Substitution of the insulin receptor transmembrane domain with the c-neu/erbB2 transmembrane domain...

Substitution of the insulin receptor transmembranedomain with that of glycophorin A inhibits insulinaction

ANNE GARDIN, COLETTE AUZAN,* ERIC CLAUSER,* TATIANA MALHERBE,DOMINIQUE AUNIS, GERARD CREMEL, AND PIERRE HUBERT1

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

ABSTRACT To study the role of transmembrane(TM) domains interactions in the activation of theinsulin receptor, we have replaced the insulin recep-tor TM domain with that of glycophorin A (GpA), anerythrocyte protein that spontaneously forms deter-gent-resistant dimers through TM–TM interactions.Insulin receptor cDNA sequences with the TM do-main replaced by that of GpA were constructed andstably transfected in CHO cells. Insulin binding tocells and solubilized receptors was not modified.Electrophoresis after partial reduction of disulfidebonds revealed an altered structure for the solublechimeric receptors, seen as an altered mobility ap-parently due to increased interactions between the bsubunits of the receptor. Insulin signaling was mark-edly decreased for cells transfected with chimericreceptors compared with cells transfected with nor-mal receptors. A decrease in insulin-induced recep-tor kinase activity was observed for solubilized chi-meric receptors. In conclusion, substitution by thenative GpA TM domain of the insulin receptorresults in structurally modified chimeric receptorsthat are unable to transmit the insulin signal prop-erly. It is hypothesized that this substitution mayimpose structural constraints that prevent the properchanges in conformation necessary for activation ofthe receptor kinase. Other mutants modifying thestructure or the membrane orientation of the glyco-phorin A TM domain are required to better under-stand these constraints.—Gardin, A., Auzan, C.,Clauser, E., Malherbe, T., Aunis, D., Cremel, G.,Hubert, P. Substitution of the insulin receptor trans-membrane domain with that of glycophorin A inhib-its insulin action. FASEB J. 13, 1347–1357 (1999)

Key Words: signaling z tyrosine phosphorylation z receptordimerization z transfection of chimeric receptors

The insulin receptor is composed of two a andtwo b disulfide-linked subunits, which form an a2b2

native structure. The ligand binding a subunit isextracellular, whereas the b subunit is an integral

membrane protein that contains a single 23 aminoacid hydrophobic transmembrane (TM)2 domainand an intracellular protein tyrosine-kinase domain.The binding of insulin to the extracellular domain ofits receptor initiates a signal transduction cascade bycausing the intracellular kinase domain to autophos-phorylate on tyrosine residues. This autophosphory-lation results in full activation of the kinase andrecruitment of SH2 and PTB domain-containingsubstrates such as proteins of the IRS and shcfamilies. Once phosphorylated, these docking pro-teins can bind other SH2 domain-containing pro-teins, which in turn activate downstream intracellu-lar enzymatic signaling cascades to achieve themultiple cellular effects of the hormone (1, 2). Thecrystal 3-dimensional structure of the soluble kinasedomain of the human insulin receptor has beenestablished both in its unphosphorylated and acti-vated tris-phosphorylated forms (3, 4). These struc-tures revealed the molecular basis for receptor acti-vation via trans-autophosphorylation and providedinsights into the mechanism of phosphotransfer andthe tyrosine kinase substrate specificity.

Although earlier studies suggested that the TMdomain does not play a major role in the signaltransduction process in the insulin receptor (5),opposing evidence has recently accumulated. Somemodifications in the TM domain have been found toalter receptor internalization (6), negative cooperat-ivity (7), and insulin signaling. Mutations resemblinga well-characterized activating mutation in the TMsegment of the proto-oncogene tyrosine-kinase c-neu/erbB-2, as well as substitution of the insulinreceptor TM domain by that of c-neu/erbB-2, lead to

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

2 Abbreviations: BSA, bovine serum albumin; CHO, Chi-nese hamster ovary; DTT, dithiothreitol; GpA, glycophorin A;hIR, human insulin receptors; LDS, lithium dodecyl sulfate;PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinyli-dene difluoride; SDS, sodium dodecyl sulfate; TM, transmem-brane; WGA, wheat germ agglutinin.

13470892-6638/99/0013-1347/$02.25 © FASEB

complete or partial activation of the insulin receptor(8–10). Although the precise mechanism by whichthese modifications confer increased receptor activa-tion is not known, a model implying increaseddimerization and oligomerization by direct TM–TMinteractions between ab dimers has been proposed.Dimerization and oligomerization are thought to bethe primary events leading to activation of the intra-cellular tyrosine kinase of growth factor receptors(11, 12).

To study further the role of TM domain inter-actions in the activation of the insulin receptor, wehave substituted the insulin receptor TM domainwith that of glycophorin A (GpA), an erythrocytemembrane protein unrelated to tyrosine-kinasereceptors. GpA spontaneously forms detergent-resistant dimers through TM–TM interactions.Residues in the TM domain of GpA responsible forthese interactions have been characterized by site-directed mutagenesis, and a specific dimerization-driving amino acid pattern has been defined inthis domain (13). We have thus characterized thestructural and functional properties of chimericinsulin receptors containing the wild-type andmutated TM domain of GpA.

