The membrane proximal disulfides of the EGF receptor extracellular domain are required for high affinity binding and signal transduction but do not play a role in the localization of the receptor to lipid rafts

Abstract

The EGF receptor is a transmembrane receptor tyrosine kinase that is enriched in lipid rafts. Subdomains I, II and III of the extracellular domain of the EGF receptor participate in ligand binding and dimer formation. However, the function of the cysteine-rich subdomain IV has not been elucidated. In this study, we analyzed the role of the membrane-proximal portion of subdomain IV in EGF binding and signal transduction. A double Cys-->Ala mutation that breaks the most membrane-proximal disulfide bond (Cys600 to Cys612), ablated high affinity ligand binding and substantially reduced signal transduction. A similar mutation that breaks the overlapping Cys596 to Cys604 disulfide had little effect on receptor function. Mutation of residues within the Cys600 to Cys612 disulfide loop did not alter the ligand binding or signal transducing activities of the receptor. Despite the fact that the C600,612A EGF receptor was significantly impaired functionally, this receptor as well as all of the other receptors with mutations in the region of residues 596 to 612 localized normally to lipid rafts. These data suggest that the disulfide-bonded structure of the membrane-proximal portion of the EGF receptor, rather than its primary sequence, is important for EGF binding and signaling but is not involved in localizing the receptor to lipid rafts.

Modulating the Structure of EGFR with UV Light: New Possibilities in Cancer Therapy

“…Ligand binding requires a change in the relative positions of domains I and III and dimerization occurs upon interaction of subdomains II of two EGFR monomers [1] , [5] , [55] , [56] . It is believed that domain IV has a role in high affinity binding and signal transduction [6] . Considering that the mechanisms of EGF binding and posterior EGFR dimerization can involve all four domains of sEGFR, it is not surprising that UV induced conformational changes will most likely impair binding to EGF.…”

“…The extracellular domain of EGFR (sEGFR, soluble extracellular region of EGFR) comprises 4 sub-domains: 2 large homologous binding domains (I and III), and 2 homologous furin-like cysteine rich domains (II and IV). Domains I, II and III have been found to be directly involved in ligand binding and dimer formation that precede the mechanism of signal transduction by RTKs [1] , [5] , [6] . Cancer progression has been correlated with the increase in the number of EGFR molecules on the cell surface [7] .…”

Regulation of Redox Signaling by Selenoproteins

“…Many GPCRs form homodimers and heterodimers, which enhance signaling and mediate transactivation between receptors [ 30 ]. Dimer formation in a model yeast GPCR has recently been shown to be accomplished by disulfide bridges [ 31 ], and ligand-induced disulfide-mediated dimerization is correlated with activation of growth hormone receptor [ 32 ] and the receptor tyrosine kinases for discoidin [ 33 ] and epidermal growth factor [ 34 ]. However, a role of ROS in receptor dimer formation has not been established.…”

Caenorhabditis elegans SDF-9 Enhances Insulin/Insulin-Like Signaling Through Interaction With DAF-2

“…This structural change may disrupt a disulfide bond formed by the conserved cysteine and could make the overall structure of the daf-2(m41) gene product unstable or unable to dimerize at higher temperatures. Mutations in a similar domain of the EGFR protein cause an inability to dimerize, preventing trans-autophosphorylation (Macdonald et al 2006). Lack of trans-autophosphorylation would render SDF-9 unable to modify m41 activity, so loss of SDF-9 function would not affect the m41 phenotype.…”