Specificity of insulin and insulin-like growth factor I receptors investigated using chimeric mini-receptors. Role of C-terminal 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 constructs comprising the first three domains (468 amino acids) of insulin receptor (IR) or insulin-like growth factor I receptor (IGFIR) combined with C-terminal domain from either insulin receptor (IR) (residues 704-719), IGFIR, or insulin receptor-related receptor (IRRR). The constructs were stably expressed 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 IR and IGFIR context, whereas other constructs bound ligands. The two constructs with first three domains of the IR demonstrated low specificity for ligands, all affinities 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 more than 3000 nM. When swapping the C-terminal domains in either receptor context only minor changes were observed in affinities (<3-fold), demonstrating that the carboxyl-terminal of IR and IGFIR alpha-subunits are interchangeable and suggesting that this domain is part of the common binding site.
Kristensen
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Insulin Research, Health Care Discovery, Novo Nordisk, 2880 Bagsvaerd, Denmark. clak@novo.dk
Wiberg
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Andersen
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D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D006224:Cricetinae; D015151:Immunoblotting; D008970:Molecular Weight; D017526:Receptor, IGF Type 1 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011972:Receptor, Insulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship
D011993:Recombinant Fusion Proteins; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin