©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutation of a Conserved Amino Acid Residue (Tryptophan 1173) in the Tyrosine Kinase Domain of the IGF-I Receptor Abolishes Autophosphorylation but Does Not Eliminate Biologic Function (*)

(Received for publication, July 26, 1994; and in revised form, November 16, 1994 )

Vicky A. Blakesley Hisanori Kato Charles T. Roberts Jr. Derek LeRoith (§)

From the Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The amino acid sequence of the tyrosine kinase domain of the insulin-like growth factor-I (IGF-I) receptor is 84% identical to the sequence of the analogous region of the insulin receptor. A naturally occurring mutation of the tryptophan residue at position 1200 of the insulin receptor to serine results in impaired beta subunit autophosphorylation of wheat germ agglutinin-purified receptors, severely impaired thymidine incorporation and moderately reduced glycogen synthesis; however, glucose uptake was unaffected. To study the importance of this residue in IGF-I receptor function, we mutated the analogous tryptophan residue at position 1173 of the IGF-I receptor to serine and overexpressed the mutant receptor in NIH-3T3 cells. In cell lines overexpressing this mutant IGF-I receptor, beta subunit autophosphorylation was severely reduced. Additionally, the overexpressed mutant receptors exhibited a dominant-negative effect on IGF-I-stimulated autophosphorylation of endogenous mouse IGF-I receptors. Phosphorylation of insulin receptor substrate (IRS)-1 in intact cells by the mutant IGF-I receptors was similar to the level of IRS-1 phosphorylation seen in the parental NIH-3T3 cells, but there was no obvious dominant-negative effect on IRS-1 phosphorylation. Wheat germ agglutinin-purified mutant receptors were as active in phosphorylating poly(Glu,Tyr) 4:1 as wild-type IGF-I receptors, suggesting that, in intact cells, additional factors are necessary in order for the IGF-I receptor to phosphorylate IRS-1. Thymidine incorporation was severely reduced in one clone overexpressing the mutant IGF-I receptor and abolished in a second clone. Glucose uptake in both clones was reduced to about half of that seen in a cell line overexpressing wild-type IGF-I receptors. Thus, we propose that the tryptophan residue at position 1173 of the IGF-I receptor is important in the regulation of autophosphorylation in vivo. This study again confirms that high levels of autophosphorylation are not required for mediation of all of the biologic activities of the IGF-I receptor.


INTRODUCTION

The insulin-like growth factor-I (IGF-I) (^1)receptor is a transmembrane tyrosine kinase that mediates the biological effects of IGF-I (Yarden and Ullrich, 1988) and many of the actions of IGF-II. The IGF-I receptor is structurally homologous to the insulin receptor and the insulin receptor-related receptor. The members of this receptor family are hetero-tetrameric glycoproteins consisting of two extracellular ligand-binding alpha subunits and two beta subunits with extracellular, transmembrane, intracellular juxtamembrane, tyrosine kinase, and carboxyl-terminal domains (Ullrich et al., 1985, 1986; Ebina et al., 1985; Shier and Watt, 1989).

After ligand binding, the IGF-I and insulin receptors may mediate both similar and distinct physiological functions. The mechanisms by which the IGF-I and insulin receptors mediate these distinct functions remain to be fully elucidated, and it has been difficult to reconcile the specificity of function with the structural homology of these receptors (Adamo et al., 1991). In each case, binding of ligand results in receptor autophosphorylation, association with intracellular proteins, phosphorylation of intracellular substrates, and activation of specific signaling processes that are involved in growth regulation or metabolism (Lowe, 1991). Mutants of both the IGF-I and insulin receptors that are incapable of binding ATP, or that lack the triple tyrosine cluster that constitutes the major autophosphorylation site in the tyrosine kinase domain, are not autophosphorylated and are not able to initiate the normal cascade of intracellular events that occurs subsequent to receptor activation (Ebina et al., 1987; Kato et al., 1993, 1994; Gronborg, 1993). Mutations outside the tyrosine kinase domain also modulate the ability of the IGF-I and insulin receptors to mediate mitogenic and metabolic functions. Replacement of the poorly conserved carboxyl-terminal domain of the insulin receptor by the carboxyl terminus of the IGF-I receptor resulted in decreased insulin stimulation of intact cell autophosphorylation, phosphorylation of insulin receptor substrate-1 (IRS-1), c-fos expression, ornithine decarboxylase activity, and thymidine incorporation. Insulin-stimulated glucose uptake was increased as compared to cells overexpressing wild-type insulin receptors. The reciprocal mutation (replacement of the carboxyl terminus of the IGF-I receptor with that of the insulin receptor) had only minimal effects on functions as compared to cells overexpressing the wild-type IGF-I receptor (Faria et al., 1994). These findings are consistent with the notion that the relatively nonconserved COOH-terminal domains of the IGF-I receptor and insulin receptor are important for receptor-specific signal transduction.

