Creation of novel insulin receptor constructs from fragments containing binding sites 1 or 2

Insulin signalling regulates crucial processes of metabolism, growth and differentiation, and insulin is an essential medicine for treatment of diabetes mellitus. The insulin receptor (IR) is an (αβ)2 dimeric transmembrane protein responsible for mediating the effects of insulin and, to a degree, insulin-like growth factors. It is over 1000 amino acids long, multi-domain, highly glycosylated and stabilized by multiple disulphide bridges. These considerations have limited our structural understanding of the receptor and its interactions with insulin despite its great medical importance. Insulin binding is complex: IR binding sites are contained entirely within the extracellular α chain, and each monomeric chain contains two distinct sub-sites that bind insulin to form a high-affinity crosslink. Despite being a symmetrical dimer, the full-length receptor binds only one insulin molecule with high affinity, presumably because an asymmetric fit induced by ligand forming one sub-site crosslink prevents formation of the other.
This work seeks to produce and characterize novel, high-affinity heterodimeric constructs of IR α chain modified to only contain one possible site 1-2 crosslink. To this end, expression vectors were engineered based on plasmids obtained from Novo Nordisk A/S coding for fusion proteins of IR1-593 and IR310-601.678-719/731 receptor fragments connected with poly(Gly-Ser) linkers containing protease recognition sites. Expression levels were found to be comparable to those of established IR constructs like IR(1-310). Two alternative signal peptides were tested, but did not improve protein expression or secretion. Two constructs were expressed and purified on a preparative scale and their oligomerization state and cleavage was assessed. Purification of a construct with a 3C protease site linker showed that producing two receptor fragments from a single polypeptide chain is a viable approach. If a site 1 – site 2’ limited heterodimers could be reconstituted, it would open an exciting new approach to structural studies of insulin/IR complexes.