Metabolism of photoaffinity-labeled insulin-like growth factor-I receptors by human cells in vitro.

0013-7227/90/1263-1334$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 126, No. 3 Printed in U.S.A.

Metabolism of Photoaffinity-Labeled Insulin-Like Growth Factor-I Receptors by Human Cells in Vitro* RICHARD W.FURLANETTO Department of Pediatrics, Division of Endocrinology, University of Rochester Medical Center, Rochester, New York 14642

ABSTRACT. These studies were undertaken to characterize the physiological fate of the insulin-like growth factor-I (IGFI)-type I receptor complex in MG-63, an IGF-responsive human osteosarcoma cell line. To investigate this, a photoreactive iodinated derivative of IGF-I [5-azido-2-nitrobenzoyl-125I-IGF-I (ANBz-125I-IGF-I)] was synthesized. This derivative retained biological activity and photolabeled a cell surface component on MG-63 cells with the size and binding specificity characteristic of the type I IGF receptor. To assess the stability of the photoaffinity-labeled IGF-I., receptor complex, quiescent monolayers of MG-63 cells were incubated with ANBz-125 I-IGF-I at 2 C, photolyzed, and then warmed to 37 C. At various times the monolayers were solubilized and the receptor complex was identified by quantitative autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under these conditions the photoaffinity-labeled IGF-I-receptor complex was relatively stable, with a halflife of 11 ± 2 h in five experiments. To determine if the complex undergoes internalization, its

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HE INSULIN-like growth factors (IGFs) IGF-I (1, 2) and IGF-II (3, 4) are polypeptide hormones that stimulate the replication of a wide variety of cell types (5). Although two distinct IGF receptors have been identified (6, 7), the mitogenic effect of both IGFs appears to be mediated through the same receptor, termed the type I IGF receptor (8, 9). This receptor is structurally similar to the insulin receptor (6, 7,10) and has intrinsic tyrosine kinase activity (11,12); binding of an IGF to this receptor activates this tyrosine-kinase activity and results in phosphorylation of tyrosine residues on both the receptor itself (11) and other cellular proteins (12). It is generally believed that tyrosine phosphorylation alters the biological activities of these proteins, thereby modulating the activities of key biochemical pathways in the cell, but the details of the signal transduction mechanism remain poorly understood.

Received August 23,1989. Address requests for reprints to: Dr. Richard W. Furlanetto, Department of Pediatrics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 777, Rochester, New York 14642. * This work was supported by USPHS Grant 5RO1-CA-38981 from the NCI, DHHS.

susceptibility to hydrolysis by trypsin at 2 C was measured. When cells that were labeled at 2 C were warmed to 37 C, a trypsin-insensitive band appeared within 20 min, reached a maximum by 1 h, and declined thereafter; however, an average of only 7% (5-8% in four experiments) of the total labeled receptor pool was present intracellularly at 1 h, and this declined to less than 1% by 4 h. A similar distribution of receptors was observed in cells photolabeled after incubation with ANBz-125IIGF-I at 37 C, indicating that this distribution was not the result of altered metabolism of the photolabeled receptor complex. Lysosomotropic agents inhibited degradation of the complex, but did not alter its distribution between the cell surface and interior. These results indicated that the IGF-I-type I receptor complex is relatively stable and does not undergo rapid ligandinduced down-regulation and degradation. These observations suggest a model in which the IGF-receptor complex functions while present at or near the cell surface. (Endocrinology 126: 1334-1342, 1990)

One approach that has been used to help elucidate the mechanisms by which growth factors function has been to examine the metabolic fate of the growth factorreceptor complexes (13-17). Such studies have shown that the hormone-receptor complexes for epidermal growth factor (EGF) (13), platelet-derived growth factor (PDGF) (14), and insulin (15,17) are rapidly internalized by their target cells. Internalization is responsible for the rapid ligand-induced down-regulation of cell surface receptors that is observed with these agents and appears to be essential for inactivation of the complexes and degradation of their components. It has also been postulated that internalization may play a role in signal transduction by bringing activated receptor complexes into proximity with their intracellular substrates (18, 19). We have recently shown that in MG-63, an IGFresponsive human osteosarcoma cell line, IGF-I is internalized via the type I receptor and that the internalized hormone is degraded in part in lysosomes (20). There is little information, however, regarding the physiological fate of the IGF-I-type I receptor complex in responsive

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IGF-I RECEPTOR METABOLISM cells. Although a number of studies have examined the binding of the IGFs to cultured cells and have described changes in binding resulting from hormone treatment (21-23), these studies were performed before it was appreciated that there are two IGF receptor types and that cultured cells often secrete IGF-binding proteins that mask type I receptor binding when standard monolayer binding procedures are used (20, 24, 25). As a result, it is not clear whether the changes observed were due to direct alterations in type I receptor binding or were due to changes in these other binding species. The present studies were undertaken to characterize the fate of the IGF-I-type I receptor complex in MG-63 cells in vitro. To this end a biologically active, photoreactive IGF-I analog was used to label the type I receptor in situ, and the rates of internalization and degradation of the photolabeled complex were determined. The results indicate that the photolabeled IGF-I-type I receptor complex is relatively stable and does not undergo rapid down-regulation. This relative stability of the IGF-Ireceptor complex may reflect a fundamental difference in the mechanisms by which the IGFs and other growth factors function.

