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(The FASEB Journal. 1999;13:1347-1357.)
© 1999 FASEB

Substitution of the insulin receptor transmembrane domain with that of glycophorin A inhibits insulin action

ANNE GARDIN, COLETTE AUZAN*, ERIC CLAUSER*, TATIANA MALHERBE, DOMINIQUE AUNIS, GÉRARD CRÉMEL and PIERRE HUBERT1

INSERM U. 338, 67084 Strasbourg, France; and
* INSERM U. 36, Collège de France, 3 rue d'Ulm, 75005 Paris, France

1Correspondence: INSERM U. 338, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France. E-mail: hubert{at}neurochem.u-strasbg.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
To study the role of transmembrane (TM) domains interactions in the activation of the insulin receptor, we have replaced the insulin receptor TM domain with that of glycophorin A (GpA), an erythrocyte protein that spontaneously forms detergent-resistant dimers through TM–TM interactions. Insulin receptor cDNA sequences with the TM domain replaced by that of GpA were constructed and stably transfected in CHO cells. Insulin binding to cells and solubilized receptors was not modified. Electrophoresis after partial reduction of disulfide bonds revealed an altered structure for the soluble chimeric receptors, seen as an altered mobility apparently due to increased interactions between the ß subunits of the receptor. Insulin signaling was markedly decreased for cells transfected with chimeric receptors compared with cells transfected with normal receptors. A decrease in insulin-induced receptor kinase activity was observed for solubilized chimeric receptors. In conclusion, substitution by the native GpA TM domain of the insulin receptor results in structurally modified chimeric receptors that are unable to transmit the insulin signal properly. It is hypothesized that this substitution may impose structural constraints that prevent the proper changes in conformation necessary for activation of the receptor kinase. Other mutants modifying the structure or the membrane orientation of the glycophorin A TM domain are required to better understand these constraints.—Gardin, A., Auzan, C., Clauser, E., Malherbe, T., Aunis, D., Crémel, G., Hubert, P. Substitution of the insulin receptor transmembrane domain with that of glycophorin A inhibits insulin action.


Key Words: signaling • tyrosine phosphorylation • receptor dimerization • transfection of chimeric receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
THE INSULIN RECEPTOR is composed of two {alpha} and two ß disulfide-linked subunits, which form an {alpha}2ß2 native structure. The ligand binding {alpha} subunit is extracellular, whereas the ß subunit is an integral membrane protein that contains a single 23 amino acid hydrophobic transmembrane (TM)2 domain and an intracellular protein tyrosine-kinase domain. The binding of insulin to the extracellular domain of its receptor initiates a signal transduction cascade by causing the intracellular kinase domain to autophosphorylate on tyrosine residues. This autophosphorylation results in full activation of the kinase and recruitment of SH2 and PTB domain-containing substrates such as proteins of the IRS and shc families. Once phosphorylated, these docking proteins can bind other SH2 domain-containing proteins, which in turn activate downstream intracellular enzymatic signaling cascades to achieve the multiple cellular effects of the hormone (1 , 2 ). The crystal 3-dimensional structure of the soluble kinase domain of the human insulin receptor has been established both in its unphosphorylated and activated tris-phosphorylated forms (3 , 4 ). These structures revealed the molecular basis for receptor activation via trans-autophosphorylation and provided insights into the mechanism of phosphotransfer and the tyrosine kinase substrate specificity.

Although earlier studies suggested that the TM domain does not play a major role in the signal transduction process in the insulin receptor (5) , opposing evidence has recently accumulated. Some modifications in the TM domain have been found to alter receptor internalization (6) , negative cooperativity (7) , and insulin signaling. Mutations resembling a well-characterized activating mutation in the TM segment of the proto-oncogene tyrosine-kinase c-neu/erbB-2, as well as substitution of the insulin receptor TM domain by that of c-neu/erbB-2, lead to complete or partial activation of the insulin receptor (8 9 10) . Although the precise mechanism by which these modifications confer increased receptor activation is not known, a model implying increased dimerization and oligomerization by direct TM–TM interactions between {alpha}ß dimers has been proposed. Dimerization and oligomerization are thought to be the primary events leading to activation of the intracellular tyrosine kinase of growth factor receptors (11 , 12 ).

To study further the role of TM domain interactions in the activation of the insulin receptor, we have substituted the insulin receptor TM domain with that of glycophorin A (GpA), an erythrocyte membrane protein unrelated to tyrosine-kinase receptors. GpA spontaneously forms detergent-resistant dimers through TM–TM interactions. Residues in the TM domain of GpA responsible for these interactions have been characterized by site-directed mutagenesis, and a specific dimerization-driving amino acid pattern has been defined in this domain (13) . We have thus characterized the structural and functional properties of chimeric insulin receptors containing the wild-type and mutated TM domain of GpA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Plasmid construction
The PetNdeI plasmid corresponds to the expression vector peCE containing the entire cDNA sequence of the human insulin receptor (hIR) (14) with the exception of the TM domain sequence (corresponding to amino acids Lys928 to Gln956), which was deleted by site-directed mutagenesis and replaced by a unique NdeI restriction site (CATATG). The insertion of an NdeI site in the construction resulted in the addition of a His codon upstream and a Met downstream of the TM sequence of the mutants.