In addition to mutants of the tyrosine kinase domain deficient in ATP binding or autophosphorylation, other naturally occurring mutations in the tyrosine kinase domain of the insulin receptor have been identified and have been shown to alter receptor-mediated functions. One of these, a tryptophan to serine substitution at position 1200 of the insulin receptor (numbering system of Ebina et al.(1985)) was identified in a patient with the type A syndrome of insulin resistance (Moller et al., 1990). In Chinese hamster ovary cells overexpressing this mutant insulin receptor, insulin binding and receptor internalization were normal, but autophosphorylation and phosphorylation of IRS-1 were severely impaired (Moller et al., 1990). Insulin-stimulated glucose uptake and glucose incorporation into glycogen measured in cells expressing mutant insulin receptors were increased as compared to control neomycin-resistant Chinese hamster ovary cells. However, there was no increase in insulin-stimulated thymidine incorporation over those seen in control cells (Moller et al., 1991).

In this study, the homologous tryptophan at position 1173 (numbering system of Ullrich et al., 1986) in the IGF-I receptor was mutated to serine. This tryptophan is found in a highly conserved 12-amino acid segment in the carboxyl-terminal portion of the tyrosine kinase domain. The function of this mutated IGF-I receptor was studied in NIH-3T3 cells to determine if this mutation affected subsequent intracellular signaling and, in particular, if it had a differential effect on these functions.


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases were purchased from New England Biolabs (Beverly, MA), Boehringer Mannheim (Indianapolis, IN), and Life Technologies, Inc. (Gaithersburg, MD). Cell culture media and reagents were purchased from Biofluids, Inc. (Rockville, MD) and Advanced Biotechnologies (Columbia, MD). Insulin-free bovine serum albumin (BSA, fraction V) was obtained from Armour (Kankakee, IL). Recombinant human IGF-I, monoclonal antiphosphotyrosine antibody (clone 4G10), polyclonal anti-IGF-I receptor antibody, and fetal bovine serum were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Recombinant antiphosphotyrosine RC20H horseradish peroxidase-conjugate was purchased from Transduction Laboratories (Lexington, KY). Monoiodinated I-IGF-I, [^3H]thymidine, 2-[^14C]deoxyglucose, horseradish peroxidase-conjugated anti-rabbit immunoglobulin, horseradish peroxidase-conjugated anti-mouse immunoglobulin, and the ECL detection kit were purchased from Amersham (Arlington Heights, IL). Prestained high-molecular weight protein standards were purchased from Life Technologies, Inc. and Sigma. [-P]ATP was purchased from DuPont NEN (Boston, MA). Wheat germ agglutinin-agarose was purchased from EY Laboratories, Inc. (San Mateo, CA).

Construction of the Mutant IGF-I Receptor cDNA

The wild-type human IGF-I receptor expression vector has been previously described (Kato et al., 1993). Mutation of the human IGF-I receptor cDNA at amino acid residue 1173 (numbering system used is that of Ullrich et al.(1986)) was performed by in vitro site-directed mutagenesis using the Double Take Mutagenesis Kit (Stratagene, Inc., La Jolla, CA) and a pBluescript II plasmid containing an EcoRI-BamHI fragment of the human IGF-I receptor (previously described by Kato et al.(1993)). The sequence of the bridging primer was 5`-GCCGCCACCGCGGTGGAGCTCCAATTCGCC-3` (the SacI site used for mutagenesis is underlined) and the sequence of the extension primer was 5`-AGCTCCACCGCGGTGGCGGCCGCT-3`. The sequence of the mutagenic primer was 5`-CCAGTGTGGCGATCTCACTTAAGACGACCCCGAAGGA-3` (the AflII site used for mutagenesis is underlined, the mutated serine codon is shown in italics). This mutation (W1173S) converted the tryptophan codon to a serine codon. The cDNA sequence of W1173S was confirmed by sequencing. The W1173S cDNA in pBluescript II was excised with SalI and NotI and cloned into a bovine papilloma virus-derived mammalian expression vector (pBPV; Pharmacia Biotech Inc., Piscataway, NJ) that had been linearized with XhoI and NotI.

