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Structural basis of the alkaline pH-dependent activation of insulin receptor-related receptor

Nature Structural & Molecular Biology volume 30, pages 661–669 (2023)Cite this article

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Abstract

The insulin receptor (IR) family is a subfamily of receptor tyrosine kinases that controls metabolic homeostasis and cell growth. Distinct from IR and insulin-like growth factor 1 receptor, whose activation requires ligand binding, insulin receptor-related receptor (IRR)—the third member of the IR family—is activated by alkaline pH. However, the molecular mechanism underlying alkaline pH-induced IRR activation remains unclear. Here, we present cryo-EM structures of human IRR in both neutral pH inactive and alkaline pH active states. Combined with mutagenesis and cellular assays, we show that, upon pH increase, electrostatic repulsion of the pH-sensitive motifs of IRR disrupts its autoinhibited state and promotes a scissor-like rotation between two protomers, leading to a T-shaped active conformation. Together, our study reveals an unprecedented alkaline pH-dependent activation mechanism of IRR, opening up opportunities to understand the structure–function relationship of this important receptor.

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Fig. 1: Overall structure of IRR at pH 7.
Fig. 2: The alkaline pH-sensitive motif of IRR.
Fig. 3: Overall structure of IRR at pH 9.0.
Fig. 4: Mechanism of pH-dependent activation of IRR.

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Data availability

All reagents generated in this study are available with a completed Materials Transfer Agreement. All cryo-EM maps and models reported in this work have been deposited with the PDB/EMDB database, under the entry ID: PDB 7TYJ/EMD-26181, PDB 7TYK/EMD-26183 and PDB 7TYM/EMD-26185. PDB used in this study are as follows: PDB 4ZXB, PDB 6PXV, PDB 5U8R and PDB 6PYH. Source data are provided with this paper.

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Acknowledgements

Cryo-EM data were collected at the University of Texas Southwestern Medical Center (UTSW) Cryo-Electron Microscopy Facility, funded in part by the Cancer Prevention and Research Institute of Texas (CPRIT) Core Facility Support Award RP170644. We thank D. Stoddard for facility access. This work is supported in part by grants from the National Institutes Health (R01GM136976 to X.-C.B., R35GM142937 to E.C. and a center grant P30DK063608), the Welch foundation (I-1944 to X.-C.B.), CPRIT (RP160082 to X.-C.B.) and the Alice Bohmfalk Charitable (to E.C.). X.-C.B. is Virginia Murchison Linthicum Scholars in Medical Research at UTSW.

Author information

Author notes

  1. These authors contributed equally: Liwei Wang, Catherine Hall.

Authors and Affiliations

  1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Liwei Wang, Jie Li & Xiao-chen Bai

  2. Department of Pathology and Cell Biology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA

    Catherine Hall & Eunhee Choi

  3. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xiao-chen Bai

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Contributions

E.C. and X.-C.B. designed and supervised research; L.W., C.H., J.L., E.C. and X.-C.B. performed research, analyzed data and contributed to the writing of the paper.

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Correspondence to Eunhee Choi or Xiao-chen Bai.

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Nature Structural & Molecular Biology thanks Pierre De Meyts and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Sara Osman was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Sequence alignment of human IRR, IR and IGF1R.

The proposed key pH sensitive residues and the residues located in the interfaces of active-IRR are marked.

Extended Data Fig. 2 Purification of the extracellular domain of human IRR.

(a) A representative size-exclusion chromatogram of IRR at pH 7. The peak fractions were visualized on SDS–PAGE by Coomassie staining, in the absence or presence of dithiothreitol (DTT). This experiment was repeated for at least 5 times independently with similar results. (b) A representative size-exclusion chromatogram of IRR at pH 9. The peak fractions were visualized on SDS–PAGE by Coomassie staining, in the absence or presence of DTT. This experiment was repeated for at least 5 times independently with similar results.

Extended Data Fig. 3 Cryo-EM analysis of IRR at pH 7.

(a) Representative electron micrograph. This experiment was repeated for 4,410 times independently with similar results. Scale bar: 200 Å. (b) 2D class averages of IRR at pH 7. (c) Cryo-EM maps of IRR at pH 7 colored by local resolution. Up: the cryo-EM map refined with C2 symmetry applied. Bottom: the cryo-EM map refined with symmetry expansion and C1 symmetry. (d) Gold-standard Fourier shell correlation (FSC) curve for the cryo-EM maps. Blue: before symmetry expansion. Red: after symmetry expansion. (e) Flowchart of cryo-EM data processing of the dataset of IRR at pH 7.

Extended Data Fig. 4 Representative cryo-EM densities of various parts of the structures of apo- and active-IRR.

