Most plasma membrane receptors and their ligands undergo receptor-mediated endocytosis and are taken up by cells through the formation of clathrin-coated vesicles(1, 2, 3, 4, 5) . But, while transport protein receptors such as the receptors for low density lipoproteins or transferrin are rapidly and constitutively internalized, signaling receptors such as those for insulin and epidermal growth factor internalize at rapid rates only when ligand is bound(1, 2, 3) . These two classes of receptors differ not only on the basis of their function (delivering of nutrients to the cells versus intracellular signaling) and of the ligand dependence or independence of the internalization process but also in their topography. Class I receptors, typified by the low density lipoprotein and the transferrin receptors, are present in clathrin-coated pits in their unoccupied form while class II receptors, like the insulin receptor, are preferentially found associated with surface microvilli in their unbound state(3, 6, 7) . Following entry into the cells, the intracellular pathway followed by receptors of these two classes is similar; the ligand-receptor complex is cleaved at the acidic pH of endosomes and its two components are targeted in different directions: the ligand is generally routed to lysosomes while the receptor is classically recycled back to the cell surface where it can be reused(1, 2, 3, 4, 5, 6, 7, 8, 9) .

The signals responsible for targeting receptors for endocytosis are being defined. Receptor-mediated endocytosis requires specific amino acid sequences found in the cytoplasmic tail (frequently the submembranous domains) of receptors. They have in common a propensity to form a tight turn exposing an aromatic residue (preferentially a tyrosine)(2, 5, 10, 11, 12, 13, 14, 15, 16, 17) . These sequences are recognized by proteins (probably the adaptors) that link the receptors to clathrin-coated pits (18, 19, 20, 21, 22) . Similar sequences in the juxtamembrane cytoplasmic domain of the insulin receptor (GPLY and NPEY) have been found to be necessary for endocytosis(23, 24, 25, 26, 27) . In the case of the insulin receptor, however, these sequences are not sufficient for endocytosis; rapid internalization also requires activation of the receptor tyrosine kinase, probably accounting for the observed ligand dependence of insulin receptor endocytosis(27, 28, 29, 30, 31) . The tyrosine kinase of the epidermal growth factor receptor has also been shown to be required for its internalization(32, 33) . Tyrosine kinase activation is involved in the surface redistribution of the insulin receptor from microvilli to the nonvillous surface of the cell(27, 28) , but how the receptor tyrosine kinase initiates this initial step of endocytosis is not clear. Tyrosine kinase activation could play an active role and initiate a controlled redistribution of the receptor in the direction of the clathrin-coated pits of the nonvillous domain of the cell surface. Conversely, tyrosine kinase activation could simply act to release receptors from microvilli, whereupon localization to clathrin-coated pits and endocytosis occurs without further kinase-dependent signaling.

In the present study, we have addressed this question by examining in detail the endocytotic itinerary of an insulin receptor deleted of the cytoplasmic tyrosine kinase domain and COOH terminus, thus leaving only the juxtamembrane domain intact. Biochemical and ultrastructural analyses indicate that the truncated insulin receptor does not preferentially associate with microvilli and undergoes efficient constitutive endocytosis. We conclude that the signal allowing the anchoring of the unoccupied insulin receptor on microvilli is contained within the regulatory domain of the insulin receptor and that the tyrosine kinase activation of the insulin receptor initiates insulin receptor internalization through the release of a brake maintaining it on microvilli. Moreover, the submembranous endocytotic motifs of the insulin receptor, once available to the endocytotic machinery, appear sufficient for endocytosis.

EXPERIMENTAL PROCEDURES

Materials and Cell Culture

Photoaffinity Labeling, Insulin Binding, and Internalization Studies

()

Insulin internalization was measured as described(30, 35) . Briefly, cells in 6-well dishes (10 cells/dish) were equilibrated at 37 °C in Krebs-Ringer phosphate, 10 mM HEPES, 0.2% bovine serum albumin. The cells were then exposed to 10 pMI-insulin for various periods of time, after which the cells were rinsed rapidly three times in ice-cold saline (pH 7.6) and then exposed to 1 ml of incubation medium at pH 4.0 for 6 min on ice to remove surface-bound insulin. This plus a further 1 ml of rinse (pH 4.0) were combined, and radioactivity corresponding to surface-bound insulin was determined in a counter. The rinsed cells were solubilized in detergent, and radioactivity corresponding to internalized insulin was also determined. Counts bound and internalized in the presence of excess (300 nM) unlabeled insulin were less than 10% of the counts observed with labeled insulin only and were subtracted.

