The three-dimensional (3D) structure of the intrinsically dimeric insulin receptor bound to its ligand, insulin, was determined by electron cryomicroscopy. Gold-labeled insulin served to locate the insulin-binding domain. The 3D structure was then fitted with available known high-resolution domain substructures to obtain a detailed contiguous model for this heterotetrameric transmembrane receptor. The 3D reconstruction indicates that the two alpha subunits jointly participate in insulin binding and that the kinase domains in the two beta subunits are in a juxtaposition that permits autophosphorylation of tyrosine residues in the first step of insulin receptor activation.

The photoreactive insulin derivatives N epsilon B29-(azidobenzoyl)insulin (MAB-insulin) and N alpha A1, N epsilon B29-di(azidobenzoyl)insulin (DAB-insulin) were synthesized by reacting bovine insulin with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. These derivatives were purified by ion exchange chromatography on SP-Sephadex, and their identities were established by polyacrylamide gel electrophoresis, amino acid analysis, and end-group determination. Their biological activities were measured by receptor binding assay and fat cell assay. The photoreactivity of these two derivatives was demonstrated by spectral changes and by the formation of covalent polymers of high molecular weight when exposed to light. Radioactive MAB-insulin and DAB-insulin were prepared by iodination with [125I]iodine. These radioactive derivatives were characterized for their photoreactivity, immunoreactivity, and receptor binding to liver plasma membrane. Liver plasma membrane preparations of rat, mouse, and guinea pig were incubated with these radioactive insulin derivatives and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization and reduction showed that two proteins were specifically labeled. The molecular weights of the two radioactive bands were estimated to be about 130 000 and 90 000 in all three species of animals.

Transmembrane signaling via receptor tyrosine kinases generally requires oligomerization of receptor monomers, with the formation of ligand-induced dimers or higher multimers of the extracellular domains of the receptors. Such formations are expected to juxtapose the intracellular kinase domains at the correct distances and orientations for transphosphorylation. For receptors of the insulin receptor family that are constitutively dimeric, or those that form noncovalent dimers without ligands, the mechanism must be more complex. For these, the conformation must be changed by the ligand from one that prevents activation to one that is permissive for kinase phosphorylation. How the insulin ligand accomplishes this action has remained a puzzle since the discovery of the insulin receptor over 2 decades ago, primarily because membrane proteins in general have been refractory to structure determination by crystallography. However, high-resolution structural evidence on individual separate subdomains of the insulin receptor and of analogous proteins has been obtained. The recently solved quaternary structure of the complete dimeric insulin receptor in the presence of insulin has now served as the structural envelope into which such individual domains were fitted. The combined structure has provided answers on the details of insulin/receptor interactions in the binding site and on the mechanism of transmembrane signaling of this covalent dimer. The structure explains many observations on the behavior of the receptor, from greater or lesser binding of insulin and its variants, point and deletion mutants of the receptor, to antibody-binding patterns, and to the effects on basal and insulin-stimulated autophosphorylation under mild reducing conditions.

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