From the Howard Hughes Medical Institute and
§ The Rockefeller University,
New York, New York 10021
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ABSTRACT |
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The leptin receptor (Ob-R) is alternatively spliced into at least five different RNAs designated Ob-R(a-e). Ob-R(a-d) predict receptors with a single transmembrane domain, and Ob-Re predicts a secreted form of the receptor. The presence of an ~120-kDa soluble leptin receptor in mouse plasma was confirmed by precipitation with leptin-Sepharose beads followed by immunobloting with anti-leptin receptor antibodies. The soluble leptin receptor is larger than that predicted by the primary sequence. Deglycosylation of the receptor with peptide N:glycosidase F results in a decrease in molecular mass to a size consistent with that of the primary sequence. The secreted receptor was present in plasma from wild type mice but was truncated in plasma from db3J/db3J and absent in dbPas/dbPas plasma. Although db3J/db3J mice are known to have a frameshift mutation at amino acid 625, the basis for the mutation in dbPas/dbPas mice was not known. Further studies indicated that dbPas/dbPas mice carry a duplication of exons 4 and 5 of Ob-R. This mutation introduces a premature stop codon into the protein at amino acid 281. The absence of Ob-R in db3J/db3J and dbPas/dbPas mice confirm the identify of the 120-kDa plasma protein as Ob-Re.
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INTRODUCTION |
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Leptin is an adipocyte hormone that functions as an afferent signal in a negative feedback loop regulating body weight (1). Administration of exogenous leptin to rodents results in a dose-dependent loss of adipose tissue mass (2-7). Leptin exerts its weight-reducing effects via interaction with a receptor, Ob-R, which is localized in the hypothalamus and other tissues (8, 9). Ob-R is a member of the cytokine receptor family and is alternatively spliced. Four of the splice variants, Ob-R(a-d), encode a receptor with a single transmembrane domain and a cytoplasmic region of variable length (9). Ob-Rb encodes a leptin receptor with a long intracytoplasmic region that contains several motifs known to be important for protein-protein interactions and signal transduction (9, 10). One of the splice variants, Ob-Re, does not encode a transmembrane domain and predicts a secreted form of the receptor (11).
The db gene has been shown to be allelic with Ob-R. Three mouse alleles of db are available, each of which leads to severe early onset obesity and diabetes. The C57BL/Ks db/db mutation affects splicing of Ob-R and leads to the specific loss of Ob-Rb RNA (9, 10). db3J/db3J mice carry a frameshift mutation in the amino terminus of the receptor that affects all of the splice forms (11). The nature of these mutations predicts that C57BL/Ks db/db mice should be missing only the Ob-Rb isoform and that 129 db3J/db3J mice should carry mutations of all receptor forms. As the basis for the dbPas/dbPas mutation was not previously known, the affect of this allele on receptor protein(s) could not be predicted.
Assays of the plasma levels of Ob-R protein were made in wild type and mutant mice. Specific antibodies to Ob-R were used to assay for the presence of Ob-Re, the secreted form of the leptin receptor, in plasma from wild type and mutant mice. An ~120-kDa protein corresponding to a glycosylated form of Ob-Re circulates in plasma from wild type and C57BL/Ks db/db mice. The wild type protein was absent in plasma from 129 db3J/db3J mice as well as dbPas/dbPas animals. dbPas/dbPas mice were shown to carry a duplication in the extracellular region of Ob-R that ablates expression of all of the receptor variants.
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EXPERIMENTAL PROCEDURES |
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Materials-- Recombinant mouse leptin was obtained from Amgen (Thousand Oaks, CA). Baculovirus cloning vector pMelBac was purchased from Invitrogen (Carlsbad, CA). PNGase F1 was from New England Biolabs, Inc. (Beverly, MA) ob/ob mice, db3J/db3J mice, as well as wild type C57Balb/C mice were from Jackson Labs (Bar Harbor, ME). dbPas/dbPas and lean littermates were from the Pasteur Institute (Paris, France).
Sucrose Gradient Centrifugation of Leptin in Mouse Plasma and PBS-- Continuous sucrose gradient was used for rate zonal centrifugation. Mouse recombinant leptin (Amgen, 50 ng) in a final volume of 100 µl of either PBS or ob/ob serum were loaded onto a linear gradient of 5-20% w/v sucrose in PBS (pH 7.4). The run parameters were 55,000 rpm at 5 °C for 12 h using the SW55 Ti rotor. At the end of the run, 13 fractions of 400 µl each were collected and measured by the Bausch and Lomb hand refractometer to ascertain the sucrose concentration at each separated zone. Fractions were analyzed for leptin using enzyme-linked immunosorbent assay.
