Abstract
Leptin is an adipocyte-derived hormone with potent effects on food intake and body weight. Genetically obese rodents with mutations of leptin or leptin receptor develop morbid obesity and diabetes. The receptor for leptin, OB-R, is alternatively spliced to at least five transcripts, encoding receptors designated OB-Ra, -b, -c, -d, and -e. OB-Re does not encode a transmembrane domain and is secreted. In humans, transcripts corresponding to OB-Re have not been discovered. However, soluble leptin receptor does circulate in human plasma and represents the major leptin-binding activity. In this report, we attempted to determine whether the soluble leptin receptor may also be derived from membrane-spanning receptor isoforms by ectodomain shedding. Using stable cell lines expressing both OB-Ra, the most abundant leptin receptor isoform, and OB-Rb, the signaling form of the leptin receptor, we demonstrate that soluble leptin receptor protein can indeed be generated by proteolytic cleavage of these two receptor isoforms in vitro. Experiments using adenoviruses expressing dually tagged OB-Ra or Ob-Rb also demonstrate that soluble leptin receptor may be derived from ectodomain shedding of both receptor isoforms in vivo. Because our earlier and other studies have shown that the soluble receptors modulate the levels as well as activity of leptin, our findings suggest that regulated shedding of the ectodomain of membrane-spanning leptin receptors may represent a novel mechanism of modulating leptin's biological activity.
Highlights
Leptin is an adipocyte-derived hormone of 167 amino acids [1]
Generation of Stable Cell Lines Overexpressing Membranespanning Receptors—To obtain permanent cell lines of OB-Ra and OB-Rb as reagents for leptin signal-transduction studies, we introduced cDNAs encoding OB-Ra, OB-Rb, or a control construct chloramphenicol acetyltransferase (CAT) into a 293 cell line using the Flp-In system
A robust response of luciferase activity was detected at both 6 h and 12 h after leptin addition to OB-Rb-expressing cells (Fig. 1C), demonstrating that a functional OB-R signal-transduction pathway is present in stably transfected OB-Rb cells. 125I-leptin binding to the surface of OB-Ra or OB-Rb cell lines is about 8- or 4-fold higher than that of CAT cells (Fig. 1D). 125I-leptin binding was essentially undetectable in the presence of an excess of cold leptin, demonstrating the specificity of this assay
Summary
Generation of Stable Cell Lines Overexpressing OB-Ra and OB-Rb—cDNA of Ob-Ra or Ob-Rb in the expression vector pcDNA3.1(Ϫ)/MycHisA was digested with Pme I and ligated into the same site of pcDNA5/FRT (Invitrogen). PCR products of both OB-Ra and OB-Rb digested by BglII and HindIII were ligated to pcDNA3.1(Ϫ)/MycHisA-SS digested with BamHI and HindIII In this fashion, the resulting vectors contain a FLAG epitope that is inserted between the signal peptide and the remaining coding sequences of OB-R. Upon cleavage of the signal peptide during receptor translocation to the plasma membrane, the FLAG epitope becomes the amino terminus of recombinant OB-R protein, while the c-myc epitope becomes the carboxyl terminus Final vectors of both OB-Ra and OB-Rb were sequence verified and transiently expressed in 293T cells to ensure that no cloning errors were introduced. 1 l of plasma sample from AdCMV-OB-Re-treated mice was run on SDS-PAGE directly to detect the levels of the soluble leptin receptor by Western blotting as described earlier [19]. Monoclonal antibodies against the FLAG epitope and the c-Myc epitope were from Sigma and Roche (Indianapolis, IN), respectively
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