The tethering arm of the EGF receptor is required for negative cooperativity and signal transduction.
The EGF receptor is a classical receptor-tyrosine kinase. In the absence of ligand, the receptor adopts a closed conformation in which the dimerization arm of subdomain II interacts with the tethering arm in subdomain IV. Following the binding of EGF, the receptor opens to form a symmetric, back-to-back dimer. Although it is clear that the dimerization arm of subdomain II is central to the formation of receptor dimers, the role of the tethering arm of subdomain IV (residues 561-585) in this configuration is not known. Here we use (125)I-EGF binding studies to assess the functional role of the tethering arm in the EGF receptor dimer. Mutation of the three major residues that contribute to tethering (D563A,H566A,K585A-EGF receptor) did not significantly alter either the ligand binding properties or the signaling properties of the EGF receptor. By contrast, breaking the Cys(558)-Cys(567) disulfide bond through double alanine replacements or deleting the loop entirely led to a decrease in the negative cooperativity in EGF binding and was associated with small changes in downstream signaling. Deletion of the Cys(571)-Cys(593) disulfide bond abrogated cooperativity, resulting in a high affinity receptor and increased sensitivity of downstream signaling pathways to EGF. Releasing the Cys(571)-Cys(593) disulfide bond resulted in extreme negative cooperativity, ligand-independent kinase activity, and impaired downstream signaling. These data demonstrate that the tethering arm plays an important role in supporting cooperativity in ligand binding. Because cooperativity implies subunit-subunit interactions, these results also suggest that the tethering arm contributes to intersubunit interactions within the EGF receptor dimer.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Sangeeta
S
Adak
Diana
D
DeAndrade
Linda J
LJ
Pike
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D007457:Iodine Radioisotopes; D008024:Ligands; D021962:Membrane Microdomains / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology*
D004220:Disulfides; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008024:Ligands; D066246:ErbB Receptors