Dimeric fragment of the insulin receptor alpha-subunit finds insulin with full holoreceptor affinity

The insulin receptor (IR) is a dimeric receptor, and its activation is thou ght to involve cross-linking between monomers initiated by binding of a sin gle insulin molecule to separate epitopes on each monomer, We have previous ly shown that a minimized insulin receptor consisting of the first three do mains of the human IR fused to 16 amino acids from the C-terminal of the al pha -subunit was monomeric and bound insulin with nanomolar affinity (Krist ensen, C,, Wiberg, F, C,, Schaffer, L,, and Andersen, A. S, (1998) J, Biol, Chem, 273, 17780-17786). To investigate the insulin binding properties of dimerized alpha -subunits, we have reintroduced the domains containing alph a-alpha disulfide bonds into this minireceptor, When inserting either the f irst fibronectin type III domain or the full-length sequence of exon 10, th e receptor fragments were predominantly secreted as disulfide-linked dimers that both had nanomolar affinity for insulin, similar to the affinity foun d for the minireceptor, However, when both these domains were included we o btained a soluble dimeric receptor that bound insulin with 1000-fold higher affinity (4-8 pM) similar to what was obtained for the solubilized holorec eptor (14-24 PM). Moreover, dissociation of labeled insulin from this recep tor was accelerated in the presence of unlabeled insulin, demonstrating ano ther characteristic feature of the holoreceptor, This is the first direct d emonstration showing that the alpha -subunit of LR contains all the epitope s required for binding insulin with full holoreceptor affinity.