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Optimizing transmembrane domain helicity accelerates insulin receptor internalization and lateral mobility

E Goncalves1, K Yamada, H S Thatte

  • 1Joslin Diabetes Center, Boston, MA 02215.

Proceedings of the National Academy of Sciences of the United States of America
|June 15, 1993
PubMed
Summary

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Altering the insulin receptor's transmembrane domain helix structure speeds up its internalization and lateral mobility. This suggests the natural kinked structure regulates receptor trafficking for optimal insulin signaling.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Transmembrane (TM) domains of integral membrane proteins are typically helical.
  • A Gly-Pro sequence in the insulin receptor's TM domain may disrupt helicity.
  • Previous studies show Ala substitutions enhance TM peptide helicity in lipid environments.

Purpose of the Study:

  • To investigate the role of the Gly-Pro helix-breaking motif in the insulin receptor's TM domain.
  • To assess how altered helicity impacts insulin receptor function and trafficking.
  • To understand the physiological significance of the native Gly-Pro sequence.

Main Methods:

  • Mutagenesis of Gly933 and Pro934 to Alanine (G-->A, P-->A, GP-->AA) in the human insulin receptor.
  • Stable expression of wild-type and mutant receptors in CHO cells.

Related Experiment Videos

  • Analysis of receptor assembly, processing, insulin binding, autophosphorylation, and kinase activity.
  • Measurement of insulin-stimulated receptor internalization, degradation, and lateral mobility using fluorescence photobleaching recovery.

    • Main Results:

    • Mutated and wild-type receptors showed similar assembly, processing, and insulin binding affinities.
    • Receptor autophosphorylation and kinase activity were unaffected by mutations.
    • The GP-->AA mutant exhibited a 2-fold accelerated insulin-stimulated internalization compared to wild-type.
    • Lateral mobility of the GP-->AA mutant receptor was increased 2- to 3-fold.
    • Insulin degradation, linked to endocytosis, was elevated for the GP-->AA mutant.

    Conclusions:

    • Altered helicity of the insulin receptor's TM domain directly influences its lateral mobility and endocytosis rates.
    • A kinked TM domain (Gly-Pro) in the wild-type receptor appears to retard lateral mobility and receptor internalization.
    • The conserved Gly-Pro sequence suggests a physiological advantage for controlled, submaximal receptor internalization rates.