Specificity of insulin and insulin-like growth factor I receptors investigated using chimeric mini-receptors - Role of C-teriminal of receptor alpha subunit

We have investigated the role of the C-terminal of the alpha-subunit in the insulin receptor family by characterizing chimeric mini-receptor construct s comprising the first three domains (468 amino acids) of insulin receptor (IR) or insulin-like growth factor I receptor (IGFIR) combined with C-termi nal domain from either insulin receptor (IR) (residues 704-719), IGFIR, or insulin receptor-related receptor (IRRR), The constructs were stably expres sed in baby hamster kidney cells and purified, and binding affinities were determined for insulin, IGFI, and a single chain insulin/IGFI hybrid. The C -terminal domain of IRRR was found to abolish binding in PR and IGFIR conte xt, whereas other constructs bound ligands, The two constructs with first t hree domains of the IR demonstrated low specificity for ligands, all affini ties ranging from 3.0 to 15 nM. In contrast, the constructs with the first three domains of the IGFIR had high specificity, the affinity of the novel minimized IGFIR for IGFI was 1.5 nM, whereas the affinity for insulin was m ore than 3000 nM. When swapping the C-terminal domains in either receptor c ontext only minor changes were observed in affinities (<3-fold), demonstrat ing that the carboxyl-terminal of IR and IGFIR alpha-subunits are interchan geable and suggesting that this domain is part of the common binding site.