Materials and Methods Materials The photoreactive esters 5-azido-2-nitrobenzoyl-iV-hydroxysuccinimide and 6-(4'-azido-2'-nitrophenylamino)hexanoylJV-hydroxysuccinimide and the bifunctional cross-linking reagent disuccinimidyl suberate (DSS) were obtained from Pierce Chemical Co. (Rockford, IL). Carrier-free Na 125I (100 mCi/ml) and [met/iy/-3H]thymidine (5 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). Culture media and antibiotics were purchased from Gibco (Grand Island, NY). Culture plates were obtained from Costar (Cambridge, MA), and fetal bovine serum was from K. C. Biological (Lenexa, KA). Chloramine-T was purchased from Eastman Kodak (Rochester, NY). The MAPS antibody purification system and reagents for electrophoresis were obtained from Bio-Rad Laboratories (Richmond, CA). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The human IGF-I used in these studies was purified from Cohn fraction IV-1 as previously described (8) and was greater than 95% pure based on multiple criteria. aIR-3 (26), a monoclonal antibody which blocks IGF binding to the human type I IGF receptor, was a generous gift of Dr. Steven Jacobs, Wellcome Research Laboratories (Research Triangle Park, NC); it was produced as ascites fluid and purified as previously described (8). The MG-63 human osteosarcoma cell line used in these studies was obtained from the NIA, Aging Cell Culture Repository (Camden, NJ). The cells were grown as described previously (20). In these studies cells between passages 20 and 42 were employed.

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Synthesis of iodinated photoreactive IGF-I derivatives Two photoreactive derivatives of [125I]IGF-1,5-azido-2-nitrobenzoyl-125I-IGF-I (ANBz-125I-IGF-I) and 6-(4'-azido-2'-nitrophenylamino)hexanoyl-125I-IGF-I (ANPAH-125I-IGF-I), were prepared using the commercially available iV-hydroxysuccinimide esters. IGF-I was first iodinated using limited quantities of chloramine-T as previously described (27), except that the reaction was terminated by adding a 2.7-fold molar excess of acetaminophen. Two micrograms (0.26 nmol) of [125I]IGF-I in volume of 100 n\ 0.5 M sodium phosphate, pH 7.4, were then mixed with a 60-fold molar excess (16 nmol) of the appropriate ester (freshly dissolved in 5 fi\ dimethylsulfoxide), and the mixture was incubated at room temperature in the dark for 1 h. The unreacted ester was then blocked by adding 20 n\ of a saturated solution of /-tyrosine in 0.01 N NaOH. After 5 min 0.5 ml phosphosaline buffer (0.05 M sodium phosphate and 0.15 M NaCl, pH 7.4) containing 12% BSA was added, and the mixture was chromatographed at 4 C in the dark on a 1.5 X 60cm column of Sephadex G-50 (fine) eluted with phosphosaline buffer containing 0.1% BSA. Appropriate fractions (Kav = 0.20.4) were combined, brought to 4% with BSA, and stored in the dark at —20 C. Both derivatives remained active for up to 6 weeks when stored under these conditions. Quantitation of DNA synthesis The mitogenic activities of IGF-I and its photoreactive derivative ANBz-I-IGF-I were assessed using a modification of the serum-free culture system described previously (20). Briefly, cells from stock cultures were plated into 0.3-cm2 (96-well) cluster plates in 160 n\ Dulbeccco's Minimum Essential Medium supplemented with glutamine and 10% fetal bovine serum. After 24 h this medium was removed and replaced with serum-free MCDB 104. After 48-72 h, ice-cold, basal medium [MCDB 104 with 0.1% BSA, penicillin (100 U/ml), streptomycin (100 Mg/ml), transferrin (1 Mg/ml), and dexamethasone (0.10 /^M)] containing 1 /^Ci [me£h;y/-:>H]thymidine and the desired concentration of growth factor, antibody, or dialyzed serum was added to triplicate wells. After incubating at 2 C for 90 min in the dark, the covers were removed from the culture dishes, and the cells were photolyzed by exposure to long wavelength (366 nm) UV light (model UVL-56 Blak-Ray Lamp, UVP, Inc., San Gabriel, CA) for 3 min at a distance of 10 cm. The cells were then incubated at 37 C in a humidified atmosphere of 7% CO2-93% air. After 42-44 h the cells were processed for autoradiography or scintillation counting as described previously (20). Binding and photolysis of iodinated photoreactive IGF-I derivatives For binding experiments MG-63 cells were plated into 2-cm2 (24-well) cluster plates in 1 ml Dulbecco's Minimum Essential Medium supplemented with glutamine and 10% fetal bovine serum. After 4 days this medium was removed and replaced with serum-free MCDB104. After 48 h in the serum-free medium, the monolayers were cooled to 2 C in an ice-water bath, the medium was aspirated, and 0.3 ml basal medium supplemented with the 125I-labeled photoreactive IGF-I derivative of