Trypsinization of intact cells and the determination of insulin binding activity in solubilized extracts of untreated or trypsinized cells in order to determine the relative proportions of intracellular and cell surface receptors were performed as described(35) .

Electron Microscope Autoradiography

RESULTS

Internalization of an Insulin Receptor Deleted of the Kinase and COOH-terminal Domains Encoded by Exons 17-22 (hIRex17-22)

We first examined the internalization of wild-type (WThIR) or truncated (hIRex17-22) receptors expressed in Rat-1 fibroblasts and labeled with an insulin photoaffinity probe. The cells were initially exposed to ligand in the cold so only the surface receptors would be labeled. The cells were then exposed to UV light to cross-link the ligand to the receptor and warmed at 37 °C for various periods of time. At each time point studied, cells were trypsinized to degrade any receptors still at the cell surface. As the receptors internalized they became trypsin-resistant such that internalized receptors remained intact while the receptors remaining at the plasma membrane were degraded by trypsin. Intact labeled -subunits of the receptor were then quantified after reducing gel electrophoresis and autoradiography. As can be seen in Fig. 1, before warming (time 0) greater than 95% of either normal or truncated hIR were at the cell surface and susceptible to trypsin degradation. With time at 37 °C, normal insulin receptors so labeled undergo internalization such that after 40 min, 52.6% of the receptors were trypsin-resistant. The truncated receptors did internalize more slowly, but after 40 min a significant number of them (22.2%) were intracellular. By comparison, the intracellular fraction of a noninternalizing kinase-defective receptor did not exceed 8-9% even after 60 min(30) . Labeling of receptors using this technique was performed at relatively high but not saturating insulin concentrations (2 nM) where roughly 50% receptor occupancy would be expected. Thus, the proportion of labeled receptors could not exceed 50% even assuming only a minimal degree of inefficiency of covalent coupling of the photoprobe. If nonligand-occupied hIRex17-22 were internalizing as well as the receptors labeled by the ligand, the total internalization rate of the hIRex17-22 could be comparable with that of the WThIR. We therefore examined whether unoccupied hIRex17-22 might also be undergoing constitutive endocytosis.

Figure 1: Internalization of wild-type and hIRex17-22 receptors labeled with I-NAPA-DP insulin. I-NAPA-DP insulin was bound to cells expressing wild-type (WT) hIR or the truncated mutant hIR (hIRex17-22) and the cells photoderivatized as described. After various times at 37 °C the cells were trypsinized and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A, autoradiogram showing intact hIR -subunits in treated cells. Receptors from cells not treated with trypsin are on the left (total), indicating the total number of labeled receptors. In cells trypsinized before warming (0) less than 5% of the receptors remain intact, illustrating that the labeled receptors are initially exclusively at the cell surface and that the trypsinization procedure is effective in degrading nearly all of the surface receptors. As a function of time at 37 °C, more receptors escape trypsin sensitivity as they internalize. B, quantitation of internalized receptors as a function of time at 37 °C. Two experiments such as the one illustrated above were performed, and the labeled bands visualized by autoradiography were excised and radioactivity determined in a counter. The counts in untrypsinized cells (A, total) were taken as 100%, and the percent of that total that were trypsin-resistant is plotted.

Resting Distribution of hIRex17-22 in Transfected Cells and the Effects of Insulin and Chloroquine

Figure 2: A, distribution of wild-type and hIRex17-22 receptors in unstimulated cells. Cells expressing either WThIR (solid bar) or hIRex17-22 (dotted bar) were solubilized in the absence of insulin and total receptor number determined in solution as described(30) . Cells were also trypsinized to degrade cell surface receptors and the number of trypsin-resistant (intracellular) receptors determined in solubilized extracts. The percentages of intracellular receptors (trypsin resistant/total) are plotted and are the means of three experiments (±S.E.), each performed in triplicate. Insulin binding assays were performed as binding competition curves; these revealed no significant difference in affinity between the receptors from cells trypsinized or not, so the plotted results are of tracer (10 pM) insulin binding. B, change in intracellular receptor number after insulin treatment. Cells were treated for 60 min at 37 °C with near saturating insulin (30 nM), and the cell receptor number (intracellular and total) was determined as above. Results are plotted as the percent increase in intracellular receptors shown in panel A and are the means of three experiments assayed in duplicate.

