JBC Transcription and Nuclear Factor Monoclonals

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Originally published In Press as doi:10.1074/jbc.M204036200 on September 5, 2002

J. Biol. Chem., Vol. 277, Issue 46, 43631-43637, November 15, 2002
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Role of Two Dileucine-like Motifs in Insulin Receptor Anchoring to Microvilli*

Sue ShackletonDagger§, Isabelle HamerDagger||, Michelangelo Foti, Nicole Zumwald, Christine Maeder, and Jean-Louis Carpentier

From the Department of Morphology, Faculty of Medicine, University of Geneva, CH-1211 Geneva 4, Switzerland

Received for publication, April 25, 2002, and in revised form, July 22, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the absence of ligand, the insulin receptor is maintained on microvilli on the cell surface. A dileucine motif (LL986-987) 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 (II1006-1007) 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 (LL986-987 and II1006-1007) play a crucial role.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell surface receptors are internalized by a process known as endocytosis (reviewed in Ref. 1). Endocytosed receptors fall into two groups: class I and class II (2). 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),1 the epidermal growth factor (EGF) receptor, TCR/CD3, and G-protein-coupled receptors, are only internalized after binding their respective ligands (2-6).

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). A similar preferential initial localization has also been reported for adhesion molecules (8-10) and other receptors including the EGF receptor, the glucagon receptor, and CD4 (3, 11, 12). Microvilli are enriched in cytoskeletal elements (13) and are characterized by the presence, in freeze-etched replicas, of a high concentration of "intramembrane particles" of larger size than those in nonvillous regions (7). In the case of integrins, the cytoskeletal proteins talin and alpha -actinin have been proposed to be responsible for their anchoring to microvilli (14). This anchoring accounts for the poor internalization of such receptors in the absence of their ligand (12, 15).

After ligand binding, HIR leaves microvilli and redistributes in the plane of the plasma membrane (7, 16-18). On reaching the nonvillous domain of the cell surface, HIR segregates into clathrin-coated pits via two types of motifs present within the juxtamembrane region of the cytoplasmic tail. The first type are the tyrosine-based motifs, NPEY and GPLY, which are exposed after ligand binding and receptor activation (19-21) and bind to the µ2 subunit of the adaptor protein, AP2, present in clathrin-coated pits (22). The second motif is a dileucine sequence (LL986-987), which does not require activation of the receptor to enable association with clathrin-coated pits and may also associate with AP2 molecules (23-25). Clathrin-coated pits pinch off from the cell surface, resulting in endocytosis of the associated receptors (26).

Whereas the processes of association with clathrin-coated pits and endocytosis of HIR are now well understood at the molecular level, little is known about the interactions maintaining the unoccupied receptor on microvilli and the mechanism for its release upon receptor activation.

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, although not sufficient, for anchoring to microvilli and that other sequences located downstream of position 1000 are also necessary (24).

The aim of the present study was to characterize the exact motif(s) required for anchoring of HIR to microvilli. On the basis of experiments involving the use of chimeric and truncated HIR, we conclude that exon 17 of the molecule (encoding amino acids 966 to 1047) contains all the sequences necessary for anchoring HIR to microvilli. Moreover, in addition to the dileucine motif LL986-987 described previously, the amino acid sequence extending between position 1000 and 1012 plays a key role in this anchoring, and a diisoleucine motif (II1006-1007) present within these 12 amino acids is of particular importance. These two dileucine-like motifs (LL986-987 and II1006-1007), which in the three-dimensional structure of HIR are in close vicinity, may participate in or form together a domain involved in the binding of HIR to cytoskeletal elements.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of CR1-HIR Chimeric Receptors and HIR Truncation Mutants-- Chimeric receptors were constructed in two steps. First, cDNAs encoding the whole cytoplasmic domain or fragments corresponding to exon 16 or 17 of the HIR were amplified by PCR using Pfu DNA polymerase (Stratagene, La Jolla, CA), the HIR cDNA as template, and oligonucleotide primers to incorporate AflII and BglII restriction sites at the 5' and 3' ends, respectively. The resulting PCR products were digested with the appropriate restriction endonucleases and cloned into pCR1-wt, a plasmid described previously (27, 28), so as to introduce the HIR fragments in-frame with the sequence coding for the extracellular and transmembrane domains of CR1. To introduce the alanine substitution of the dileucine at position 986-987 in the HIR cDNA (AA1), the plasmid pBPV-HIR AA1 (23) was used as template in the above-mentioned procedures. All constructs were verified by sequencing.