The two chimeric receptors Pet IR-GpA and Pet IR-GpAmut were constructed with complementary oligonucleotides phosphorylated and annealed to form linkers. These linkers represented the wild-type TM sequence of glycophorin A (Pet IR-GpA) and a Val80-> Trp mutant of this sequence for Pet IR-GpAmut (numbering according to ref 15 ). The linker of the control plasmid, Pet IR TM+2, was representative of the entire TM sequence of hIR, with the adjunction of a carboxyl-terminal His and an amino-terminal Met. These three linkers were introduced in the NdeI site after linearization of the PetNde1 plasmid. The correct positions and orientations of the linkers were verified by sequencing.

Transfection of cDNAs and selection of cell lines
Chinese hamster ovary (CHO) cells were cultured in Ham's F12 medium containing 10% fetal calf serum, 1 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Subconfluent cells were transfected with 1 µg pSV2neo and 10 µg of each of the constructions described above by the calcium phosphate precipitation procedure. Cells were selected with 750 µg/ml of the antibiotic G418 (Life Technologies, Inc., Gaithersburg, Md.). Monoclonal cell lines were obtained by limiting dilution and screened by [125I]insulin binding for high levels of recombinant IR expression. Alternatively, G418-resistant cells were collected and subjected to fluorescence-activated cell sorting with human insulin receptor-specific monoclonal antibody B6 (Immunotech, Marseille, France) to separate cells expressing an equivalent number of receptors.

125I-Insulin binding
Transfected cells were grown to confluency in 24-well dishes and incubated overnight in Ham's F12 medium containing 0.1% bovine serum albumin (BSA), 10 mM HEPES. Binding was performed at 15°C for 2 h in the presence of ~20,000 cpm [125I]insulin (Amersham, Little Chalfont, U.K.) and various concentrations of unlabeled insulin (0 to 10-7 M). Thereafter, the cells were washed and the cell-associated radioactivity was counted in a gamma counter (Wallac 1261). All binding experiments were performed in duplicate. Data obtained were analyzed with the program LIGAND (16) .

35S-Amino acid labeling of cell proteins
After a 1 h incubation in serum-free, Cys and Met-free medium, transfected cells were labeled with [35S]Cys-[35S]Met mix (Amersham) for 30 min and chase was initiated by replacing the culture medium. After various times, cells were lysed in Triton X-100 lysis buffer (Tris 20 mM pH 7.4, NaCl 150 mM, EDTA 10 mM, Triton X-100 1%, BSA 0.1%, containing protease inhibitors) and immunoprecipitated with anti-insulin receptor antibodies (clone B6, Immunotech). Immune complexes were collected on protein A-Sepharose beads (Pierce, Rockford, Ill.), which were washed extensively and analyzed by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Solubilization and wheat germ agglutinin (WGA) purification of recombinant receptors
Briefly, insulin receptors were solubilized from transfected CHO cells and purified by WGA-agarose (E.Y. labs., San Mateo, Calif.) chromatography (17) . Insulin binding assays of solubilized receptors were performed according to the polyethylene glycol precipitation method.

Structural studies of solubilized receptors
The subunit structure of solubilized and lectin-purified insulin receptors from transfected cells was studied by gradient gel lithium dodecyl sulfate-PAGE (LDS-PAGE) after treatment of 32P-labeled receptors with various concentrations of the disulfide-reducing agent dithiothreitol (DTT). Briefly, samples of mutant or wild-type receptors were incubated in 150 mM HEPES, pH 7.6, 50 mM NaCl, 0.1% Triton with or without insulin for 30 min at 4°C, and autophosphorylation was initiated by adding the same buffer containing 4 mM MnCl2, 8 mM MgCl2, and 15 µM [{gamma}-32P]ATP (Amersham). The reaction was stopped after 30 min in ice-cold sample buffer containing NaF (80 mM) and EDTA (30 mM), LDS instead of SDS, and different concentrations of DTT (0 to 50 mM). Samples were analyzed by electrophoresis at 4°C in gradient slab gels (3–15% acrylamide resolving gel). After electrophoresis, gels were dried and analyzed on a PhosphorImager (Fuji BAS 1000). The structure of the solubilized receptors was also assessed by nondenaturating PAGE in the presence of Triton X-100, as described by Flörke et al. (18) . This nondenaturating method allows for protein separation according to size and shape. Briefly, samples of mutant or wild-type receptors were [32P]-labeled as above except that the reaction was terminated by adding cold sample buffer containing Triton X-100 instead of LDS. Electrophoresis was performed at 4°C overnight in gradient slab gels (3.5–25% acrylamide, acrylamide/bis ratio 100). Gels were dried and analyzed as above.

Metabolic and mitogenic actions of insulin on parental and transfected CHO cells
To study glycogen synthesis, transfected and parental CHO cells were grown to confluence into 12-well dishes and exposed to increasing concentrations of insulin for 60 min at 37°C, then incubated in the presence of 5 µM [14C]glucose (NEN, Frankfurt/Main, Germany) for 3 h. Total glycogen was precipitated with ethanol, as described previously (19) , and the amount of radioactivity incorporated was determined using a scintillation counter (Wallac 1409).

To determine thymidine uptake into DNA, transfected and parental CHO cells were grown in 12-well plates in Ham's F12 medium containing 10% fetal calf serum. When cells had reached 75% confluence, they were incubated for 72 h in serum-free medium containing 0.1% BSA and 10 mM HEPES, pH 7.4. Incubation in the same medium containing increasing concentrations of insulin (0 to 10-7 M) was continued for 15 h at 37°C before a 45 min pulse with [3H]thymidine (Amersham). Cells were washed with ice-cold PBS and DNA was precipitated with 10% TCA, washed twice with 5% TCA, solubilized in 1N NaOH, neutralized, and counted for radioactivity.

