Visualization of ligand-induced transmembrane signaling in the full-length human insulin receptor

Abstract

Insulin receptor (IR) signaling plays a critical role in the regulation of metabolism and growth in multicellular organisms. IRs are unique among receptor tyrosine kinases in that they exist exclusively as covalent (αβ)₂ homodimers at the cell surface. Transmembrane signaling by the IR can therefore not be based on ligand-induced dimerization as such but must involve structural changes within the existing receptor dimer. In this study, using glycosylated full-length human IR reconstituted into lipid nanodiscs, we show by single-particle electron microscopy that insulin binding to the dimeric receptor converts its ectodomain from an inverted U-shaped conformation to a T-shaped conformation. This structural rearrangement of the ectodomain propagates to the transmembrane domains, which are well separated in the inactive conformation but come close together upon insulin binding, facilitating autophosphorylation of the cytoplasmic kinase domains.

Graphical abstract

Figure 1.

IR reconstitution into nanodiscs and activity assay. (A) Left: Schematic cartoon showing the ectodomain (ECD), transmembrane domain (TMD), and tyrosine kinase domain (TKD). Right: Structural model of the full-length human (αβ)₂ IR assembled from available crystal structures of the tyrosine kinase (Hubbard et al., 1994) and extracellular domain (Croll et al., 2016) as well as the nuclear magnetic resonance solution structure of the transmembrane domain (Li et al., 2014). (B) Silver-stained native PAGE of glycosylated full-length human IR reconstituted into MSP1E3D1 nanodiscs. (C) SDS-PAGE of nanodisc-embedded IRs under nonreducing (−DTT) and reducing conditions (+DTT). (D) Activity assay showing that both CHAPS-solubilized and nanodisc-embedded IRs are autophosphorylated upon exposure to insulin. IRα, IR α subunit; IRβ, IR β subunit; IR(αβ)₂, mature IR; pIRβ, phosphorylated IRβ; pro-IRαβ, unprocessed intracellular form of IR. Experimental details are provided in the In vitro phosphorylation assay and SDS-PAGE and Western blots sections in Materials and methods as well as Figs. S1 and S2.

Figure 2.

Schematic illustration of the conformations assigned to IRs in nanodiscs. (A and B) Representative class averages (A) and schematic drawings (B) of IRs in nanodiscs adopting different conformations: U-shaped dimer (ectodomain in red) in one (1U) or two nanodiscs (2U), T-shaped dimer (ectodomain in blue) in one (1T and II) or two nanodiscs (2U), and L-shaped monomer (ectodomain in dark gray) in one nanodisc (L). (C) Models of how the nanodisc-embedded IRs may look in solution. Because the tyrosine kinase domains are flexibly tethered to the transmembrane domains, their locations are somewhat speculative, especially in the 2T conformation. Note, however, that extra density in the class averages of the 1T and II conformations may represent tyrosine kinase domain dimers.

Figure 3.

Selected EM averages of IRs reconstituted into MSP1E3D1 nanodiscs and ligand-induced IR activation. (A) In the absence of insulin, IRs reconstituted into one nanodisc (1U; top) or two nanodiscs (2U; bottom) and adopted a U-shaped conformation. (B) Upon insulin addition, all IRs in a single nanodisc adopted the T-shaped conformation (1T; middle), whereas IRs in two nanodiscs adopted either the U-shaped (top) or T-shaped conformation (2T; bottom). (C) In the presence of insulin, all IRs reconstituted into a single nanodisc. Most of them adopted a T-shaped conformation (top), whereas some showed an II-shaped conformation (II), likely representing a different view of IRs in the T-shaped conformation (bottom). The first three averages show a globular density that may represent dimerized tyrosine kinase domains (TKDs; arrows). (D) Upon insulin addition, all IRs showed a T-shaped conformation. The side length of individual panels is 47.7 nm. (E) Quantification of IR populations with a particular ectodomain (ECD) conformation at given insulin concentrations. (F) Cartoon illustrating the ligand (green) binding–induced conformational change in the ectodomain and its coupling to the transmembrane domains (TMDs) with concomitant activation of the tyrosine kinase domains by autophosphorylation (red asterisks). Our experimental data imply that binding of one ligand is sufficient to induce the transition of the IR ectodomain from the U-shaped to the T-shaped conformation.