HIR truncation mutants were created by PCR amplification using a forward primer (HIR-AvrII, 5'-CCGAGGACCCTAGGCCATCTCG-3') to incorporate the unique AvrII restriction enzyme site and reverse primers to incorporate a stop codon and SalI restriction enzyme site immediately 3' to the last codon of each truncation to be made. After restriction enzyme digestion, each fragment was used to replace the full-length HIR AvrII-SalI fragment in pSP64 (Promega). The entire cDNA was then excised and cloned into the expression vector pCIneo (Promega).

HIR-AA2, -AA3, and -AA4 mutants were generated by PCR-based mutagenesis involving two steps of PCR. Two PCR fragments were produced, overlapping at the site of the mutation. The first product was amplified using HIR-AvrII, described above, and a reverse primer to introduce the mutation (AA2, 5'-GCAATGCCAGGGACGCCGCCAAGGGTGAGGCA-3'; AA3, 5'-GGACACCACTCCCGCGGCGCGCACCACGTG-3'; AA4, 5'-CTGGCCCTTGGACGCCGCTCCCAGGAGGCG-3'; mutated bases are in bold). The second product was amplified using a forward primer that was the reverse of the mutant primers described above and a reverse primer to incorporate a BstXI site (HIR-BstXI, 5'-AGGATTATTCTCAGCCTCTCC-3'). The two products were combined, and a second PCR was performed upon them using the outer primers HIR-AvrII and HIR-BstXI. The resulting product was digested with AvrII and BstXI and used to replace the wild-type sequence of pSP64 containing the EcoRI-SalI fragment of HIR. This EcoRI-SalI fragment was then excised and cloned into pCIneo-HIR.

Cell Culture and Transfection-- CHO cells were cultured in Ham's F-12 medium supplemented with 10% fetal calf serum and incubated at 37 °C, 5% CO2. Cells were seeded onto 10-cm Petri dishes and transfected by calcium phosphate precipitation with either a mixture of 1 µg of pRSV-Neo and 5 µg of pCR1-HIR chimeric constructs or 10 µg of pCI-HIR constructs. After selection for resistance to the antibiotic G418 (Invitrogen), stable transfectants were isolated by fluorescence-activated cell-sorting analysis, as described previously (24). Cell surface expression was verified by measuring the binding of a monoclonal antibody (3D9) against CR1 (29) labeled with [125I]iodine (Amersham Biosciences) using IODO-GEN (Pierce).

Internalization Assays-- To study the internalization of chimeric receptors in CHO cells, we used the whole IgG or Fab fragments of 3D9 labeled with [125I]iodine. Fab fragments were produced by papain digestion and purified on an immobilized protein A column (Pierce).

Confluent CHO cells, grown in 35-mm Petri dishes, were incubated at 4 °C for 2 h in 1 ml of CHO buffer (100 mM HEPES, pH 7.4, 120 mM NaCl, 1.2 mM MgSO4, 1 mM EDTA, 15 mM sodium acetate, 10 mM glucose, and 1% bovine serum albumin) containing a tracer amount of 125I-labeled antibody (IgG or Fab fragments). Cells were then warmed at 37 °C for various periods of time. At each time point studied, the incubation medium was removed, and the cells were washed three times with ice-cold phosphate-buffered saline containing 1% bovine serum albumin. Cells were then subjected to three 5-min washes with 3 ml of the same buffer (but at pH 1.5) to remove the ligands present at the cell surface. Finally, cells were lysed with 1 ml of 1 N NaOH for 15 min. Acid washes and solubilized cells were counted in a gamma counter. Results were expressed as the percentage of radioactivity remaining associated with the cells at the end of the acid washes versus total radioactivity (radioactivity recovered in the acid washes and in solubilized cells). Values obtained after 2 h at 4 °C were subtracted from values calculated at all times points at 37 °C.

