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Endocrinology Vol. 139, No. 4 1618-1629
Copyright © 1998 by The Endocrine Society

ARTICLES

Analysis of the Juxtamembrane Dileucine Motif in the Insulin Receptor1

Carol Renfrew Haft, Maria de La Luz Sierra, Isabelle Hamer, Jean-Louis Carpentier and Simeon I. Taylor

Diabetes Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (C.R.H., M.d.L.L.S., S.I.T.), Bethesda, Maryland 20892; and the Department of Morphology, University of Geneva (I.H., J.-L.C.), Geneva, Switzerland

Address all correspondence and requests for reprints to: Simeon I. Taylor, M.D., Ph.D., National Institutes of Health, Building 10, Room 9S-213, 10 Center Drive, MSC-1829, Bethesda, Maryland 20892-1829.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WHEN INSULIN binds its receptor, this triggers a redistribution of ligand-receptor complexes from microvilli to the nonvillous region of the cell surface. This redistribution requires ligand-induced autophosphorylation of the ß-subunit of the insulin receptor and kinase activation (1, 2). The second step in endocytosis, interaction of the ligand-receptor complexes with clathrin-coated pits, involves other structures in the intracellular domain (3, 4). Subsequently, insulin-receptor complexes are internalized into the cell, and both insulin and its receptors are either recycled back to the cell surface or targeted to lysosomes for degradation. In many cell types, insulin stimulates receptor endocytosis and accelerates the rate of receptor degradation. This process, called down-regulation, results in a decrease in the number of cell surface receptors and may be important for attenuating signals initiated by insulin binding (5, 6, 7).

Over the past few years, progress has been made toward understanding the signals involved in internalization and down-regulation of transmembrane receptors. Efficient internalization of transmembrane receptor molecules requires one or more signal sequences in the cytoplasmic domain of the protein (8, 9). To date, two different types of internalization signals have been described: tyrosine-based motifs and dileucine-based motifs (10, 11, 12). These sorting motifs are often clustered together in the juxtamembrane domain of the receptor molecule in close apposition to the plasma membrane (13, 14). For the insulin receptor, the sequences GPLY (residues 950–953)2 and NPEY (residues 957–960) have been reported by several laboratories to be required for rapid endocytosis (15, 16). These tyrosine-based motifs are thought to form type I turns that then interact with clathrin-associated adaptins (17). In addition, extensive site-directed mutagenesis of residues including and surrounding the tyrosine-based motifs suggests that additional information required for rapid endocytosis is found in the juxtamembrane domain of the receptor (18). We have recently identified a six-amino acid dileucine-containing sequence (EKITLL, residues 982–987) that is also involved in mediating efficient internalization of the receptor (19, 20).

The type I insulin-like growth factor I (IGF-I) receptor is another member of the tyrosine kinase family of receptors that includes the insulin receptor and the insulin receptor-related receptor (21, 22). Like the insulin receptor, the IGF-I receptor undergoes ligand-stimulated internalization, and the integrity of the juxtamembrane domain of the IGF-I receptor is required for rapid endocytosis (23, 24). However, it has been suggested that the IGF-I receptor is internalized more slowly than the insulin receptor (25). Interestingly, the dileucine motif found in the juxtamembrane domain of the insulin receptor is not conserved in the IGF-I receptor. In place of the EKITLL motif found in the juxtamembrane domain in the insulin receptor, the IGF-I receptor contains the hexapeptide sequence EKITMS. The substitution of MetSer for LeuLeu is notable because there are so few amino acid differences in the juxtamembrane domains of these two growth factor receptors (69% identical; 84% similar over 55 amino acids; Fig. 1Go) (26).



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  		Figure 1. Amino acid sequence alignment of the juxtamembrane domains of the insulin and IGF-I receptors. A best-fit alignment is shown for the juxtamembrane domain sequences of the insulin receptor (IR; amino acids 940–994) and the IGF-I receptor (IGF-IR; amino acids 928–979). Two previously identified tyrosine-based internalization motifs are shown in boldface (15, 16) as well as the dileucine-containing motif of the IR and the homologous amino acid sequence in the IGF-IR. Vertical bars are shown between identical amino acids, double dots mark conservative amino acid substitutions with best-fit matrix scores greater than 0.5, and single dots mark conservative amino acid substitutions with positive best-fit scores less than 0.5 (53, 54).

