Mynarcik D C, Williams P F, Schaffer L, Yu G Q, Whittaker J
Department of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794, USA.
J Biol Chem. 1997 Jul 25;272(30):18650-5. doi: 10.1074/jbc.272.30.18650.
Insulin and insulin-like growth factor 1 (IGF-1) are peptides that share nearly 50% sequence homology. However, although their cognate receptors also exhibit significant overall sequence homology, the affinity of each peptide for the non-cognate receptor is 2-3 orders of magnitude lower than for the cognate receptor. The molecular basis for this discrimination is unclear, as are the molecular mechanisms underlying ligand binding. We have recently identified a major ligand binding site of the insulin receptor by alanine scannning mutagenesis. These studies revealed that a number of amino acids critical for insulin binding are conserved in the IGF-1 receptor, suggesting that they may play a role in ligand binding. We therefore performed alanine mutagenesis of these amino acids to determine whether this is the case. cDNAs encoding alanine-substituted secreted recombinant IGF-1 receptors were expressed in 293 EBNA cells, and the ligand binding properties of the expressed proteins were evaluated. Mutation of Phe701 resulted in a receptor with undetectable IGF-1 binding; alanine substitution of the corresponding amino acid of the insulin receptor, Phe714, produces a 140-fold reduction in affinity for insulin. Mutation of Asp8, Asn11, Phe58, Phe692, Glu693, His697, and Asn698 produces a 3.5-6-fold reduction in affinity for IGF-1. In contrast, alanine mutation of the corresponding amino acids of the insulin receptor with the exception of Asp12 produces reductions in affinity that are 50-fold or greater. The affinity of insulin for these mutants relative to wild type receptor was similar to that of their relative affinity for IGF-1 with two exceptions; the IC50 values for insulin binding to the mutants of Arg10, which has normal affinity for IGF-1, and His697, which has a 6-fold reduction in affinity for IGF-1, were both at least 2 orders of magnitude greater than for wild type receptor. The Kd values for insulin of the corresponding alanine mutants of the insulin receptor, Arg14 and His710, are 2-3 orders of magnitude greater than for wild type receptor. However, in contrast, the relative affinity of des(25-30)[PheB25 alpha-carboxamide]insulin for these IGF-1 receptor mutants is reduced only 4- and 50-fold, respectively.
胰岛素和胰岛素样生长因子1（IGF-1）是序列同源性近50%的肽类。然而，尽管它们的同源受体也表现出显著的整体序列同源性，但每种肽对非同源受体的亲和力比对同源受体的亲和力低2至3个数量级。这种区分的分子基础尚不清楚，配体结合的分子机制也不清楚。我们最近通过丙氨酸扫描诱变确定了胰岛素受体的一个主要配体结合位点。这些研究表明，许多对胰岛素结合至关重要的氨基酸在IGF-1受体中是保守的，这表明它们可能在配体结合中起作用。因此，我们对这些氨基酸进行了丙氨酸诱变，以确定是否如此。编码丙氨酸取代的分泌型重组IGF-1受体的cDNA在293 EBNA细胞中表达，并对表达蛋白的配体结合特性进行了评估。Phe701突变导致受体对IGF-1的结合无法检测到；胰岛素受体相应氨基酸Phe714的丙氨酸取代使对胰岛素的亲和力降低了140倍。Asp8、Asn11、Phe58、Phe692、Glu693、His697和Asn698的突变使对IGF-1的亲和力降低了3.5至6倍。相比之下，胰岛素受体相应氨基酸（除Asp12外）的丙氨酸突变导致亲和力降低50倍或更多。胰岛素对这些突变体相对于野生型受体的亲和力与其对IGF-1的相对亲和力相似，但有两个例外；胰岛素与对IGF-1具有正常亲和力的Arg10突变体以及对IGF-1亲和力降低6倍的His697突变体结合的IC50值均比对野生型受体至少高2个数量级。胰岛素受体相应丙氨酸突变体Arg-14和His710对胰岛素的Kd值比对野生型受体高2至3个数量级。然而，相比之下，des(25-30)[PheB25α-羧酰胺]胰岛素对这些IGF-1受体突变体的相对亲和力仅分别降低了4倍和50倍。