In vitro MAP kinase and PtdIns 3-kinase assays
Transfected cells, grown to confluence in 100 mm dishes, were deprived of serum and incubated for 10 min at 37°C with or without insulin. MAP kinase activity was assayed as incorporation of [32P] into myelin basic protein after immunoprecipitation of cell lysates with anti-erk-2 antibody (UBI) essentially as described (20) . Activity of PtdIns 3-kinase was measured as incorporation of [32P] into phosphatidylinositol after immunoprecipitation of cell lysates with antiphosphotyrosine antibody (clone 4G10, UBI) as described (21) .

Phosphorylation in intact cells and immunoblotting
Intact confluent cells grown in serum-free medium overnight were incubated with different concentrations of insulin for 10 min at 37°C. Cells were then solubilized in electrophoresis sample buffer containing protease and phosphatase inhibitors; proteins were resolved by reducing SDS-PAGE and transferred to a nitrocellulose membrane according to the procedure of Towbin (22) . After blocking, the blot was incubated with antiphosphotyrosine monoclonal antibody 4G10 (UBI), followed by antimouse immunoglobulin-horseradish peroxidase (Amersham). Detection of phosphotyrosine containing proteins with the ECL detection kit (Amersham) was then performed according to the manufacturer's instructions. This procedure was slightly modified for immunoblotting with antibodies against the {alpha} (Santa Cruz Biothechnology) and ß subunits of the insulin receptor: polyvinylidene difluoride (PVDF; Amersham) was used instead of nitrocellulose; methanol and SDS concentrations were reduced; blocking was performed with powdered milk; and peroxidase-coupled protein A (Zymed, San Francisco, Calif.) was used as secondary reagent.

In vitro autophosphorylation and kinase activity of recombinant receptors
The insulin-stimulated autophosphorylation of its receptor was assayed as incorporation of [32P] from [{gamma}-32P]ATP (Amersham) in the 95kDa ß subunit. Various quantities of receptor preparations, diluted so that their [125I]insulin binding activities were equivalent, were [32P]-labeled as described above for structural studies. The reaction was stopped by boiling in sample buffer containing phosphatase inhibitors (NaF, Na vanadate, Na pyrophosphate, and EDTA). Autophosphorylation was visualized and quantified after denaturing SDS-PAGE with a Fuji BAS 1000 PhosphorImager. To demonstrate the equivalence of the receptor quantities used in these experiments, aliquots of the same receptor preparations were submitted to Western blotting after electrophoresis using monoclonal antibodies against the carboxyl-terminal sequence of the ß subunit of the human insulin receptor (gifts of Dr. Bentley Cheatham and Dr. Kenneth Siddle) (23) .

Kinase activity assay was performed using poly(GluTyr) (4:1) as exogenous substrate. Lectin-purified receptors were preincubated with or without insulin for 30 min at 4°C. Phosphorylation reactions were performed as for autophosphorylation in the presence of 0.2 mg/ml poly(Glu,Tyr) and [{gamma}-32P]ATP. After 30 min incubation at room temperature, the reaction was stopped by applying the samples to 2 x 2 cm phosphocellulose strips and immersing these immediately in 10% trichloroacetic acid, 10 mM sodium pyrophosphate. After two washes with 5% trichloroacetic acid, 10 mM sodium pyrophosphate, the papers were counted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Construction and expression of recombinant receptors
We have constructed and expressed three hIR mutants in which sequences of the TM domain have been replaced by others. As shown in Fig. 1 A, the first mutant receptor (IR-GpA) contains the wild-type TM sequence of glycophorin A. The second mutant receptor (IR-GpAmut) has the TM sequence of glycophorin A with a point mutation Val(80) -> Trp, which has been shown to have a disruptive effect on GpA dimer formation (13) . This mutation was chosen to allow for the use of the Trp residue in fluorescence studies. Both these chimeric receptors contain two additional amino acids flanking the TM domain (His and Met) that correspond to the nucleotides added to generate a unique NdeI insertion site (Fig. 1B ). As a control of the possible effect of these extra amino acids on receptor function, the third mutant receptor (IR TM+2) contains the IR wild-type TM domain plus the two extra amino acids. Stably transfected cell lines expressing each of the kinase mutant receptors, CHO IR-GpA, CHO IR-GpAmut, and CHO IR TM+2, were cloned before performing the present studies. The structures and functions of these mutant receptors were compared with that of the wild-type IR, which is overexpressed in the cell line CHO IR (24) . Parental or mock-transfected (with pSV2neo only) CHO cells were also used as controls in the functional studies.



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  		Figure 1. Mutated transmembrane sequences. A) Part of the wild-type human insulin receptor sequence (IR), starting with Asn 926, is shown at the top using the one-letter amino acid code, with the entire transmembrane domain sequence shown in boldface characters. IR-GpA, IR-GpA mut, and IR TM+2 transmembrane sequences are shown below. The seven residues important for glycophorin A dimerization are underlined in the GpA sequence. The mutation Val -> Trp in the IR-GpAmut sequence is doubly underlined, as well as the two supplementary flanking amino acids in the IR TM+2 sequence. B) Nucleotide and amino acid sequences of the wild-type human insulin receptor (nucleotides 2901–3087) and the plasmid PebNde1, where the entire sequence of the insulin receptor transmembrane domain has been replaced by a NdeI restriction site.

