Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor β-subunit

Jonathan M. Backer, C. Ronald Kahn, Deborah A. Cahill, Axel Ullrich, Morris F. White

Research output: Contribution to journalArticle

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Abstract

The juxtamembrane region of the insulin receptor (IR) β-subunit contains an unphosphorylated tyrosyl residue (Tyr960) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M. F., Livingston, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A., and Kahn, C. R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXT is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IRF960, in which Tyr960 has been substituted with phenylalanine, and IRΔ960, in which 12 amino acids (Ala954-Asp965), including the putative consensus sequence NPXT, were deleted. Although the in vivo autophosphorylation of IRF960 and IRΔ960 was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IRF960 cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IRΔ960 cells was 20-30% of wild-type. However, insulin internalization in the CHO/IRΔ960 cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IRA1018). The degradation of insulin was equally impaired in CHO/IRΔ960 and CHO/IRA1018 cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXT may play a general role in directing the internalization of cell surface receptors.

Original languageEnglish (US)
Pages (from-to)16450-16454
Number of pages5
JournalJournal of Biological Chemistry
Volume265
Issue number27
StatePublished - Sep 25 1990
Externally publishedYes

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Insulin Receptor
Cricetulus
Amino Acid Sequence
Ovary
Insulin
Amino Acids
Cells
Phosphorylation
LDL Receptors
Cell Surface Receptors
Substrates
Cell membranes
Phenylalanine
Consensus Sequence
Endocytosis
Tyrosine
Phosphotransferases
Cell Membrane
Degradation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor β-subunit. / Backer, Jonathan M.; Kahn, C. Ronald; Cahill, Deborah A.; Ullrich, Axel; White, Morris F.

In: Journal of Biological Chemistry, Vol. 265, No. 27, 25.09.1990, p. 16450-16454.

Research output: Contribution to journalArticle

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abstract = "The juxtamembrane region of the insulin receptor (IR) β-subunit contains an unphosphorylated tyrosyl residue (Tyr960) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M. F., Livingston, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A., and Kahn, C. R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXT is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IRF960, in which Tyr960 has been substituted with phenylalanine, and IRΔ960, in which 12 amino acids (Ala954-Asp965), including the putative consensus sequence NPXT, were deleted. Although the in vivo autophosphorylation of IRF960 and IRΔ960 was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IRF960 cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IRΔ960 cells was 20-30{\%} of wild-type. However, insulin internalization in the CHO/IRΔ960 cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IRA1018). The degradation of insulin was equally impaired in CHO/IRΔ960 and CHO/IRA1018 cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXT may play a general role in directing the internalization of cell surface receptors.",
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N2 - The juxtamembrane region of the insulin receptor (IR) β-subunit contains an unphosphorylated tyrosyl residue (Tyr960) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M. F., Livingston, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A., and Kahn, C. R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXT is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IRF960, in which Tyr960 has been substituted with phenylalanine, and IRΔ960, in which 12 amino acids (Ala954-Asp965), including the putative consensus sequence NPXT, were deleted. Although the in vivo autophosphorylation of IRF960 and IRΔ960 was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IRF960 cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IRΔ960 cells was 20-30% of wild-type. However, insulin internalization in the CHO/IRΔ960 cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IRA1018). The degradation of insulin was equally impaired in CHO/IRΔ960 and CHO/IRA1018 cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXT may play a general role in directing the internalization of cell surface receptors.

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