Analysis of the Juxtamembrane Dileucine Motif in the Insulin Receptor1

• Published in: Endocrinology (Philadelphia) Vol. 139; no. 4; pp. 1618 - 1629
• Main Authors: Haft, Carol Renfrew, de La Luz Sierra, Maria, Hamer, Isabelle, Carpentier, Jean-Louis, Taylor, Simeon I
• Format: Journal Article
• Language: English
• Published: Endocrine Society 01-04-1998
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/endo.139.4.5939

Dileucine-containing motifs are involved in trans-Golgi
sorting, lysosomal targeting, and internalization. Previously, we have
shown that the dileucine motif (EKITLL, residues 982–987) in the
juxtamembrane region of the insulin receptor is involved in receptor
internalization. Substitution of alanine residues for
Leu986 and Leu987 led to a 3- to 5-fold
decrease in the ability of the receptors to mediate insulin uptake. In
the current study, we show that mutation of the same motif to
Met986Ser987, the sequence found in the
homologous position in the type I insulin-like growth factor receptor,
did not affect insulin uptake. Therefore, we inquired whether the
sequence EKITMS as an isolated motif could mediate the targeting of a
reporter molecule to endosomes and then lysosomes, as was shown
previously with the EKITLL motif of the normal receptor. Chimeric
molecules containing Tac antigen fused to different hexapeptide
sequences showed distinct patterns of subcellular localization by
immunofluorescence microscopy. Tac-EKITLL and Tac-EKITAA were found
predominantly in lysosomes and the plasma membrane, respectively. In
contrast, Tac-EKITMS was found at the plasma membrane, in the
trans-Golgi network, and in endosomes, but only small
amounts were found in lysosomes. Thus, the dileucine motif (EKITLL)
plays an important role in directing endocytosis of the intact insulin
receptor and in mediating efficient endocytosis and lysosomal targeting
as an isolated motif. Substitution of AA for LL inhibits endocytosis
and lysosomal targeting in both systems. In contrast, substitution of
MS for LL permits rapid endocytosis in the intact receptor, but
mediates modest endocytosis and very little targeting to lysosomes as
an isolated motif. Our observations support the idea that sorting
signals are recognized at multiple steps in the cell, and that specific
amino acid substitutions may differentially affect each of these
sorting steps.