All receptors were normally expressed at the cell surface, as indicated by insulin binding experiments (see below) and by indirect immunofluorescence and flow cytometry that used an antibody directed against the {alpha} subunit of the human insulin receptor (data not shown).

Binding parameters of recombinant receptors
Scatchard analyses were used to estimate relative affinities of the mutant receptors as well as receptor expression levels. Isolated clones expressed variable amounts of receptors (0.2–4 106 receptors/cell) compared with ~104 receptors/cell for parental CHO cells or cells mock-transfected with pSV2neo only. Scatchard curves for 125I-insulin binding were curvilinear for all the cell lines, and estimated high-affinity KA values were identical (5.3 109 ± 1.2 109 M-1, 4.5 109 ± 1.6 109 M-1, 3.9 109 ± 0.7 109 M-1, and 4.9 109 ± 1.5 109 M-1 for CHO IR, CHO IR-GpA, CHO IR-GpAmut, and CHO IR TM+2 cells, respectively). Insulin binding analyses were also conducted with solubilized receptor preparations; again, the high-affinity KA values for all receptors were indistinguishable (data not shown).

Metabolic labeling of wild-type and chimeric receptors in transfected cells
The biosynthesis and processing of the IR transmembrane mutants were analyzed using pulse-chase experiments on metabolically labeled transfected CHO cells. Figure 2 indicates that all receptors are initially synthesized as a ~190 kDa proreceptor. Two hours after the beginning of the chase, the proreceptor started to be cleaved into its two mature subunits ({alpha} at 135 kDa and ß at 95 kDa), which were the prominent forms after 6 h. The three modified receptors exhibited normal patterns of biosynthesis both in terms of subunit structure and rates of processing as compared with the wild-type IR.



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  		Figure 2. Metabolic labeling of chimeric and wild-type insulin receptors. Transfected CHO cells were labeled for 1 h with [35S]methionine and [35S]cysteine in methionine/cysteine-free medium and then grown for the indicated chase times in complete medium. At each time, cells were lysed and the receptors immunoprecipitated. Metabolically labeled insulin receptors were then analyzed by denaturing SDS-PAGE and identified by autoradiography. The labeled proreceptors (pro), {alpha} subunits, and ß subunits are indicated.

Structural studies of solubilized and WGA-purified receptors by nondenaturing PAGE
To determine whether the modifications introduced in the TM domain of the insulin receptor modified its structure, we performed gradient slab gel electrophoresis of insulin receptors solubilized from the different transfected cell lines and partially purified by WGA-agarose chromatography.

We took advantage of the differential sensitivity of the ß–ß and {alpha}–ß disulfide bonds to chemical reduction with DTT and performed SDS-PAGE of 32P-labeled solubilized receptors in the presence of different concentrations of DTT. Figure 3 shows (upper panel) the reduction profile of wild-type receptors with the predominance of an {alpha}ß form (~450 kDa) at a low concentration of DTT (< 1 mM), the appearance of an {alpha}ß form (~230 kDa) at 1–2 mM DTT, and the complete reduction of phosphorylated receptors to the ß monomer (95 kDa) at DTT > 10 mM. The same reduction profile was observed for receptors solubilized from the IR-GpA CHO cells (Fig. 3 , lower panel), except for the appearance of a novel band for DTT concentrations above 2 mM. This band had an apparent molecular mass of ~190 kDa, which is likely to correspond to a ß form. This observation is consistent with the hypothesis that the introduction of the GpA TM domain would induce increased interactions between ß subunits. As the phosphorylated band with an apparent mass of ~190 kDa may also correspond to the precursor form of the receptor, its identity was assessed by immunoblotting with antibodies directed vs. the {alpha} and ß subunits of the insulin receptor. Figure 4 shows that the lower ~190 kDa band was recognized by both anti-{alpha} and anti-ß subunits antibodies, indicating that this band contains some proreceptor in the two preparations. However, there was a significant difference in intensity of this band labeled with the anti-ß subunit antibody between the chimeric (IR-GpA) and wild-type (IR) receptors, whereas no difference was observed after anti-{alpha} antibody labeling. This is a strong indication that in the chimeric IR-GpA receptors, the ~190 kDa band also contains a large proportion of ß2 dimers.



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  		Figure 3. Structural studies of solubilized mutant receptors, dimerization assay. Solubilized and lectin-purified insulin receptors from IR-GpA CHO and IR CHO cells were phosphorylated in the presence of insulin and [{gamma}-32P]ATP, and the reaction was stopped by addition of LDS sample buffer containing the indicated concentrations of dithiothreitol (DTT). Electrophoresis was run at 4°C overnight, and the gels were dried and submitted to autoradiography and PhosphorImager analysis. An image representative of four experiments with different receptor preparations is shown. Positions of molecular weight markers are indicated at left, and positions of the {alpha}2ß2, {alpha}ß, ß, and ~190 kDa forms are at right.



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  		Figure 4. Dimerization assay, Western blot. Solubilized and lectin-purified insulin receptors from IR-GpA CHO and IR CHO cells were incubated in the presence of ATP without insulin, and the reaction was stopped by addition of LDS sample buffer containing 5 mM DTT. Electrophoresis was run at 4°C and samples were transferred onto PVDF membranes. Western blot analysis was performed with antibodies directed against the {alpha} or ß subunits of the human insulin receptor; revelation was performed with the ECL kit. The experiment presented here was performed with two different receptor preparations, each from IR-GpA CHO and IR CHO cells; the same membrane was probed first with the anti-{alpha} subunit antibody and reprobed with the anti-ß subunit antibody after stripping of the first reagents. Molecular masses of protein standards are indicated at left, and the position of the {alpha}ß and ß2/proreceptor forms of the insulin receptor are shown at right.

