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.
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.
Macdonald
Jennifer
J
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Box 8231, St. Louis, MO 63110, USA.
Li
Zhengzhe
Z
Su
Wanwen
W
Pike
Linda J
LJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D021962:Membrane Microdomains / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D018076:DNA, Complementary; D004220:Disulfides; D011994:Recombinant Proteins; D066246:ErbB Receptors