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IGF-I RECEPTOR METABOLISM

interest (4.6-8.2 nM), with or without other additives as indicated, was added using a red light for illumination (Safe Light, Eastman Kodak). After incubating for 90 min at 2 C in the dark, the plastic covers were removed from the dishes, and the monolayers were photolyzed by exposure to long wavelength UV light. The usual photolysis conditions were 3 min at a distance of 10 cm for the ANBz derivative or 15 min at 3.5 cm for the ANPAH derivative (see below). After photolysis the monolayers were aspirated and washed once with 0.3 ml icecold basal medium. Depending on the experimental design, the monolayers were then either solubilized with 200 jul electrophoresis sample buffer [60 mM Tris, pH 6.8, containing 1% glycerol, 0.3% sodium dodecyl sulfate (SDS), 0.015% bromophenol blue, 1 mM phenylmethylsulfonylfluoride, 10 tig/m\ tosylphenylalanine chloromethyl ketone, 300 tig/ml benzamidine, 0.3 U/ml aprotinin, and 10 ng/ral leupeptin], or 300 n\ basal medium, with or without other additives, were added and the monolayers were incubated at 37 C. In some experiments the monolayers were not photolyzed after the incubation at 2 C, but, instead, the desired additions were made, and the (unphotolyzed) monolayers were incubated at 37 C in the dark. At the specified times the monolayers were removed from the incubator, photolyzed as described above, washed, and either solubilized with electrophoresis sample buffer or trypsinized as described below. This experimental design was termed postincubation photolysis. The appropriate conditions for photolysis were determined by exposing aqueous solutions of the photoreactive AMiydroxysuccinimide esters to long wavelength UV light for various times and from various distances and monitoring the change in absorbance at the absorbance maximum of the nitrophenylazide groups (320 nm for the ANBz analog and 260 nm for the ANPAH analog). These studies indicated that the ANBz moiety is much more susceptible to photolysis than is the ANPAH moiety. In fact, under the conditions usually employed in photolysis experiments (i.e. 3-min exposure to long wavelength UV light at a distance of 10 cm) (15, 16, 19, 28), greater than 90% of the ANBz ester underwent photolysis, while less than 10% of the ANPAH ester reacted; 90% photolysis of the ANPAH ester required approximately 15 min of exposure at 3.5 cm (not shown). Subsequent studies with the homologous photoreactive [125I] IGF-I derivatives showed a similar difference in reactivity: ANBz-125I-IGF-I was near maximally crosslinked after 3 min of exposure at 10 cm, while maximal crosslinking with ANPAH-125I-IGF-I required more than 15 min of exposure at a distance of 3.5 cm (not shown). In four experiments maximal cross-linking of ANBz-125I-IGF-I averaged 6.5% (range, 5.9-7.8%) of the total bound hormone. Internalization and degradation of the photoaffinity-labeled IGF-I-receptor complex To assess degradation of the photoaffinity-labeled IGF-Ireceptor complex, monolayers that were photoaffinity labeled at 2 C were incubated at 37 C in fresh basal medium, with or without other additives, for times ranging from 20 minutes to 24 h. At the specified times the monolayers were removed from the incubator, washed twice with ice-cold phosphosaline buffer, solubilized with 200 /A sample buffer, and stored at 4 C until

