Insulin and insulin-like growth factor-1 binding specificity is determined by distinct regions of their cognate receptors.

R Schumacher,L Mosthaf,J Schlessinger,D Brandenburg,A Ullrich

doi:10.1016/s0021-9258(18)54996-6

R Schumacher, L Mosthaf + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(18)54996-6

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Abstract

Chimeric insulin/insulin-like growth factor-1 receptors and insulin receptor alpha-subunit point mutants were characterized with respect to their binding properties for insulin and insulin-like growth factor-1 (IGF-1) and their ability to translate ligand interaction into tyrosine kinase activation in intact cells. We found that replacement of the amino-terminal 137 amino acids of the insulin receptor (IR) with the corresponding 131 amino acids of the IGF-1 receptor (IGF-1R) resulted in loss of affinity for both ligands. Further replacement of the adjacent cysteine region with IGF-1R sequences fully reconstituted affinity for IGF-1, but only marginally for insulin. Unexpectedly, replacement of the IR cysteine-rich domain alone by IGF-1R sequences created a high affinity receptor for both insulin and IGF-1. The binding characteristics of all receptor chimeras reflected the potential of both ligands to regulate the receptor tyrosine kinase activity in intact cells. Our chimeric receptor data, in conjunction with IR amino-terminal domain point mutants, strongly suggest major contributions of structural determinants in both amino- and carboxyl-terminal IR alpha-subunit regions for the formation of the insulin-binding pocket, whereas, surprisingly, the residues defining IGF-1 binding are present predominantly in the cysteine-rich domain of the IGF-1R.

Highlights

Cysteine-rich domain alone by IGF-1 receptor (IGF-1R) sequences created a high affinity receptor for both insulin and IGF1
Amino acid sequence conservation and theoretical secondary structure analysis have predicted the existence of two independently folded subdomains on either side of the insulin receptor (IR) and IGF-1R cysteine-rich domains (Bajaj et al, 1987; Schaefer et al, 1990). Based on this structural assumption, we constructed three hybrid receptors, using newly introduced restriction sites at or near the borders of these structuralsubdomains in both IR and IGF-1R cDNAs, without altering the amino acid sequence coding information at the fusion site (Fig. 1).The chimeric receptor constructscontained IGF-1R sequences encoding amino acids 1-131 (IR/IGF-1R Cl), 132-315 (IR/ IGF-1R C2), or 1-315 (IR/IGF-1R ClZ),within an IR precursor backbone that was under the control of the cytomegalovirus major immediate early promoter (Gorman et al, 1989)
Variation in the intensities of the IR a-subunit bandsmay beattributed to differences in the methionine content of wild-type and chimeric sequences (IR, 9 Met; IR/IGF-1R C1, 5 Met; IR/IGF-1R C12, 9 Met; IR/IGF-1R C2, 13Met),as well as differences in biosynthetic efficiency and processing

Summary

Preparation of Constructs

0.6pgof plasmid DNA containing the entire cells were washed carefully with phosphate-buffered saline (PBS), cDNAs of IR or IGF-1R were used as a template and subjected to 40 scraped into 0.6 ml of lysis buffer The supernatant was diluted 1:2 with HNTG (20 mM Hepes, pH 7.5, 150 mM NaC1, 0.1% Triton X-100, 10% glycerol) and incubated with protein A-Sepharose and anti-IR or anti-IGF-1R antibodies for 3 h in the cold. The protein A-Sepharose-antibody duced in the expression plasmid pCMV-HIR (Eaton et al, 1986) complex was washed three times with HNTG, whereas the containing the entire IR cDNA to exchange the corresponding wildtype IR sequences. Obtained were analyzed by the method of Scatchard (Scatchard1, 949)

Receptor Phosphorylation in Intact Cells

RESULTS

No binding

DISCUSSION

Ligand binding characteristics of ZR point mutants

KO Ins