Cell Culture and Transfection

All NIH-3T3 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO(2) at 37 °C. NIH-3T3 cells were co-transfected with 20 µg of mutant expression vector or insert-less pBPV plus 1 µg of pMCINeo (Clontech, Palo Alto, CA) in LIPOFECTIN reagent (Life Technologies, Inc.). Selection was carried out as described previously (Kato et al., 1993). Clones overexpressing IGF-I receptors were selected based on results of IGF-I binding assays as described previously (Kato et al., 1993). Stably transfected cells were maintained in DMEM supplemented with 10% fetal calf serum, antibiotics, and 500 µg/ml G418 (Geneticin; Life Technologies, Inc.). Cells were split for each experiment and cultured in serum-supplemented DMEM without G418. Serum-free medium containing 1% BSA (DMEM with 1% BSA, 20 mM Hepes, pH 7.5, and antibiotics) was used in assays of IGF-I receptor binding, thymidine incorporation, and 2-deoxyglucose uptake. Serum-free medium containing 0.1% BSA, 20 mM Hepes, pH 7.5, and antibiotics was used for receptor phosphorylation studies.

Intact Cell Tyrosine Phosphorylation

Confluent cells in 100-mm plates were serum-starved overnight and then incubated either with or without IGF-I (100 nM) for 1 min at 37 °C. The cells were washed rapidly with ice-cold phosphate-buffered saline and lysed in the presence of 50 mM Hepes, pH 7.9, 100 mM NaCl, 10 mM EDTA, 1% Triton X-100, 4 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 mM sodium fluoride. Cell lysates were cleared by centrifugation. Protein content was determined by the method of Bradford using a protein assay kit (Bio-Rad). Equal amounts of protein (up to 20 µg) were reduced by beta-mercaptoethanol and separated in a Laemmli discontinuous buffer system (Laemmli, 1970) with a 7.5% resolving gel. Resolved proteins were electrophoretically transferred to nitrocellulose membrane (Hybond, ECL, Amersham, Arlington Heights, Il). The amount of IGF-I receptor present was determined by immunoblotting with a polyclonal anti-IGF-I-receptor alpha subunit antibody at a 1:100 dilution, according to recommendations of the supplier (UBI, Lake Placid, NY). This antibody was detected by horseradish peroxidase-conjugated anti-rabbit immunoglobulin (1:5000 dilution) using an ECL system. Based on these immunoblots, equal amounts of receptor were fractionated by SDS-PAGE as described above. Tyrosine-phosphorylated proteins were immunoblotted with monoclonal anti-phosphotyrosine antibody (clone 4G10) (1:1000 dilution) and detected with horseradish peroxidase-conjugated anti-mouse immunoglobulin (1:5000 dilution) using an ECL system. Phosphorylated IRS-1 from whole cell lysates was detected using a 1:2500 dilution of the RC20H antibody according to manufacturer's specifications.

Wheat Germ Agglutinin (WGA) Purification of Solubilized Membranes

Confluent cells were washed with ice-cold phosphate-buffered saline and frozen on an ethanol/dry ice mixture. The cells were thawed on ice in the presence of freshly prepared lysis buffer (50 mM Hepes, pH 7.6, 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 100 units/ml bacitracin). The solubilized receptors were partially purified over a WGA-agarose column as described previously (Shemer et al., 1987). Typically, the third 1-ml fraction eluted from the column was used for ligand binding and phosphorylation of exogenous substrate.

Solubilized IGF-I Receptor Binding Assays

I-IGF-I binding assays were done essentially as described previously (Shemer et al., 1987) using 25 µl of freshly prepared WGA-purified receptors (20 µg/µl). The assay was incubated for 16 h at 4 °C. Bound/free counts were used to calculate the appropriate dilution factor for receptor preparations in each to achieve equivalent ligand-binding concentrations. Receptor preparations were stored at -70 °C.

