Perez H D, Vilander L, Andrews W H, Holmes R
Department of Immunology, Berlex Biosciences, Richmond, California 94804.
J Biol Chem. 1994 Sep 9;269(36):22485-7.
Recently, we reported the domain requirements for the binding of formyl peptide to its specific receptor. Based on experiments using receptor chimeras, we also postulated an importance for the amino-terminal domain of the receptor in ligand binding (Perez, H. D., Holmes, R., Vilander, L., Adams, R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295). We have begun to perform a detailed analysis of the regions within the formyl peptide receptor involved in ligand binding. To address the importance of the receptor amino-terminal domain, we substituted (or inserted) hydrophilic sequences within the amino-terminal domain, expressed the receptors, and determined their ability to bind ligand. A stretch of nine amino acids next to the initial methionine was identified as crucial for receptor occupancy. A peptide containing such a sequence specifically completed binding of the ligand to the receptor. Alanine screen mutagenesis of the second extracellular domain also identified amino acids involved in ligand binding as well as a disulfide bond (Cys98 to Cys176) crucial for maintaining the binding pocket. These studies provide evidence for a novel mechanism involved in regulation of receptor occupancy. Binding of the ligand induces conformational changes in the receptor that result in the apposition of the amino-terminal domain over the ligand, providing a lid to the binding pocket.
最近，我们报道了甲酰肽与其特异性受体结合的结构域要求。基于使用受体嵌合体的实验，我们还推测了受体氨基末端结构域在配体结合中的重要性（佩雷斯，H.D.，霍姆斯，R.，维兰德，L.，亚当斯，R.，曼萨纳，W.，乔利，D.，安德鲁斯，W.H.（1993年）《生物化学杂志》268卷，2292 - 2295页）。我们已开始对甲酰肽受体中参与配体结合的区域进行详细分析。为了阐明受体氨基末端结构域的重要性，我们在氨基末端结构域内替换（或插入）亲水性序列，表达这些受体，并确定它们结合配体的能力。起始甲硫氨酸旁边的一段九个氨基酸被确定为受体占据的关键。含有这样一个序列的肽特异性地完成了配体与受体的结合。对第二个细胞外结构域进行丙氨酸扫描诱变也确定了参与配体结合的氨基酸以及对于维持结合口袋至关重要的二硫键（半胱氨酸98至半胱氨酸176）。这些研究为受体占据调节中涉及的一种新机制提供了证据。配体的结合诱导受体构象变化，导致氨基末端结构域与配体并列，为结合口袋提供一个盖子。
