Leptin Receptor Activation Depends on Critical Cysteine Residues in Its Fibronectin Type III Subdomains

Abstract

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 ligandinduced 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 membranebound 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.The 16-kDa cytokine-like hormone leptin has been identified as one of the key players in the control of body weight. The product of the ob gene is produced and secreted mainly by adipocytes (1, 2), and plasma protein levels positively correlate with body fat energy stores. Leptin reduces food intake and stimulates energy expenditure by activating its receptor in specific hypothalamic nuclei (3-5). Spontaneous mutations that lead to a functional defect in either leptin (6) or its receptor (7-9) result in a complex syndrome that includes morbid obesity, hypothermia, infertility, hyperglycemia, decreased insulin sensitivity, and hyperlipidemia. Besides serving a weight-regulating function, leptin also plays a role in other processes including metabolism, reproduction, hematopoiesis, and immunity (10 -13).The leptin receptor (LR), 1 the product of the db gene (14), is a member of the class I cytokine receptor family. The extracellular (EC) domain is composed of two so-called cytokine receptor homology (CRH) domains, a membrane distal CRH1 and a membrane proximal CRH2. Both domains are separated by an immunoglobulin-like (Ig) domain and are followed by two fibronectin type III (FNIII) domains proximal to the membrane. The CRH2 domain is necessary and sufficient for leptin binding (15). Despite the lack of any binding affinity for the ligand, the two FNIII and the Ig domains are needed for receptor activation (15, 16). Because of alternative splicing and ectodomain shedding, the LR can exist as six isoforms: a LR long ...

Excessive nitric oxide attenuates leptin-mediated signal transducer and activator of transcription 3 activation

“…All isoforms except LEPR-e have a similar extracellular ligand-binding domain, but differ in their intracellular domains. Recently, it was reported that leptin receptor activation depends on critical cysteine residues located in the extracellular domain (Zabeau et al, 2005). In connection with this, we found that ascorbic acid partially recovered the NO-mediated inhibition (almost complete inhibition by accumulated NOx concentration for 30 min of 300 μM) of leptin signaling (Fig.…”

Mapping of Binding Site III in the Leptin Receptor and Modeling of a Hexameric Leptin·Leptin Receptor Complex

“…Couturier and Jockers (37) used a bioluminescence resonance energy transfer assay to show that 60% of the LR population exists as dimers and that leptin induces conformational reorganization of these preformed LR complexes. At least part of the LR population exists as disulfide-linked dimers at the cell surface, even in the absence of leptin, and both the isolated CRH2 domain and the two fibronectin type III domains can form disulfidelinked dimers (13). In the hexameric model complex, the C termini of the CRH2 domains of LR1 and LR2 approach each other.…”

“…2) Ligand-independent clustering of the LR has been demonstrated in solution (13,27) and at the cell surface (13,(37)(38)(39). Couturier and Jockers (37) used a bioluminescence resonance energy transfer assay to show that 60% of the LR population exists as dimers and that leptin induces conformational reorganization of these preformed LR complexes.…”

“…The membrane-proximal CRH domain (CRH2) was identified as the main high affinity binding site of the LR (10 -12), whereas the other CRH domain (CRH1) seems to be not essential for leptin binding and receptor activation (10,12). Although the Ig-like domain and the two fibronectin type III modules are not prerequisites for high affinity leptin binding (10), they are essential for LR activation (12,13), but the underlying mechanism has not been fully resolved.…”

Monitoring leptin activity using the chicken leptin receptor

“…The two WS!WS residues suggested to contribute to ligand-recognition in cytokine receptors, and the cytokine receptor homology (CRH) domains, implicated in leptin binding, were well conserved. The C residues characteristic of the fibronectin type II modules (Zabeau et al 2005) are indicated. Thus, despite the low sequence similarity among these sequences, all of the known functional motifs and domains of LEPR were conserved in terms of both sequence and position.…”