Insulin binding to its receptor induces a conformational change in the receptor C-terminus.
Antibodies against peptides corresponding to sequences in the C-terminus of the insulin receptor beta-subunit were used to approach the putative role of this receptor domain in signal generation. Two sequences were chosen and correspond to peptide C1, comprising amino acids 1309-1326, and peptide C2, comprising amino acids 1294-1317. The two antibodies produced distinct immunoprecipitation patterns as a function of the insulin receptor form and recognized changes in the insulin receptor molecule induced by ligand binding and autophosphorylation. Both antipeptides, anti-C1 and anti-C2, showed an important decrease in their recognition capacity for the receptor occupied by insulin when compared to the empty receptor. Further, anti-C1 had a lower affinity for the phosphorylated receptor compared to the unphosphorylated receptor and failed to recognize a fraction of the phosphoreceptor population. In contrast, anti-C2 had similar affinities for the phosphorylated and unphosphorylated receptors but was unable to interact with part of the unphosphorylated receptors. Finally, using immunoblotting of the receptor to analyze the denatured molecules, we showed that the phosphorylation-induced changes detected by anti-C1 are retained, suggesting that they are likely not of a conformational nature. In contrast, the insulin-induced changes in the receptor molecule disappear with receptor denaturation which points to their reversible nature. We conclude from these data that (i) antipeptides against the receptor C-terminal sequence are able to distinguish between phosphorylated and unphosphorylated receptor forms and (ii) binding of insulin to its receptor leads to a reversible, phosphorylation-independent, and possibly conformational change at the level of the receptor C-terminal domain.
Baron
V
V
Institut National de la Santé et de la Recherche Médicale, INSERM U145, Faculté de Médecine, Nice, France.
Gautier
N
N
Komoriya
A
A
Hainaut
P
P
Scimeca
J C
JC
Mervic
M
M
Lavielle
S
S
Dolais-Kitabgi
J
J
Van Obberghen
E
E
D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D002478:Cells, Cultured; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011972:Receptor, Insulin / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D007328:Insulin; D011972:Receptor, Insulin