Identification of determinants that confer ligand specificity on the insulin receptor.
We have previously shown, using truncated soluble recombinant receptors, that substituting the 62 N-terminal amino acids of the alpha subunit from the insulin-like growth factor I receptor (IGFIR) with the corresponding 68 amino acids from the insulin receptor (IR) results in a chimeric receptor with an approximately 200-fold increase in affinity for insulin and only a 5-fold decrease in insulin-like growth factor I (IGFI) affinity (Kjeldsen, T., Andersen, A. S., Wiberg, F. C., Rasmussen, J. S., Schäffer, L., Balschmidt, P., Møller, K. B., and Møller, N. P. H. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4404-4408). We demonstrate that these 68 N-terminal amino acids of the IR also confer insulin affinity on the intact IGFI holoreceptor both in the membrane-bound state and when solubilized by Triton X-100. Furthermore, this domain can be subdivided into two regions (amino acids 1-27 and 28-68 of the IR alpha subunit) that, when replacing the corresponding IGFIR sequences, increases the insulin affinity of truncated soluble receptor chimeras 8- and 20-fold, respectively, with only minor effects on the IGFI affinity. Within the latter of these two regions, we found that amino acids 38-68 of the IR, representing 13 amino acid differences from IGFIR, confer the same 20-fold increase in insulin affinity on the IGFIR. Finally, the amino acids from position 42 to 50 are not responsible for this increase in insulin affinity. We thus propose that at least two determinants within the 68 N-terminal amino acids of the insulin receptor are involved in defining the ligand specificity of the insulin receptor, and that one or a combination of the remaining seven amino acid differences between position 38 and 68 are involved in conferring insulin affinity on the insulin receptor.
Andersen
A S
AS
Novo Nordisk A/S, Bagsvaerd, Denmark.
Kjeldsen
T
T
Wiberg
F C
FC
Vissing
H
H
Schäffer
L
L
Rasmussen
J S
JS
De Meyts
P
P
Møller
N P
NP
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D011972:Receptor, Insulin / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D008024:Ligands; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011993:Recombinant Fusion Proteins; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin