Tsuda T, Ikeda Y, Taniguchi N
Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Biol Chem. 2000 Jul 21;275(29):21988-94. doi: 10.1074/jbc.M003400200.
To elucidate a role(s) of Asn-linked sugar chain(s) in the function of epidermal growth factor receptor (EGFR), a series of the EGFR mutants were prepared in which potential glycosylation sites in the domain III were eliminated by site-directed mutagenesis. Although the wild-type and mutants of Asn-328, Asn-337, and Asn-389 underwent autophosphorylation in response to epidermal growth factor (EGF), the Asn-420 --> Gln mutant was found to be constitutively tyrosine-phosphorylated. This abnormal ligand-independent phosphorylation of the mutant appears to be due to a ligand-independent spontaneous oligomer formation, as shown by a cross-linking experiment using the purified soluble extracellular domain (sEGFR). As revealed by the dissociation of the Asn-420 --> Gln sEGFR oligomer by simple dilution, it seems likely that the equilibrium is shifted toward oligomer formation to an unusual degree. Furthermore, it was also found that the mutation caused a loss of the ability to bind EGF. These findings suggest that the sugar chain linked to Asn-420 plays a crucial role in EGF binding and prevents spontaneous oligomerization of the EGFR, which may otherwise lead to uncontrollable receptor activation, and support the view of a specific role of an Asn-linked sugar chain in the function of a glycoprotein.
为阐明天冬酰胺连接的糖链在表皮生长因子受体（EGFR）功能中的作用，制备了一系列EGFR突变体，其中通过定点诱变消除了结构域III中的潜在糖基化位点。尽管野生型以及天冬酰胺328、天冬酰胺337和天冬酰胺389的突变体在表皮生长因子（EGF）刺激下会发生自磷酸化，但天冬酰胺420突变为谷氨酰胺的突变体被发现组成性地发生酪氨酸磷酸化。如使用纯化的可溶性细胞外结构域（sEGFR）进行的交联实验所示，该突变体这种异常的非配体依赖性磷酸化似乎是由于非配体依赖性自发寡聚体形成所致。通过简单稀释使天冬酰胺420突变为谷氨酰胺的sEGFR寡聚体解离，这表明平衡可能向寡聚体形成方向异常偏移。此外，还发现该突变导致结合EGF的能力丧失。这些发现表明，与天冬酰胺420连接的糖链在EGF结合中起关键作用，并可防止EGFR自发寡聚化，否则可能导致受体的不可控激活，这支持了天冬酰胺连接的糖链在糖蛋白功能中具有特定作用的观点。