Phosphorylation of Exogenous Substrate

Tyrosine kinase activity was assayed as described by Shemer et al.(1987). Briefly, aliquots of WGA-purified receptor preparations were stimulated at 4 °C overnight in the absence or presence of IGF-I (100 nM) in a total volume of 30 µl of buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 0.04% Triton X-100, and 0.01% BSA). Phosphorylation was initiated by addition of the artificial substrate poly(Glu,Tyr) 4:1 (Sigma), 5 µCi of [-P]ATP (final activity 6 µCi/nmol, specific activity 6000 Ci/mmol), 50 µM ATP, 1 mM CTP, and 50 mM magnesium chloride to a total volume of 90 µl. The reaction was continued for 30 min at 24 °C. The reaction was stopped by spotting 70 µl of reaction mixture on a 4-cm^2 Whatman No. 3MM filter paper and immediately immersing in ice-cold 10% trichloroacetic acid and 10 mM sodium pyrophosphate. After extensive washing the filter paper was dried and the filter-bound radioactivity was determined by counting in a liquid scintillation counter. Nonspecific filter-bound radioactivity was measured on a sample containing WGA-purified receptors, buffer, and radiolabeled ATP.

In Vitro Autophosphorylation

Equivalent ligand-binding amounts of WGA-purified receptors from WT, b13, and b14 were assayed for beta subunit autophosphorylation in vitro. The WGA-purified receptors were diluted in a Hepes/NaCl buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 50 mM MgCl(2), 0.04% Triton X-100, and 0.01% BSA) and stimulated for 1 h at 24 °C in the presence or absence of 100 nM IGF-I. Autophosphorylation was initiated by the addition of 50 µM ATP and 200 µM CTP. The reaction was continued for 15 min at 24 °C. The reaction was stopped by addition of Laemmli loading buffer (Laemmli, 1970). The proteins were resolved by 7.5% SDS-PAGE and transferred to a nitrocellulose membrane as described above. Tyrosylphosphorylated proteins were detected by immunoblotting with a monoclonal antiphosphotyrosine antibody as described above for intact cell tyrosine phosphorylation.

Thymidine Incorporation

Cell monolayers in 12-well plates were grown to quiescence in serum-free DMEM with 1% BSA for 24 h. Different concentrations of IGF-I in fresh serum-free DMEM (1% BSA) were added and the cells were incubated for an additional 16 h. The medium was replaced with fresh serum-free DMEM (1% BSA) containing 1 µCi/well of [methyl-^3H]thymidine, and incubated for an additional hour. The cells were rinsed twice with ice-cold phosphate-buffered saline, twice with ice-cold 5% trichloroacetic acid, and twice with ice-cold 95% ethanol. The cells were lysed in 0.3 ml of 1 N NaOH, neutralized with 0.3 ml of HCl, and counted in a liquid scintillation counter. Triplicate wells were solubilized with 0.03% SDS, and protein content was measured as described above.

2-Deoxyglucose Uptake

Confluent cell monolayers in 12-well plates were washed twice with Krebs-Ringer phosphate buffer containing 1% BSA and 20 mM Hepes, pH 7.4 (DG buffer). Fresh DG buffer containing varying concentrations of IGF-I was added and the cells incubated for 20 h. The cells were washed twice with DG buffer and incubated in DG buffer for 40 min. Deoxy-D-[1-^14C]glucose (0.5 µCi/well) and 2-deoxy-D-glucose (0.2 mM final concentration) were added for the final 10 min of incubation. The cells were rinsed three times with ice-cold DG buffer containing 0.2 mM phloretin, solubilized with 0.03% SDS, and counted in a liquid scintillation counter. Triplicate wells were solubilized with 0.03% SDS and protein content was measured as described above.


RESULTS

Cell Lines

NIH-3T3 mouse fibroblast cells overexpressing wild-type or mutant IGF-I receptors were obtained as described under ``Experimental Procedures.'' Two separate clones overexpressing the mutant IGF-I receptor in which the tryptophan at position 1173 was converted to serine were used for this study and designated W1173Sb13 and W1173Sb14 (abbreviated designations are b13 and b14). A new clone (NWTb3) overexpressing wild-type IGF-I receptors was used in this study, and will be designated WT. All clones overexpressing IGF-I receptors had similar numbers of cell-surface IGF-I receptors as determined by Scatchard analysis (Table 1) (Scatchard, 1949). Neo cells are NIH-3T3 cells co-transfected with a neomycin-resistant plasmid and the pBPV vector. The particular clone (pNeo1) used was selected for neomycin resistance, and the presence of pBPV was determined by a slot-blot analysis (data not shown).