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electrophoresis. The susceptibility of the complex to proteolysis by trypsin was used to distinguish internalized receptors (trypsin insensitive) from those remaining on the cell surface (trypsin sensitive) (14-16). At the specific times, monolayers were removed from the 37 C incubator, cooled to 2 C, washed twice with icecold phosphosaline buffer, and trypsinized by adding 300 n\ ice-cold phosphosaline containing 100 Mg/ml trypsin (bovine pancreas, type XIII; tosylphenylalanine chloromethyl ketone treated; 11,500 benzoylarginine ethyl ester units/mg) and incubating at 2 C for 2 h. Trypsinization was terminated by adding 300 n\ ice-cold phosphosaline buffer containing 200 fig/ ml soybean trypsin inhibitor. Cells still adhering to the plate were loosened by scraping, the well was washed with 300 /ul phosphosaline, and the trypsin, trypsin inhibitor, and wash solutions were combined. To limit possible internalization of trypsin by the cells, care was taken to precool the cells and to maintain them at 2 C during these procedures. The cells were then pelleted by centrifugation at 400 X g for 10 min at 10 C, solubilized using 200 n\ sample buffer, and stored at 4 C until electrophoresis. Polyacrylamide gel electrophoresis (PAGE) and autoradiography The solubilized proteins were analyzed by SDS-PAGE using the discontinuous buffer system of Laemmli (29) as previously described (8, 20). After electrophoresis the gels were stained with Coomassie brilliant blue R-250, destained, dried, and autoradiographed at -70 C using X-Omat AR film (Eastman Kodak) and Cronex Lightning Plus intensifying screens (DuPont Industries, Wilmington, DE). Quantitative densitometric analysis of the autoradiograms was performed using an LKB Ultroscan XL Densitometer (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) with a beam width of 56 mm. In some experiments the radiolabeled bands were excised from the gel, and the radioactivity quantitated by y-counting. The two procedures yielded similar results.

Results Biological activity of ANBz-I-IGF-I Studies of mitogenic activity were restricted to the more highly photoreactive ANBz derivative. To determine if this derivative retained biological activity, the nonradioactive analog ANBz-I-IGF-I was synthesized, and its potency in stimulating DNA synthesis was measured. On a molar basis ANBz-I-IGF-I was approximately half as potent as native IGF-I in stimulating DNA synthesis (Fig. 1). However, at saturating concentrations this analog stimulated DNA synthesis to a level similar to that observed with the native hormone, indicating that it has full agonist activity. Identification of type I IGF receptor using photoreactive IGF-I derivatives To evaluate the usefulness of the photoreactive [125I] IGF-I derivatives for studying type I receptor metabo-


IGF-I RECEPTOR METABOLISM 100

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FIG. 1. Biological activity of ANBz-I-IGF-I. Quiescent monolayers of MG-63 cells were cooled to 2 C, the medium was aspirated, and 0.3 ml fresh medium (MCDB 104) containing 0.1% BSA, transferrin (1 ng/ ml), dexamethasone (0.10 nM), penicillin (100 U/ml), streptomycin (100 Mg/ml), and graded concentrations of IGF-I (•) or ANBz-I-IGF-I (O) was added to triplicate wells. After incubating for 90 min at 2 C in the dark all monolayers were photolyzed by exposure to 366 nm UV light for 3 min at a distance of 10 cm. [3H]Thymidine (1 jtCi) was then added to each well, and the monolayers were incubated at 37 C in a humidified atmosphere of 7% CO2-93% air. After 42 h the cells were processed for scintillation counting. The values shown are the mean ± SD of triplicate samples.

lism, monolayers of quiescent MG-63 cells were photoaffinity labeled with the derivatives at 2 C, and the labeled proteins were analyzed by SDS-PAGE and autoradiography. Using ANBz-125I-IGF-I, a single band with an apparent Mr of about 330,000 was observed when the electrophoresis was performed in the absence of reducing agent (not shown); when the sample was reduced with 100 mM dithiothreitol (DTT) before electrophoresis the band at 330,000 mol wt (Mr) disappeared and a major band of 130,000 Mr and a minor band of 100,000 Mr were observed (Fig. 2, lane A). Inclusion of 100 nM unlabeled IGF-I in the binding medium eliminated binding to both bands (Fig. 2, lane B); inclusion of either 900 nM insulin (Fig. 2, lane C) or 100 nM «IR-3 (Fig. 2, lane D) partially inhibited binding to both bands. This binding specificity identifies the photolabeled species as the type I IGF receptor and distinguishes it from the closely related insulin receptor (7, 8, 20). The band of 130,000 Mr in the reduced gels is characteristic of the binding (a) subunit of the type I IGF receptor and is also observed when [125I] IGF-I is cross-linked to MG-63 cells using the bifunctional cross-linking reagent DSS (Fig. 2, lane E). The band of 100,000 Mr is due to labeling of the /5-subunit of the type I IGF receptor; a similar band is observed when photoreactive insulin analogs are used to label insulin receptors (16, 17) and has been attributed to labeling of the 0-subunit by these analogs. In some experiments a faint band of 47,000 Mr was also observed (i.e. Fig. 5, lanes C and D); this band is due to binding

