Ballinger M D, Jones J T, Lofgren J A, Fairbrother W J, Akita R W, Sliwkowski M X, Wells J A
Department of Protein Engineering, Genentech, Incorporated, South San Francisco, California 94080, USA.
J Biol Chem. 1998 May 8;273(19):11675-84. doi: 10.1074/jbc.273.19.11675.
Heregulins (HRGs) are epidermal growth factor (egf) domain containing polypeptide growth factors that bind and activate several members of the ErbB receptor family. Although HRG can bind to ErbB3 and ErbB4 homodimers, the highest affinity and most intracellularly active receptor complexes are hetero-oligomers containing ErbB2. The HRGbeta egf domain was displayed on the surface of M13 phage to facilitate mutagenic analysis and optimize for binding to a homodimeric ErbB3-immunoglobulin (IgG) fusion. Nine libraries were constructed in which virtually the entire sequence was randomized in stretches of four to six amino acids. These were selected separately for binding to immobilized ErbB3-IgG. Analysis of the resulting sequences revealed some areas that diverged radically from the wild-type, whereas others showed strong conservation. The degree of wild-type conservation correlated strongly with the functional importance of the residues as determined by alanine scanning mutagenesis (Jones, J. T., Ballinger, M. D., Pisacane, P. I., Lofgren, J. A., Fitzpatrick, V. D., Fairbrother, W. J., Wells, J. A., and Sliwkowski, M. X. (1998) J. Biol. Chem. 273, 11667-11674). Some variants from several libraries showed significant improvements in binding affinity to the ErbB3-IgG. These optimized segments were combined in various ways in the same molecule to generate variants (containing up to 16 mutations) that had >50-fold higher affinity than wild-type HRGbeta. The optimized variants stimulated ErbB2 phophorylation on MCF7 cells at levels similar to wild-type. This indicates wild-type affinity is optimized for potency and that factors other than affinity for ErbB3 are limiting. These variants showed enhanced affinity toward the ErbB4 homodimer, suggesting these receptors use very similar binding determinants despite them having 65% sequence identity.
Heregulins（HRGs）是一类含有表皮生长因子（EGF）结构域的多肽生长因子，它们能够结合并激活ErbB受体家族的多个成员。尽管HRG可以与ErbB3和ErbB4同二聚体结合，但亲和力最高且细胞内活性最强的受体复合物是含有ErbB2的异源寡聚体。将HRGβ EGF结构域展示在M13噬菌体表面，以利于进行诱变分析，并优化其与同二聚体ErbB3 - 免疫球蛋白（IgG）融合蛋白的结合。构建了9个文库，其中几乎整个序列在4至6个氨基酸的片段中是随机化的。分别选择这些文库用于与固定化的ErbB3 - IgG结合。对所得序列的分析揭示了一些与野生型有显著差异的区域，而其他区域则表现出很强的保守性。野生型保守程度与通过丙氨酸扫描诱变确定的残基功能重要性密切相关（琼斯，J.T.，巴林杰，M.D.，皮萨卡内，P.I.，洛夫格伦，J.A.，菲茨帕特里克，V.D.，费尔布拉泽，W.J.，韦尔斯，J.A.，和斯利夫科夫斯基，M.X.（1998年）《生物化学杂志》273，11667 - 11674）。来自几个文库的一些变体在与ErbB3 - IgG的结合亲和力方面有显著提高。这些优化片段以各种方式组合在同一分子中，以产生亲和力比野生型HRGβ高50倍以上的变体（含有多达16个突变）。优化后的变体在MCF7细胞上刺激ErbB2磷酸化的水平与野生型相似。这表明野生型亲和力是为效力而优化的，并且对ErbB3的亲和力以外的因素是限制性的。这些变体对ErbB4同二聚体表现出增强的亲和力，表明尽管这些受体的序列同一性为65%，但它们使用非常相似的结合决定簇。
