Leucine-973 is a crucial residue differentiating insulin and IGF-1 receptor signaling
Hirofumi Nagao, … , Matthias Mann, C. Ronald Kahn
Hirofumi Nagao, … , Matthias Mann, C. Ronald Kahn
Published December 22, 2022
Citation Information: J Clin Invest. 2023;133(4):e161472. https://doi.org/10.1172/JCI161472.
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Research Article Endocrinology Metabolism

Leucine-973 is a crucial residue differentiating insulin and IGF-1 receptor signaling

Abstract

Insulin and IGF-1 receptors (IR and IGF1R) are highly homologous and share similar signaling systems, but each has a unique physiological role, with IR primarily regulating metabolic homeostasis and IGF1R regulating mitogenic control and growth. Here, we show that replacement of a single amino acid at position 973, just distal to the NPEY motif in the intracellular juxtamembrane region, from leucine, which is highly conserved in IRs, to phenylalanine, the highly conserved homologous residue in IGF1Rs, resulted in decreased IRS-1/PI3K/Akt/mTORC1 signaling and increased Shc/Gab1/MAPK cell cycle signaling. As a result, cells expressing L973F-IR exhibited decreased insulin-induced glucose uptake, increased cell growth, and impaired receptor internalization. Mice with knockin of the L973F-IR showed similar alterations in signaling in vivo, and this led to decreased insulin sensitivity, a modest increase in growth, and decreased weight gain when mice were challenged with a high-fat diet. Thus, leucine-973 in the juxtamembrane region of the IR acts as a crucial residue differentiating IR signaling from IGF1R signaling.

Authors

Hirofumi Nagao, Weikang Cai, Bruna B. Brandão, Nicolai J. Wewer Albrechtsen, Martin Steger, Arijeet K. Gattu, Hui Pan, Jonathan M. Dreyfuss, F. Thomas Wunderlich, Matthias Mann, C. Ronald Kahn

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Figure 6

Effects of the L973F substitution on IR signaling in mice.

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Effects of the L973F substitution on IR signaling in mice. (A) Schematic...
(A) Schematic representation of L973F-IR modification in the genome of C57BL/6N mice by CRISPR-mediated gene editing. There were also 2 silent point mutations inserted in codons for glycine-962 and proline-963 to generate a new ApaI restriction site for genotyping. (B) Genotyping analysis of L973F mutation knockin mice. WT mouse (–/–) produces a 658 bp fragment. Homozygous knockin mouse (+/+) produces 516 bp and 141 bp fragments. Heterozygous knockin mouse (+/–) produces 658 bp, 516 bp, and 141 bp fragments. (C) IR levels in liver tissue from male WT and L973F-IR mice (7 months old) by immunoblotting using antibodies specific for IRβ from Santa Cruz Biotechnology Inc. (sc-711) and Cell Signaling Technology (CST3025). Mice were injected with either saline or 2 units insulin via the vena cava 10 minutes before sacrifice. CST3025 detects residues surrounding tyrosine-972 in the juxtamembrane region of IRβ. SC-711 antibody detects the C-terminus of IRβ. (D) Quantification of IRβ levels in liver tissues from male WT and L973F-IR mice. The level of each protein in the control mice was set at 1. Saline-injected mice are represented by white circles and insulin-injected mice by black circles. Data are represented as mean ± SEM (n = 6–9 per group). ***P < 0.001, WT versus L973F-IR mice, unpaired Student’s t test. (E) Analysis of insulin signaling in gastrocnemius muscle of WT and L973F-IR mice (7-month-old males) administered insulin (2 U) or saline 10 minutes prior to sacrifice. Immunoblot analysis was performed with the indicated antibodies. (F) Quantification of p-IRβ, p-IRS1Y608, p-AktS473, p-mTORS2481, p-ShcY239/240, and p-ERKT202/Y204. Results are represented as mean ± SEM. **P < 0.01; ***P < 0.001, basal versus insulin. #P < 0.05; ##P < 0.01; ###P < 0.001, WT versus L973F-IR mice, 2-way ANOVA.