Quantitative Electron Microscope Autoradiography-- Cell monolayers were incubated at 4 °C for 2 h in the presence of 1 ml of CHO buffer containing a tracer amount of 125I-labeled 3D9 mAb (IgG). Cells were then washed three times with ice-cold phosphate-buffered saline containing 1% bovine serum albumin, fixed, dehydrated, processed for electron microscope (EM) autoradiography, and quantified as described previously (30). For each clone studied, two separate experiments were performed, three Epon blocks were prepared, and three sections were cut from each block. For each condition, 2000 grains within a distance of 250 nm from the plasma membrane were analyzed. Grains were considered to be associated with microvilli if their center was <250 nm from these surface domains (18).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Exon 17 of HIR Contains the Sequences Required for Association with Microvilli-- Previous studies have suggested that, in addition to the dileucine motif at position 986-987, (LL986-987), sequences C-terminal to amino acid 1000 are required for maintaining the unoccupied HIR on microvilli (24).

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 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). These chimeric receptors were then stably expressed into CHO cells.


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  		Fig. 1.   Schematic representation of HIR mutants. Amino acid positions corresponding to full-length HIR are shown above each construct. Internalization motifs, dileucine motifs, and alanine substitutions are shown in bold underneath.

Anchoring to microvilli prevents the HIR from gaining access to endocytic gates that form only on planar membranes. Internalization of the HIR via clathrin-coated pits is thus directly correlated with the propensity of the receptor to leave microvilli (32). We measured the ability of the chimera to internalize by an acid wash procedure after tagging of cell surface-expressed chimeric molecules with 125I-labeled anti-CR1 antibody (3D9). When exon 16 (which includes the two tyrosine-based internalization motifs) was present as cytoplasmic domain (CR1-HIRex16), a rapid and sustained internalization of the chimeric molecule was observed, reaching maximal values of 32%. By contrast, in the presence of exon 17 alone (CR1-HIRex17), internalization of the chimeric molecule was significantly reduced (Fig. 2A), despite presence of the dileucine motif LL986-987 that plays a key role in association with clathrin-coated pits (23, 24). Similar results were obtained using either the whole anti-CR1 antibody or its Fab fragments (Fig. 2, A and B); therefore, all further experiments were carried out using only the whole antibody.


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  		Fig. 2.   Internalization of CR1-HIR chimeric receptors in CHO cells. Confluent cells were incubated for 2 h at 4 °C in the presence of a tracer amount of 125I-labeled anti-CR1 antibody 3D9 (the whole IgG (A) or the Fab fragments (B)) and then warmed at 37 °C to allow internalization of ligand-receptor complexes. Internalization of 125I-labeled 3D9 was quantified at each time point as described under "Experimental Procedures." Results are expressed as the percentage of radioactivity associated with cell lysates versus total radioactivity and represent the mean ± S.E. of four independent experiments. triangle , CR1-HIRex16; black-triangle, CR1-HIRex17.

To determine whether the internalization defect of CR1-HIRex17 is related to a reduced access to the internalization gates due to the fact that this chimeric receptor is maintained on microvilli, we analyzed the ability of the chimeric molecules to associate with microvilli. Quantitative EM autoradiographic analysis revealed no preferential association of 125I-labeled anti-CR1 with microvilli in cells expressing CR1-HIRex16 (Fig. 3). In contrast, in cells expressing CR1-HIRex17, more than 70% of the autoradiographic grains were associated with microvilli. Because ~50% of the cell surface is occupied by microvilli, these observations indicate that CR1-HIRex17 exhibits preferential association with microvilli and suggest that the sequence motif(s) required for association of HIR with microvilli is (are) located within exon 17. 


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  		Fig. 3.   Association of CR1-HIR chimeric molecules with microvilli. A, electron microscopic view of microvilli at the surface of CHO cells. Cells were incubated in the presence of 125I-insulin for 2 h at 4 °C. The radioactive ligand was revelead by autoradiography and appears as black autoradiographic grains. B, cell monolayers were incubated at 4 °C for 2 h in the presence of 1 ml of CHO buffer containing a tracer amount of 125I-labeled 3D9 (whole IgG) and then processed for EM autoradiography as described under "Experimental Procedures." Results are expressed as the percentage of autoradiographic grains associated with microvilli versus the total number of grains and represent the mean ± S.E. of four independent experiments totaling >400 autoradiographic grains/condition.