MATERIALS AND METHODS

Plasmid construction

The PetNdeI plasmid corresponds to the expression vectorpeCE containing the entire cDNA sequence of the humaninsulin receptor (hIR) (14) with the exception of the TMdomain sequence (corresponding to amino acids Lys928 toGln956), which was deleted by site-directed mutagenesis andreplaced by a unique NdeI restriction site (CATATG). Theinsertion of an NdeI site in the construction resulted in theaddition of a His codon upstream and a Met downstream ofthe TM sequence of the mutants.

The two chimeric receptors Pet IR-GpA and Pet IR-GpAmut

were constructed with complementary oligonucleotides phos-phorylated and annealed to form linkers. These linkersrepresented the wild-type TM sequence of glycophorin A (PetIR-GpA) and a Val803 Trp mutant of this sequence for PetIR-GpAmut (numbering according to ref 15). The linker ofthe control plasmid, Pet IR TM12, was representative of theentire TM sequence of hIR, with the adjunction of a carboxyl-terminal His and an amino-terminal Met. These three linkerswere introduced in the NdeI site after linearization of thePetNde1 plasmid. The correct positions and orientations ofthe linkers were verified by sequencing.

Transfection of cDNAs and selection of cell lines

Chinese hamster ovary (CHO) cells were cultured in Ham’sF12 medium containing 10% fetal calf serum, 1 mM glu-tamine, 100 U/ml penicillin, and 100 mg/ml streptomycin.Subconfluent cells were transfected with 1 mg pSV2neo and10 mg of each of the constructions described above by thecalcium phosphate precipitation procedure. Cells were se-lected with 750 mg/ml of the antibiotic G418 (Life Technol-ogies, Inc., Gaithersburg, Md.). Monoclonal cell lines were

obtained by limiting dilution and screened by [125I]insulinbinding for high levels of recombinant IR expression. Alter-natively, G418-resistant cells were collected and subjected tofluorescence-activated cell sorting with human insulin recep-tor-specific monoclonal antibody B6 (Immunotech, Mar-seille, France) to separate cells expressing an equivalentnumber of receptors.

125I-Insulin binding

Transfected cells were grown to confluency in 24-well dishesand incubated overnight in Ham’s F12 medium containing0.1% bovine serum albumin (BSA), 10 mM HEPES. Bindingwas performed at 15°C for 2 h in the presence of ;20,000cpm [125I]insulin (Amersham, Little Chalfont, U.K.) andvarious concentrations of unlabeled insulin (0 to 1027 M).Thereafter, the cells were washed and the cell-associatedradioactivity was counted in a gamma counter (Wallac 1261).All binding experiments were performed in duplicate. Dataobtained 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-freemedium, transfected cells were labeled with [35S]Cys-[35S]Met mix (Amersham) for 30 min and chase was initiatedby replacing the culture medium. After various times, cellswere 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 immunoprecipitatedwith anti-insulin receptor antibodies (clone B6, Immuno-tech). Immune complexes were collected on protein A-Sepharose beads (Pierce, Rockford, Ill.), which were washedextensively 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 transfectedCHO cells and purified by WGA-agarose (E.Y. labs., SanMateo, Calif.) chromatography (17). Insulin binding assays ofsolubilized receptors were performed according to the poly-ethylene glycol precipitation method.

Structural studies of solubilized receptors

The subunit structure of solubilized and lectin-purified insu-lin receptors from transfected cells was studied by gradientgel lithium dodecyl sulfate-PAGE (LDS-PAGE) after treat-ment of 32P-labeled receptors with various concentrations ofthe disulfide-reducing agent dithiothreitol (DTT). Briefly,samples of mutant or wild-type receptors were incubated in150 mM HEPES, pH 7.6, 50 mM NaCl, 0.1% Triton with orwithout insulin for 30 min at 4°C, and autophosphorylationwas initiated by adding the same buffer containing 4 mMMnCl2, 8 mM MgCl2, and 15 mM [g-32P]ATP (Amersham).The reaction was stopped after 30 min in ice-cold samplebuffer containing NaF (80 mM) and EDTA (30 mM), LDSinstead of SDS, and different concentrations of DTT (0 to 50mM). Samples were analyzed by electrophoresis at 4°C ingradient slab gels (3–15% acrylamide resolving gel). Afterelectrophoresis, gels were dried and analyzed on a Phospho-rImager (Fuji BAS 1000). The structure of the solubilizedreceptors was also assessed by nondenaturating PAGE in thepresence of Triton X-100, as described by Florke et al. (18).This nondenaturating method allows for protein separation

1348 Vol. 13 August 1999 GARDIN ET AL.The FASEB Journal

according to size and shape. Briefly, samples of mutant orwild-type receptors were [32P]-labeled as above except thatthe reaction was terminated by adding cold sample buffercontaining Triton X-100 instead of LDS. Electrophoresis wasperformed at 4°C overnight in gradient slab gels (3.5–25%acrylamide, acrylamide/bis ratio 100). Gels were dried andanalyzed as above.

