Activity-dependent conformational transitions of the insulin receptor–related receptor

Oleg V Batishchev,Natalia V Kuzmina,Andrey A Mozhaev,Alexander S Goryashchenko,Ekaterina D Mileshina,Alexander N Orsa,Eduard V Bocharov,Igor E Deyev,Alexander G Petrenko

doi:10.1016/j.jbc.2021.100534

Abstract

The insulin receptor (IR), insulin-like growth factor 1 receptor (IGF-1R), and insulin receptor-related receptor (IRR) form a mini family of predimerized receptor-like tyrosine kinases. IR and IGF-1R bind to their peptide agonists triggering metabolic and cell growth responses. In contrast, IRR, despite sharing with them a strong sequence homology, has no peptide-like agonist but can be activated by mildly alkaline media. The spatial structure and activation mechanisms of IRR have not been established yet. The present work represents the first account of a structural analysis of a predimerized receptor-like tyrosine kinase by high-resolution atomic force microscopy in their basal and activated forms. Our data suggest that in neutral media, inactive IRR has two conformations, where one is symmetrical and highly similar to the inactive Λ/U-shape of IR and IGF-1R ectodomains, whereas the second is drop-like and asymmetrical resembling the IRR ectodomain in solution. We did not observe complexes of IRR intracellular catalytic domains of the inactive receptor forms. At pH 9.0, we detected two presumably active IRR conformations, Γ-shaped and T-shaped. Both of conformations demonstrated formation of the complex of their intracellular catalytic domains responsible for autophosphorylation. The existence of two active IRR forms correlates well with the previously described positive cooperativity of the IRR activation. In conclusion, our data provide structural insights into the molecular mechanisms of alkali-induced IRR activation under mild native conditions that could be valuable for interpretation of results of IR and IGF-IR structural studies.

Highlights

Oligomerization of the receptor upon ligand binding [1]
Similar drop-like conformation of the ectodomain was recently detected for the ligand-bound active form of IGF1R [12]. This conformation shows a small distance between the fibronectin domains of the two receptor subunits indicating that FnIII-2 and FnIII-3 domains are most critical for insulin receptor–related receptor (IRR) activity
The samples were incubated with Tris-HCl buffer of a neutral or alkaline pH and further analyzed by blotting with antibodies against phosphorylated forms of IRR (Fig. 2B)

Summary

Introduction

Oligomerization of the receptor upon ligand binding [1]. a small set of RTKs, representing the most commonly expressed insulin receptor (IR) and named the insulin receptor family, preexists in the membrane as disulfide-linked dimers of two subunits, called αβ halfreceptors and, requires different mechanisms of activation and signal transduction across the plasma membrane [4]. Intensive studies of IR and IGF-1R by structural methods such as X-ray crystallography, cryo-electron microscopy (cryoEM), and small-angle X-ray scattering reveals details of ligand binding and subsequent protein remodeling [6,7,8,9,10,11,12,13] Both receptors show inactive Λ-shape of the ectodomain region transforming into active predominantly T-like conformation for IR or Γ-shape of IGF-1R upon interaction with insulin or IGF-1, respectively. Similar drop-like conformation of the ectodomain was recently detected for the ligand-bound active form of IGF1R [12] This conformation shows a small distance between the fibronectin domains of the two receptor subunits indicating that FnIII-2 and FnIII-3 domains are most critical for IRR activity. They are located at the interface between the L1C and fibronectin domains within the IRR dimer that explains their role in the IRR function and implies that L1C domains are components of the pH sensing mechanism of the IRR [21]

Results

Discussion

Conclusion