Because exon 17 includes a dileucine motif (LL986-987) that plays a role at different stages of HIR internalization, the role of this dileucine motif was further investigated in the present experimental system. The substitution of these two leucines for alanines in a chimeric receptor carrying exon 17 of HIR as cytoplasmic domain (CR1-HIRex17-AA1) resulted in only a slight increase in the rate of internalization of the receptor when compared with the internalization of receptors carrying the wild-type exon 17 sequence (Fig. 4A). By contrast, this mutation caused a drastic reduction in the level of association of the receptor with microvilli (Fig. 4B), confirming the requirement for LL986-987 in the association of HIR with microvilli. The small increase in the level of internalization of CR1-HIRex17-AA1 highlights once more the importance of LL986-987 for the segregation of the receptor into clathrin-coated pits.


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  		Fig. 4.   CR1-HIRex17 dileucine mutant internalization and association with microvilli. A, confluent cells were incubated for 2 h at 4 °C in the presence of a tracer amount of 125I-labeled 3D9 (whole IgG) and then warmed at 37 °C to allow internalization of ligand-receptor complexes. Internalization of 125I-labeled 3D9 was quantified at each time point as described under "Experimental Procedures." Results are the mean ± S.E. of four independent experiments. black-triangle, CR1-HIRex17; triangle , CR1-HIRex17-AA2. B, cell monolayers were incubated at 4 °C for 2 h in the presence of 1 ml of CHO buffer containing a tracer amount of 125I-labeled 3D9 and then processed for EM autoradiography as described under "Experimental Procedures." Results are expressed as the percentage of autoradiographic grains associated with microvilli versus the total number of grains and represent the mean ± S.E. of four independent experiments totaling >400 autoradiographic grains/condition.

An Amino Acid Sequence Contained within Amino Acids 1000-1012 Is Involved in the Anchoring of HIR to Microvilli-- To further delineate the sequence motif contained within exon 17 that is required for the anchoring of HIR to microvilli and to test the physiological relevance of the experiments carried out with chimeric molecules, three truncations mutants of the wild-type HIR molecule were produced terminating at positions 1000 (HIR-Delta 1000), 1012 (HIR-Delta 1012), and 1047 (HIR-Delta 1047), respectively (Fig. 1). These constructs were stably expressed in CHO cells.

The level of internalization of these truncated receptors was measured in the absence of insulin using 125I-labeled anti-HIR antibody 83-14 as a tag. This antibody has been shown not to activate HIR (31). As reported previously, the constitutive internalization of HIR-Delta 1000 was rapid and of similar magnitude to the internalization recorded for the wild-type receptor in the presence of insulin (Ref. 24; Fig. 5). In contrast to HIR-Delta 1000, HIR-Delta 1012 and HIR-Delta 1047 exhibited a low level of internalization, almost reduced to those observed for the constitutive internalization of wild-type HIRs (Fig. 5).


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  		Fig. 5.   Constitutive internalization of HIR truncation mutants. Confluent cells were incubated for 2 h at 4 °C in the presence of a tracer amount of 125I-labeled anti-HIR antibody (83-14 IgG) and then warmed at 37 °C to allow internalization of ligand-receptor complexes. Internalization of 125I-labeled 83-14 was quantified at each time point as described under "Experimental Procedures." Results are expressed as the percentage of radioactivity associated with cell lysates versus total radioactivity and represent the mean ± S.E. of four independent experiments. , HIR; black-triangle, HIR-Delta 1000; open circle , HIR-Delta 1012; , HIR-Delta 1047.

In terms of association with microvilli, an inverse correlation with the internalization data was observed: receptors with a high degree of internalization (HIR-Delta 1000) displayed a poor association with microvilli, whereas those with a low degree of internalization (HIR-Delta 1012 and HIR-Delta 1047) exhibited a preferential association with microvilli (Fig. 6).