Receptor distribution after treatment of cells with the lysosomotropic drug chloroquine that blocks receptor recycling was also investigated and was also consistent with the truncated hIRex17-22 constitutively undergoing an endocytotic itinerary. As shown in Fig. 3, after exposure to chloroquine but no insulin for 24 h, hIRex17-22 cells had lost 20.3 ± 5.4% of their surface insulin binding compared with a negligible loss (1.0 ± 3.6%) of binding in cells expressing normal receptors. There was a concomitant increase in the fraction of intracellular trypsin-resistant receptors after chloroquine treatment of the truncated but not normal receptors, comparable with what was seen with cells expressing normal receptors but after insulin treatment (Fig. 3).

Figure 3: Effect of chloroquine treatment on insulin receptor distribution in the absence of insulin. Left, cells expressing WThIR (solid bar) or hIRex17-22 (dotted bar) were exposed to 100 µM chloroquine for 1 h. Cell surface insulin binding was then assayed and compared with that of control cells incubated in the absence of chloroquine. Results are the means of two independent experiments, each assayed in triplicate. Right, cells treated with or without chloroquine were either trypsinized or not, and the percentage of intracellular (trypsin-resistant) receptors was determined in solubilized extracts of cells as described under ``Experimental Procedures.'' Results are the means of two experiments, each assayed in quintuplicate.

Morphological Tracking of I-insulin Internalization Pathway in hIRex17-22 Cells

Figure 4: I-insulin internalization in Rat-1 fibroblasts transfected with WThIR or hIRes17-22. Results presented are the average of the analysis of four different Epon blocks. For each time point and each cell line = 600-800 autoradiographic grains were quantitated. Results are expressed as a percent of the total number of grains associated with the cells whose centers were >250 nm from the plasma membrane.

As previously observed in isolated rodent hepatocytes, human monocytes and various cultured cells(38, 39, 40, 41) , I-insulin preferentially associated with microvilli on the surface of wild-type cells at 4 °C, demonstrating that the unoccupied insulin receptor was preferentially localized on these surface domains in cultured Rat-1 fibroblasts (Fig. 5). Warming of the incubation medium to 37 °C resulted in a redistribution of the radioactive hormone-hIR complex in the direction of the nonvillous domain of WT cells (Fig. 5). By contrast, in hIRex17-22 cells, I-insulin did not preferentially associate with microvilli at 4 °C (Fig. 5), and at 37 °C, the labeled material remained localized on the nonvillous regions of the cell surface at all time points studied (Fig. 5).

Figure 5: Surface redistribution of I-insulin in Rat-1 fibroblasts transfected with WThIR or hIRex17-22. Results presented are the average of the analysis of four different Epon blocks. For each time point and each cell line, 600-800 autoradiographic grains were quantitated. Results are expressed as a percent of the total number of grains associated with the cell surface (±250 nm from the plasma membrane) whose centers were within 250 nm of microvilli.

The redistribution of I-insulin on the surface of WT cells was accompanied by a progressive sequestration of the radioactively labeled material in clathrin-coated pits. By 2 h at 4 °C less than 4% were associated with these surface differentiations while by 30 min this value reached 14.5% (Fig. 6A). By contrast, in hIRex17-22 cells, by 2 h of incubation at 4 °C and before warming, 13.7% of surface-bound I-insulin was already present in clathrin-coated pits, and this association of the radiolabeled ligand with clathrin-coated pits remained practically constant with time at 37 °C. When the quantification was restricted to autoradiographic grains present on the nonvillous domain of the cell surface and the grains present on this surface domain at all time points were pooled, the propensity of occupied WThIR and hIRex17-22 insulin receptors to anchor to clathrin-coated pits was identical (Fig. 6B).

Figure 6: Association of I-insulin with clathrin-coated pits. A, percent of autoradiographic grains present on the total surface of Rat-1 fibroblasts transfected with WThIR or hIRex17-22 that are associated with clathrin-coated pits. B, percent of the autoradiographic grains present on the nonvillous surface of Rat-1 fibroblasts transfected with WThIR or hIR ex17-22 that are associated with clathrin-coated pits. Results presented are the average of the analysis of four different Epon blocks. Results are expressed as a percent of the total number of grains associated with the cell surface (±250 nm from the plasma membrane) whose centers were within 250 nm from a clathrin-coated pit. In B, values are the mean of the values obtained at the 5, 15, and 30 min time points ± S.E. (n = 3).