26.6 FIG. 2. Photoaffinity labeling of type I IGF receptors on MG-63 cells. Quiescent monolayers of MG-63 cells were cooled to 2 C and incubated in the dark with 4.8 nM ANBz-125I-IGF-I (lanes A-D and F) or 3.5 nM [126I]IGF-I (lane E) in the absence (lane A, E, and F) or presence of 100 nM IGF-I (lane B), 900 nM insulin (lane C), or 100 nM aIR-3 (lane D). After 90 min the monolayers were photolyzed by exposure to 366 nm UV light for 3 min at a distance of 10 cm. The samples in lanes E and F were then treated with 0.2 mM DSS. All monolayers were washed, solubilized with SDS-PAGE sample buffer, reduced with 100 mM DTT, and electrophoresed on a 5-15% linear gradient acrylamide gel. An autoradiogram of the fixed dried gel is shown. The numbers to the left show the positions and sizes (in thousands) of the Mr standards.

of the analog to IGF-binding protein(s) produced by MG63 cells (20). It is noteworthy that the very large Mr complexes observed when the bifunctional reagent DSS is used as the cross-linking agent (Fig. 2, lane E) are not observed when cross-linking is accomplished by photolabeling (Fig. 2, lane A); however, very large Mr complexes are observed when the photolabeled receptor is subsequently cross-linked using DSS (Fig. 2, lane F). This indicates that the large Mr bands observed with DSS are due to the formation of aggregates containing the type I receptors and not to binding of [125I] IGF-I to other high Mr IGF receptor forms, such as the type II IGF/mannose-6phosphate receptor. Similar results were obtained with ANPAH-125I-IGFI, except that the more intense photolysis conditions described above were necessary for optimal labeling (not shown). Internalization and degradation of the photoaffinity labeled IGF-I-receptor complex To assess the stability of the IGF-I-receptor complex under mitogenic conditions, MG-63 cells photolabeled with ANBz-125I-IGF-I at 2 C were transferred to 37 C and at various times solubilized and examined by SDSPAGE. Incubation at 37 C resulted in a time-dependent decrease in the intensities of both the 130,000 and



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IGF-I at 37 C in the dark for various times and then photolyzed and harvested. Using this procedure, labeled bands of 130,000 and 100,000 Mr were again observed (Fig. 5A). Trypsin treatment of these cells resulted in the near-quantitative loss of both of these bands and the appearance of a band of 54,000 Mr (fig. 5, lane B). On prolonged exposure bands of 120,000 and 82,000 Mr were also visible in the trypsin-treated cells (not shown). The near-quantitative loss of the 130,000 Mr band in the trypsinized cells indicates that under steady state conditions very few IGF-I-receptor complexes are present intracellularly; this finding corroborates the results of the preincubation photolysis experiments described above.

100,000 Mr bands (Figs. 3A and 4). Analysis of the data from five consecutive experiments as a first order rate process gave a half-life for the 130,000 Mr receptor band of 10.9 ± 1.6 h (mean ± SD; Fig. 4, inset). In a limited series of experiments, similar results were observed with ANPAH-125I-IGF-I (not shown). To determine if the photoaffinity-labeled IGF-I-type I receptor complex is internalized by MG-63 cells, its resistance to proteolysis by trypsin was determined. Trypsin treatment of cells maintained at 2 C resulted in the quantitative loss of the 130,000 Mr band and the appearance of major bands of 82,000 and 54,000 Mr and minor bands of 120,000 and 26,000 Mr or smaller (Fig. 3B, time zero); the quantitative loss of the 130,000 Mr band confirms that at the beginning of the 37 C incubation all of the photolabeled receptors were present on the cell surface. Incubation of the cells at 37 C resulted in the appearance of a trypsin-insensitive band of 130,000 Mr; this band was detectable as early as 20 min after transfer to 37 C, reached a maximum at approximately 1 h, and declined thereafter (Fig. 3B). In four experiments an average of 6.7% (range, 5.1-7.6%) of the initially labeled cell surface receptor pool was present in the intracellular pool after 1 h at 37 C. This declined to less than 1% by 4 h, but remained detectable as long as 18 h in some experiments (Fig. 3B). It is possible that the internalization of the photoaffinity-labeled IGF receptor is different from that of the native receptor. To evaluate this possibility postincubation photolysis experiments were performed; in these experiments the cells were incubated with ANBz-125!-

Effects of lysosomotropic agents on the metabolism of photoaffinity-labeled IGF-I-receptor complex We have previously shown that lysosomotropic agents inhibit the degradation of IGF-I and stimulate a timedependent intracellular accumulation of the hormone by MG-63 cells (20). To determine if these agents also affect the metabolism of the IGF-I-type I receptor complex, both pre- and postincubation photolysis experiments were performed. In the preincubation photolysis experiments, cells that had been photolabeled at 2 C were incubated with specific lysosomotropic agents at 37 C and at appropriate times were processed and examined by SDS-PAGE. Under these conditions, chloroquine (200 fiM), methylamine (40 mM), and monensin (50 IJ.M) each slowed the degradation of the photoaffinity-labeled re-

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FIG. 3. Time course of photoaffinity-labeled IGF-I-type I receptor complex degradation (A) and internalization (B). MG-63 cells photolabeled at 2 C were washed and incubated at 37 C. At the times indicated, duplicate monolayers were removed from the incubator. One monolayer was immediately solubilized (A); the other was cooled to 2 C, treated with trypsin (100 fig/ml) for 2 h, and then solubilized (B). All samples were reduced with 100 mM DTT and electrophoresed on 5-15% gradient acrylamide gels. Autoradiograms of the fixed dried gels are shown. The autoradiogram shown in A was exposed for 28 h while that shown in B was exposed for 161 h.