 
In the present work, we demonstrated that substitution of MetSer for Leu986Leu987 in the full-length insulin receptor did not impair endocytosis of the mutant receptor. This raised the question of whether the MetSer sequence was capable of functioning as a dileucine motif even though it lacked leucine residues. We addressed this question using the original assay for a dileucine motif, i.e. the ability to target a Tac chimera to lysosomes (12). We constructed chimeric molecules consisting of Tac fused to either the IGF-I receptor juxtamembrane domain sequence, EKITMS, or the insulin receptor juxtamembrane domain sequence, EKITLL. In addition, we constructed a chimera with the EKITAA sequence that lacks the ability to target the chimera to lysosomes. These chimeras were constructed to isolate the hexapeptide sequences from other targeting motifs in the cytoplasmic domain of the full-length receptor (e.g. tyrosine-based motifs and other dileucine motifs). Interestingly, the EKITMS chimera did not possess the full activity of the EKITLL sequence to target the chimeric molecule efficiently to lysosomes. A substantial fraction of the EKITMS chimeras was detected in endosomes and the trans-Golgi network (TGN), whereas the EKITAA chimera was found almost exclusively at the plasma membrane.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and medium
NIH-3T3 cells transfected with wild-type (WT) and mutant insulin receptors were maintained as previously described (27). HeLa cells were maintained in DMEM (high glucose, 4.5 mM glucose; Biofluids, Gaithersburg, MD), supplemented with 10% (vol/vol) FBS, 4 mM glutamine, 100 U/ml penicillin, and streptomycin at 37 C in 5% CO2.

Construction of juxtamembrane dileucine pair mutants of the human insulin receptor complementary DNA (cDNA)
A plasmid containing a PstI/EcoRI fragment of the human insulin receptor cDNA (nucleotides 2744–4037) (28, 29) was digested with BanII and XhoI to remove a 41-nucleotide fragment. A double stranded insert was formed from the following oligonucleotide primers to introduce the MetSer for LeuLeu substitutions (mutations denoted by underliningnucleotide sequence): 5'-TCGAGAGAAGATCACCATGTCTCGAG-AGCTGGGGCAGGGCT (nucleotides 3070–3111 of the sense strand) and 5'-CTGCCCAGCTCTCGAGACATGGTGATCTTCTC (nucleotides 3074–3108 of the antisense strand). The double stranded insert was then substituted for the WT BanII/XhoI fragment. The construction was verified by determination of its nucleotide sequence. Introduction of an AlaAla pair for the same juxtamembrane leucine residues has been described previously (19).

A plasmid containing the full-length insulin receptor cDNA was then digested with SspI and the combination of SspI and BstXI. The subclones were also cut with SspI and BstXI, and the 441-nucleotide fragments containing the mutant sequences were isolated. After ligation of each mutant fragment into the WT insulin receptor cDNA, the complete insulin receptor cDNAs encoding either the AlaAla pair or the MetSer pair were ligated into a bovine papilloma virus-based expression vector (Pharmacia, Piscataway, NJ) in which insulin receptor cDNA expression is driven by the murine metallothionein promoter (27, 30). The constructions were verified by restriction digestion and by determination of the nucleotide sequences.

Expression of receptors by transfection of cDNAs into NIH-3T3 cells
NIH-3T3 cells (~2 x 106 cells) were stably transfected with a bovine papilloma virus-based expression vector encoding WT human insulin receptor and the various mutant receptors as described previously (27). Expression of insulin receptors was assayed by measuring [125I]insulin binding and/or immunoblotting (31). We have chosen to use this expression vector in NIH-3T3 cells because this method has consistently provided the highest level of stable expression among the methods we have investigated in our laboratory.

Construction of chimeras
Double stranded inserts containing the insulin receptor juxtamembrane dileucine motif or the corresponding mutant motifs followed by a stop codon were ligated into a modified version of Tac-DKQTLL(CD3{gamma}) as previously described (19). The sequences of the inserts are available upon request. The constructions were verified by restriction digestion and determination of their nucleotide sequence.

Transient transfection of chimeras into HeLa cells
Two to 3 h before the start of the transfection, subconfluent cultures of HeLa cells were placed in 9 ml fresh growth medium (see above). For each 10-cm plate, 10–15 µg DNA were mixed with 64 µl 2 M CaCl2 and brought to a final volume of 0.5 ml with water. The DNA/calcium phosphate mixture was then slowly added to 0.5 ml 2 x HEPES-buffered saline (280 mM NaCl, 1.5 mM Na2HPO4·7H2O, and 50 mM HEPES, pH 7.05). After 20 min at room temperature, 1 ml of the DNA complex was added dropwise to each plate. After 14–18 h, the cells were washed twice with PBS and then placed in standard growth medium for 40–48 h before experiments were performed. We have chosen to express the chimeras in HeLa cells because this facilitates high quality morphological studies and allows for comparisons with previous published work using Tac chimeras (12).