Metabolic and mitogenic actions of insulin on parental andtransfected CHO cells

To study glycogen synthesis, transfected and parental CHOcells were grown to confluence into 12-well dishes andexposed to increasing concentrations of insulin for 60 min at37°C, then incubated in the presence of 5 mM [14C]glucose(NEN, Frankfurt/Main, Germany) for 3 h. Total glycogen wasprecipitated with ethanol, as described previously (19), andthe amount of radioactivity incorporated was determinedusing a scintillation counter (Wallac 1409).

To determine thymidine uptake into DNA, transfected andparental CHO cells were grown in 12-well plates in Ham’s F12medium containing 10% fetal calf serum. When cells hadreached 75% confluence, they were incubated for 72 h inserum-free medium containing 0.1% BSA and 10 mM HEPES,pH 7.4. Incubation in the same medium containing increas-ing concentrations of insulin (0 to 1027 M) was continued for15 h at 37°C before a 45 min pulse with [3H]thymidine(Amersham). Cells were washed with ice-cold PBS and DNAwas precipitated with 10% TCA, washed twice with 5% TCA,solubilized in 1N NaOH, neutralized, and counted for radio-activity.

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°Cwith or without insulin. MAP kinase activity was assayed asincorporation of [32P] into myelin basic protein after immu-noprecipitation of cell lysates with anti-erk-2 antibody (UBI)essentially as described (20). Activity of PtdIns 3-kinase wasmeasured as incorporation of [32P] into phosphatidylinositolafter immunoprecipitation of cell lysates with antiphosphoty-rosine antibody (clone 4G10, UBI) as described (21).

Phosphorylation in intact cells and immunoblotting

Intact confluent cells grown in serum-free medium overnightwere incubated with different concentrations of insulin for 10min at 37°C. Cells were then solubilized in electrophoresissample buffer containing protease and phosphatase inhibi-tors; proteins were resolved by reducing SDS-PAGE andtransferred to a nitrocellulose membrane according to theprocedure of Towbin (22). After blocking, the blot wasincubated with antiphosphotyrosine monoclonal antibody4G10 (UBI), followed by antimouse immunoglobulin-horse-radish peroxidase (Amersham). Detection of phosphoty-rosine containing proteins with the ECL detection kit (Am-ersham) was then performed according to the manufacturer’sinstructions. This procedure was slightly modified for immu-noblotting with antibodies against the a (Santa Cruz Bioth-echnology) and b subunits of the insulin receptor: polyvinyli-dene difluoride (PVDF; Amersham) was used instead ofnitrocellulose; methanol and SDS concentrations were re-duced; blocking was performed with powdered milk; andperoxidase-coupled protein A (Zymed, San Francisco, Calif.)was used as secondary reagent.

In vitro autophosphorylation and kinase activity ofrecombinant receptors

The insulin-stimulated autophosphorylation of its receptorwas assayed as incorporation of [32P] from [g-32P]ATP (Am-ersham) in the 95kDa b subunit. Various quantities of recep-tor preparations, diluted so that their [125I]insulin bindingactivities were equivalent, were [32P]-labeled as describedabove for structural studies. The reaction was stopped byboiling in sample buffer containing phosphatase inhibitors(NaF, Na vanadate, Na pyrophosphate, and EDTA). Auto-phosphorylation was visualized and quantified after denatur-ing SDS-PAGE with a Fuji BAS 1000 PhosphorImager. Todemonstrate the equivalence of the receptor quantities usedin these experiments, aliquots of the same receptor prepara-tions were submitted to Western blotting after electrophoresisusing monoclonal antibodies against the carboxyl-terminalsequence of the b 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 werepreincubated with or without insulin for 30 min at 4°C.Phosphorylation reactions were performed as for autophos-phorylation in the presence of 0.2 mg/ml poly(Glu,Tyr) and[g-32P]ATP. After 30 min incubation at room temperature,the reaction was stopped by applying the samples to 2 3 2 cmphosphocellulose strips and immersing these immediately in10% trichloroacetic acid, 10 mM sodium pyrophosphate.After two washes with 5% trichloroacetic acid, 10 mM sodiumpyrophosphate, the papers were counted.

RESULTS

Construction and expression of recombinantreceptors

We have constructed and expressed three hIR mu-tants in which sequences of the TM domain havebeen replaced by others. As shown in Fig. 1A, thefirst mutant receptor (IR-GpA) contains the wild-type TM sequence of glycophorin A. The secondmutant receptor (IR-GpAmut) has the TM sequenceof glycophorin A with a point mutation Val(80) 3Trp, which has been shown to have a disruptiveeffect on GpA dimer formation (13). This mutationwas chosen to allow for the use of the Trp residue influorescence studies. Both these chimeric receptorscontain two additional amino acids flanking the TMdomain (His and Met) that correspond to the nucle-otides added to generate a unique NdeI insertion site(Fig. 1B). As a control of the possible effect of theseextra amino acids on receptor function, the thirdmutant receptor (IR TM12) contains the IR wild-typeTM domain plus the two extra amino acids. Stablytransfected cell lines expressing each of the kinasemutant receptors, CHO IR-GpA, CHO IR-GpAmut,and CHO IR TM12, were cloned before performingthe present studies. The structures and functions ofthese mutant receptors were compared with that ofthe wild-type IR, which is overexpressed in the cellline CHO IR (24). Parental or mock-transfected

1349TM DOMAIN INACTIVATION OF INSULIN RECEPTORS

(with pSV2neo only) CHO cells were also used ascontrols in the functional studies.