Tyrosine Phosphorylation of IGF-I Receptors and IRS-1 in Intact Cells

IGF-I stimulation of tyrosine phosphorylation of the IGF-I receptor and other proteins in intact cells was studied by Western blot analysis using antiphosphotyrosine antibodies. A typical immunoblot of whole cell lysates for cells not stimulated or stimulated with IGF-I (100 nM) for 1 min at 37 °C is shown in Fig. 1A. The membrane was blotted with a monoclonal antiphosphotyrosine antibody (clone 4G10) and phosphotyrosyl proteins were detected as described under ``Experimental Procedures.'' Similar numbers of IGF-I receptors from WT, b13, and b14 clones were applied to each lane. Receptors were detected in independent gels immunoblotted with an anti-IGF-I receptor alpha subunit antibody (data not shown). The amount of protein from Neo cells was approximately that loaded for b13 and b14. Neo cells exhibited low levels of tyrosine phosphorylation of the beta subunit of the IGF-I receptor in the basal state that increased upon stimulation by IGF-I. The level of tyrosine phosphorylation of the beta subunit of the IGF-I receptor after stimulation in WT cells was markedly elevated as compared to Neo cells. Two clones overexpressing the mutant IGF-I receptor (b13 and b14), however, exhibited less IGF-I-stimulated autophosphorylation than did Neo, which expressed much fewer IGF-I receptors. To evaluate the tyrosine phosphorylation of IRS-1, cells were stimulated with IGF-I as described above, equal amounts of protein from the whole cell lysates were fractionated by SDS-PAGE and, after transfer to a membrane, were immunoblotted with RC20H (an antiphosphotyrosine antibody conjugated to horseradish peroxidase). A typical immunoblot using RC20H is shown in Fig. 1B. The ability of IGF-I to stimulate phosphorylation of a 185-kDa (IRS-1) protein was reduced in cells expressing the mutant receptors (clones b13 and b14) as compared to WT. The level of IRS-1 phosphorylation in the b13 and b14 clones was approximately equal to that seen in Neo.


Figure 1: Tyrosine phosphorylation in intact cells. Whole cells were stimulated with IGF-I (0 nM or 100 nM) as described under ``Experimental Procedures.'' Protein content in cleared whole-cell lysates was determined. All samples were separated by 7.5% SDS-PAGE. The positions of protein molecular weight standards are indicated in each panel. Each panel is representative of three individual experiments. Panel A, equal amounts of IGF-I receptors from WT, b13, and b14 cells were applied to the gel. pNeo1 was loaded as the amount of protein similar to that loaded for b13 and b14 extracts. The transferred proteins were immunoblotted with antiphosphotyrosine antibody (monoclonal, mouse) and detected by a second antibody conjugated to horseradish peroxidase. Panel B, equal amounts of cleared whole cell lysate were separated by SDS-PAGE. After transfer to a nitrocellulose membrane the proteins were immunoblotted with RC20H (monoclonal antiphosphotyrosine antibody conjugated to horseradish peroxidase).



Autophosphorylation by Wheat Germ Agglutinin-purified IGF-I Receptors

The IGF-I-stimulated autophosphorylation of WGA-purified receptors was investigated using two independent preparations of each receptor. The results of a typical assay are shown in Fig. 2. A 1-min exposure of the Western blot is presented in panel A. Basal (unstimulated) phosphorylation of WGA-purified receptors from WT cells was detectable. IGF-I-stimulated autophosphorylation was severalfold higher than basal for the WT receptors. There was no evidence of IGF-I-stimulated autophosphorylation of mutant receptors in either clone as detected by a 1-min exposure or a 15-min exposure of the Western blot (panel B).


Figure 2: Autophosphorylation of WGA-purified IGF-I receptors. Equal amounts of WGA-purified receptors, as determined by binding assays, were unstimulated or stimulated for 1 h at 24 °C. The activated receptors were then treated with 50 µM ATP and 200 µM CTP for 15 min at 24 °C. The tyrosylphosphorylated proteins were detected by Western blotting with a monoclonal antiphosphotyrosine antibody following resolution through a 7.5% SDS-PAGE and electrotransfer to a nitrocellulose membrane. One of two experiments is shown. Each experiment used independently prepared WGA-purified receptors. The positions of protein molecular weight standards are indicated in Panel A. Panel A, Kodak X-AR film was exposed for 1 min against the Western blot. Panel B, Kodak X-AR film was exposed for 15 min against the same nitrocellulose filter as in panel A.