The mechanism of activation of the insulin receptor tyrosine kinase differs from that of other receptor tyrosine kinases in that the receptor is constitutively dimeric. Its activation involves conformational changes, which have been detected by a variety of techniques [summarized in (25) ]. One method used to demonstrate such conformational changes has been nondenaturing Triton X-100 PAGE, which allows for the separation of proteins according to molecular size and shape. Flörke et al. (18) have shown that the native {alpha}ß form of insulin receptors possesses an apparent Stokes radius of ~9.5 nm, which decreases to ~7.9 nm upon insulin binding. Phosphorylated insulin receptors turn back to a 9.5 nm form when insulin dissociates. As it had recently been shown that the presence of the TM domain is required for this change in conformation to occur in constructs containing the receptor cytoplasmic domain (26) , we used this method to study the conformation of receptors solubilized from the transfected CHO cell lines after phosphorylation. Figure 5 shows a typical profile of molecular forms in the presence and absence of insulin, without cross-linking. For IR receptors (at right), the high Stokes radius form was always predominant (high/low forms ratio > 9 according to PhosphorImager analysis of the gels). In contrast, the IR-GpA receptors existed predominantly as a low Stokes radius form, even in the absence of insulin (at left), with a constant ratio between high and low forms of ~1.5. IRTM+2 receptors had a profile identical to the IR receptors, whereas the IR-GpAmut receptors displayed an intermediate profile (high/low forms ratio ~3 in the presence of insulin). Taken together, these results indicate that the replacement of the insulin receptor TM domain with that of glycophorin A had a major effect on the receptor structure.



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  		Figure 5. Structural studies of solubilized mutant receptors: Triton X-100 native electrophoresis. Solubilized and lectin-purified insulin receptors from chimeric and wild-type IR transfected cells were phosphorylated, as described in Fig. 3 , and analyzed in the Triton X-100 polyacrylamide gel electrophoresis gradient gel system of Flörke (18) . Electrophoresis was run at 4°C overnight; the gels were dried and submitted to autoradiography and PhosphorImager analysis. A representative image of four experiments with different receptor preparations is shown. Positions of marker proteins (Thyr., thyroglobulin, molecular mass 669 kDa, Stokes radius 8.6 nm; Ferr., Ferritin, molecular mass 440 kDa, Stokes radius 6.3 nm) are indicated at left and positions of the two forms of insulin receptors (high and low Stokes radius) at right.

Insulin signaling in transfected cells
Next we examined the consequences of TM domain substitutions on insulin cellular actions in transfected CHO cells. Figure 6 A shows the insulin dose-response curve for the incorporation of [14C]glucose into glycogen in different clones of CHO IR-GpA cells compared with CHO IR and mock-transfected cells. CHO IR-GpA cells were less responsive to hormonal stimulation than CHO IR cells expressing wild-type receptors. Maximal stimulation factor above basal was 2.2 for untransfected cells and 3.5 for the CHO IR cell line. Cells expressing the IR-GpA chimeric receptor were nearly insensitive to insulin, as maximal stimulation was close to 1.5-fold. EC50 for CHO IR-GpA cells was intermediate between that of CHO IR and CHO cells for different CHO IR-GpA clones expressing different amounts of receptors (~1–3 nM vs. ~0.5 nM and ~12 nM, respectively). Similarly, CHO IR cells were hypersensitive to insulin for the incorporation of [3H]thymidine (EC50 ~0.4 nM, maximal stimulation fivefold) as compared with parental CHO cells (EC50 ~4 nM, maximal stimulation 2.7-fold) (Fig. 6B ). Again, the CHO IR-GpA cells did not respond to insulin (EC50 ~0.3 nM, maximal stimulation twofold), whereas the CHO IR-GpAmut cells showed intermediate stimulation (EC50 ~0.8 nM, maximal stimulation 3.4-fold) and the CHO IRTM+2 cells were indistinguishable from the CHO IR cells.



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  		Figure 6. Biological effects of insulin in CHO cells transfected with the mutant insulin receptors. A) Glycogen synthesis. Cells were exposed for 1 h to the indicated concentrations of insulin, after which [14C]glucose was added for 3 h at 37°C. Glycogen was precipitated with ethanol and counted for radioactivity. Data are expressed as stimulation factor over basal level in the absence of insulin, as mean ± SE of four experiments performed in triplicate with mock-transfected (neo, filled squares) CHO cells, cells transfected with the wild-type human insulin receptor cDNA (IR, filled circles), and two different monoclonal cell lines transfected with the chimeric construct containing the TM domain of glycophorin A (IR-GpA 9 and IR-GpA 33, diamonds). The two chimeric cell lines differed by their receptor levels (120,000 and 600,000 binding sites per cell for GpA 9 and GpA 33 cells, respectively, as compared to 350,000 sites per cell for the IR-CHO cells). Basal levels in the absence of insulin were similar for all cell lines. B) Thymidine incorporation. Serum-deprived cells were incubated for 15 h with the indicated concentrations of insulin, after which [3H]thymidine was added for 45 min at 37°C. Incorporation of thymidine into DNA was measured as trichloroacetic acid-precipitable radioactivity. Data are expressed as stimulation factor over basal level in the absence of insulin, as mean ± SE of 2–5 experiments performed in triplicate with mock-transfected (neo, filled squares) CHO cells, cells transfected with the wild-type human insulin receptor cDNA (IR, filled circles) and its variant containing the two supplementary flanking amino acids (IRTM+2, filled triangles), and cells transfected with the chimeric construct containing the TM domain of glycophorin A (IR-GpA, diamonds) and its Val -> Trp mutant (IR-GpAmut, triangles). Basal levels in the absence of insulin were similar for all cell lines. All transfected cell lines contained a similar amount of insulin receptors (~350,000 sites/cell, as judged by insulin binding assay).