Immunofluorescence microscopy
HeLa cells (~1 x 104/chamber) were grown overnight on two-chambered glass slides (Nunc, Naperville, IL). The cells were then fixed for 15 min in 2% (vol/vol) formaldehyde in PBS at 25 C, and immunofluorescent staining was carried out using an anti-Tac monoclonal antibody as previously described (19). For colocalization studies, the following polyclonal antibodies were used: anti-ßCOP (coatomer protein) antibody (1:500), provided by Dr. J. Lippincott-Schwartz (NIH, Bethesda, MD); antiendoplasmic reticulum (anti-ER) antibodies (1:100), provided by Dr. D. Louvard (Pasteur Institute, Paris, France); antihuman transferrin antibody (1:500; Sigma Chemical Co., St. Louis, MO); or antilysosome-associated membrane protein I (anti-LAMPI) antibody (1:100), provided by Dr. M. Fukuda (Cancer Research Center, La Jolla, CA). After the appropriate washes, cells were incubated for 1 h with fluorescein isothiocyanate (FITC) swine antirabbit IgG (1:50 dilution; Dako, Carpenteria, CA). Samples were then mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and examined under a Zeiss Axiophot microscope (Zeiss, New York, NY) using x40–100 oil lenses. For transferrin uptake studies, cells were first incubated for 10 min at 37 C with iron-loaded transferrin [10 µg/ml in DMEM and 0.2% (wt/vol) BSA; Sigma Chemical Co.), washed three times, and then fixed (32). For antibody uptake studies, cells were incubated for 30 min at 37 C with anti-Tac antibody [2 µg/ml in DMEM and 0.5% (wt/vol) BSA], provided by Dr. Thomas Waldmann. The cells were then washed and fixed (33). The above immunofluorescence protocol was then followed. Photomicrographs were taken using identical conditions for exposure, development, and printing.

Measurement of [125I]insulin internalization by NIH-3T3 cells expressing WT or mutant insulin receptors
To determine the internalization rate constant for WT and mutant receptors, transfected cells were grown to confluence in six-well plates. The cells were washed once with PBS and then incubated at 37 C for 2–10 min in 1 ml internalization buffer [RPMI 1640 medium, 25 mM HEPES, and 0.1% (wt/vol) BSA] containing 0.1 ng/ml [125I]insulin (20,000-50,000 dpm/ml). At 2-min intervals, the cells were placed on ice, washed twice with ice-cold PBS to remove unbound insulin, and washed twice with PBS, pH 3.0, containing 0.1% (wt/vol) BSA to remove any surface-bound radioactivity. The residual cell-associated radioactivity (internalized insulin) was then quantified after dissolving the acid-washed cells in 1 ml 1 N NaOH (19, 31). The internalization rate constant for each receptor was determined during a 2- to 10-min incubation period as previously described (34, 35). The data reflect the mean ± the SE for three experiments, each performed in triplicate. The rate constant for the WT receptor was compared with those of the dileucine mutants using unpaired Student’s t test. P < 0.05 was considered significant.

Insulin receptor and endogenous substrate phosphorylation
Transfected NIH-3T3 cells were grown to confluence in 10-cm dishes. After 12–18 h of serum starvation, cells were incubated in the absence or presence of insulin (0–10-6 M) for 1 min at 37 C. The incubation medium was then removed, and the cell monolayers were rapidly frozen with liquid nitrogen. The cells were solubilized, and proteins containing phosphotyrosine were then detected by immunoblotting or immunoprecipitation with an {alpha}-subunit-specific antibody (B7/B10), followed by immunoblotting as described previously (36, 37). Insulin receptors were also detected by immunoblotting with an {alpha}-subunit-specific antibody (Upstate Biotechnology, Lake Placid, NY).

