An irregularity in the transmembrane domain helix correlates with the rate of insulin receptor internalization.
Internalization of insulin and its receptor via receptor-mediated endocytosis is an important step in insulin-induced signal transduction. To investigate the structural determinants underlying the enhanced internalization rate observed for the insulin receptor transmembrane mutant Gly933-Pro934-->Ala-Ala (GP-->AA), we have designed and chemically synthesized two peptides, IR(TM)-GP and IR-(TM)-AA, corresponding respectively to the N-terminal portion of the wild-type and the mutant insulin receptor TM segment containing these sites. Conformational studies by circular dichroism (CD) spectroscopy on these two peptides in their monomeric states revealed that peptide IR(TM)-GP forms an irregular helix in the membrane-mimetic environments of sodium dodecyl sulfate (SDS) micelles with a possible "kink" in the helix imposed by its Gly-Pro sequence, while peptide IR(TM)-AA assumes largely classical alpha-helical structure under corresponding conditions. The helical pattern of peptide IR(TM)-AA was maintained at elevated temperatures, while the shape of the CD curve for peptide IR(TM)-GP was found to alter as a function of temperature. At higher concentrations, both peptides formed high molecular weight aggregates in SDS micelles, as demonstrated by SDS-PAGE gels, but peptide IR(TM)-AA was shown to aggregate more readily and more extensively than peptide IR(TM)-GP. Fluorescent dye-leakage experiments indicated that peptide IR(TM)-GP produces an enhanced disruption of the membrane bilayer in phosphatidylglycerol vesicles vs that induced by IR(TM)-AA.(ABSTRACT TRUNCATED AT 250 WORDS)
Li
S C
SC
Division of Biochemistry Research, Hospital for Sick Children, Toronto, Ontario, Canada.
Deber
C M
CM
Shoelson
S E
SE
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry*; D002942:Circular Dichroism; D004705:Endocytosis; D007700:Kinetics; D046911:Macromolecular Substances; D008823:Micelles; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D017433:Protein Structure, Secondary; D011972:Receptor, Insulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature
D046911:Macromolecular Substances; D008823:Micelles; D010446:Peptide Fragments; D011972:Receptor, Insulin