Functional properties of two naturally occurring isoforms of the human insulin receptor in chinese hamster ovary cells

Yoshihiko Yamaguchi, Jeffrey S. Flier, Atsushi Yokota, Heike Benecke, Jonathan M. Backer, David E. Moller

Research output: Contribution to journalArticle

124 Citations (Scopus)

Abstract

We and others have previously demonstrated that the human insulin receptor messenger RNA (mRNA) is alternatively spliced such that the 36-nucleotide sequence encoded by exon 11 of the receptor gene is included (Ex11+) or excluded (Ex11-). Although both Ex11- and Ex11+ insulin receptors which differ in the presence or absence of 12 amino acids in the carboxy-terminal α-subunit have been demonstrated to function as insulin receptors when independently overexpressed and studied, the possibility that subtle functional differences between the two isoforms exist has received limited attention. Given that the relative abundance of the two mRNA transcripts is highly regulated in a tissue-specific manner, differences in the functional properties of the two receptor variants might contribute to tissue-specific differences in insulin receptor function and insulin action that are known to exist. To address this hypothesis, we transfected cDNAs encoding the two receptor isoforms into Chinese hamster ovary (CHO) cells and prepared several stable CHO cell lines expressing high numbers of Ex11-or Ex11+ receptors. Several functional properties of the expressed insulin receptors were compared in parallel with the following results: 1) steady state binding of insulin to cells expressing the Ex11- isoform exhibited higher (≈2-fold) affinity; 2) using two different methods, a significant difference in receptor-mediated insulin internalization was noted such that the Ex11- isoform displayed a higher (≈25% increase in the rate constant, Ke) rate of internalization; 3) partially purified Ex11- and Ex11+ receptors displayed similar maximal and insulin dose-response characteristics for receptor autophosphorylation and kinase activity toward an exogenous substrate (poly Glu-Tyr, 4:1); 4) the ability of expressed Ex11- and Ex11+ receptors to couple to a metabolic (glucose incorporation into glycogen) and mitogenic (thymidine incorporation into DNA) action of insulin was not discernibly different. Thus, when expressed in CHO cells, the two alternatively spliced isoforms of the insulin receptor have subtle differences in insulin binding affinity and the kinetics of ligand-stimulated internalization that would be expected to influence the pattern of insulin receptor expression and signaling in vivo in a tissue-specific manner.

Original languageEnglish (US)
Pages (from-to)2058-2066
Number of pages9
JournalEndocrinology
Volume129
Issue number4
StatePublished - Oct 1991
Externally publishedYes

Fingerprint

Insulin Receptor
Cricetulus
Ovary
Protein Isoforms
Insulin
Messenger RNA
human INSR protein
Glycogen
Thymidine
Exons
Phosphotransferases
Complementary DNA
Ligands
Amino Acids
Glucose
Cell Line
DNA
Genes

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Yamaguchi, Y., Flier, J. S., Yokota, A., Benecke, H., Backer, J. M., & Moller, D. E. (1991). Functional properties of two naturally occurring isoforms of the human insulin receptor in chinese hamster ovary cells. Endocrinology, 129(4), 2058-2066.

Functional properties of two naturally occurring isoforms of the human insulin receptor in chinese hamster ovary cells. / Yamaguchi, Yoshihiko; Flier, Jeffrey S.; Yokota, Atsushi; Benecke, Heike; Backer, Jonathan M.; Moller, David E.

In: Endocrinology, Vol. 129, No. 4, 10.1991, p. 2058-2066.

Research output: Contribution to journalArticle

Yamaguchi, Y, Flier, JS, Yokota, A, Benecke, H, Backer, JM & Moller, DE 1991, 'Functional properties of two naturally occurring isoforms of the human insulin receptor in chinese hamster ovary cells', Endocrinology, vol. 129, no. 4, pp. 2058-2066.
Yamaguchi, Yoshihiko ; Flier, Jeffrey S. ; Yokota, Atsushi ; Benecke, Heike ; Backer, Jonathan M. ; Moller, David E. / Functional properties of two naturally occurring isoforms of the human insulin receptor in chinese hamster ovary cells. In: Endocrinology. 1991 ; Vol. 129, No. 4. pp. 2058-2066.
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abstract = "We and others have previously demonstrated that the human insulin receptor messenger RNA (mRNA) is alternatively spliced such that the 36-nucleotide sequence encoded by exon 11 of the receptor gene is included (Ex11+) or excluded (Ex11-). Although both Ex11- and Ex11+ insulin receptors which differ in the presence or absence of 12 amino acids in the carboxy-terminal α-subunit have been demonstrated to function as insulin receptors when independently overexpressed and studied, the possibility that subtle functional differences between the two isoforms exist has received limited attention. Given that the relative abundance of the two mRNA transcripts is highly regulated in a tissue-specific manner, differences in the functional properties of the two receptor variants might contribute to tissue-specific differences in insulin receptor function and insulin action that are known to exist. To address this hypothesis, we transfected cDNAs encoding the two receptor isoforms into Chinese hamster ovary (CHO) cells and prepared several stable CHO cell lines expressing high numbers of Ex11-or Ex11+ receptors. Several functional properties of the expressed insulin receptors were compared in parallel with the following results: 1) steady state binding of insulin to cells expressing the Ex11- isoform exhibited higher (≈2-fold) affinity; 2) using two different methods, a significant difference in receptor-mediated insulin internalization was noted such that the Ex11- isoform displayed a higher (≈25{\%} increase in the rate constant, Ke) rate of internalization; 3) partially purified Ex11- and Ex11+ receptors displayed similar maximal and insulin dose-response characteristics for receptor autophosphorylation and kinase activity toward an exogenous substrate (poly Glu-Tyr, 4:1); 4) the ability of expressed Ex11- and Ex11+ receptors to couple to a metabolic (glucose incorporation into glycogen) and mitogenic (thymidine incorporation into DNA) action of insulin was not discernibly different. Thus, when expressed in CHO cells, the two alternatively spliced isoforms of the insulin receptor have subtle differences in insulin binding affinity and the kinetics of ligand-stimulated internalization that would be expected to influence the pattern of insulin receptor expression and signaling in vivo in a tissue-specific manner.",
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