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IGF-I RECEPTOR METABOLISM 100

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FIG. 4. Time course of photoaffinity-labeled IGF-I-receptor complex degradation. The 130,000 Mr band in the autoradiogram shown in Fig. 3A was scanned using an LKB densitometer. The intensity of this band at each time is expressed relative to that of the band at zero time (O). The line is the best fit for the data from five consecutive experiments. The inset shows the data from the five experiments plotted as a first order rate process.

Ceptor complex; surprisingly, however, these agents had little effect on the size of the intracellular (i.e. trypsin insensitive) IGF-I-receptor complex pool. For example, in the experiment shown in Fig. 6, 200 /zM chloroquine decreased degradation of the labeled receptor complex from 29% to 8% while increasing the intracellular receptor complex pool size by less than 2%. In contrast, in isolated adipocytes chloroquine is reported to increase the intracellular insulin receptor pool size by 30% (15, 16). To further evaluate the effects of chloroquine on IGFI-receptor metabolism, postincubation photolysis experiments were performed. In these experiments quiescent monolayers were incubated at 37 C with ANBz-125I-IGFI in the presence of 200 /*M chloroquine for times ranging up to 6 h and were then photolyzed (Fig. 5, lanes C and D). Under these conditions the previously identified receptor bands of 130,000 and 100,000 Mr as well as new bands of 7,000, 40,000-47,000, and 70,000-82,000 Mr were observed (Fig. 5, lane C). The band at 7,000 Mr is uncross-linked IGF-I whose degradation was inhibited by chloroquine; the band at 40,000-47,000 Mr is due to labeling of IGF-I-binding proteins present on these cells (see below); the nature of the broad band at 70,00082,000 Mr is unknown. The intensities of all bands were increased in the chloroquine-treated cells (compare Fig. 5, lanes A and C); this increase averaged 1.4-fold in three experiments and can be attributed at least in part to the increased stability (resulting in a higher effective concentration) of IGF-I in the presence of chloroquine (20). Trysinization of the chloroquine-treated cells markedly decreased the intensity of the 130,000 and 100,000 Mr bands and resulted in the appearance of a band of 54,000 Mr (Fig. 5, lane D). The near-quantitative loss of

26.6

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FIG. 5. Postincubation photolysis of MG-63 cells in the absence and presence of chloroquine. Quiescent monolayers of MG-63 cells were incubated at 37 C in the dark in basal medium containing 5.8 nM ANBz-125I-IGF-I without (lanes A and B) or with (lanes C and D) 200 HM chloroquine (CQ). After 4 h the monolayers were photolyzed by exposure to 366 nm UV light for 3 min at a distance of 10 cm. One plate from each group was immediately solubilized (lanes A and C). The remaining plates were cooled to 2 C and treated with trypsin for 2 h before solubilization. All samples were then reduced with 100 mM DTT and electrophoresed on a 5-15% gradient gel. An autoradiogram of the fixed dried gel is shown. t h e 130,000 M r b a n d in t h e trypsinized cells indicates

that under steady state conditions chloroquine has no appreciable effect on the size of the intracellular IGF-Ireceptor complex pool and supports the results obtained in the preincubation labeling experiments described above. Trypsinization of the chloroquine-treated cells also substantially decreased the intensity of the 47,000 Mr band (Fig. 5, lane D). The size and trypsin sensitivity of this band indicate that it is due to labeling of IGFbinding protein(s) present on the cell surface. The marked increase in labeling of this band in the chloroquine-treated cells (compare Fig. 5, lanes A and C) suggests that chloroquine increases the stability of the binding protein or prevents its release from the cell surface. The cellular location of the unidentified band of Mr 80,000, prominent in the chloroquine-treated cells, could not be determined, since trypsinization can generate a band of similar size (Figs. 2B and 6).