Quantitative electron microscopic autoradiography
After incubation at 37 C in the presence of [125I]insulin (10-11 M), cells were fixed, dehydrated, and quantified as previously described (1). For each time point studied, three Epon blocks were prepared and sectioned. About 450–600 grains were analyzed from all morphologically intact cells. Grains within 250 nm of the plasma membrane were considered associated with the cell surface. Grains inside the cytoplasm and more than 250 nm from the plasma membrane were considered internalized. Grains present at the plasma membrane fell into the following classes: microvilli, clathrin-coated pits, nonvillous nonclathrin-coated pit segments, and unclassifiable. Grains were considered associated with microvilli or clathrin-coated pits if the center was less than 250 nm from the surface domain.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of insulin receptors mutated in the dileucine sequence of their juxtamembrane domain
To investigate the importance of the dileucine pair at positions 986 and 987 in the juxtamembrane domain of the insulin receptor, we mutated the LeuLeu residues to MetSer and AlaAla, and expressed the various recombinant receptors in NIH-3T3 cells. Metabolic labeling studies confirmed that the mutant receptors were synthesized, processed, and transported to the plasma membrane normally (data not shown) (19). We used immunoblotting to estimate the number of receptors expressed in each cell line. As judged by an antibody directed against the {alpha}-subunit, the cells expressing the mutant receptors (Met986Ser987 and Ala986Ala987) contained approximately equal numbers of receptors. In contrast, the cells expressing the WT receptors (Leu986Leu987) contained 2- to 3-fold more receptors. Furthermore, all three receptors appeared to bind insulin with similar affinities. When we plotted [125I]insulin binding as a function of the free insulin concentration, half-maximal inhibition of [125I]insulin binding was obtained at free insulin concentrations of 0.9, 1.1, and 1.0 nM for the WT, Ala986Ala987, and Met986Ser987-mutant receptors, respectively (data not shown). Moreover, in the presence of tracer concentrations of [125I]insulin (50 pg/ml), the ratios of bound/free insulin were 0.54 ± 0.01 (Ala986Ala987), 0.70 ± 0.01 (Met986Ser987), and 1.38 ± 0.14 (WT) (19). If one assumes that all three receptors bind insulin with the same affinity constant, then these binding data imply that the ratios of WT receptors to mutant receptors are 2.6 and 2.0 for the Ala986Ala987 and Met986Ser987 mutant receptors, respectively. These ratios are entirely consistent with the results of the immunoblotting studies, thus supporting the hypothesis that the affinity of insulin binding (Ke, as defined in Ref.38) is normal in the two mutant receptors.

Phosphorylation of insulin receptors and endogenous substrates in intact cells
To assess the in situ autophosphorylation of the MetSer mutant insulin receptor, intact cells were incubated in the presence or absence of insulin. Phosphorylated insulin receptors were detected by immunoblotting with antiphosphotyrosine antibody. Both the MetSer mutant and the WT receptor molecules underwent rapid phosphorylation after the addition of 100 nM insulin (Fig. 2AGo). When the amount of ß-subunit phosphorylation was normalized for the total receptor number present in each sample, as determined by Western blot analyses, the MetSer mutant showed no significant change in the amount of ß-subunit phosphorylation compared with the WT receptor. Furthermore, the MetSer mutant receptors exhibited normal tyrosine kinase activity toward endogenous substrates. Two prominent phosphorylated bands were visualized in cells expressing both WT and mutant receptors after insulin addition (Fig. 2BGo). The 95-kDa band corresponds to the ß-subunit of the insulin receptor, and the 185-kDa band corresponds to pp185/insulin receptor substrate-1 and/or -2 (39). Neither ß-subunit nor insulin receptor substrate-1 and/or -2 phosphorylation was detectable in the parental cell line (data not shown) (40).



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  		Figure 2. Phosphorylation of insulin receptors and endogenous substrates stimulated by insulin in intact cells. NIH-3T3 cells expressing either WT or mutant (Met986Ser987) insulin receptors were incubated in the absence or presence of insulin for 1 min at 37 C. A, A portion of the cell lysate was immunoprecipitated with an antiinsulin receptor antibody directed against the {alpha}-subunit, followed by Western blotting with an antiphosphotyrosine antibody. A portion of lysate was also used to determine the total amount of receptor present in each sample by Western blotting with antibody directed against the {alpha}-subunit of the receptor. B, Western blots of whole cell lysates were probed with an antiphosphotyrosine antibody. The positions of insulin receptor substrate-1 and the insulin receptor precursor (prec) {alpha}- and ß-subunits are indicated. The total amount of cell surface receptor was estimated by quantifying the {alpha}-subunit. The amount of phosphorylated receptor was determined by quantifying the tyrosine-phosphorylated ß-subunit as described in Materials and Methods. Autoradiograms representative of two experiments are shown. Note that we adjusted the loading of the gel so as to load approximately equal numbers of WT and MetSer mutant receptors: 40 µl extract from cells expressing MetSer mutant receptor, and 20 µl extract from cells expressing WT receptor.

 
Internalization of WT and mutant receptors
To investigate whether the juxtamembrane domain mutation affected events on the cell surface, we analyzed quantitatively the surface localization and redistribution of the various receptors. As shown in Fig. 3AGo, both the WT and MetSer mutant receptors associated normally and preferentially with microvilli after a 2-h incubation at 4 C in the presence of [125I]insulin. At 37 C, both receptors redistributed toward the nonvillous domain of the cell surface (Fig. 3AGo) and segregated similarly in coated pits (Fig. 3BGo).