We also investigated the insulin-induced activation of two different intermediary enzymes involved in insulin action, MAP kinase and PtdIns 3-kinase. As shown in Fig. 7 A, the basal activity of MAP kinase was maximally stimulated ~5.5-fold by insulin in CHO IR cells, whereas a smaller, 2.5-fold stimulation was observed in CHO neo cells. In cells expressing IR-GpA chimeric receptors, a 2.4-fold increase was observed, whereas the CHO IR-GpAmut displayed a maximal stimulation of 3.8-fold. In the five cell lines, 10% fetal calf serum stimulated MAP kinase activity to the same extent, indicating the absence of alteration in the signaling pathway (data not shown). For insulin stimulation of PtdIns 3-kinase activity (Fig. 7B ), a similar pattern was observed since maximal activation factors of 1.8-, 4.8-, 2.2-, and 3.6-fold were found for CHO neo, CHO IR, CHO IR-GpA, and CHO IR-GpAmut cells, respectively. CHO IRTM+2 cells responses were identical to those of normal CHO IR cells (not shown).



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  		Figure 7. Insulin signaling in CHO cells transfected with the mutant insulin receptors. A) Map kinase activity was assayed using myelin basic protein as substrate in anti-erk-2 immunoprecipitates of control and transfected CHO cells stimulated with the indicated concentrations of insulin. After SDS-PAGE, quantification was performed with a Fuji BAS-1000 PhosphorImager; results are depicted as the level of stimulation over basal (mean ±SE of four experiments). B) PtdIns 3-kinase activity was assayed using PtdIns as substrate in antiphosphotyrosine immunoprecipitates of control and transfected CHO cells stimulated with the indicated concentrations of insulin. After TLC, quantification was performed with a Fuji BAS-1000 PhosphorImager; results are depicted as the level of stimulation over basal (mean ±SE of four experiments).

Phosphorylation of the insulin receptor and its major substrate after insulin stimulation was studied by Western blotting analysis of whole cell extracts with specific monoclonal antiphosphotyrosine antibodies. Figure 8 shows a representative tyrosine phosphorylation pattern of cellular extracts of IR and IR-GpA CHO cells after 10 min stimulation with increasing concentrations of insulin. Tyrosine phosphorylation of two main protein bands, with apparent molecular masses of 95 kDa (insulin receptor ß subunit) and 180 kDa (IRS-1), was stimulated by insulin. Although the two cell lines contained equivalent amounts of insulin receptors, major differences were apparent: maximal phosphorylation of the two bands was higher, and a lower insulin concentration was required to observe phosphorylation in the IR CHO cells. IRS 1 and receptor ß subunit tyrosine phosphorylation were already apparent at 0.1 nM and 1 nM, respectively, in the IR CHO cells, and a reduced sensitivity of at least 10-fold was observed for IR-GpA CHO cells.



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  		Figure 8. Insulin effect on tyrosine phosphorylation of cellular proteins in IR CHO and IR-GpA CHO cells. Cells were serum starved overnight and treated with the indicated concentrations of insulin for 10 min. Cells were lysed in electrophoresis sample buffer; equal amounts of protein were submitted to SDS-PAGE, followed by blotting with antiphosphotyrosine monoclonal antibody 4G10 and revelation with ECL. A typical experiment of four is depicted. Molecular masses of protein standards are indicated at left; the position of IRS-1 and the ß subunit of the insulin receptor are shown at right.

These results indicate that the receptors containing the TM domain of GpA are unable to normally transduce the insulin signals in transfected CHO cells. This defect is not due to clonal variation, since it was observed with all clones tested as well as with polyclonal cell lines established by fluorescence-activated cell sorting. The two supplementary amino acids flanking the TM domain appear to be neutral with respect to all insulin actions tested, since the CHO IRTM+2 cells behaved similarly to the CHO IR cells. The CHO IR-GpAmut cells showed intermediate characteristics in their responses to insulin, indicating that the disruption of dimer formation by this Val -> Trp mutation [which was seen in a GpA TM domain-nuclease chimeric protein (13) ] may not be sufficient for restoring proper function of the insulin receptor or may not be fully operative when inserted in the insulin receptor protein (see Discussion).

Since the signaling defect was already seen as a decreased tyrosine phosphorylation of the insulin receptor, we also studied autophosphorylation and tyrosine kinase activity of the solubilized wild-type and recombinant receptors in vitro. IR-GpA receptors displayed major abnormalities, as their basal autophosphorylation was markedly higher and their insulin-stimulated autophosphorylation markedly lower than those for the wild-type IR receptors (data not shown). Similar alterations were also evident for the phosphorylation of the synthetic substrate poly(Glu-Tyr) when using equal amounts of receptor preparations (Fig. 9 ). IR-GpA receptors were able to maximally phosphorylate this substrate by 2.2-fold, compared with 4-fold for IR receptors. Again, IRTM+2 receptors were identical to the IR preparation, and the IR-GpAmut receptors were intermediate between IR-GpA and IR receptors (maximal stimulation 3.1-fold).