Discussion The present study was undertaken to elucidate the fate of the IGF-I-type I receptor complex during IGF-I-stimulated mitogenesis. To investigate this, photoreactive iodinated IGF-I analogs were synthesized and used to



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CQ(200/iM) Trypsin

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FIG. 6. Preincubation photolysis of MG-63 cells in the absence and presences of chloroquine. Quiescent monolayers of MG-63 cells were incubated at 2 C in the dark in basal medium containing 5.8 nM ANBz125 I-IGF-I. After 90 min the monolayers were photolyzed and washed once, and fresh basal medium without (lanes A and B) or with (lanes C and D) 200 nM chloroquine (CQ) was added. The plates were then incubated at 37 C. After 4 h one plate from each group was solubilized (lanes A and C); the remaining plates were cooled to 2 C and trypsinized for 2 h before solubilization. All samples were reduced with 100 mM DTT and electrophoresed on a 5-15% gel. An autoradiogram of the fixed dried gel is shown.

label MG-63, an IGF-responsive human osteosarcoma cell line, in situ. One derivative, ANBz-125I-IGF-I, was found to be particularly photoreactive and to retain biological activity. This derivative photolabeled a cell surface component on MG-63 cells with the size and binding specificity characteristic of the type I IGF receptor. Under mitogenic conditions this photolabeled complex was relatively stable, with a half-life of about 11 h; moreover, under these conditions the majority of the complex remained susceptible to hydrolysis by trypsin, indicating that it remained distributed on the cell surface and did not undergo substantial ligand-induced intracellular sequestration. Lysosomotropic agents inhibited degradation of the complex, but did not affect its distribution between the cell surface and interior. These findings suggest that the IGF-I-type I receptor complex functions while located at or near the cell surface and that the complex does not undergo rapid ligand-induced down-regulation. This study is the first to specifically examine the fate of the IGF-I-type I receptor complex under mitogenic conditions. Previous studies which employed [125I]IGF binding to monolayer cells to monitor receptor metabolism (21-23) are difficult to interpret because monolayer cells can express multiple IGF-binding species which may mask type I receptor binding (6, 7, 20, 24, 25). In

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the present study this difficulty was overcome by using a photoaffinity-labeling procedure which allowed the type I IGF receptor to be specifically identified, and its metabolism examined directly. It is noteworthy that a previous study (28) employed ANPAH-125I-IGF-I to investigate IGF receptor metabolism in rat chondrocytes in vitro, but in that study only the type II IGF/mannose6-phosphate receptor was labeled by the analog. The type II IGF receptor on MG-63 cells has a low affinity for IGF-I (20) and was not labeled with either of the photoreactive IGF-I analogs used in this study. A major finding of this study is that during mitogenesis the IGF-I-type receptor complex remains at the cell surface and does not undergo rapid IGF-induced downregulation. Although this result could be due to aberrant metabolism of the covalently modified receptor, this seems unlikely, since similar results were obtained regardless of whether the photolabeling was performed before or after the 37 C incubation with the analog. As susceptibility to hydrolysis by trypsin was used to distinguish cell surface from internalized receptors, internalization of trypsin by the cells could also lead to spurious results; to limit this, care was taken to precool the monolayers and all solutions to 2 C and to maintain them at this temperature during the trypsinization procedure. While temperature-independent internalization of the enzyme has not been rigorously excluded, this possibility seems unlikely, especially since some trypsininsensitive complex was readily detectable during the first few hours of incubation at 37 C. As discussed below, it is likely that internalized IGF-I-receptor complexes are recycled back to the cell surface. If recycling were temperature independent, then the internal pool would not be detected using these procedures; however, there is no evidence to support a temperature-independent recycling mechanism. We conclude, therefore, that during mitogenesis the majority of the IGF-receptor complex remains present on the cell surface and is not sequestered internally. The apparent stability of the IGF-I-type I receptor complex in MG-63 cells and its continued presence at the cell surface are in marked contrast to the results reported for the PDGF- and EGF-receptor complexes. Bishayee et al. (30) have investigated PDGF receptor metabolism by MG-63 cells and have shown that in these cells the complex is rapidly endocytosed and is degraded with a half-life of less than an hour. Similar results have been reported for PDGF receptors in other cell types (14, 31). Using a photoaffinity-labeling technique similar to that used in the present studies, Das and Fox (13) have shown that in Swiss 3T3 cells the EGF-receptor complex is rapidly endocytosed. Although in this cell type the complex is also rapidly degraded (t./2 = ~1 h), in many other cell types the endocytosed EGF receptor undergoes