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  		Figure 3. Surface redistribution of [125I]insulin in NIH-3T3 cells expressing WT or mutant insulin receptors. The results presented are the mean ± SE of the analysis of sections from each of seven different Epon blocks (n = 7). Three blocks were obtained from one experiment, and the remaining four blocks were obtained from two separate experiments (two blocks from each experiment). Cells were incubated at 37 C in the presence of [125I]insulin for the indicated periods and then processed for EM autoradiography. A shows the number of grains associated with the microvilli (expressed as a percentage of the total number of grains) as a function of time. B shows the number of grains associated with clathrin-coated pits (expressed as a function of the number of grains associated with the nonvillous surface). Because the association with clathrin-coated pits did not change over time, we pooled the values obtained at 0, 5, 15, and 30 min.

 
Next we examined the delivery of mutant and WT receptors into endosomes. NIH-3T3 cells expressing WT or mutant receptors were incubated with [125I]insulin for 0–10 min at 37 C. The cells were then chilled to 4 C and washed with acidic PBS (pH 3) to determine the amount of acid-dissociable radioactivity (i.e. surface-bound insulin) and the amount of residual cell-associated radioactivity (i.e. internalized insulin). Figure 4Go shows the ratio of intracellular to cell surface [125I]insulin as a function of time for the mutant and WT receptor molecules. Over a 10-min time frame, when receptor recycling and degradation were negligible, MetSer mutant receptors internalized similar amounts of ligand as the WT receptor. In contrast, a mutant in which the dileucine pair was changed to a pair of alanine residues internalized approximately 80% less ligand than the WT receptor. Using the data shown in Fig. 4Go, we then determined the rate constant for insulin receptor internalization for each cell line from the slope of a plot of internalized insulin vs. the integral of the surface-bound ligand measured at each time point (34, 35) The slope was calculated by linear regression (41) using experimental data; the regression coefficients were greater than 0.95. Cells expressing WT insulin receptors internalized 0.01 nM insulin rapidly, with a rate constant of ke = 0.159 ± 0.068 min-1. The rate constant for MetSer mutant receptors was slightly, but not significantly, lower, with a value of ke = 0.133 ± 0.078 min-1. In contrast, the AlaAla mutant receptors internalized insulin with a rate 5-fold slower (ke = 0.031 ± 0.012 min-1) than that estimated for WT receptors. Similar results were obtained with three independent clones of cells expressing the mutant receptors.



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  		Figure 4. Insulin internalization by NIH-3T3 cells expressing WT or mutant insulin receptors. NIH-3T3 cells expressing either WT or mutant insulin receptors were exposed to [125I]insulin (0.01 nM) for 2–10 min at 37 C. At the indicated times after insulin addition, surface-bound and internalized [125I]insulin were determined as described in Materials and Methods. The ratio of the intracellular insulin to surface-bound insulin is plotted as a function of time for NIH-3T3 cells expressing either WT (solid squares), AlaAla mutant (Ala986Ala987; solid triangles), or MetSer mutant insulin receptors (Met986Ser987; open triangles). The results are the mean ± SE of three experiments, each performed in triplicate.

 
Pulse-chase labeling studies showed that there were no significant differences in the turnover of the WT and mutant receptor molecules after extended insulin treatment (data not shown) (19).

Localization of Tac chimeras
In the context of the full-length insulin receptor molecule, the EKITMS sequence permits normal receptor endocytosis and degradation. There are at least two possible explanations for this observation. It is possible that the isolated EKITMS sequence is a functional equivalent of the dileucine motif (EKITLL) as a sorting signal. Alternatively, there may be other sequences in the insulin receptor that are capable of substituting for the EKITLL targeting sequence. To address this question, we separated the hexapeptide sequences from other potential internalization motifs present in the intact receptors by constructing chimeric proteins. The chimeras consisted of the human Tac antigen (interleukin-2R, {alpha}-chain) (42) fused to the various hexapeptide motifs. After transient transfection of HeLa cells with the Tac chimeras, the subcellular localization of the chimeric molecules was evaluated by indirect immunofluorescence microscopy. As shown previously in rat basophilic leukemia cells (19), the chimeras containing the WT insulin receptor sequence (Tac-EKITLL) were associated with intracellular vesicular structures (Fig. 5AGo), whereas molecules expressing the mutated sequence (Tac-EKITAA) were localized to the plasma membrane (Fig. 5BGo). In contrast, cells transiently transfected with chimeras containing the IGF-I receptor sequence (Tac-EKITMS) showed a heterogeneous pattern. Staining was detected both at the plasma membrane and intracellularly (Fig. 5CGo). When the immunostaining was carried out in the absence of detergent, no specific signal was observed for Tac-EKITLL, and the intracellular signal for Tac-EKITMS was eliminated. However, the cell surface staining observed for the Tac-EKITAA and Tac-EKITMS was unchanged (data not shown).



