Leptin receptor activation depends on critical cysteine residues in its fibronectin type III subdomains.
The leptin receptor (LR) complex is composed of a single subunit belonging to the class I cytokine receptor family and exists as a preformed complex. The extracellular portion contains two cytokine receptor homology (CRH) domains, separated by an Ig-like domain and followed by two membrane-proximal fibronectin type III (FNIII) domains. The mechanisms underlying ligand-induced receptor activation are still poorly understood. LRs can exist as disulfide-linked dimers at the cell surface, even in the absence of leptin. We evaluated the role of the two unpaired cysteine residues (Cys-672 and Cys-751) in the FNIII domains in receptor clustering, leptin binding, and biological activity. Although mutation of cysteine on position 751 to serine has hardly any effect on ligand binding and receptor activation, the C672S mutant exhibits a marked reduction in STAT3-dependent signaling. The double mutant was completely devoid of biological activity, although leptin binding remained unaffected. Mutation of both residues resulted in complete loss of disulfide bridge formation of FNIII domains in solution. In contrast, no difference was observed in ligand-independent oligomerization of the membrane-bound receptor, suggesting a role for cysteines in the CRH2 domain in formation of the preformed LR complex. We propose a model wherein leptin-induced clustering of two preformed dimers forms the activated LR complex. Disulfide bridge formation involving Cys-672 and Cys-751 may be necessary for JAK activation and hence signaling.
Zabeau
Lennart
L
Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Flanders Interuniversity Institute for Biotechnology, VIB09, Ghent University, Belgium.
Defeau
Delphine
D
Iserentant
Hannes
H
Vandekerckhove
Joël
J
Peelman
Frank
F
Tavernier
Jan
J
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016000:Cluster Analysis; D003545:Cysteine / Q000737:chemistry*; D016207:Cytokines / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism; D017353:Gene Deletion; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D047468:Immunoprecipitation; D020738:Leptin / Q000737:chemistry; D008024:Ligands; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D054411:Receptors, Leptin; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000737:chemistry; D014162:Transfection
D016207:Cytokines; D004268:DNA-Binding Proteins; D004220:Disulfides; D005353:Fibronectins; C515448:LEPR protein, human; D020738:Leptin; D008024:Ligands; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C494087:Stat3 protein, mouse; D015534:Trans-Activators; C501364:fibronectin type III like peptide, human; C515447:leptin receptor, mouse; D003545:Cysteine