Phosphorylation of an Exogenous Substrate by Wheat Germ Agglutinin-purified IGF-I Receptors

To test if the mutant IGF-I receptor tyrosine kinase was capable of phosphorylating an exogenous substrate, WGA-purified receptors were prepared from lysates of b13 and b14 cells, and compared to receptors purified from WT cells. Equal numbers of receptors, as determined by I-IGF-I binding, were used for these assays. These partially purified receptors were stimulated in vitro with IGF-I and then allowed to phosphorylate poly(Glu,Tyr) 4:1 in the presence of radiolabeled ATP. A typical experiment is presented in Fig. 3. In each of two experiments performed, 100 nM IGF-I-stimulated poly(Glu,Tyr) 4:1 phosphorylation was about 2-3-fold using WGA-purified receptors from all three cell lines. Phosphorylation with 3 nM IGF-I stimulated receptors was approximately equivalent with all receptors.


Figure 3: In vitro tyrosine kinase activity. Equal amounts of WGA-purified receptors, as determined by binding assays, were unstimulated or stimulated overnight with 3 or 100 nM IGF-I. The activated receptors were then assayed for their ability to phosphorylate the exogenous substrate poly(Glu,Tyr) 4:1. One of the two experiments is shown. Each data point was done in triplicate and represents the mean ± S.E. after subtraction of nonspecific filter-bound counts. The receptors were unstimulated (hatched bars), stimulated with 3 nM IGF-I (solid bars), or stimulated with 100 nM IGF-I (shaded bars).



IGF-I Stimulation of Thymidine Incorporation

The effect of increasing concentrations of IGF-I on thymidine incorporation measured in NIH-3T3 cells overexpressing normal human IGF-I receptors or mutant receptors is shown in Fig. 4. In one of the mutant clones (b13), there was no increase in IGF-I-stimulated thymidine incorporation over that seen in the parental cell transfected with a plasmid encoding a neomycin-resistance gene (Neo). WT cells had a maximal response 3.75-fold higher than the maximal response for Neo or b13 cells. The second mutant clone (b14) had significantly impaired thymidine incorporation as compared to WT. The maximal response for b14 was about one-half that seen with WT cells. The IGF-I concentration for half-maximal response in the b14 clone was increased by 3-fold as compared to that for WT (3 times 10Mversus 1 times 10M IGF-I). The stimulation of thymidine incorporation by serum in either of the mutant cell lines was not significantly diminished as compared to WT. In paired experiments, serum-stimulated incorporation/µg of protein was 96-149% of that for WT cells (data not shown).


Figure 4: IGF-I stimulation of thymidine incorporation. Subconfluent monolayers of cells were made quiescent in serum-free media for 24 h and then stimulated with the indicated concentration of IGF-I or 10% fetal bovine serum for 16 h at 37 °C. [^3H]Thymidine (1 µCi/ml) was added and the incubation was continued for 1 h. Incorporated counts were measured as described under ``Experimental Procedures.'' All assays were carried out in triplicate and the standard error for each cell line in one experiment was within 5%. The data shown are the means ± S.E. of three independent experiments with the exception of Neo, which was an average of four experiments. Where not shown, S.E. bars are smaller than the size of the symbol. For each cell line, the relative stimulation with respect to stimulation by serum was calculated as follows: percent of serum stimulation = (incorporation in the presence of IGF-I - basal incorporation)/(incorporation in the presence of serum - basal incorporation) times 100. Although absolute responses varied between experiments, maximal responses when corrected for protein showed less variability. Values for serum stimulation were: Neo (times, 3176, 3400, 3126, and 2851 cpm/µg protein), WT (bullet; 2919, 3537, and 3113 cpm/µg protein), b13 (box; 6117 and 2455 cpm/µg protein), and b14 (circle; 4031 and 2368 cpm/µg protein). Within the same experiment, counts incorporated in the presence of serum/µg of protein for each mutant cell line as compared to counts incorporated in the presence of serum/µg of protein for WT ranged from 96 to 149%.