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  		Figure 9. Insulin stimulation of poly (Glu, Tyr) phosphorylation by solubilized mutant insulin receptors. Phosphorylation of the synthetic substrate poly (Glu 4, Tyr 1) in the presence of [{gamma}-32P]ATP was catalyzed by equal amounts of WGA-purified IR (circles), IR-GpA (diamonds), IR-GpAmut (open triangles), and IRTM+2 (filled triangles) that had been incubated with the insulin concentrations shown. The reaction was stopped by spotting samples onto phosphocellulose papers in 10% TCA. Papers were then extensively washed in 5% TCA, dried, and incorporated radioactivity was measured by Cerenkov counting. The results of 5 separate experiments are shown (mean ±SE).

Taken together, these results show that introduction of the GpA TM domain in the insulin receptor provokes apparent structural changes and causes defects in insulin signaling due to a reduced activation of the receptor tyrosine kinase activity by insulin.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
Our understanding of the conformational changes generated by the insulin signal and how this signal is propagated through the receptor protein and across the membrane is limited. Contrary to the early assumption that the TM domain of the insulin receptor (and other receptor tyrosine kinases in general) is only necessary to anchor the protein in the membrane, several studies have shown that modifications of this TM domain can affect the activity of various members of this receptor family. The first experimental evidence for the active role of the TM sequence in this class of membrane proteins came from the study of the neu/erbB2 proto-oncogene mutations in chemically induced glioblastoma in rats. The oncogenic allele of neu contains a point mutation [Val(664) -> Glu] in the predicted TM sequence of its product, which causes increased tyrosine kinase activity and increased aggregation of the receptor protein (27 , 28 ). A similar point mutation (Gly -> Arg) was discovered in the transmembrane domain of the fibroblast growth factor receptor-3; this mutation causes achondroplasia, a common form of dwarfism (29 , 30 ). Again it was shown that the molecular consequence of this mutation is the constitutive activation of the tyrosine kinase of the receptor (31) . Chimeric receptors comprising the extracellular domain of the epidermal growth factor receptor, the kinase domain of c-ros, and the TM domain of either the epidermal growth factor receptor or c-ros had opposite effects on cell growth when transfected in NIH 3T3 cells (32) . Recent data suggest that the transmembrane domain of gp130, the signal-transducing subunit of the IL-6 receptor, is necessary for signal transduction and determines its interaction with Janus kinases (33) . Mutations resembling that of oncogenic neu/erbB2 have also been introduced in the epidermal growth factor receptor (34) and insulin-like growth factor 1 receptor (35) , resulting in constitutive activation of the receptors.

Replacement of the insulin receptor TM domain by that of the oncogenic form of the product of c-neu/erbB-2 induces ligand-independent activation of the insulin receptor and modulation of the hormone actions (8 , 10 ). The fact that introduction of this dimerizing TM sequence in the insulin receptor resulted in constitutive activation led further support to the evidence that interactions between {alpha}ß receptor halves and receptor transphosphorylation are important for tyrosine kinase activation of the insulin receptor. The replacement of the TM domain of the PDGF ß receptor by the mutated neu/erbB2 domain also led to constitutive activation of this receptor (36) . These data demonstrate that the introduction of the dimerizing TM sequence of neu/erbB2 in different members of the tyrosine kinase receptor family invariably provokes major alterations in their biological activity.

To further characterize the role of TM–TM domains interactions in the mechanism of activation of the insulin receptor, we designed chimeric receptors containing another well-characterized dimerizing TM sequence from an unrelated protein. GpA is an erythrocyte integral membrane protein with one transmembrane {alpha}-helix, which forms dimers that are stable even in the detergent SDS. Dimerization of GpA is driven by specific interactions between the transmembrane {alpha}-helices. This domain is also able to promote stable dimerization of heterologous proteins, and a chimeric protein containing the TM domain of GpA and the carboxyl terminus of staphylococcal nuclease was used to characterize by mutagenesis the residues responsible for helix-helix association (13 , 15 ). Finally, the 3-dimensional structure of the dimeric TM domain of GpA has been established (37) .

All mutant receptors were expressed at the cell surface in the CHO cells. Glycosylation and maturation occurred normally in all mutant receptors, as evidenced by normal apparent molecular masses of the two subunits and similar processing of the proreceptor into {alpha} and ß subunits. When electrophoresis of [32P]-labeled solubilized receptors was run at low temperature in the presence of increasing concentrations of the disulfide-reducing agent DTT, normal reduction from the native {alpha}2ß2 form to {alpha}ß monomers and to ß subunits was observed for all receptors. However, a novel band at ~190 kDa was seen for the IR-GpA receptors and to a lesser extent for the IR-GpAmut receptors (not shown). This novel band was identified as a ß2 form due to noncovalent dimer formation between the 95 kDa ß subunits, induced by the GpA TM sequence. It is surprising that this ~190 kDa form was not more prominent, since the chimeric receptors represented 80–90% of the total insulin receptors in transfected CHO cells. This is probably related to the intrinsic difficulties of preserving noncovalent interactions between subunits during the steps of sample phosphorylation, partial reduction, and subsequent electrophoresis. We found that boiling or freezing the protein samples abolished this ß2 form.

