Involvement of dileucine motifs in the internalization and degradation of the insulin receptor.
Dileucine motifs have been shown to be involved in trans Golgi sorting, lysosomal targeting, and internalization of a number of proteins. The insulin receptor contains four dileucine pairs in its cytoplasmic domain. To determine if these insulin receptor sequences can serve as lysosomal sorting sequences, chimeric molecules expressing the Tac antigen fused to each isolated insulin receptor motif were constructed. A chimera containing the juxtamembrane dileucine motif (EKITLL), which closely resembles the sequences originally identified in the gamma- and delta-chains of the T cell receptor (DKQTLL and EVQALL), was shown to sort to lysosomes by immunofluorescence microscopy, as did a chimera expressing the dileucine motif (GGKGLL) found in the tyrosine kinase domain. Chimeras expressing either a second tyrosine kinase domain sequence (HVVRLL) or the carboxyl-terminal sequence (EIVNLL) localized to both lysosomes and the plasma membrane. In contrast, chimeras expressing two other potential sorting signals found in the cytoplasmic tail of the insulin receptor (NARDII and KNGRIL) localized predominantly to the plasma membrane. Exclusively cell surface staining was also seen for a chimera expressing a mutant motif (EKITAA), where the leucine residues were mutated to alanines. When the alanine pair was introduced into the juxtamembrane domain of the intact insulin receptor and the mutant receptor expressed in NIH-3T3 cells, we found that the mutation did not impair insulin binding or receptor tyrosine kinase activity. However, the Ala-Ala mutant internalized insulin 5-fold slower than the wild-type receptor. Taken together, these findings suggest that the dileucine motif found in the juxtamembrane domain of the insulin receptor is involved in receptor internalization and that other insulin receptor sequences may mask the potential lysosomal targeting signals in the intact molecule.
Haft
C R
CR
Diabetes Branch, NIDDKD, National Institutes of Health, Bethesda, Maryland 20892-1770.
Klausner
R D
RD
Taylor
S I
SI
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D007930:Leucine; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D051381:Rats; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D015375:Receptors, Interleukin-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D011972:Receptor, Insulin; D007930:Leucine