Characterization of the functional insulin binding epitopes of the full-length insulin receptor

被引:39
|
作者
Whittaker, J
Whittaker, L
机构
[1] Case Western Reserve Univ, Dept Nutr, Cleveland, OH 44106 USA
[2] Case Western Reserve Univ, Dept Biochem, Cleveland, OH 44106 USA
关键词
D O I
10.1074/jbc.M411320200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1 - 470) and at the C terminus of the alpha subunit (amino acids 705 - 715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the K-d of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg(14), Phe(64), Phe(705), Glu(706), Tyr(708), Asn(711), and Val(715) inactivated the receptor. Alanine mutation of Asn(15) resulted in a 20-fold decrease in affinity, whereas mutations of Asp(12), Gln(34), Leu(36), Leu(37), Leu(87), Phe(89), Tyr(91), Lys(121), Leu(709), and Phe(714) all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn(15), Leu(37), Asp(707), Leu(709), Tyr(708), Asn(711), Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn(15), Gln(34), and Phe(89) on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.
引用
收藏
页码:20932 / 20936
页数:5
相关论文
共 50 条
  • [21] PURIFICATION OF INSULIN-RECEPTOR WITH FULL BINDING-ACTIVITY
    FUJITAYAMAGUCHI, Y
    CHOI, S
    SAKAMOTO, Y
    ITAKURA, K
    DIABETES, 1983, 32 : A43 - A43
  • [22] LIGAND-BINDING KINETICS OF A SOLUBLE FULL-LENGTH MURINE ERYTHROPOIETIN RECEPTOR
    AVEDISSIAN, LS
    POOLA, I
    SPIVAK, JL
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1995, 216 (01) : 62 - 68
  • [23] Are androgen receptor variants a substitute for the full-length receptor?
    Lu, Ji
    Van der Steen, Travis
    Tindall, Donald J.
    NATURE REVIEWS UROLOGY, 2015, 12 (03) : 137 - 144
  • [24] Are androgen receptor variants a substitute for the full-length receptor?
    Ji Lu
    Travis Van der Steen
    Donald J. Tindall
    Nature Reviews Urology, 2015, 12 : 137 - 144
  • [25] Purification of functional full-length liver X receptor β produced in Escherichia coli
    Toresson, G
    Schuster, GU
    Steffensen, KR
    Bengtsson, M
    Ljunggren, J
    Dahlman-Wright, K
    Gustafsson, JÅ
    PROTEIN EXPRESSION AND PURIFICATION, 2004, 35 (02) : 190 - 198
  • [26] Conformational states of the full-length glucagon receptor
    Yang, Linlin
    Yang, Dehua
    de Graaf, Chris
    Moeller, Arne
    West, Graham M.
    Dharmarajan, Venkatasubramanian
    Wang, Chong
    Siu, Fai Y.
    Song, Gaojie
    Reedtz-Runge, Steffen
    Pascal, Bruce D.
    Wu, Beili
    Potter, Clinton S.
    Zhou, Hu
    Griffin, Patrick R.
    Carragher, Bridget
    Yang, Huaiyu
    Wang, Ming-Wei
    Stevens, Raymond C.
    Jiang, Hualiang
    NATURE COMMUNICATIONS, 2015, 6 : 1 - 13
  • [27] Full-length nuclear receptor allosteric regulation
    Choi, Woong Jae
    Haratipour, Zeinab
    Blind, Raymond D.
    JOURNAL OF LIPID RESEARCH, 2023, 64 (08)
  • [28] Characterization of a second ligand binding site of the insulin receptor
    Hao, Caili
    Whittaker, Linda
    Whittaker, Jonathan
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2006, 347 (01) : 334 - 339
  • [29] Purification of a full-length recombinant glucocorticoid receptor
    Okamoto, K
    Suematsu, N
    Isohashi, F
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, 2003, 790 (1-2): : 349 - 353
  • [30] Cloning, expression and functional characterization of the full-length murine ADAMTS-13
    Bruno, K
    Völkel, D
    Plaimauer, B
    Antoine, G
    Pable, S
    Motto, DG
    Lemmerhirt, HL
    Dorner, F
    Zimmermann, K
    Scheiflinger, F
    JOURNAL OF THROMBOSIS AND HAEMOSTASIS, 2005, 3 (05) : 1064 - 1073