This structural alteration induced by the introduction of the GpA TM domain led to major modifications in insulin signaling. All the actions of the hormone studied here (MAP kinase activity, PtdIns 3-kinase activity, glycogen synthesis. and thymidine incorporation) were markedly reduced in IR-GpA CHO cells, whereas mutated IR-GpAmut CHO cells displayed intermediary responses. This was clearly related to major abnormalities in the tyrosine kinase activity of the receptor. Some disparity was noted between in vivo and in vitro phosphorylation assays, especially on the level of basal autophosphorylation in the absence of insulin, which was elevated in the chimeric receptors in vitro without any significant change in the basal levels of biological effects and cell protein tyrosine phosphorylation. This was also observed for other TM domain chimeric constructs of insulin receptors (10) , and possibly is accounted for by regulatory mechanisms in intact cells such as phosphatases. It is also intriguing that the mutated IR-GpAmut CHO cells displayed intermediary responses in all the functional assays, although mutagenesis data of the GpA TM would predict full activity. This Val(80) -> Trp mutation is located at the middle of the dimerizing sequence of GpA and was found to be totally disruptive of helix-helix association in the GpA/nuclease chimeric protein in SDS micelles (13) . However, other mutations at this position were not as effective in disrupting dimer formation, and this precise mutation may still allow for significant packing interactions when inserted in a different protein or in a protein localized in a natural membrane. To support this, structure-based analysis (38) of this mutation in the GpA TM domain has shown that it provokes only a mild steric clash at the dimer interface (K. R. MacKenzie and D. M. Engelman, personal communication). Furthermore, using the ToxR transcription activator system, Langosch et al. (39) have shown that a Val(80) -> Ala mutation was not as effective in disrupting dimerization as was predicted from the detergent studies.

Why does introduction of the dimerizing TM domain of GpA in the insulin receptor lead to an inhibition of insulin signaling instead of activation, as was seen with the neuVal->Glu TM domain? Similar results were observed when the GpA TM domain was introduced into the neu/erbB2 protein, where strong dimerization was observed but without transforming activity, clearly demonstrating that dimerization of a tyrosine-kinase receptor is not by itself sufficient for its activation (40) . It thus seems that not all dimerizing TM domains are equivalent in their ability to activate receptor tyrosine-kinases, and this may be related to the known differences in structure of the TM domains themselves. It has been established that the GpA TM domain promotes a right-handed interaction of {alpha}-helices (37) , whereas the neu/erbB2 TM domain adopts a left-handed coiled-coil structure (41) . Thus, we suggest that these two different dimerizing TM domain sequences may impose very different geometries of interaction between intracytoplasmic kinase domains, thereby favoring (erbB2 TM domain) or impeding (GpA TM domain) the mechanism of transphosphorylation between dimerized partners. In the case of the insulin receptor-GpA TM domain chimeras studied here, it is also possible that such a modified orientation of the kinase domains leads to intermolecular transphosphorylation between {alpha}2ß2 holoreceptors instead of the normal intramolecular transphosphorylation between the two {alpha}ß receptor halves of the receptor. It has been shown that intermolecular phosphorylation between insulin holoreceptors is unable to stimulate substrate kinase activity (42) . Such a mechanism may be operative in our chimeric receptors, which do not signal while exhibiting detectable autophosphorylation after insulin stimulation in vitro. Another possibility is that the abnormal geometry imposed by the GpA TM domain may impair either the phosphorylation of key tyrosine residues or the normal interaction of phosphotyrosines with SH2/PTB domains proteins, or both. To test our geometrical hypothesis, we have undertaken the construction and characterization of other modifications of the GpA TM domain inserted in the insulin receptor. For example, addition or substraction of one amino acid of the GpA TM domain may reorient its dimerization interface, and therefore the tyrosine kinase monomers, modifying their interactions during activation and oligomerization.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
Introduction of the GpA TM sequence in the insulin receptor led to changes in the receptor structure. The chimeric receptors displayed altered tyrosine kinase activation by insulin, with a decreased autophosphorylation and substrate phosphorylation. This, in turn, led to an almost complete abolition of insulin responsiveness in cells transfected with the chimeric IR-GpA receptors. Studies of the sites of phosphorylation of the chimeric receptors and of their interactions with SH2/PTB signaling proteins, together with the characterization of other mutations, should provide further insight into the mechanism of transphosphorylation of insulin receptors and into the role of their TM domain.


   ACKNOWLEDGMENTS
 
We wish to thank Ms. Caroline Waltzinger (Illkirch, France) for her help with the fluorescence-activated cell sorter, and Drs. Bentley Cheatham (Boston, Mass.) and Kenneth Siddle (Cambridge, U.K.) for the gift of anti-insulin receptor antibodies. We also thank the Eli Lilly Co. (Indianapolis, Ind.) for the gift of recombinant human insulin. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and by grants from the Association pour la Recherche sur le Cancer and the Ligue Nationale contre le Cancer.


   FOOTNOTES
 
2 Abbreviations: BSA, bovine serum albumin; CHO, Chinese hamster ovary; DTT, dithiothreitol; GpA, glycophorin A; hIR, human insulin receptors; LDS, lithium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; TM, transmembrane; WGA, wheat germ agglutinin.

Received for publication October 5, 1998. Revision received February 24, 1999.
   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
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