All receptors were normally expressed at the cellsurface, as indicated by insulin binding experiments(see below) and by indirect immunofluorescenceand flow cytometry that used an antibody directedagainst the a subunit of the human insulin receptor(data not shown).

Binding parameters of recombinant receptors

Scatchard analyses were used to estimate relativeaffinities of the mutant receptors as well as receptorexpression levels. Isolated clones expressed variableamounts of receptors (0.2–4 106 receptors/cell)compared with ;104 receptors/cell for parentalCHO cells or cells mock-transfected with pSV2neoonly. Scatchard curves for 125I-insulin binding werecurvilinear for all the cell lines, and estimated high-affinity KA values were identical (5.3 109 6 1.2 109

M21, 4.5 109 6 1.6 109 M21, 3.9 109 6 0.7 109 M21,and 4.9 109 6 1.5 109 M21 for CHO IR, CHOIR-GpA, CHO IR-GpAmut, and CHO IR TM12 cells,respectively). Insulin binding analyses were also con-ducted with solubilized receptor preparations; again,the high-affinity KA values for all receptors wereindistinguishable (data not shown).

Metabolic labeling of wild-type and chimericreceptors in transfected cells

The biosynthesis and processing of the IR transmem-brane mutants were analyzed using pulse-chase ex-periments on metabolically labeled transfected CHOcells. Figure 2 indicates that all receptors are initiallysynthesized as a ;190 kDa proreceptor. Two hoursafter the beginning of the chase, the proreceptorstarted to be cleaved into its two mature subunits (aat 135 kDa and b at 95 kDa), which were theprominent forms after 6 h. The three modifiedreceptors exhibited normal patterns of biosynthesisboth in terms of subunit structure and rates ofprocessing as compared with the wild-type IR.

Structural studies of solubilized and WGA-purifiedreceptors by nondenaturing PAGE

To determine whether the modifications introducedin the TM domain of the insulin receptor modifiedits structure, we performed gradient slab gel electro-phoresis of insulin receptors solubilized from thedifferent transfected cell lines and partially purifiedby WGA-agarose chromatography.

We took advantage of the differential sensitivity ofthe b–b and a–b disulfide bonds to chemical reduc-tion with DTT and performed SDS-PAGE of 32P-labeled solubilized receptors in the presence ofdifferent concentrations of DTT. Figure 3 shows(upper panel) the reduction profile of wild-typereceptors with the predominance of an ab form(;450 kDa) at a low concentration of DTT (, 1mM), the appearance of an ab form (;230 kDa) at1–2 mM DTT, and the complete reduction of phos-phorylated receptors to the b monomer (95 kDa) atDTT . 10 mM. The same reduction profile wasobserved for receptors solubilized from the IR-GpACHO cells (Fig. 3, lower panel), except for theappearance of a novel band for DTT concentrations

Figure 2. Metabolic labeling of chimeric and wild-type insulinreceptors. 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 incomplete medium. At each time, cells were lysed and thereceptors immunoprecipitated. Metabolically labeled insulinreceptors were then analyzed by denaturing SDS-PAGE andidentified by autoradiography. The labeled proreceptors(pro), a subunits, and b subunits are indicated.

Figure 1. Mutated transmembrane sequences. A) Part of thewild-type human insulin receptor sequence (IR), starting withAsn 926, is shown at the top using the one-letter amino acidcode, with the entire transmembrane domain sequenceshown in boldface characters. IR-GpA, IR-GpA mut, and IRTM12 transmembrane sequences are shown below. The sevenresidues important for glycophorin A dimerization are under-lined in the GpA sequence. The mutation Val 3 Trp in theIR-GpAmut sequence is doubly underlined, as well as the twosupplementary flanking amino acids in the IR TM12 sequence.B) Nucleotide and amino acid sequences of the wild-typehuman insulin receptor (nucleotides 2901–3087) and theplasmid PebNde1, where the entire sequence of the insulinreceptor transmembrane domain has been replaced by a NdeIrestriction site.

