Expression and characterization of a putative high affinity human soluble leptin receptor

Endocrinology. 1997 Aug;138(8):3548-54. doi: 10.1210/endo.138.8.5343.

Abstract

Leptin, a circulating 16-kDa protein secreted by adipocytes, decreases body weight by reducing food intake and enhancing energy utilization. Leptin receptors that share homology to the glycoprotein gp130 have been recently cloned. In addition, differentially spliced leptin receptor messenger RNAs have been identified. Functional mutations in either the leptin or leptin receptor gene cause obesity. In the present study, expression of the full length human leptin receptor complementary DNA encoding the long cytoplasmic domain of leptin receptor in COS7 cells resulted in high affinity membrane binding of 125I-leptin (Ki approximately 200 pM); no detectable binding was present in the medium. In addition, we expressed the extracellular domain of human leptin receptor in COS7 cells and identified a soluble leptin receptor in the conditioned medium that binds human and mouse leptin with high affinity comparable with the full length membrane receptor. Transfected COS7 cells expressing the soluble leptin receptor also demonstrated modest specific 125I-leptin binding in whole cells, presumably due to association of the soluble leptin receptor to cell membrane proteins. Data from cross-linking studies identified two specific bands in the 125I-leptin/soluble leptin receptor complex with molecular masses of approximately 130-150 kDa and 300 kDa. The 130-150 kDa molecular mass was confirmed in Western blot analysis and Coomassie staining of the purified soluble receptor and probably represents the glycosylated form of the receptor. The 300-kDa band most likely represents a homodimer of the soluble leptin receptor complex because HPLC gel filtration analysis of the 125I-leptin/soluble leptin receptor complex identified a single peak corresponding to a molecular mass of approximately 340 kDa. The soluble leptin receptor antagonized 125I-leptin binding to the membrane receptor, suggesting its potential utility as a functional tool for determining the role of endogenous leptin.

MeSH terms

  • Animals
  • Base Sequence
  • Binding, Competitive
  • Blotting, Western
  • COS Cells
  • Carrier Proteins / biosynthesis*
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism
  • Chromatography, High Pressure Liquid
  • DNA, Complementary / analysis
  • DNA, Complementary / chemistry
  • DNA, Complementary / genetics
  • Gene Expression Regulation
  • Humans
  • Iodine Radioisotopes
  • Leptin
  • Mice
  • Molecular Sequence Data
  • Molecular Weight
  • Proteins / metabolism
  • Radioligand Assay
  • Receptors, Cell Surface*
  • Receptors, Leptin
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Solubility
  • Transfection

Substances

  • Carrier Proteins
  • DNA, Complementary
  • Iodine Radioisotopes
  • Leptin
  • Proteins
  • Receptors, Cell Surface
  • Receptors, Leptin
  • Recombinant Proteins
  • leptin receptor, human
  • leptin receptor, mouse