Role of the phenylalanine B25 side chain in directing insulin interaction with its receptor. Steric and conformational effects.
Role of the phenylalanine B25 side chain in directing insulin interaction with its receptor. Steric and conformational effects.
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DOI：
10.1016/s0021-9258(17)38396-5
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发表时间：
1986-06
期刊：
影响因子：
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通讯作者：
S. Nakagawa;H. Tager
中科院分区：
文献类型：
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作者：
S. Nakagawa;H. Tager
为了了解B25位氨基酸取代胰岛素生物活性降低的原因以及PheB 25侧链在指导胰岛素-受体相互作用中的重要性，我们制备了多种胰岛素类似物，并研究了它们与离体犬肝细胞的相互作用以及它们通过离体大鼠脂肪细胞刺激葡萄糖氧化的能力。半合成类似物分为三种结构类型：（a）其中胰岛素B链的COOH-末端5、6或7个残基已缺失的类似物，但其中B链的COOH-末端残基已通过α-羧酰胺化衍生化;（B）其中PheB 25已被非天然芳族或天然L-氨基酸取代的类似物;和（c）类似物，其中胰岛素B链的COOH-末端5个残基已缺失，并且其中残基B25已被选定的α-羧酰胺化氨基酸取代。我们的结果表明：（a）胰岛素残基B26-B30可以缺失而不降低生物学效价，而残基B25-B30和B24-B30的缺失引起效价的显著和累积性降低;（B）用Leu或Ser取代胰岛素中的PheB 25导致生物学效价甚至低于当残基B25-B30缺失时观察到的生物学效价的类似物;（c）在B25位的萘基（1）-丙氨酸或萘基（2）-丙氨酸的侧链主体在胰岛素与受体相互作用期间是良好耐受的，而高苯丙氨酸的侧链主体不是;和（d）由Ala、Ser、Leu或高苯丙氨酸取代胰岛素PheB 25引起的降低的生物效力部分或全部逆转，通过缺失COOH末端残基B26-B30。另外的实验表明，通过将细胞与胰岛素或[萘基（2）-丙氨酸B25]胰岛素孵育，而不是与其中PheB 25被丝氨酸、亮氨酸或高苯丙氨酸取代的类似物孵育，受体结合的125 I标记的胰岛素从分离的肝细胞中解离的速率增强;然而，在所研究的所有情况下，残基B26-B30的缺失导致类似物增强受体结合胰岛素的解离速率。我们的结论是：（a）空间位阻涉及的B链的COOH-末端结构域在指导胰岛素与其受体的相互作用中起着重要作用;（B）该结构域的初始负效应在反映受体和PheB 25侧链的β-芳香环相互作用的位点填充后被逆转。（400字处截断摘要）
To gain an understanding of the causes of decreased biological activity in insulins bearing amino acid substitutions at position B25 and the importance of the PheB25 side chain in directing hormone-receptor interactions, we have prepared a variety of insulin analogs and have studied both their interactions with isolated canine hepatocytes and their abilities to stimulate glucose oxidation by isolated rat adipocytes. The semisynthetic analogs fall into three structural classes: (a) analogs in which the COOH-terminal 5, 6, or 7 residues of the insulin B-chain have been deleted, but in which the COOH-terminal residue of the B-chain has been derivatized by alpha-carboxamidation; (b) analogs in which PheB25 has been replaced by unnatural aromatic or natural L-amino acids; and (c) analogs in which the COOH-terminal 5 residues of the insulin B-chain have been deleted and in which residue B25 has been replaced by selected alpha-carboxamidated amino acids. Our results showed that (a) insulin residues B26-B30 can be deleted without decrease in biological potency, whereas deletion of residues B25-B30 and B24-B30 causes a marked and cumulative decrease in potency; (b) replacement of PheB25 in insulin by Leu or Ser results in analogs with biological potency even less than that observed when residues B25-B30 are deleted; (c) the side chain bulk of naphthyl(1)-alanine or naphthyl(2)-alanine at position B25 is well tolerated during insulin interactions with receptor, whereas that of homophenylalanine is not; and (d) the decreased biological potency attending substitution of insulin PheB25 by Ala, Ser, Leu, or homophenylalanine is reversed, in part or in total, by deletion of COOH-terminal residues B26-B30. Additional experiments showed that the rate of dissociation of receptor-bound 125I-labeled insulin from isolated hepatocytes is enhanced by incubating cells with insulin or [naphthyl(2)-alanineB25]insulin, but not with analogs in which PheB25 is replaced by serine, leucine, or homophenylalanine; deletion of residues B26-B30, however, results in analogs that enhance the rate of dissociation of receptor-bound insulin in all cases studied. We conclude that (a) steric hindrance involving the COOH-terminal domain of the B chain plays a major role in directing the interaction of insulin with its receptor; (b) the initial negative effect of this domain is reversed upon the filling of a site reflecting interaction of the receptor and the beta-aromatic ring of the PheB25 side chain.(ABSTRACT TRUNCATED AT 400 WORDS)