DISCUSSION

The requirement of receptor tyrosine kinase activation and autophosphorylation for tyrosine kinase receptor internalization is widely accepted(28, 29, 30, 31, 32, 33, 42, 43, 44) . In the case of the insulin receptor, this activation governs the first step of receptor internalization: the ligand-dependent induction of receptor redistribution from microvilli to the nonvillous domain of the cell surface(27, 28) . A question is whether this process is active or passive. The ``active'' hypothesis would involve activation of enzyme(s) (probably via phosphorylation reactions) inducing a directed redistribution of the receptors on the cell surface and controlling their segregation in the internalization gates: the clathrin-coated pits. The ``passive'' process would imply that a brake is keeping the receptors out of the endocytotic machinery. In this case, ligand binding would result, via kinase activation and receptor autophosphorylation, in the release of this block to internalization. In the case of this passive hypothesis, two further possibilities exist: either the process is mediated via phosphorylation of a specific substrate(s) that mediates internalization or autophosphorylation of the receptor molecule itself constitutes the signal(s) for internalization through unmasking of endocytotic signal sequences. The present study addresses these questions. Data presented demonstrate that (a) the process is occurring through the release of a brake: receptor kinase activation and autophosphorylation free the receptor from constraints maintaining it on microvilli of the cell surface; (b) the signal sequences contained in exon 16 are entirely sufficient to promote clathrin-coated pit-mediated internalization of insulin receptors, which have access to the nonvillous domain of the cell surface; (c) these sequences are not uncovered by kinase activation; and (d) the ``code'' maintaining the unoccupied receptors on microvilli is contained within exons 17-21 of the receptor.

In its unoccupied state, the insulin receptor preferentially localizes on microvilli(27, 28, 38, 39, 40, 41) . This may facilitate its optimal interaction with circulating insulin. What maintains the receptor on these thin digitations of the cell surface remains unknown, but an interaction with cytoskeleton elements particularly rich in the submembrane domain of these regions is highly probable. Taken together with previous observations, present data indicate that the receptor site involved in this potential interaction is contained neither in the juxtamembrane domain nor in the COOH-terminal tail (27, 28) but in the regulatory domain of the cytoplasmic tail of the receptor and that it is abolished by kinase activation. The regulatory domain contains dileucine motifs that have been shown to control endocytosis of several receptors including the insulin receptor(45, 46) . ()Studies are in progress to determine their exact role in the initial stages of insulin receptor internalization and especially in the anchoring of the unoccupied receptor on microvilli.

We observed that the truncated receptor with a cytoplasmic tail containing only the juxtamembrane domain of the receptor but no kinase domain did not preferentially associate with microvilli in its unoccupied form. Rather, the unoccupied truncated receptor showed the same propensity as an activated normal hIR to associate with the nonvillous domain of the cell surface, arguing against an active insulin-induced surface redistribution of the receptor mediated by kinase activation and receptor autophosphorylation. Moreover, taken together with previous observations that kinase-inactive or autophosphorylation-deficient insulin receptors preserve their capacity to associate with clathrin-coated pits(27, 47) , present data showing that hIRex17-22 receptors exhibit full capacity to be segregated in clathrin-coated pits strengthen the argument that kinase activation is not required for clathrin-coated pit association and that the signal sequences allowing insulin receptor anchoring in clathrin-coated pits are not unmasked by kinase activation.

Whether the activated tyrosine kinase phosphorylates a specific substrate(s) that mediates the release from microvilli or whether autophosphorylation of the receptor molecule itself constitutes the signal for this release remains an open question. In support of the first possibility are the recent observations that ligand-induced internalization of two tyrosine kinase receptors (platelet-derived growth factor receptor and fibroblast growth factor receptor) depends on the recruitment, at the autophosphorylation site of the receptor, of specific regulatory proteins implicated in the initiation of a phosphorylation cascade (phosphatidylinositol 3-kinase and phospholipase C, respectively)(42, 43) . In the case of the insulin receptor, neither IRS-1 nor phosphatidylinositol 3-kinase, two substrates that play key roles in the early stages of postreceptor insulin signal transduction, seem involved in mediating insulin receptor internalization(24, 27, 48) . On the other hand, the presence in the cytosol of unidentified specific factors required for kinase-dependent internalization of insulin receptors was recently proposed(44) . Thus, a dichotomy in the signals initiated by kinase activation that mediate biological action and internalization is highly probable. Strengthening this argument is our recent observation that insulin receptor internalization is not required for transmission of insulin's biological actions(49, 50) .

