Role of the exon 11 of the insulin receptor gene on insulin binding identified by anti-peptide 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 for insulin, thus suggesting that the sequence encoded by exon 11 may be important for insulin binding. To further investigate this issue, we generated polyclonal antibodies against C-terminal peptides of the two 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) directed against a sequence in the carboxy-terminal region of the alpha-subunit which is common to HIR-A and HIR-B. Antibodies were characterized for their ability to immunoprecipitate the receptor and to inhibit [125I]insulin binding to both isoforms. We found that PA-13 immunoprecipitates both the HIR-A and the HIR-B, PA-12 immunoprecipitates exclusively the HIR-B, and PA-11 fails to precipitate both isoforms. Interestingly, 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 dissociation rate of insulin, thus suggesting that antibody affects cooperative interactions among binding sites. We conclude that the sequence encoded by exon 11 may play a role in modulating the binding of insulin to the receptor and negative cooperativity.
Sesti
G
G
Dipartimento di Medicina Interna, Università di Roma Tor Vergata, Rome, Italy.
Tullio
A N
AN
Marini
M A
MA
Manera
E
E
Borboni
P
P
Accili
D
D
Longhi
R
R
Fusco
A
A
Lauro
R
R
Montemurro
A
A
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D005091:Exons / Q000235:genetics*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D007328:Insulin; D011972:Receptor, Insulin