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  		Fig. 6.   Association of HIR truncation mutants with microvilli. Cell monolayers were incubated at 4 °C for 2 h in the presence of 1 ml of CHO buffer containing a tracer amount of 125I-labeled 83-14 and then processed for EM autoradiography as described under "Experimental Procedures." Results are expressed as the percentage of autoradiographic grains associated with microvilli versus the total number of grains and represent the mean ± S.E. of four independent experiments totaling >400 autoradiographic grains/condition. The dashed line represents a random cell surface distribution.

These results suggest that the sequence(s) required for anchoring of HIR to microvilli is (are) located between amino acids 1000 and 1012.

Role of the Diisoleucine Motif II1006-1007 in Anchoring HIR to Microvilli-- The 12-amino acid stretch extending between positions 1000 and 1012 contains a diisoleucine motif at position 1006-1007. Analogous motifs have been shown to be involved in various steps of receptor trafficking, including association with microvilli and segregation into clathrin-coated pits, and in intracellular sorting (23-25, 33-49). To determine whether the diisoleucine motif plays a role in anchoring to microvilli, alanine substitution of the two isoleucines (HIR-AA2) was performed. As shown in Fig. 7, the constitutive internalization of this mutated receptor was significantly increased. Similar results were obtained when the receptor was tagged with 125I-insulin (Fig. 7).


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  		Fig. 7.   Constitutive internalization of HIR-AA2 mutant. Confluent cells were incubated for 2 h at 4 °C in the presence of a tracer amount of 125I-labeled anti-HIR antibody (83-14 IgG) or 125I-insulin and then warmed at 37 °C to allow internalization of ligand-receptor complexes. Internalization of 125I-ligand was quantified at each time point as described under "Experimental Procedures." Results are expressed as the percentage of radioactivity associated with cell lysates versus total radioactivity and represent the mean ± S.E. of four independent experiments. black-square, HIR + 83-14; black-triangle, HIR-AA2 + 83-14; , HIR + insulin; triangle , HIR-AA2 + insulin.

The association of HIR-AA2 with microvilli in the absence of insulin was measured after a 2-h incubation at 4 °C with 125I-labeled anti-HIR antibody 83-14. The level of association was significantly lower than that observed for wild-type HIR and similar to values obtained for HIR truncated at position 1000 (37-38%) (Figs. 6 and 8), indicating that in addition to dileucine LL986-987, II1006-1007 is required for binding of HIR to microvilli.


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  		Fig. 8.   Association of HIR dileucine motif mutants with microvilli. Cell monolayers were incubated at 4 °C for 2 h in the presence of 1 ml of CHO buffer containing a tracer amount of 125I-labeled 83-14 and then processed for EM autoradiography as described under "Experimental Procedures." Results are expressed as the percentage of autoradiographic grains associated with microvilli versus the total number and represent the mean ± S.E. of four independent experiments totaling >400 autoradiographic grains/condition. The dashed line represents a random cell surface distribution.

As a control, other dileucine-type motifs present downstream of position 1007 were analyzed for their ability to participate in anchoring to microvilli. As illustrated in Fig. 8, neither the two leucines at position 1050-1051 (HIR-AA3) nor the two valines at position 1053-1054 (HIR-AA4) had any influence on the anchoring of HIR in its unoccupied form with microvilli.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we provide evidence demonstrating that specific amino acid sequences within the cytoplasmic juxtamembrane domain of HIR mediate association of the inactivated receptors with plasma membrane protrusions called microvilli. Observation of the trafficking and surface distribution of a panel of chimeric and truncated HIR molecules allowed us to determine that exon 17 of HIR (amino acids 966 to 1047) contains all the determinants mediating anchoring to microvilli. Generation of chimeric and truncated HIR mutants established that in addition to a previously characterized dileucine motif, LL986-987, the integrity of another dileucine-like motif (II1006-1007) located 20 amino acids downstream of LL986-987 is crucial for association of HIR with villous structures. The two motifs are necessary for HIR anchoring to microvilli, but each one is not sufficient by itself to fulfill the function. The two motifs have no additive function and, due to their close proximity, may participate in or form an anchoring site.