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above 2 mM. This band had an apparent molecularmass of ;190 kDa, which is likely to correspond to ab form. This observation is consistent with the hy-pothesis that the introduction of the GpA TM do-main would induce increased interactions between bsubunits. As the phosphorylated band with an appar-ent mass of ;190 kDa may also correspond to theprecursor form of the receptor, its identity wasassessed by immunoblotting with antibodies directedvs. the a and b subunits of the insulin receptor.Figure 4 shows that the lower ;190 kDa band wasrecognized by both anti-a and anti-b subunits anti-bodies, indicating that this band contains someproreceptor in the two preparations. However, therewas a significant difference in intensity of this bandlabeled with the anti-b subunit antibody between thechimeric (IR-GpA) and wild-type (IR) receptors,whereas no difference was observed after anti-aantibody labeling. This is a strong indication that in

the chimeric IR-GpA receptors, the ;190 kDa bandalso contains a large proportion of b2 dimers.

The mechanism of activation of the insulin recep-tor tyrosine kinase differs from that of other receptortyrosine kinases in that the receptor is constitutivelydimeric. Its activation involves conformationalchanges, which have been detected by a variety oftechniques [summarized in (25)]. One method usedto demonstrate such conformational changes hasbeen nondenaturing Triton X-100 PAGE, whichallows for the separation of proteins according tomolecular size and shape. Florke et al. (18) haveshown that the native ab form of insulin receptorspossesses an apparent Stokes radius of ;9.5 nm,which decreases to ;7.9 nm upon insulin binding.Phosphorylated insulin receptors turn back to a 9.5nm form when insulin dissociates. As it had recentlybeen shown that the presence of the TM domain isrequired for this change in conformation to occur inconstructs containing the receptor cytoplasmic do-main (26), we used this method to study the confor-mation of receptors solubilized from the transfectedCHO cell lines after phosphorylation. Figure 5 showsa typical profile of molecular forms in the presence

Figure 3. Structural studies of solubilized mutantreceptors, dimerization assay. Solubilized andlectin-purified insulin receptors from IR-GpACHO and IR CHO cells were phosphorylated inthe presence of insulin and [g-32P]ATP, and thereaction was stopped by addition of LDS samplebuffer containing the indicated concentrationsof dithiothreitol (DTT). Electrophoresis was runat 4°C overnight, and the gels were dried andsubmitted to autoradiography and PhosphorIm-ager analysis. An image representative of fourexperiments with different receptor prepara-tions is shown. Positions of molecular weightmarkers are indicated at left, and positions of thea2b2, ab, b, and ;190 kDa forms are at right.

Figure 4. Dimerization assay, Western blot. Solubilized andlectin-purified insulin receptors from IR-GpA CHO and IRCHO cells were incubated in the presence of ATP withoutinsulin, and the reaction was stopped by addition of LDSsample buffer containing 5 mM DTT. Electrophoresis was runat 4°C and samples were transferred onto PVDF membranes.Western blot analysis was performed with antibodies directedagainst the a or b subunits of the human insulin receptor;revelation was performed with the ECL kit. The experimentpresented here was performed with two different receptorpreparations, each from IR-GpA CHO and IR CHO cells; thesame membrane was probed first with the anti-a subunitantibody and reprobed with the anti-b subunit antibody afterstripping of the first reagents. Molecular masses of proteinstandards are indicated at left, and the position of the ab andb2/proreceptor forms of the insulin receptor are shown atright.

Figure 5. Structural studies of solubilized mutant receptors:Triton X-100 native electrophoresis. Solubilized and lectin-purified insulin receptors from chimeric and wild-type IRtransfected cells were phosphorylated, as described in Fig. 3,and analyzed in the Triton X-100 polyacrylamide gel electro-phoresis gradient gel system of Florke (18). Electrophoresiswas run at 4°C overnight; the gels were dried and submittedto autoradiography and PhosphorImager analysis. A repre-sentative image of four experiments with different receptorpreparations 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 insulinreceptors (high and low Stokes radius) at right.

1351TM DOMAIN INACTIVATION OF INSULIN RECEPTORS

and absence of insulin, without cross-linking. For IRreceptors (at right), the high Stokes radius form wasalways predominant (high/low forms ratio . 9 ac-cording to PhosphorImager analysis of the gels). Incontrast, the IR-GpA receptors existed predomi-nantly as a low Stokes radius form, even in theabsence of insulin (at left), with a constant ratiobetween high and low forms of ;1.5. IRTM12 recep-tors had a profile identical to the IR receptors,whereas the IR-GpAmut receptors displayed an inter-mediate profile (high/low forms ratio ;3 in thepresence of insulin). Taken together, these resultsindicate that the replacement of the insulin receptorTM domain with that of glycophorin A had a majoreffect on the receptor structure.