2-Deoxyglucose Uptake

The effect of increasing concentrations of IGF-I on stimulation of long-term (20-h) glucose uptake was examined in NIH-3T3 cells overexpressing mutant or wild-type human IGF-I receptors (Fig. 5). Both mutant clones and Neo had higher basal (unstimulated) long-term glucose uptake than did WT (Neo, 331 cpm/µg of protein; b13, 446 cpm/µg of protein; b14, 396 cpm/µg of protein; and WT, 228 cpm/µg of protein). The maximal fold stimulation for Neo was about 1.4; for WT, about 2.2; for b13, about 1.7; and for b14, about 1.7. The estimation of maximal stimulation of glucose uptake by IGF-I presented here for the two IGF-I receptor mutants may be an underestimation because of the greater than 2-fold higher basal uptake as compared to WT. In addition, there was a shift in sensitivity to IGF-I stimulation with both of these mutant clones as compared to WT. The IGF-I concentration for half-maximal stimulation for WT was about 3 times 10M IGF-I, whereas for both b13 and b14 it was about 5 times 10M IGF-I.


Figure 5: IGF-I stimulation of 2-deoxyglucose uptake. Confluent monolayers of cells were incubated in the presence of the indicated concentration of IGF-I for 20 h and washed as indicated under ``Experimental Procedures.'' Uptake was measured after a 10-min incubation in the presence of 2-deoxy-D-[^14C]glucose (0.5 µCi/well) and 0.2 mM 2-deoxy-Dglucose. The results shown are the means ± S.E. of three experiments, with the exception of WT which is an average of four experiments, and b14, which was from two experiments. Where not shown, S.E. bars are smaller than the size of the symbol. Additionally, each assay point for each experiment was done in triplicate with a standard error for each cell line within one experiment less than 5%. The results shown are for Neo (times), WT (bullet), b13 (box), and b14 (circle).




DISCUSSION

The molecular basis for the differential physiologic functions of the insulin and IGF-I receptors has not been fully elucidated. Although the two receptors share remarkable structural homology and are capable of associating with some of the same proteins in the tyrosine kinase signal transduction cascade, ligand stimulation of these receptors can elicit different effects on cellular metabolism and growth in vivo. In the present study, we have investigated the regulatory function of an amino acid residue in the tyrosine kinase domain of the IGF-I receptor. The tryptophan residue at position 1173 in the IGF-I receptor is highly conserved in other members of the protein-tyrosine kinase family, particularly the insulin, epidermal growth factor, and platelet-derived growth factor receptors, and the abl oncogene (Hanks et al., 1988). In addition, in both tyrosine and serine/threonine kinases, there are two invariant amino acids NH(2)-terminal to this tryptophan. Aspartic acid is 9 amino acids away and glycine is 4 amino acids to the NH(2)-terminal side. Replacement of tryptophan 1173 with serine severely reduced IGF-I-stimulated autophosphorylation of the beta subunit in intact cells. This autophosphorylation was significantly less than that seen in parental cells (Neo) with 20-fold fewer IGF-I receptors, indicating that these mutant receptors exert a dominant-negative effect on autophosphorylation of the endogenous mouse IGF-I receptors. In vitro autophosphorylation of the mutant receptors was also significantly less than that exhibited by wild-type receptors. Phosphorylation of an endogenous substrate, IRS-1, was impaired, but not to the same extent as autophosphorylation; presumably, this degree of phosphorylation is sufficient to mediate certain of the biological responses studied. Furthermore, there is no evidence of the mutant receptors exerting a dominant-negative effect on phosphorylation of IRS-1 by the endogenous mouse IGF-I receptors. The in vitro kinase activity of the mutant receptors, measured as the ability to phosphorylate poly(Glu,Tyr) 4:1, was equivalent to that of the wild-type receptor, suggesting that, in intact cells, additional factors are involved in the ability of the receptor to phosphorylate endogenous substrates such as IRS-1. The tryptophan residue at position 1173 in the IGF-I receptor is homologous to tryptophan 1200 in the insulin receptor. A naturally occurring mutation in the insulin receptor at position 1200 (numbering system of Ebina et al., 1985) has been described (Moller et al., 1990). Substitution of the homologous tryptophan in the insulin receptor in a patient with type A insulin resistance illustrated that this residue is also important for insulin-stimulated autophosphorylation in transformed lymphocytes (Iwanishi et al., 1993). Phosphorylation, using radiolabeled ATP, of insulin-stimulated WGA-purified receptors was significantly diminished in the mutant insulin receptor. It has not yet been determined if this loss of autophosphorylation is exclusively on tyrosine residues or if serine/threonine kinase activity is also affected.