Affinity Purification of the Soluble Leptin Receptor-- CNBr-activated Sepharose 4B from Amersham Pharmacia Biotech was coupled to recombinant mouse leptin from Amgen (Thousand Oaks, CA) following manufacturer's instructions. Coupled resin was kept at 4 °C in PBS (Life Technologies, Inc.) as a 50% slurry (v/v) in the presence of 0.02% sodium azide. About 1 mg of leptin was coupled for every ml of Sepharose. To affinity purify the soluble leptin receptor, mouse plasma was first prepared following an eye bleed procedure. Blood was mixed with EDTA to give a final concentration of 2 mM, which was centrifuged for 7 min at 4,000 × g. The resulting supernatant was used directly for leptin binding. In a typical experiment, 12.5 µl of leptin beads was incubated with 100 µl of mouse plasma diluted in 150 µl of PBS. Incubation was allowed overnight at 4 °C, and beads were then washed three times in cold PBS. The pellet was reconstituted in 1× SDS sample buffer and loaded onto an 8% gel. Bound receptor was analyzed with antipeptide antibodies of Ob-R. Peptide antibodies against Ob-R were generated by Research Genetics (Huntsville, AL). Two antibodies were used to detect the receptor: antibody A (corresponding to amino acids 145-158 of mouse Ob-R, amino acid sequences EPLPKNPFKNYDSK) and antibody B (corresponding to amino acids 465-484 of mouse Ob-R, amino acid sequences HRRSLYCPDSPSIHPTSEPK).
Generation of Ob-Re Expression Vector in SF9 Cells-- Ob-Re cDNA was subcloned into the vector pMelBac (Invitrogen, CA), a polyhedrin promoter-based transfer cloning vector of baculovirus. This vector is designed to direct expression of recombinant proteins through the secretory pathway to the extracellular medium. The vector contains a signal sequence for honeybee melittin, which is efficiently secreted by SF9 cells, the host for the baculovirus. To subclone Ob-Re into pMelBac, the endogenous signal sequence of the leptin receptor was not used. It was replaced by the honeybee melittin signal sequence to achieve efficient secretion of the receptor. Ob-R sequence starts at amino acid residue 28 and ends at residue 805 of Ob-Re (GenBankTM accession number U49110) containing 778 amino acids of Ob-Re sequence. Inserts were obtained by PCR using primers that contain restriction sites at both ends for subcloning into pMelBac. Large scale expression of recombinant protein was produced in suspension culture. Supernatant from productive stocks was used directly in the assay for leptin binding. Signal for Ob-R can be obtained directly from 1 µl of total supernatant. In a binding experiment, 15 µl of the supernatant was used for incubation with the leptin-Sepharose.
Deglycosylation of Ob-Re from Baculovirus and Mouse Plasma-- Binding of leptin-Sepharose to mouse plasma and baculovirus supernatant was as described above. At the last wash with PBS, sample was divided into two halves, and pellet was saved for the deglycosylation reaction. Bound protein on leptin-Sepharose beads were denatured by boiling in denaturation buffer for 10 min followed by treatment with or without 1 µl of PNGase F (500 units/µl; New England Biolabs, MA) at 37 °C for 3 h. At the end of the incubation, equal volume of 2× sample buffer was added to each tube, and the entire reaction was loaded onto a 7% SDS-PAGE gel and blotted with anti-receptor antibody A.
Reverse Transcription-PCR Analysis of
dbPas/dbPas and Wild Type Mice Hypothalamic
First Strand cDNA--
RNA was purified from adult hypothalami of
wild type and dbPas/dbPas
mice using the Trizol reagent (Life Technologies). 1 µg of total RNA
was reverse-transcribed into first-strand cDNA using random primers
and SuperScript II Rnase H reverse transcriptase (Life
Technologies, MD). Heat-denatured sample was used directly for reverse
transcriptase-PCR analysis. To analyze the mutation of Lepr in
dbPas/dbPas mouse, three
pairs of primers spanning about 1 kilobase, each overlapping each
other, were used to amplify wild type and mutant cDNA. Primer
sequences: F1(CL086), ATGATGTGTCAGAAATTCTATG; R1(CL053), AACATCTTGTGTGGTAAAG; F2(CL049), TCAGGAGTCTGGAGTGACTGG; R2(CL005), CTTTTACATCCATGACAAGCG; F3 (CL042), GAGAATAACCTTCAATTCCAGATTC; and
R3(CL062), GGTGTGAAATTAACAGTGTTC. Cycle parameters are 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min 30 s
for a total of 40 cycles. PCR products were purified using Centricon-30
from Amicon (Beverly, MA) by diluting the sample with H2O
and spinning at 4 °C until sample contains DNA at a concentration of
0.1 µg/µl. Sequencing of PCR products was performed by the
sequencing facility at the Protein DNA Technology Center of The
Rockefeller University.
