Dimeric fragment of the insulin receptor alpha-subunit binds insulin with full holoreceptor affinity.
The insulin receptor (IR) is a dimeric receptor, and its activation is thought to involve cross-linking between monomers initiated by binding of a single insulin molecule to separate epitopes on each monomer. We have previously shown that a minimized insulin receptor consisting of the first three domains of the human IR fused to 16 amino acids from the C-terminal of the alpha-subunit was monomeric and bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Schäffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780-17786). To investigate the insulin binding properties of dimerized alpha-subunits, we have reintroduced the domains containing alpha-alpha disulfide bonds into this minireceptor. When inserting either the first fibronectin type III domain or the full-length sequence of exon 10, the receptor fragments were predominantly secreted as disulfide-linked dimers that both had nanomolar affinity for insulin, similar to the affinity found for the minireceptor. However, when both these domains were included we obtained a soluble dimeric receptor that bound insulin with 1000-fold higher affinity (4-8 pm) similar to what was obtained for the solubilized holoreceptor (14-24 pm). Moreover, dissociation of labeled insulin from this receptor was accelerated in the presence of unlabeled insulin, demonstrating another characteristic feature of the holoreceptor. This is the first direct demonstration showing that the alpha-subunit of IR contains all the epitopes required for binding insulin with full holoreceptor affinity.
Insulin Research, Novo Nordisk A/S, 2880 Bagsvaerd, Denmark.
J
J
Brandt
A S
AS
Andersen
C
C
Kristensen
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D019281:Dimerization; D007328:Insulin / Q000378:metabolism*; D011485:Protein Binding; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D007328:Insulin; D011972:Receptor, Insulin