Insulin signaling in transfected cells

Next we examined the consequences of TM domainsubstitutions on insulin cellular actions in trans-fected CHO cells. Figure 6A shows the insulin dose-response curve for the incorporation of [14C]glucoseinto glycogen in different clones of CHO IR-GpAcells compared with CHO IR and mock-transfectedcells. CHO IR-GpA cells were less responsive tohormonal stimulation than CHO IR cells expressingwild-type receptors. Maximal stimulation factorabove basal was 2.2 for untransfected cells and 3.5 forthe CHO IR cell line. Cells expressing the IR-GpAchimeric receptor were nearly insensitive to insulin,as maximal stimulation was close to 1.5-fold. EC50 forCHO IR-GpA cells was intermediate between that ofCHO IR and CHO cells for different CHO IR-GpAclones expressing different amounts of receptors(;1–3 nM vs. ;0.5 nM and ;12 nM, respectively).Similarly, CHO IR cells were hypersensitive to insulinfor the incorporation of [3H]thymidine (EC50 ;0.4nM, maximal stimulation fivefold) as compared withparental CHO cells (EC50 ;4 nM, maximal stimula-tion 2.7-fold) (Fig. 6B). Again, the CHO IR-GpA cellsdid not respond to insulin (EC50 ;0.3 nM, maximalstimulation twofold), whereas the CHO IR-GpAmut

cells showed intermediate stimulation (EC50 ;0.8nM, maximal stimulation 3.4-fold) and the CHOIRTM12 cells were indistinguishable from the CHOIR cells.

We also investigated the insulin-induced activationof two different intermediary enzymes involved ininsulin action, MAP kinase and PtdIns 3-kinase. Asshown in Fig. 7A, the basal activity of MAP kinase wasmaximally stimulated ;5.5-fold by insulin in CHO IRcells, whereas a smaller, 2.5-fold stimulation wasobserved in CHO neo cells. In cells expressingIR-GpA chimeric receptors, a 2.4-fold increase wasobserved, whereas the CHO IR-GpAmut displayed amaximal stimulation of 3.8-fold. In the five cell lines,10% fetal calf serum stimulated MAP kinase activity

Figure 6. Biological effects of insulin in CHO cells transfectedwith the mutant insulin receptors. A) Glycogen synthesis. Cellswere exposed for 1 h to the indicated concentrations of insulin,after which [14C]glucose was added for 3 h at 37°C. Glycogenwas precipitated with ethanol and counted for radioactivity. Dataare expressed as stimulation factor over basal level in theabsence of insulin, as mean 6 se of four experiments performedin triplicate with mock-transfected (neo, filled squares) CHOcells, cells transfected with the wild-type human insulin receptorcDNA (IR, filled circles), and two different monoclonal cell linestransfected with the chimeric construct containing the TMdomain 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 GpA33 cells, respectively, as compared to 350,000 sites per cell for theIR-CHO cells). Basal levels in the absence of insulin were similarfor all cell lines. B) Thymidine incorporation. Serum-deprivedcells were incubated for 15 h with the indicated concentrationsof insulin, after which [3H]thymidine was added for 45 min at37°C. Incorporation of thymidine into DNA was measured astrichloroacetic acid-precipitable radioactivity. Data are ex-pressed as stimulation factor over basal level in the absence ofinsulin, as mean 6 se of 2–5 experiments performed in triplicatewith mock-transfected (neo, filled squares) CHO cells, cellstransfected with the wild-type human insulin receptor cDNA(IR, filled circles) and its variant containing the two supplemen-tary flanking amino acids (IRTM12, filled triangles), and cellstransfected with the chimeric construct containing the TMdomain of glycophorin A (IR-GpA, diamonds) and its Val3Trpmutant (IR-GpAmut, triangles). Basal levels in the absence ofinsulin were similar for all cell lines. All transfected cell linescontained a similar amount of insulin receptors (;350,000sites/cell, as judged by insulin binding assay).

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to the same extent, indicating the absence of alter-ation in the signaling pathway (data not shown). Forinsulin stimulation of PtdIns 3-kinase activity (Fig.7B), a similar pattern was observed since maximalactivation factors of 1.8-, 4.8-, 2.2-, and 3.6-fold werefound for CHO neo, CHO IR, CHO IR-GpA, andCHO IR-GpAmut cells, respectively. CHO IRTM12

cells responses were identical to those of normalCHO IR cells (not shown).

Phosphorylation of the insulin receptor and itsmajor substrate after insulin stimulation was studiedby Western blotting analysis of whole cell extractswith specific monoclonal antiphosphotyrosine anti-bodies. Figure 8 shows a representative tyrosinephosphorylation pattern of cellular extracts of IRand IR-GpA CHO cells after 10 min stimulation withincreasing concentrations of insulin. Tyrosine phos-phorylation of two main protein bands, with appar-ent molecular masses of 95 kDa (insulin receptor bsubunit) and 180 kDa (IRS-1), was stimulated byinsulin. Although the two cell lines contained equiv-alent amounts of insulin receptors, major differenceswere apparent: maximal phosphorylation of the twobands was higher, and a lower insulin concentrationwas required to observe phosphorylation in the IRCHO cells. IRS 1 and receptor b subunit tyrosinephosphorylation were already apparent at 0.1 nMand 1 nM, respectively, in the IR CHO cells, and areduced sensitivity of at least 10-fold was observed forIR-GpA CHO cells.

