Fusion of insulin receptor ectodomains to immunoglobulin constant domains reproduces high-affinity insulin binding in vitro

Joseph Bass*, Takeshi Kurose, Mohammad Pashmforoush, Donald F. Steiner

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

34 Scopus citations


A unique feature of the insulin receptor is that it is dimeric in the absence of ligand. Dimerization of two adjacent transmembrane domain (TMD) α helices has been shown to be critical in receptor kinase activation. Moreover, previous work has suggested that the TMD is involved in stabilizing the high-affinity binding site; soluble receptors expressed after simple truncation at the ectodomain-TMD junction have reduced affinity for insulin. To further examine this issue, we have replaced the TMD and intracellular domain of the soluble human insulin receptor (HIRs) with constant domains from immunoglobulin Fc and λ subunits (HIRs-Fc and HIRs- λ). Studies of receptor biosynthesis and binding characteristics were performed following transient transfection of receptor cDNAs into human embryonal kidney 293 cells. Each hybrid receptor was initially synthesized as a single chain proreceptor, followed by cleavage into α- and β-Fc or β-λ subunits. The majority of secreted protein appeared in the cell medium as fully processed heterotetramer. Fc fragments released from HIRs-Fc by papain digestion and analyzed by nonreducing SDS-polyacrylamide gel electrophoresis were dimeric. Furthermore, dissociation constants for both chimeras were similar to those for the full-length holoreceptor (wild-type receptor, K(d1) = 200 pM and K(d2) = 2 nM; HIRs-Fc, K(d1) = 200 pM and K(d2) = 40 nM; and HIRs-λ, K(d1) = 200 pM and K(d2) = 5 nM). These results extend previous observations that dimerization of the membrane-proximal ectodomain is necessary to maintain an intact high-affinity insulin-binding site.

Original languageEnglish (US)
Pages (from-to)19367-19375
Number of pages9
JournalJournal of Biological Chemistry
Issue number32
StatePublished - 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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