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  		Figure 5. Immunofluorescence microscopy of HeLa cells expressing Tac-insulin receptor chimeras. HeLa cells were transiently transfected with Tac-EKITLL (A), Tac-EKITAA (B), and Tac-EKITMS (C). Forty-eight hours after transfection, the cells were fixed, permeabilized, and stained for immunofluorescence with anti-Tac antibody and rhodamine-conjugated second antibody.

 
We next carried out a series of colocalization studies to define the subcellular localization of the various Tac chimeras. Tac-EKITLL showed extensive colocalization with LAMPI (43), an endogenous HeLa cell protein that is located in late endosomes and lysosomes (Fig. 6Go, A and B). In contrast, Tac-EKITMS showed only slight colocalization with LAMPI (Fig. 6Go, C and D). Tac-EKITAA was not detectable in LAMPI-containing structures (data not shown). When cells were incubated for 10 min at 37 C with diferric transferrin (10 µg/ml) before fixation to load early endosomes with tracer, a different colocalization pattern was seen. Tac-EKITMS showed extensive colocalization with transferrin (Fig. 7Go, A and B), whereas Tac-EKITAA (Fig. 7Go, C and D) and Tac-EKITLL (Fig. 7Go, D and E) showed only small amounts of colocalization with early endosomes. Compared with the distribution of an ER marker in double immunofluorescence labeling experiments, the punctate intracellular stainings for Tac-EKITMS and Tac-EKITLL were distinct from the diffuse reticular structures labeled by an ER-specific antibody (Fig. 8Go, A and B, data not shown). However, double labeling for Tac and the coatomer protein, ßCOP, a marker for the intermediate compartment and TGN (44, 45, 46), showed partial colocalization with both Tac-EKITLL and Tac-EKITMS (Fig. 8Go, C–F).



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  		Figure 6. Colocalization of Tac chimeras with endogenous LAMPI. HeLa cells were transiently transfected with Tac-EKITLL (A and B) or Tac-EKITMS (C and D). Forty-eight hours after transfection, the cells were fixed, permeabilized, and processed for immunofluorescence. Endogenous LAMPI was stained with polyclonal anti-LAMPI antibody (left), and the Tac chimeras were stained with monoclonal anti-Tac antibody (right). FITC-conjugated swine antirabbit and rhodamine-conjugated donkey antimouse second antibodies were used.

 


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  		Figure 7. Colocalization of Tac chimeras with transferrin-loaded endosomes. HeLa cells were transiently transfected with Tac-EKITMS (A and B), Tac-EKITAA (C and D), and Tac-EKITLL (E and F). Forty-eight hours after transfection, the cells were incubated for 10 min at 37 C with 10 µg/ml diferric transferrin to label early endocytic compartments. The cells were then washed, fixed, permeabilized, and processed for immunofluorescence. Transferrin was stained with polyclonal antitransferrin antibody (left), and the Tac chimeras were stained with monoclonal anti-Tac antibody (right). FITC-conjugated swine antirabbit and rhodamine-labeled donkey antimouse second antibodies were used. Arrows point to representative collections of vesicles where transferrin and the various Tac chimeras colocalize. Arrowheads point to vesicular structures that contain Tac-EKITLL, but lack transferrin.

 


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  		Figure 8. Colocalization of Tac chimeras with endogenous ER antigens and ßCOP. HeLa cells were transiently transfected with Tac-EKITMS (A–D) or Tac-EKITLL (E and F). Forty-eight hours after transfection, the cells were washed, fixed, permeabilized, and processed for immunofluorescence. Tac chimeras (B, D, and F) were stained with anti-Tac monoclonal antibody followed by rhodamine antimouse IgG (right). Endogenous HeLa cell ßCOP (C and E) was stained with an anti-ßCOP polyclonal antibody and endoplasmic reticulum (ER) antigens were stained with anti-ER (A) followed by FITC antirabbit IgG (left). Arrows point to representative structures where ßCOP and Tac-EKITMS or Tac-EKITLL chimeras colocalize. Arrowheads point to vesicular structures that contain the designated chimeras, but lack ßCOP.

 
Figure 9Go summarizes the subcellular localizations of the various Tac-hexapeptide chimeras obtained by quantifying which organelles contained detectable immunofluorescence signal for each chimera. Four or five independent experiments were examined. {chi}2 analyses of the data showed that the distributions obtained for Tac-EKITAA, Tac-EKITMS, and Tac-EKITLL were significantly different from each other with P <= 0.001.



