Ligand-independent dimerization of the extracellular domain of the leptin receptor and determination of the stoichiometry of leptin binding.
The leptin receptor is a class I transmembrane protein with either a short or a long cytoplasmic domain. Using chemical cross-linking we have analyzed the binding of leptin to its receptor. Cross-linking of radiolabeled leptin to different isoforms of the leptin receptor expressed on COS-1 cells reveals leptin receptor monomer, homodimer, and oligomer complexes. Cotransfection of the long and short form of the leptin receptor did not provide any evidence for the formation of heterodimer complexes. Soluble forms consisting of either the entire extracellular domain or the two cytokine receptor homologous domains of the leptin receptor were purified to homogeneity from recombinant baculovirus-infected insect cells by leptin affinity chromatography. Gel filtration chromatography showed that these proteins exist in a dimeric form. Analysis of the complex formed between soluble leptin receptor and leptin by native polyacrylamide gel electrophoresis, and data obtained from the amino acid composition of the complex provide direct evidence that the extracellular domain of the leptin receptor binds leptin in a 1:1 ratio.
Devos
R
R
Roche Research Gent, F. Hoffmann-La Roche & Co., B-9000 Gent, Belgium.
Guisez
Y
Y
Van der Heyden
J
J
White
D W
DW
Kalai
M
M
Fountoulakis
M
M
Plaetinck
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D003432:Cross-Linking Reagents; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D007700:Kinetics; D020738:Leptin; D008969:Molecular Sequence Data; D009765:Obesity; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011506:Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D000906:Antibodies; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; C515448:LEPR protein, human; D020738:Leptin; D010446:Peptide Fragments; D011506:Proteins; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011994:Recombinant Proteins