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RESULTS |
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Soluble Leptin Receptor (Ob-Re) Circulates in Mouse Plasma-- Previous data have indicated mouse and human leptin sediment with a higher molecular mass than that predicted of a 16-kDa protein, suggesting that leptin circulates bound to other proteins (12, 13). To confirm this, either human or mouse plasma was loaded on a 5-20% sucrose gradient. The gradient fractions were assayed using enzyme-linked immunosorbent assay (Fig. 1) (12, 13). These data confirmed that a fraction of endogenous human leptin sediments on a sucrose gradient in lower fractions than leptin diluted in PBS (data not shown). When recombinant leptin was added to plasma from ob/ob mice (which are leptin-deficient), its sedimentation was shifted (Fig. 1). The apparent molecular mass of the complex is ~100-kDa (within the limits of resolution of the sucrose gradient). Although the nature of the leptin binding protein(s) was not known, soluble leptin receptor was a possible candidate.
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DISCUSSION |
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Leptin circulates in mouse and human plasma either as a free 16-kDa protein or bound to other proteins (12, 13). The identity of these leptin-binding proteins has not been previously elucidated, although a secreted form of the leptin receptor was a logical candidate. The data presented here confirm that soluble leptin receptor is present in mouse plasma. However, the data do not exclude the possibility that proteins other than Ob-Re also bind to leptin.
Several consensus sequences for glycosylation are contained in the Ob-R coding sequence (8). The size of the soluble leptin receptor is larger than that predicted by its amino acid sequence, suggesting that it is glycosylated. The truncation of Ob-Re after PNGase F treatment confirms that it is glycosylated. This conclusion is supported by the observation that after PNGase F treatment, baculovirus and native Ob-Re migrate to the same position by SDS-PAGE. The identify of this 120-kDa band as Ob-Re is also confirmed by the absence of the wild type protein in plasma from dbPas/dbPas and db3J/db3J mice. The db3J/db3J mutation was previously predicted to encode a 625-amino acid secreted protein (11). The observation that Ob-Re is truncated in plasma from its mutant supports this. The mutation in dbPas/dbPas mice is shown here to result from a duplication resulting in the synthesis of a 281-amino acid protein. (Fig. 5). Ob-Re is not detectable in plasma from dbPas/dbPas mice. This suggests that in this mutant, Ob-Re mRNA is not efficiently translated or that its protein may be degraded. Alternatively, the truncated protein may not bind to the leptin-Sepharose.
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The observation that leptin-Sepharose resin precipitates Ob-R suggests that soluble receptor may be present in excess relative to leptin in mouse plasma. This is surprising given the observation that only a portion of mouse and human leptin sediments with the apparent molecular mass of free leptin (12, 13). It may be that the leptin-Sepharose displaces leptin already bound to Ob-Re in plasma.
The function of Ob-Re remains to be determined. Soluble forms of other cytokine receptors are also found to circulate in plasma (15). In most cases the circulating receptor acts to chelate the ligand and acts as an inhibitor. This is also possible with leptin. Alternatively, soluble Ob-R could play a role in other aspects of leptin function such as reuptake after filtration by the kidney or in transport across the blood brain barrier (16, 17). Available data indicate that the hypothalamus is an important target of leptin action and that a saturable transport system is responsible for leptin access to its site of action in the brain (18, 19). Further studies will be required to determine whether Ob-Re plays a role in these aspects of leptin's function. The availability of recombinant receptor expressed in baculovirus will facilitate these studies.
It is as yet unclear whether the affinity of leptin for membrane-bound and soluble receptor in vivo is similar. A recent report did show that when the entire extracellular region of Ob-R is expressed in COS7 cells, it binds to leptin with similar affinity as that of the full-length transmembrane receptor (20). It is also not known whether baculovirus Ob-Re binds to leptin with an affinity similar to that of membrane-bound receptor expressed in mammalian cells (21).
With the identification of the dbPas/dbPas mutation, the molecular nature of all the rodent db (and fa) alleles is known. Three of the mutations truncate the receptor in the extracellular region, whereas the db mutation in C57BL/Ks mice is the result of a splicing mutation. This mutation specifically alters splicing of Ob-Rb, highlighting the importance of this receptor variant. Further studies of the other receptor forms (Ob-R(a-d)) will be required to evaluate their functions.
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ACKNOWLEDGEMENTS |
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We would like to thank Amgen for providing us with recombinant leptin, Dr. Jean-Louis Guenet (Pasteur Institute, Paris) for providing us with dbPas/dbPas mice, Drs. Jeff Winick, Jeff Halaas, and Jason Montez for stimulating discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Howard Hughes Medical Institute, c/o The Rockefeller University, 1230 York Ave., Box 305, New York, NY 10021. Tel: 212-327-8800; Fax: 212-327-7420; E-mail: friedj{at}rockvax.rockefeller.edu.
1 The abbreviations used are: PNGase F, N-glycosidase F; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Lepr, leptin receptor; PBS, phosphate-buffered saline.
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REFERENCES |
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