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  		Figure 9. Quantitation of the subcellular distribution of Tac chimeras. HeLa cells were transiently transfected with expression vectors encoding each of three Tac-hexapeptide chimeras, and immunofluorescence staining was performed with anti-Tac antibody. Each transfected cell was classified according to which organelles were stained (plasma membrane, endosomes, lysosomes, and/or TGN). Staining was scored as positive if a signal was visually detectable. At the time the pictures were scored, the observer was blinded with respect to the identity of the construct. The number of cells counted for each construct (n) is indicated in the upper righthand corner of each panel.

 
Internalization of Tac chimeras
The immunofluorescence studies demonstrated that a large fraction of Tac-EKITLL molecules are located in lysosomes. Although we did not detect immunofluorescent staining at the plasma membrane, it is possible that the steady state level of the Tac-EKITLL chimera at the plasma membrane is below the limit of detection due to extremely rapid endocytosis. Therefore, we incubated living cells transfected with the various Tac chimeras in the presence of anti-Tac antibody for 30 min at 37 C. Under these conditions, antibody can bind to and be internalized by the chimeras even if they only appear transiently on the cell surface. After washing, fixing, and permeabilizing the cells, the cell-associated anti-Tac antibody was visualized by immunofluorescence using a labeled secondary antibody. When cells transfected with Tac-EKITAA were examined, they exhibited bright surface labeling and small amounts of vesicular staining near the cell periphery (Fig. 10Go, A and A'). The Tac-EKITMS chimeras had cell surface staining that was not as extensive as that seen with Tac-EKITAA (Fig. 10BGo). In addition, endosomal labeling was more extensive in cells expressing the EKITMS chimera (Fig. 10BGo'). In contrast, with cells expressing Tac-EKITLL, the anti-Tac antibody was seen in endosomal and lysosomal structures that were often found in the perinuclear region of the cell; the chimera was not detectable on the cell surface (Fig. 10Go, C and C'). These findings demonstrate that at least a portion of the Tac-EKITLL chimeras was delivered to the cell surface, where it then bound antibody and was rapidly internalized and delivered to lysosomes. However, although Tac-EKITMS is internalized from the cell surface, it does so more slowly than Tac-EKITLL. Furthermore, once internalized into endosomes, Tac-EKITMS is not efficiently targeted to lysosomes.



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  		Figure 10. Surface distribution and endocytosis of Tac chimeras. Forty-eight hours after transfection of HeLa cells with Tac-EKITAA, Tac-EKITMS, and Tac-EKITLL, living cells were incubated for 30 min at 37 C with 2 µg/ml anti-Tac monoclonal antibody. The cells were then washed, fixed, and permeabilized, and the surface-bound or endocytosed anti-Tac monoclonal antibody was detected with rhodamine antimouse IgG. Two focal planes are shown for each field of cells examined (A–C, a plane near the surface of the cell; A'–C', a plane deeper in the cell). All pictures were taken using identical conditions for exposure, development, and printing. Tac-EKITAA was found predominantly at the cell surface (A and A'), whereas Tac-EKITLL was found exclusively in intracellular structures resembling late endosomes and lysosomes (C and C'). In contrast, Tac-EKITMS was found both at the plasma membrane (B) and in vesicular structures resembling early endosomes (B').

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sequence similarities in the juxtamembrane domains of the insulin receptor family of tyrosine kinases
Comparison of the amino acid sequences of the insulin receptor with the homologous IGF-I receptor reveals that the juxtamembrane domains are highly conserved (69% identical; 84% similar over 55 amino acids) (26). One of the few nonconservative substitutions (i.e. MetSer for Leu986Leu987) is located in the juxtamembrane dileucine motif that we had previously demonstrated to be required for rapid endocytosis of the insulin receptor (19, 20). Because the insulin receptor has been reported to undergo 3-fold more rapid endocytosis than the IGF-I receptor (25), we inquired whether the substitution of MetSer for LeuLeu in the EKITLL sequence of the insulin receptor might account for the reduction of the endocytosis rate. However, the MetSer substitution did not affect the endocytosis of the receptor, as measured by uptake of [125I]insulin or by quantification of both constitutive and insulin-stimulated internalization of receptors. The ability to substitute Met for the first Leu was not surprising, in that MetLeu has been shown to function as an internalization signal in the major histocompatibility complex-associated invariant chain (14). However, substitution of Ser (a hydrophilic amino acid residue) for the second Leu in the insulin receptor juxtamembrane dileucine motif is clearly a nonconservative substitution. In any case, preservation of the ability of the sequence motif, EKITMS in the juxtamembrane domain of the IGF-I receptor, to direct a protein toward endocytosis suggests that there are evolutionary constraints that conserved the functionality of this targeting sequence.