When insulin receptors have access to the nonvillous domain of the cell, their internalization becomes relatively nonspecific and similar to that of most receptors that are constitutively internalized whether a ligand is bound or not (i.e. transferrin receptor, low density lipoprotein receptor, . . . ). In these conditions, the internalization sequence(s) identified in the juxtamembrane domain of the insulin receptor and which is (are) analogous to the ones present in most of these receptors (tight turn exposing an aromatic amino acid) is (are) sufficient to promote a rapid and efficient internalization of the receptor. Based on the slow internalization of insulin mediated by the hIRex17-22 receptor, we had earlier concluded that the receptor did not undergo endocytosis at a rate equivalent to the complete receptor(25) . In these experiments, however, it is difficult to compare ligand-independent or -constitutive endocytosis with ligand-triggered endocytosis because the measure of endocytosis is the ligand itself. If endocytosis is constitutive, ligand-occupied receptors will not be preferentially internalized, so the internalization rate will appear lower than ligand-dependent endocytosis because those unoccupied receptors that are concomitantly internalized are not being scored. Biochemical comparisons of the resting distribution of hIRex17-22 and WThIR between the surface and the inside of the cells and of the influence of chloroquine and insulin on these distributions confirm this interpretation of the data. (a) A larger fraction of receptors was internal in hIRex17-22 cells than in hIR cells, indicating a significant continuous turnover of receptors in hIRex17-22 cells but not in hIR cells; (b) a blockade of receptor recycling by chloroquine increased the intracellular fraction of hIRex17-22 receptor but had no effect on unoccupied hIR receptors, which is also in favor of a constitutive internalization recycling of hIRex17-22 receptors; and (c) insulin had no effect on the distribution of hIRex17-22 receptors while it led to an increase in the proportion of internal hIR receptors confirming that insulin receptor internalization was essentially ligand-dependent in hIR cells.

Our observations disagree with those recently published by Smith et al.(51, 52) who claimed that insulin-induced kinase activation and autophosphorylation did not control the surface redistribution of the insulin receptor but were required for the receptor to concentrate in clathrin-coated pits. Since in both cases the mutations studied were very similar and Rat-1 fibroblasts were used as the host cells, the most likely explanation for the differing conclusions is in the morphological probe used. Large electron-dense probes (i.e. ferritin or colloidal gold) have frequently raised suspicions about the reliability of results generated due to the fact that these large molecules could perturb the subcellular distribution of the bound molecule and to the difficulty in producing a conjugate that preserved the full biological properties of the native ligand. In the present work as well as in previous ones (for review see (6) and (7) ), we have tracked, by quantitative electron microscope autoradiography, monoiodinated I-human insulin, a ligand with full biochemical and biological activation and whose use is widely accepted as valid.

In conclusion, taken together with previous observations(26, 27, 28) , data presented allow us to propose the following ordered sequence of events, including ligand-dependent and ligand-independent steps, leading to insulin receptor internalization in target cells.

1) In its unoccupied and unstimulated state, the insulin receptor preferentially associates with microvilli on the cell surface. This preferential association is dependent on the integrity of the cytoplasmic tail of the receptor, but neither the juxtamembrane domain, nor the COOH-terminal domain, nor kinase activation and receptor autophosphorylation play a role in this association.

2) Insulin binding releases the constraint maintaining the receptor on microvilli and does so via receptor kinase activation and autophosphorylation of the three tyrosine residues present in the regulatory domain.

3) The insulin-receptor complex, freely mobile on the cell surface, next associates with the internalization gates, the clathrin-coated pits, via signal sequences contained in the juxtamembrane domain of the cytoplasmic tail of the receptor. These sequences, which are constitutively unmasked (neither kinase activation nor specific autophosphorylation of the three tyrosines of the kinase domain is required to uncover them), are sufficient to promote rapid and efficient internalization of the receptor.

4) The subsequent invagination, budding, and pinching off to form clathrin-coated vesicles would then proceed as with all classes of receptors employing this pathway(53) .

FOOTNOTES

*

This work has been supported by Grant 31.34093.92 from the Swiss National Science Foundation and the Juvenile Diabetes Foundation (to J.-L. C.) and by the Research Service of the Veterans Administration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed. Tel.: 41-22-7025201; Fax: 41-22-7025260.

()

The abbreviations used are: hIR, human insulin receptor; WThIR, wild-type hIR; hIRex17-22, hIR with domains encoded by exons 17-22 deleted; WT cells, cells expressing wild-type hIR; NAPA-DP, 2-nitro-4-azidophenylacetyl-des-Phe.

()

C. Renfrew, personal communication.

ACKNOWLEDGEMENTS

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