An increasing amount of data is accumulating to indicate that villous protrusions of the plasma membrane concentrate transmembrane proteins involved in processes such as adhesion and signaling (7-10, 14, 16, 18, 50-54). We have demonstrated previously that HIR preferentially segregates on microvillous domains in IM9 lymphocytes and hepatocytes, and similar results have been reported for the EGF receptor (3, 7, 11, 17, 18). However, the functional relevance of this localization and the mechanisms mediating the sorting of proteins to these plasma membrane domains are still unclear. In the case of HIR, such localization may favor the binding of soluble circulating ligands and therefore might represent a particular cell surface domain involved in receptor signaling. Recent data support this hypothesis because mutated HIR with a defect in ligand-induced internalization (where the receptors remain on microvilli and do not redistribute in response to insulin binding) transduces most insulin metabolic signals at an even higher level than normally internalized receptor (55). With regard to the mechanisms mediating the sorting of HIR to villous domains, our previous studies (24) have shown that a dileucine motif, LL986-987, located in exon 17, within the juxtamembrane region of HIR cytoplasmic tail, is required for efficient anchoring of the inactive receptor to microvilli. However, this sequence is not sufficient by itself because a truncation mutant, HIR-Delta 1000, which retains the dileucine motif LL986-987, does not reside preferentially on microvilli (24). Our analysis of sequences downstream of amino acid 1000 now suggests the involvement of another dileucine-like motif, II1006-1007, in HIR anchoring to microvilli. The sequence encompassing these two dileucine-like motifs in HIR cytoplasmic tail appears to be necessary and sufficient to mediate association with microvilli and thus represents, to our knowledge, the first molecular determinant identified as a sorting motif targeting transmembrane molecules to cytoskeleton-enriched cell surface protrusions, i.e. microvilli. Eventually, independent mutations of each of these dileucine-like motifs totally abrogate HIR preferential association with microvilli, indicating that each motif is necessary and that the two motifs have no additive function.

The crystal structure of HIR tyrosine kinase domain has been solved in both the inactive and activated states (56, 57) and includes the distal region of the juxtamembrane domain. This domain forms a beta -sheet composed of five parallel beta -strands. The two dileucine-like motifs we found crucial for HIR anchoring to microvilli, LL986-987 and II1006-1007, are located within beta -sheets 1 and 2, respectively, and, interestingly, lie in close proximity to one another on the same face of the protein (Fig. 9). Whether these dileucine-like motifs participate in or together form the binding motif or whether they are stabilizing a protein-protein interaction is not clear. In either case, these leucines/isoleucines side chains are critical because binding to microvilli is abolished by alanine substitution of these residues.


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  		Fig. 9.   Crystallographic structure of the HIR tyrosine kinase domain (57). The dileucine or diisoleucine motifs mutated in this study are highlighted.

It is now well established that dileucine-like motifs represent key sorting determinants involved in protein-protein interactions during protein trafficking processes. More precisely, dileucine-like motifs have been shown to mediate coupling between adaptor complexes, such as AP1, AP2, AP3, and GGA2, and proteins sorted from the plasma membrane (CD4, CD3, beta 2-adrenergic receptor, CXCR4, HIRs, Nef, Fc receptors, and interleukin 2 and interleukin 13 receptors (23, 24, 33-39, 41, 45-47)), from endosomes (MPR46, EGF receptor, Nef (42-45)), and from the trans-Golgi network (CI-MPR, env, and Nef (39, 40, 45, 48, 49)). Although the direct demonstration of dileucine motifs as a binding motif has been provided only in the case of AP1 (25), preservation of the integrity of these motifs appears to be necessary for specific membrane-bound proteins to ensure their correct sorting along the secretory and/or the endocytic pathway. In the case of HIR, the dileucine motif LL986-987 has been demonstrated to be important for receptor association with clathrin-coated pits components and subsequent targeting to lysosomes (23, 24). By contrast, mutation of the diisoleucine motif, II1006-1007, does not inhibit HIR internalization but prevents association of the receptor with microvilli. These observations suggest that this motif is not involved in interaction with adaptor complexes but, together with the dileucine motif LL986-987, displays affinities for cytoskeletal proteins responsible for the association of HIR with microvilli. A dileucine motif has similarly been previously shown to promote interactions during the viral encapsidation process with two proteins, Vpr and Vpx, totally unrelated to adaptor complexes directing protein trafficking (58).