The substitution of MetSer for LeuLeu does not explain the difference in the rates at which the receptors for insulin and IGF-I undergo endocytosis. Thus, it is likely that other structural differences between the two receptors provide an explanation for the observed differences in the rates of endocytosis. In this regard, it is noteworthy that the insulin receptor has three additional dileucine motifs in its cytoplasmic domain (19). Although two of these are conserved in the IGF-I receptor, the C-terminal dileucine motif (EIVNLL, amino acids 1246–1251) is not present in the IGF-I receptor.

Comparison of observations in full-length receptor and Tac chimeras
We used two complementary experimental approaches in this manuscript. First, we analyzed the effects of mutations in the juxtamembrane dileucine motif in the full-length insulin receptor. This provides insight into the function of the sequence in a physiologically relevant context, but is limited by interactions with other targeting motifs elsewhere in the cytoplasmic domain of the receptor. This approach led to the conclusion that the Met986Ser987 substitution did not impair endocytosis of the full-length insulin receptor. Second, we have assayed the ability of hexapeptide sequences to target chimeric proteins to lysosomes. Although this is an artificial experimental system, it has the advantage of isolating a specific motif from other sequences in the insulin receptor. This approach demonstrated that MetSer does not substitute for LeuLeu in the dileucine motif when the hexapeptide sequence is placed at the C-terminus of the Tac antigen (interleukin-2 receptor, {alpha}-chain). In the context of the Tac chimera, MetSer does not function as efficiently as LeuLeu in mediating endocytosis and targeting of the chimeric protein to lysosomes. Nevertheless, the Met986Ser987 substitution allowed for more efficient internalization of both the full-length receptor and the Tac chimera than the corresponding Ala986Ala987 substitution. The differences in sorting efficiencies seen with the three hexapeptide motifs are probably due to differences in the specificity and avidity with which these sequences interact (either directly or indirectly) with the plasma membrane adaptor complex (AP2), the TGN adaptor complex (AP1), and/or the recently described adaptor complex (AP3), which may serve as an adaptor complex on endosomes (17, 47, 48, 49, 50). According to this interpretation, our findings could be explained by assuming that the motif EKITLL has a high affinity interaction with AP1, and thus, much of Tac-EKITLL would be directly targeted from the TGN to lysosomes. The small amount of Tac-EKITLL that reaches the plasma membrane would then be recognized by AP2, rapidly internalized into endosomes, and subsequently targeted to lysosomes, presumably via high affinity interactions with a adaptor complex on endosomes, perhaps AP3 (50, 51). In contrast, Tac-EKTIAA and Tac-EKITMS would have lower affinity interactions with AP1 than Tac-EKITLL and thus leave the TGN and travel to the plasma membrane. Tac-EKITAA would then be internalized slowly from the plasma membrane due to weak interactions with AP2. In contrast, we propose that Tac-EKITMS has a higher affinity interaction with AP2 and thus would be internalized into endosomes more rapidly than Tac-EKITAA. However, in light of the fact that very little Tac-EKITMS is found in lysosomes, it is possible that the MS substitution weakened the interactions with the adaptor complex on endosomes. Although several tyrosine-based motifs have been shown to bind differentially to the µ-chains of the AP1, AP2, and AP3, to date no direct interaction between dileucine-containing motifs and the µ-chains has been demonstrated (17, 47, 48, 49, 50). Perhaps, dileucine motifs interact with other components of the adaptor complexes or with molecules such as Nef HIV early protein that are thought to serve as connectors between adaptor complexes and receptor molecules (52).

In conclusion, it is likely that dileucine motifs as well as tyrosine-based signals interact with multiple adaptor molecules. The localization of a protein within the cell is, therefore, determined by the net effect of interactions of its various targeting sequences with multiple adaptor complexes. Mutations would thus have differential effects on each of these binding interactions, leading to complex effects of mutations upon protein trafficking.


   Acknowledgments
 
We are grateful to Dr. Axel Ullrich for the generous gift of insulin receptor cDNA. In addition, we thank Drs. Juan Bonifacino, Julie Donaldson, Mickey Marks, and Hagai Agmon-Snir for advice and helpful discussions, and Mrs. G. Porcheron-Berthet for skilled technical assistance. Finally, we thank Dr. Valarie Barr for critical reading of the manuscript.


   Footnotes
 
1 This work was supported in part by Grant 3100–043409-95 from the Swiss National Science Foundation. Back

2 The numbering system used throughout the text for describing the amino acid positions in the insulin receptor are based on the cDNA that lacks exon 11 (29 ). Back

Received June 16, 1997.


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 Introduction
 Materials and Methods
 Results
 Discussion
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