BiaCore analysis of leptin-leptin receptor interaction: evidence for 1:1 stoichiometry.
Leptin is a hormonal protein involved in energy homeostatis that acts to inhibit food intake, to stimulate energy expenditure, and to influence insulin secretion, lipolysis, and sugar transport. Its action is mediated by a specific receptor whose activation is highly controversial. As a member of the cytokine receptor superfamily, it has been predicted to be activated by ligand-induced dimerization. However, recent evidence has indicated that this receptor exists as a dimer in both ligand-free and ligand-bound states. Here, the BiaCore has been used to measure the kinetics and stoichiometry of the interaction between the leptin and its receptor. Human or mouse receptor chimeras comprising two receptor extracellular domains fused to the Fc region of IgG(1) were captured on to the sensor via protein G. Kinetic data fitted to the simplest 1/1 model. The observed stoichiometry at ligand saturation was 1:1. Analyzing the binding mode and the reaction stoichiometry allowed us to conclude that the leptin receptor dimerization is not induced by ligand binding. This contradicts the common paradigm of cytokine receptor activation. Furthermore, data demonstrated a high-affinity interaction. The KD was 0.23+/-0.08 nM, with ka = (1.9 +/- 0.4) x 10(6) M(-1)s(-1) and kd = (4.4 +/- 0.6) x 10(-4) s(-1) for human leptin with its cognate receptor. Similar results were observed for the affinity of different species of leptin binding to mouse leptin receptor.
Mistrík
P
P
Division of Biochemistry and Molecular Biology, IBLS, Wolfson Building, Level 2, University of Glasgow, Glasgow G12 8QQ, UK.
Moreau
F
F
Allen
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007700:Kinetics; D020738:Leptin / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D054411:Receptors, Leptin; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
C515448:LEPR protein, human; D020738:Leptin; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; C515447:leptin receptor, mouse