These results indicate that the receptors contain-ing the TM domain of GpA are unable to normallytransduce the insulin signals in transfected CHOcells. This defect is not due to clonal variation, sinceit was observed with all clones tested as well as withpolyclonal cell lines established by fluorescence-activated cell sorting. The two supplementary aminoacids flanking the TM domain appear to be neutralwith respect to all insulin actions tested, since theCHO IRTM12 cells behaved similarly to the CHO IRcells. The CHO IR-GpAmut cells showed intermediatecharacteristics in their responses to insulin, indicat-ing that the disruption of dimer formation by thisVal 3 Trp mutation [which was seen in a GpA TM

Figure 8. Insulin effect on ty-rosine phosphorylation of cellu-lar proteins in IR CHO and IR-GpA CHO cells. Cells wereserum starved overnight andtreated with the indicated con-centrations of insulin for 10min. Cells were lysed in electro-phoresis sample buffer; equalamounts of protein were sub-mitted to SDS-PAGE, followedby blotting with antiphosphoty-rosine monoclonal antibody 4G10 and revelation with ECL. A typical experiment of four is depicted. Molecular masses ofprotein standards are indicated at left; the position of IRS-1 and the b subunit of the insulin receptor are shown at right.

Figure 7. Insulin signaling in CHO cells transfected with themutant insulin receptors. A) Map kinase activity was assayedusing myelin basic protein as substrate in anti-erk-2 immuno-precipitates of control and transfected CHO cells stimulatedwith the indicated concentrations of insulin. After SDS-PAGE,quantification was performed with a Fuji BAS-1000 Phospho-rImager; results are depicted as the level of stimulation overbasal (mean6se of four experiments). B) PtdIns 3-kinaseactivity was assayed using PtdIns as substrate in antiphospho-tyrosine immunoprecipitates of control and transfected CHOcells stimulated with the indicated concentrations of insulin.After TLC, quantification was performed with a Fuji BAS-1000PhosphorImager; results are depicted as the level of stimula-tion over basal (mean6se of four experiments).

1353TM DOMAIN INACTIVATION OF INSULIN RECEPTORS

domain-nuclease chimeric protein (13)] may not besufficient for restoring proper function of the insulinreceptor or may not be fully operative when insertedin the insulin receptor protein (see Discussion).

Since the signaling defect was already seen as adecreased tyrosine phosphorylation of the insulinreceptor, we also studied autophosphorylation andtyrosine kinase activity of the solubilized wild-typeand recombinant receptors in vitro. IR-GpA recep-tors displayed major abnormalities, as their basalautophosphorylation was markedly higher and theirinsulin-stimulated autophosphorylation markedlylower than those for the wild-type IR receptors (datanot shown). Similar alterations were also evident forthe phosphorylation of the synthetic substrate poly-(Glu-Tyr) when using equal amounts of receptorpreparations (Fig. 9). IR-GpA receptors were able tomaximally phosphorylate this substrate by 2.2-fold,compared with 4-fold for IR receptors. Again,IRTM12 receptors were identical to the IR prepara-tion, and the IR-GpAmut receptors were intermediatebetween IR-GpA and IR receptors (maximal stimula-tion 3.1-fold).

Taken together, these results show that introductionof the GpA TM domain in the insulin receptor pro-

vokes apparent structural changes and causes defects ininsulin signaling due to a reduced activation of thereceptor tyrosine kinase activity by insulin.

DISCUSSION

Our understanding of the conformational changesgenerated by the insulin signal and how this signal ispropagated through the receptor protein and acrossthe membrane is limited. Contrary to the earlyassumption that the TM domain of the insulinreceptor (and other receptor tyrosine kinases ingeneral) is only necessary to anchor the protein inthe membrane, several studies have shown that mod-ifications of this TM domain can affect the activity ofvarious members of this receptor family. The firstexperimental evidence for the active role of the TMsequence in this class of membrane proteins camefrom the study of the neu/erbB2 proto-oncogenemutations in chemically induced glioblastoma inrats. The oncogenic allele of neu contains a pointmutation [Val(664) 3 Glu] in the predicted TMsequence of its product, which causes increasedtyrosine kinase activity and increased aggregation ofthe receptor protein (27, 28). A similar point muta-tion (Gly 3 Arg) was discovered in the transmem-brane domain of the fibroblast growth factor recep-tor-3; this mutation causes achondroplasia, acommon form of dwarfism (29, 30). Again it wasshown that the molecular consequence of this muta-tion is the constitutive activation of the tyrosinekinase of the receptor (31). Chimeric receptorscomprising the extracellular domain of the epider-mal growth factor receptor, the kinase domain ofc-ros, and the TM domain of either the epidermalgrowth factor receptor or c-ros had opposite effectson cell growth when transfected in NIH 3T3 cells(32). Recent data suggest that the transmembranedomain of gp130, the signal-transducing subunit ofthe IL-6 receptor, is necessary for signal transductionand determines its interaction with Janus kinases(33). Mutations resembling that of oncogenic neu/erbB2 have also been introduced in the epidermalgrowth factor receptor (34) and insulin-like growthfactor 1 receptor (35), resulting in constitutive acti-vation of the receptors.

