the sequence in is an but is a based on the this was was to be in the free of this revealed not to be contained an N-glycosylation sequence, peptides, and had the sequence of (residues an N-glycosylation of the a than After with the of which is to the The observed in HPLC is to and the that was in a free contained two peptides, (residues and (residues a two contain four disulfide with the two an of this that two were through a disulfide shows of indicating the for and a signal at from of the disulfide were determined from contained the sequence N (residues this to the DNA sequence, at be in the free in this were determined after as was no was detected at this contained two (residues and (residues containing of cystine knots in the receptor. disulfide in not be determined from this Further digestion of this with thermolysin the (residues (residues and (residues and were to be N-glycosylation as determined by sequence shows sequence analysis of analysis of this the third not significant that is not to but to The complete disulfide of this was determined by digestion as analysis of 1 10 were 2 was was was was 10 were was N in a to the sequence as but contained N-terminal amino a (residues with free analysis of two (residues and (residues the presence of a disulfide between and This was by the two (residues and (residues using to that and are contained the two (residues and (residues and the molecular of this was to the of a disulfide of A (residues contained a free of this was not to its had the sequence (residues by contained a free contained two in a (residues and both residues by in the free the second was previously detected as in the In order to the disulfide of cystine knots in receptor was digested with in the presence of 5 mm The map in 7 several key for disulfide and of selected are in Tryptic contained in two peptides, of which be in the free of the human receptor is by in both and were determined by analysis after digestion of this with in a of (residues (residues and (residues the to and significant of T. PubMed Scopus Google M. C. J. Protein 1994; PubMed Scopus Google Scholar). this a of two (residues and which significant of at to and that was to was to be a free Cys-containing from the the N the the in a analysis of 1 10 were 2 detected to as as detected to 10 were detected to as in a contained two and was digested with to obtain peptides, in which residues are at the T. PubMed Scopus Google Scholar, M. C. J. Protein 1994; PubMed Scopus Google Scholar). the two Cys-containing were as containing (residues and (residues and which contained (residues and (residues a in of a signal of which is to the was no significant was detected at upon a significant at the to and of was approximately the presence of that not contain the was to be analysis of 1 2 as as observed as in a a sequence (residues in which an is This contained residues as be in the free the was in low we a after of soluble OB-R. shows the HPLC map of the The contained a sequence (residues analysis revealed that a of to a the two disulfide the was with 10 at 25 °C for 5 min, followed by with in order to a disulfide 10 an HPLC after and of analysis of the revealed that and were detected as as between and contained four and the contained including additional the presence of disulfide and was to be This was by direct of from The was with and was digested with followed by The were purified by HPLC as in 10 contained the sequence (residues a of at 7 to This a presence of disulfide of the was modified with a of contained two and indicating a disulfide as by analysis of and from or are as as detected to detected to detected to detected to as as as as as as as as as from or are detected to as in a of and and of Cys-containing from and of map of The was and digested with and as described under 2, and as and analysis of 1 and was was was in a of both and information on the of the OB-R. of the upon no detection of obtained in sequence in the Cys-containing have N-glycosylation The were by sequence analysis and of other or were with in order to obtain shows the N-glycosylation of the human receptor and a with the sequence was found to be modified by and two were and Asn624 at the second WSXWS motif was found to the WSXWS motif been to be important in the of receptor or at its ligand binding region T. M. M. T. EMBO J. 1991; 10: PubMed Scopus Google Scholar), N-glycosylation at Asn624 the of OB-R. of 20 N-glycosylation be by overall structural of the extracellular domain of human OB-R disulfide 10 free and N-glycosylation is in disulfide and N-glycosylation are not with cytokine receptors including M. T. T. J. S. 1996; PubMed Scopus Google N-glycosylation of human or shows the in which N sequence in shows the in which N sequence in OB-R. in a In the of the human and the extracellular domain of the receptor several and in the human receptor This to that four be in the free This revealed that of residues are in the free that and are in the free Although 10 free residues are in the human of are to that are within the protein or from by or of free were not in this OB-R an N-glycosylation motif, as an sequence, the is in the disulfide This sequence was found in several proteins including protein and M. T. T. J. S. 1996; PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and are two in OB-R. other N-glycosylation containing have been for proteins including immunoglobulin and factor in which the or are found to be J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. C. J. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). contained a or at the second or for or the receptor and gp-130 have significant sequence homology to the OB-R (6Tartaglia L.A. Dembski M. Weng X. Deng N Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3267) Google Scholar). The domains of and other receptors a WSXWS motif, which is to an important in and is between two and Y. Protein PubMed Scopus Google D. C. A. Clark T. A. T. J. Exp. Med. 1997; PubMed Scopus Google Scholar). sequence motif, been to be important in the region of receptor M. J. A. D. J. S. H. J. J. 1996; PubMed Scopus Google Scholar). A sequence was found in (residues Although OB-R two repeating domains, the disulfide of two domains were found to be from each other in that the of the C-terminal contained two free a from the N-terminal which not have free cysteines. that domains I and have for ligand interaction or other for this or of the receptor be for of the