IGF-I RECEPTOR METABOLISM efficient recycling (32). The metabolism of the insulin-receptor complex has been investigated in a number of cell types and with a wide variety of experimental techniques, including photoaffinity labeling (15-17, 32, 33). While isolated adipocytes the photoaffinity-labeled insulin-receptor complex has a half-life of only about 2 h (16), in most other cell types, including isolated hepatocytes (33), H4 hepatoma cells (17), and 3T3-L1 adipocytes (34), the complex is relatively stable, with a half-life of 6 h (34) or more (17, 33). Thus, in most cell types the stability of the insulin-receptor complex is similar to that observed for the IGF-I-receptor complex in MG-63 cells. The finding that ligand binding does not dramatically alter the distribution of the IGF-I-receptor complex between the cell surface and interior does not exclude significant internalization of the complex, provided that an efficient recycling mechanism also exists. Indeed, studies in both the insulin and EGF receptor systems indicate that receptor down-regulation is a dynamic process in which the steady state distribution of receptors is determined by the relative rates of internalization and recycling (32-37). The finding that in MG-63 cells IGFI is internalized by a type I receptor-dependent process (20) strongly suggests that the receptor itself is also internalized; if this is so, the relative stability of the complex and the absence of a large intracellular receptor complex pool suggest that in this system internalized receptors undergo relatively rapid and efficient recycling back to the cell surface. The observation that lysosomotropic agents do not substantially alter the distribution of the IGF-I-receptor complex between the plasma membrane and cell interior indicates that these agents do not affect the recycling process. This is in contrast to the adipocyte insulin-receptor system, where these agents cause intracellular accumulation of the complex by inhibiting its recycling. The findings that the IGF-receptor complex is relatively stable and that it does not undergo rapid quantitative down-regulation may have functional significance. PDGF induces rapid degradation of its receptor, and only a transient exposure to PDGF is required to initiate DNA synthesis (38). In contrast, kinetic studies of IGF action (38, 39), including studies in MG-63 cells (Furlanetto, R. W., and R. Womer, unpublished observations), indicate that IGF pathway activity is required during most of the prereplicative (Gl) phase of the cell cycle. Such prolonged exposure to IGF would not be expected to be necessary if its receptor underwent rapid degradation. Similarly, it has been postulated that internalization of the EGF-receptor complex is essential to bring the activated complex into proximity with its intracellular substrates (18). The observation that the IGF-Ireceptor complex remains on the cell surface suggests,

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then, that its substrates are located at or near the plasma membrane. In summary, we have used a photoreactive IGF-I analog to study the physiological fate of the IGF-I-type I receptor complex in the MG-63 human osteosarcoma cell line. The results indicate that under conditions where IGF-I stimulates DNA synthesis, the IGF-I-receptor complex is relatively stable, with a half-life of about 11 h; moreover, under these conditions most of the complex remains distributed on the cell surface and does not undergo rapid ligand-induced down-regulation. These observations suggest a model for IGF action in which the IGF-receptor complex functions while present at or near the cell surface.

Acknowledgements The author would like to thank Drs. K. Frick, J. N. Livingston, and R. Womer for their helpful suggestions, Mr. J. N. DiCarlo for his expert technical assistance, and Ms. W. Roesch for her help in preparing this manuscript.

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Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells.

A unique receptor-independent mechanism by which insulinlike growth factor I regulates the availability of insulinlike growth factor binding proteins in normal and transformed human fibroblasts.

insulinlike growth factor II receptors.

Expression of functional growth hormone receptors in human granulosa cells.

Nerve growth factor receptors on human melanoma cells in culture.

In Vitro Metabolism of Flucetosulfuron by Human Liver Microsomes.

Metabolism of androgens in vitro by human foetal skin.

In vitro metabolism of thyroxine by rat and human hepatocytes.

The metabolism of 2'-deoxycoformycin by L1210 cells in vitro.

Growth and responsiveness of human granulocytic leukemic cells in vitro.

Presence of epidermal growth factor, platelet-derived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro.

Delivery of iron to human cells by bovine transferrin. Implications for the growth of human cells in vitro.

Constitutive phosphorylation of the receptor for insulinlike growth factor I in cells transformed by the src oncogene.

Overexpression of the human insulinlike growth factor I receptor promotes ligand-dependent neoplastic transformation.

Growth control of human colon cancer cells by vitamin D and calcium in vitro.

Autocrine stimulation by erythropoietin and autonomous growth of human erythroid leukemic cells in vitro.

Insulinlike growth factor I: a potent mitogen for human osteogenic sarcoma.

Alterations in the growth rate and metabolism of Chinese hamster cells in vitro (38530).

Up-regulation of thrombomodulin by activation of histamine H1-receptors in human umbilical-vein endothelial cells in vitro.

Clonal growth in vitro by mouse Kupffer cells.

Microencapsulation of human cells: its effects on growth of normal and tumour cells in vitro.

Regulation of Schwann cell surface and truncated nerve growth factor receptors in vitro by axonal components.

Differentiation of population of peripheral blood lymphocytes into cells bearing sheep erythrocyte receptors in vitro by human thymic extract.

In vitro analysis of transport and metabolism of 4'-thiothymidine in human tumor cells.