(a) Representative cryo-EM density of each domain of apo-IRR. (b) Representative cryo-EM density of each domain of active-IRR.

Extended Data Fig. 5 Sequence alignment of IRR.

The proposed key pH-sensitive residues are marked.

Extended Data Fig. 6 Effects of mutations at the interface between L1 and FnIII-2 domains in the apo-IRR.

(a) IR phosphorylation induced by insulin in 293FT cells expressing IR WT or the indicated mutants. (b) Quantification of the Western blot data shown in (a) (Mean ± SD). Each experiment was repeated three times. Significance calculated using two-tailed students t-test. *p = 0.0252. (c) Histidine residues at the L1/FnIII-2 interface in the apo-IRR. (d) IRR phosphorylation induced by alkaline media in 293FT cells expressing IRR WT or the indicated mutants. (e) Quantification of the Western blot data at pH 9.0 shown in (d) (Mean ± SD). Each experiment was repeated three times. Significance calculated using two-tailed student t-test. No significant difference was found. (f) The expression of mature (IRRβ) and proreceptor forms of IRR in 293FT cells expressing IRR wild-type (WT) or the indicated mutants. (g) Quantification of mature forms of IRR of the Western blot data (f) (Mean ± SD). Each experiment was repeated three times. Significance calculated using two-tailed students t-test. (h) Quantification of proreceptor forms of IRR of the Western blot data (f) (Mean ± SD). Each experiment was repeated three times. Significance calculated using two-tailed students t-test.

Source data

Extended Data Fig. 7 Cryo-EM analysis of IRR at pH 9.

(a) Representative electron micrograph. This experiment was repeated for 7,195 times independently with similar results. Scale bar: 200 Å. (b) 2D class averages of IRR at pH 9. (c) Cryo-EM map of IRR at pH 9 colored by local resolution, shown in two orthogonal views. (d) Gold-standard Fourier shell correlation (FSC) curve for the cryo-EM map. (e) Flowchart of cryo-EM data processing of the dataset of IRR at pH 9.

Extended Data Fig. 8 Three interfaces between two protomers stabilize the active IRR dimer.

(a) IRR phosphorylation induced by alkaline media in 293FT cells expressing IRR WT or the indicated mutants. Mutants were designed to abolish interaction at Interface I (L489E; L554E; K117E) or Interface II (R448A; H360A). (b) IRR phosphorylation induced by alkaline media in 293FT cells expressing IRR WT or the indicated mutants. Mutants were designed to abolish interaction at Interface III. Quantification of (a) and (b) is shown in Fig. 3f. (c) The locations of acidic residues from the pH-sensitive motif of IRR in the active state.

Source data

Extended Data Fig. 9 The protonation status of interface histidine does not affect the structural stability of a T-shaped IRR.

(a) Histidine residues at the protomer-protomer interface of the T-shaped, active structure of IRR. (b) IRR phosphorylation induced by alkaline media in 293FT cells expressing IRR WT or the indicated mutants. (c) Quantification of the Western blot data at pH 9.0 shown in (b) (Mean ± SD). Each experiment was repeated three times. Significance calculated using two-tailed student t-test. (Note that the quantification of H360A is the same as that in Fig. 3f).

Source data

Extended Data Fig. 10 The effect of insulin on IRR activation in different pH conditions.

(a) IRR phosphorylation induced by alkaline media in 293FT cells expressing IRR WT or indicated point mutants, which abolish site-1 insulin binding. pH treatment with serum-free DMEM at 7.4, 8.0, or 9.0; insulin treatment at 100 nM. (b) IRR phosphorylation induced by alkaline media in 293FT cells expressing IRR WT or indicated point mutants, which abolish site-2 insulin binding. pH treatment with serum-free DMEM at 7.4, 8.0, or 9.0; insulin treatment at 100 nM. (c) Quantification of Western blot data shown in (a) and (b) (Mean ± SD). WT, n = 6 independent experiments; E484K, n = 3; R486E, n = 3; R476E, n = 3; T535A, n = 3. Relative pY-IRR/Total IRR levels are normalized to IRR WT phosphorylation with pH 9.0 treatment, no insulin. No significant difference was found between + and – insulin conditions at the same pH for any mutant.

Source data

Supplementary information

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 8

Unprocessed western blots.

Source Data Extended Data Fig. 9

Statistical source data.

Source Data Extended Data Fig. 9

Unprocessed western blots.

Source Data Extended Data Fig. 10

Statistical source data.

Source Data Extended Data Fig. 10

Unprocessed western blots.

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Wang, L., Hall, C., Li, J. et al. Structural basis of the alkaline pH-dependent activation of insulin receptor-related receptor. Nat Struct Mol Biol 30, 661–669 (2023). https://doi.org/10.1038/s41594-023-00974-0

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