Buchegger F, Trowbridge I S, Liu L F, White S, Collawn J F
Department of Cancer Biology, The Salk Institute, San Diego, CA, USA.
Eur J Biochem. 1996 Jan 15;235(1-2):9-17. doi: 10.1111/j.1432-1033.1996.0009u.x.
Chimeric human/chicken transferrin receptors have been constructed using the polymerase chain reaction. Different regions of the 671-residue external domain of the human transferrin receptor were replaced by the corresponding sequences from the chicken transferrin receptor. As chicken transferrin receptors do not bind human transferrin, functional analysis of such chimeric receptors provides an approach to define the ligand-binding site of the human transferrin receptor. Four of 16 chimeric human/chicken transferrin receptors expressed in chick embryo fibroblasts were efficiently transported to the plasma membrane and displayed on the cell surface. Studies of the four chimeric receptors indicated that binding of human transferrin was abolished if the carboxy terminal 192 amino acids of the human transferrin receptor (residues 569-760) were replaced with the corresponding region from the chicken transferrin receptor. Further, a chimeric receptor in which the carboxy-terminal 72 residues were derived from the chicken transferrin receptor exhibited a 16-fold decrease in binding affinity for human transferrin. In contrast, analysis of the other two chimeric receptors showed that 340 amino acids of the human transferrin receptor external domain more proximal to the transmembrane region (residues 151-490) could be replaced with the corresponding region from the chicken transferrin receptor without loss of high-affinity ligand binding. In contrast, two mAbs against the human transferrin receptor external domain, B3/25 and D65.3, that do not compete with transferrin binding, do not bind the chimeric transferrin receptors in which the membrane proximal part is replaced by chicken sequences, while they do bind the two other chimeric transferrin receptors with high affinity. These data indicate that sequence differences in the carboxy-terminal region of human and chicken transferrin receptor external domains are important for the species specificity of transferrin binding and imply that this portion of the human transferrin receptor is critical for ligand binding.
利用聚合酶链反应构建了人/鸡嵌合转铁蛋白受体。人转铁蛋白受体671个氨基酸残基的胞外结构域的不同区域被鸡转铁蛋白受体的相应序列所取代。由于鸡转铁蛋白受体不结合人转铁蛋白，对这类嵌合受体的功能分析为确定人转铁蛋白受体的配体结合位点提供了一种方法。在鸡胚成纤维细胞中表达的16种人/鸡嵌合转铁蛋白受体中有4种被有效地转运到质膜并展示在细胞表面。对这4种嵌合受体的研究表明，如果人转铁蛋白受体的羧基末端192个氨基酸（第569 - 760位残基）被鸡转铁蛋白受体的相应区域取代，人转铁蛋白的结合就会被消除。此外，一种羧基末端72个残基来源于鸡转铁蛋白受体的嵌合受体对人转铁蛋白的结合亲和力降低了16倍。相比之下，对另外两种嵌合受体的分析表明，人转铁蛋白受体胞外结构域中更靠近跨膜区域的340个氨基酸（第151 - 490位残基）可以被鸡转铁蛋白受体的相应区域取代，而不会丧失高亲和力配体结合能力。相反，两种针对人转铁蛋白受体胞外结构域的单克隆抗体B3/25和D65.3，它们不与转铁蛋白结合竞争，不结合膜近端部分被鸡序列取代的嵌合转铁蛋白受体，而它们确实能高亲和力地结合另外两种嵌合转铁蛋白受体。这些数据表明，人和鸡转铁蛋白受体胞外结构域羧基末端区域的序列差异对转铁蛋白结合的物种特异性很重要，并暗示人转铁蛋白受体的这一部分对配体结合至关重要。
