ROLE OF THE EXON-11 OF THE INSULIN-RECEPTOR GENE ON INSULIN BINDING IDENTIFIED BY ANTIPEPTIDE ANTIBODIES

The insulin receptor exists in two isoforms differing by the absence ( HIR-A) or presence (HIR-B) of 12 amino acids in the C-terminus of the alpha-subunit as a consequence of alternative splicing of exon 11. It was shown that the two isoforms exhibit different binding affinities f or insulin, thus suggesting that the sequence encoded by exon 11 may b e important for insulin binding. To further investigate this issue, we generated polyclonal antibodies against C-terminal peptides of the tw o HIR alpha-subunit variants. Herein, we characterized two antibodies, PA-11 and PA-12, directed against the C-terminus or the N-terminus of the sequence encoded by exon 11, respectively, and one (PA-13) direct ed against a sequence in the carboxy-terminal region of the alpha-subu nit which is common to HIR-A and HIR-B. Antibodies were characterized for their ability to immunoprecipitate the receptor and to inhibit [I- 125]insulin binding to both isoforms. We found that PA-13 immunoprecip itates both the HIR-A and the HIR-B, PA-12 immunoprecipitates exclusiv ely the HIR-B, and PA-11 fails to precipitate both isoforms. Interesti ngly, PA-12 inhibits specifically insulin to the HIR-B, whereas other PAs fail to affect insulin binding to either isoforms. Furthermore, PA -12 linearises the Scatchard plot of binding data, and retards the dis sociation rate of insulin, thus suggesting that antibody affects coope rative interactions among binding sites. We conclude that the sequence encoded by exon 11 may play a role in modulating the binding of insul in to the receptor and negative cooperativity.