Functional studies of IGF-I receptors with mutated tryptophan residues showed that thymidine incorporation was severely reduced in one mutant clone and abolished in the second clone. Glucose uptake in both clones was only reduced to about half of that seen with NIH-3T3 cells overexpressing the wild-type IGF-I receptor. Functional studies performed on Chinese hamster ovary cells transfected with the mutant insulin receptor (Trp-1200 to Ser) revealed severely impaired thymidine incorporation and moderately reduced glycogen synthesis but unaffected glucose uptake when compared to wild-type insulin receptors (Moller et al., 1991).

The importance of tyrosine autophosphorylation has been studied in both the insulin receptor and the IGF-I receptor. A cluster of 3 tyrosines located 21 amino acids downstream of the COOH terminus of the catalytic loop are the first tyrosine residues to be phosphorylated following ligand binding (Yarden and Ullrich, 1988). Mutation of all 3 tyrosines in the triple tyrosine cluster of the insulin or IGF-I receptor results in severe reduction of the tyrosine kinase activity (Ellis et al., 1986; Zhang et al., 1991; Murakami and Rosen, 1991; Wilden et al., 1990, 1992a, 1992b; Debant et al., 1988; Rafaeloff et al., 1991; Gronborg et al., 1993; Kato et al., 1994). Thymidine incorporation and glucose uptake were diminished in Chinese hamster ovary cells expressing mutant insulin receptors and NIH-3T3 cells expressing mutant IGF-I receptors, suggesting an essential requirement of autophosphorylation of the triple tyrosine cluster in biological activities (Ellis et al., 1986; Zhang et al., 1991; Murakami and Rosen, 1991; Gronborg et al., 1993; Kato et al., 1994). Mutation of only 2 of the 3 tyrosines in the cluster within the insulin receptor renders measurable but significantly reduced insulin-stimulated kinase activity in intact cells (Yonezawa and Roth, 1991). Recently it has been suggested that the autophosphorylation domain contains sequences that inhibit autophosphorylation of tyrosine residues (Filipek and Soderling, 1993). Consistent with this hypothesis, mutation of a conserved amino acid (Arg-1152 to Gln) just downstream from the last tyrosine in the autophosphorylation cluster resulted in increased insulin-independent kinase activity with diminished insulin-stimulated tyrosine kinase activity (Formisano et al., 1993).

Unlike our results in the IGF-I mutant receptor, the solubilized mutant insulin receptor was incapable of phosphorylating an exogenous substrate, and phosphorylation of IRS-1 in intact cells was undetectable (Moller et al., 1991). The difference in these results may be due to the use of a different exogenous substrate in the first case and the sensitivity of antibodies used to detect phosphorylated proteins in the second, or these findings may suggest that the IGF-I and insulin receptor beta subunits are inherently different.

Our observations on the substitution of Trp-1173 suggest that the functional defect in the receptor is similar to those previously reported in the tyrosine kinase domain; that is, incomplete autophosphorylation. However, unlike mutations of the glycine-rich segment with its proximal triad of charged amino acids and mutations of the ``catalytic loop,'' some biologic function is maintained despite severely reduced tyrosine autophosphorylation. The retention of certain biologic functions is more akin to the pattern seen in mutations of the triple tyrosine cluster in the autophosphorylation site. The mechanism by which this differentiation of function occurs may be of at least two types. First, a mutation causing disruption of conformation in the unstimulated state or conformational change after ligand stimulation may block specific signal transduction. Second, phosphorylation of specific residues may be essential for specific signal transduction.

In conclusion, based on our present study and observations on mutations in this region of the insulin receptor, we propose that the region surrounding Trp-1173 of the tyrosine kinase domain of the IGF-I receptor is an important regulator of autophosphorylation in vivo. Furthermore, the phenotype of cells expressing this mutation demonstrates once again that high levels of autophosphorylation are not required for mediation of all the biologic activities of the IGF-I receptor.


FOOTNOTES

*
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: Diabetes Branch, NIDDK, National Institutes of Health, Bldg. 10, Rm. 8S-239, Bethesda, MD 20892. Tel.: 301-496-8090; Fax: 301-480-4386.

(^1)
The abbreviations used are: IGF, insulin-like growth factor; IRS-1, insulin receptor substrate-1; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; WGA, wheat germ agglutinin.


ACKNOWLEDGEMENTS

We thank Dr. Steven Jacobs (Burroughs Welcome Co., Research Triangle, NC) for the human IGF-I receptor cDNA and Dr. Simeon Taylor for critical reading of this manuscript.


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