Cr. Haft et al., INVOLVEMENT OF DILEUCINE MOTIFS IN THE INTERNALIZATION AND DEGRADATION OF THE INSULIN-RECEPTOR, The Journal of biological chemistry, 269(42), 1994, pp. 26286-26294
Dileucine motifs have been shown to be involved in trans Golgi sorting
, lysosomal targeting, and internalization of a number of proteins. Th
e insulin receptor contains four dileucine pairs in its cytoplasmic do
main. To determine if these insulin receptor sequences can serve as ly
sosomal sorting sequences, chimeric molecules expressing the Tac antig
en fused to each isolated insulin receptor motif were constructed. A c
himera con taining the juxtamembrane dileucine motif (EKITLL), which c
losely 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 d
omain. Chimeras expressing either a second tyrosine kinase domain sequ
ence (HVVRLL) or the carboxyl-terminal sequence (EIVNLL) localized to
both lysosomes and the plasma membrane. In contrast, chimeras expressi
ng two other potential sorting signals found in the cytoplasmic tail o
f the insulin receptor (NARDII and KNGRIL) localized predominantly to
the plasma membrane. Exclusively cell surface staining was also seen f
or a chimera expressing a mutant motif (EKITAA), where the leucine res
idues were mutated to alanines. When the alanine pair was introduced i
nto the juxtamembrane domain of the intact insulin receptor and the mu
tant receptor expressed in NM-3T3 cells, we found that the mutation di
d not impair insulin binding or receptor tyrosine kinase activity. How
ever, the Ala-Ala mutant internalized insulin 5-fold slower than the w
ild-type receptor. Taken together, these findings suggest that the dil
eucine motif found in the juxtamembrane domain of the insulin receptor
is involved in receptor internalization and that other insulin recept
or sequences may mask the potential lysosomal targeting signals in the
intact molecule.