In the present study, we provide evidence suggesting that dileucine-like motifs are involved in protein-protein interactions between transmembrane signaling receptors and cytoskeletal elements. Indeed, the localization of HIR on microvilli is likely to reflect its tight coupling to the cytoskeleton. Microvilli are actin-filled cell extensions enriched in a panel of actin-associated cytoskeletal proteins (13, 59). In this regard, cytoskeletal proteins such as talin and alpha -actinin have been proposed to participate in the villous localization of a series of adhesion molecules (8, 10, 14, 53). Other candidates are the members of the ERM (ezrin-moesin-radixin) family (60). Unfortunately, except for the EGF receptor, which has been shown to bind actin (61), little information is available on the potential cytoskeletal partner(s) responsible for signaling receptor localization on microvilli, and even less information is available regarding the determinants leading to such localization. Potential cytoskeletal protein candidates interacting with HIR and the role of the diisoleucine motif, II1006-1007, in this context are currently under investigation.

Interestingly, release of HIR from microvilli requires activation of the intrinsic tyrosine kinase that leads to autophosphorylation of the receptor on multiple tyrosines and the subsequent internalization of the receptor through the clathrin-coated pits endocytic gates (17). The present study, together with our previous results, indicates that both tyrosine motifs and the LL986-987 dileucine motif, but not the II1006-1007 diisoleucine motif, act as sorting signals for endocytosis. Thus, HIR, like other membrane proteins including the M6PR (62), contains more than one sorting motif within its cytoplasmic tail. Some of these motifs appear to be functionally redundant. For example NPEY/GPLY tyrosine motifs and LL986-987 dileucine motif are required for internalization of the receptors, whereas the LL986-987 dileucine motif and the II1006-1007 diisoleucine motif are required for anchoring to microvilli. Conversely, the same motif can be important for different functions, as is the case for the LL986-987 dileucine motif, which is required for both internalization and anchoring to microvilli. It is also noted that the tyrosine-based motifs play a role not only in receptor trafficking but also in the binding of major docking proteins involved in receptor signaling i.e. IRS-1, IRS-2, and Shc (63, 64). The multiplicity of these sorting motifs may reflect a need for higher avidity of binding or may represent a combinatorial method to allow specificity for a larger variety of target sites. For example, different motifs may act synergistically or be exposed/masked in different circumstances such as before or after receptor activation. Alternatively, slight conformational changes induced by receptor activation may lead to competition of one motif for different partners, thus explaining the dual role of the LL986-987 dileucine motif in both internalization and anchoring of the receptor to microvilli.

In conclusion, we have demonstrated that the sequence motif required for anchoring of the unoccupied HIR is contained within exon 17, encoding amino acids 966-1047 in the cytoplasmic tail of the receptor. Within this sequence, two dileucine-like motifs, LL986-987 and II1006-1007, are crucial to allow anchoring of HIR to microvilli. These findings strongly suggest that dileucine-like motifs represent molecular determinants mediating protein-protein interactions between transmembrane signaling receptors and cytoskeletal elements.

    ACKNOWLEDGEMENTS

We thank G. Porcheron and C. Giroud for technical assistance.

    FOOTNOTES

* This work was supported by Grants 31.53686.98 and 31.65392.01 from the Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Both authors contributed equally to this work.

§ Present address: Dept. of Genetics, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom.

|| Present address: Dept. Chimie Physiologique, Faculté Universitaire Notre-Dame de la Paix, 61 rue de Bruxelle, Namur B-5000 Belgium.

To whom correspondence should be addressed: Dept. of Morphology, Faculty of Medicine, CMU, Rue Michel Servet 1, CH-1211 Geneva 4, Switzerland. Tel.: 41-22-7025201; Fax: 41-22-7025220; E-mail: Jean- Louis.Carpentier@medecine.unige.ch.

Published, JBC Papers in Press, September 5, 2002, DOI 10.1074/jbc.M204036200

    ABBREVIATIONS

The abbreviations used are: HIR, human insulin receptor; EGF, epidermal growth factor; CHO, Chinese hamster ovary; AP, adaptor protein; EM, electron microscope; CR1, complement receptor 1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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