Abstract
In the absence of ligand, the insulin receptor is maintained on microvilli on the cell surface. A dileucine motif (LL(986-987)) is necessary but not sufficient for this anchoring, which also required the presence of additional sequence(s) downstream of position 1000. The aim of the present study was to identify this (these) additional sequence(s). First, exons 16 or 17 were fused to the extracellular and transmembrane domains of complement receptor 1 and stably expressed in Chinese hamster ovary cells. Results obtained indicate that exon 17 is sufficient for anchoring to microvilli. Second, analysis of insulin receptor mutants truncated within exon 17 demonstrated that whereas receptors truncated at position 1000 showed no preferential association with microvilli, receptors truncated at position 1012 displayed a level of association identical to that of the full-length insulin receptor. Third, mutation of a diisoleucine motif (II(1006-1007)) present within this 12-amino acid stretch abrogated the preferential association of the receptor with microvilli. These results indicate that the domain required for association of insulin receptor with microvilli is contained within the region encoded by exon 17 and that, within this sequence, two dileucine-like motifs (LL(986-987) and II(1006-1007)) play a crucial role.
Highlights
Cell surface receptors are internalized by a process known as endocytosis
To delimit the cytoplasmic domain involved in this anchoring to a specific exon and evaluate the role of this potential domain in the absence of other human insulin receptor (HIR) domains, we produced chimeric receptors (Fig. 1) by fusing individual exons encoding the juxtamembrane region of the cytoplasmic domain of HIR to the exoplasmic and transmembrane domains of CR1, a receptor previously shown to lack any preferential association with specific domains of the cell surface [31]
Generation of chimeric and truncated HIR mutants established that in addition to a previously characterized dileucine motif, LL986–987, the integrity of another dileucinelike motif (II1006–1007) located 20 amino acids downstream of LL986–987 is crucial for association of HIR with villous structures
Summary
Cell surface receptors are internalized by a process known as endocytosis (reviewed in Ref. 1). Class I receptors, including transferrin and low density lipoprotein receptors, are constitutively internalized even in the absence of their cargo molecules, whereas class II receptors, including signaling receptors such as the human insulin receptor (HIR), the epidermal growth factor (EGF) receptor, TCR/CD3, and G-protein-coupled receptors, are only internalized after binding their respective ligands [2,3,4,5,6] It has been known for more than 20 years that, in the absence of ligand, HIR preferentially associates with thin digitations on the cell surface known as microvilli [7]. We and others have previously provided evidence that the dileucine motif (LL986–987), in addition to being involved in binding to clathrin-coated pits, is required, not sufficient, for anchoring to microvilli and that other sequences located downstream of position 1000 are necessary [24]
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