Herrera R, Petruzzelli L, Thomas N, Bramson H N, Kaiser E T, Rosen O M
Proc Natl Acad Sci U S A. 1985 Dec;82(23):7899-903. doi: 10.1073/pnas.82.23.7899.
Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the beta subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the beta subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor; it was not detected when the receptor was autophosphorylated prior to addition of AbP4. AbP4 did not inhibit activity of the related epidermal growth factor (EGF)-receptor tyrosine protein kinase nor did it inhibit the activity of cAMP-dependent kinase or protein kinase C. The observation that an antibody directed to residues 952-967 of the proreceptor neutralizes the protein kinase activity of the beta subunit suggests that this region may play a critical role in the function of the hormone-dependent, protein tyrosine-specific kinase activity of the insulin receptor.
通过用源自前体受体β亚基的cDNA预测氨基酸序列的化学合成肽免疫兔子，制备了两种免疫沉淀人胰岛素受体的位点特异性抗体。针对羧基末端的抗体（AbP5）和针对酪氨酸-960周围区域的抗体（AbP4）在免疫印迹中特异性识别受体的β亚基。两种抗体都免疫沉淀了125I标记的胰岛素受体复合物和自身磷酸化的受体。虽然两种抗体都不抑制胰岛素与受体的结合，但AbP4抑制胰岛素依赖性自身磷酸化和外源底物磷酸化。AbP4的抑制作用取决于受体的磷酸化状态；在添加AbP4之前受体自身磷酸化时未检测到抑制作用。AbP4不抑制相关的表皮生长因子（EGF）受体酪氨酸蛋白激酶的活性，也不抑制cAMP依赖性激酶或蛋白激酶C的活性。针对前体受体952-967位残基的抗体中和β亚基的蛋白激酶活性这一观察结果表明，该区域可能在胰岛素受体的激素依赖性蛋白酪氨酸特异性激酶活性的功能中起关键作用。