Replacement of the insulin receptor TM domainby that of the oncogenic form of the product ofc-neu/erbB-2 induces ligand-independent activationof the insulin receptor and modulation of the hor-mone actions (8, 10). The fact that introduction ofthis dimerizing TM sequence in the insulin receptorresulted in constitutive activation led further supportto the evidence that interactions between ab recep-tor halves and receptor transphosphorylation areimportant for tyrosine kinase activation of the insu-

Figure 9. Insulin stimulation of poly (Glu, Tyr) phosphoryla-tion by solubilized mutant insulin receptors. Phosphorylationof the synthetic substrate poly (Glu 4, Tyr 1) in the presenceof [g-32P]ATP was catalyzed by equal amounts of WGA-purified IR (circles), IR-GpA (diamonds), IR-GpAmut (opentriangles), and IRTM12 (filled triangles) that had been incu-bated with the insulin concentrations shown. The reactionwas stopped by spotting samples onto phosphocellulose pa-pers in 10% TCA. Papers were then extensively washed in 5%TCA, dried, and incorporated radioactivity was measured byCerenkov counting. The results of 5 separate experiments areshown (mean6se).

1354 Vol. 13 August 1999 GARDIN ET AL.The FASEB Journal

lin receptor. The replacement of the TM domain ofthe PDGF b receptor by the mutated neu/erbB2domain also led to constitutive activation of thisreceptor (36). These data demonstrate that theintroduction of the dimerizing TM sequence ofneu/erbB2 in different members of the tyrosinekinase receptor family invariably provokes majoralterations in their biological activity.

To further characterize the role of TM–TM do-mains interactions in the mechanism of activation ofthe insulin receptor, we designed chimeric receptorscontaining another well-characterized dimerizingTM sequence from an unrelated protein. GpA is anerythrocyte integral membrane protein with onetransmembrane a-helix, which forms dimers that arestable even in the detergent SDS. Dimerization ofGpA is driven by specific interactions between thetransmembrane a-helices. This domain is also able topromote stable dimerization of heterologous pro-teins, and a chimeric protein containing the TMdomain of GpA and the carboxyl terminus of staph-ylococcal nuclease was used to characterize by mu-tagenesis the residues responsible for helix-helixassociation (13, 15). Finally, the 3-dimensional struc-ture of the dimeric TM domain of GpA has beenestablished (37).

All mutant receptors were expressed at the cellsurface in the CHO cells. Glycosylation and matura-tion occurred normally in all mutant receptors, asevidenced by normal apparent molecular masses ofthe two subunits and similar processing of the pro-receptor into a and b subunits. When electrophore-sis of [32P]-labeled solubilized receptors was run atlow temperature in the presence of increasing con-centrations of the disulfide-reducing agent DTT,normal reduction from the native a2b2 form to abmonomers and to b subunits was observed for allreceptors. However, a novel band at ;190 kDa wasseen for the IR-GpA receptors and to a lesser extentfor the IR-GpAmut receptors (not shown). This novelband was identified as a b2 form due to noncovalentdimer formation between the 95 kDa b subunits,induced by the GpA TM sequence. It is surprisingthat this ;190 kDa form was not more prominent,since the chimeric receptors represented 80–90% ofthe total insulin receptors in transfected CHO cells.This is probably related to the intrinsic difficulties ofpreserving noncovalent interactions between sub-units during the steps of sample phosphorylation,partial reduction, and subsequent electrophoresis.We found that boiling or freezing the protein sam-ples abolished this b2 form.

This structural alteration induced by the introduc-tion of the GpA TM domain led to major modifica-tions in insulin signaling. All the actions of thehormone studied here (MAP kinase activity, PtdIns3-kinase activity, glycogen synthesis. and thymidine

incorporation) were markedly reduced in IR-GpACHO cells, whereas mutated IR-GpAmut CHO cellsdisplayed intermediary responses. This was clearlyrelated to major abnormalities in the tyrosine kinaseactivity of the receptor. Some disparity was notedbetween in vivo and in vitro phosphorylation assays,especially on the level of basal autophosphorylationin the absence of insulin, which was elevated in thechimeric receptors in vitro without any significantchange in the basal levels of biological effects andcell protein tyrosine phosphorylation. This was alsoobserved for other TM domain chimeric constructsof insulin receptors (10), and possibly is accountedfor by regulatory mechanisms in intact cells such asphosphatases. It is also intriguing that the mutatedIR-GpAmut CHO cells displayed intermediary re-sponses in all the functional assays, although mu-tagenesis data of the GpA TM would predict fullactivity. This Val(80) 3 Trp mutation is located atthe middle of the dimerizing sequence of GpA andwas found to be totally disruptive of helix-helixassociation in the GpA/nuclease chimeric protein inSDS micelles (13). However, other mutations at thisposition were not as effective in disrupting dimerformation, and this precise mutation may still allowfor significant packing interactions when inserted ina different protein or in a protein localized in anatural membrane. To support this, structure-basedanalysis (38) of this mutation in the GpA TM domainhas shown that it provokes only a mild steric clash atthe dimer interface (K. R. MacKenzie and D. M.Engelman, personal communication). Furthermore,using the ToxR transcription activator system, Lan-gosch et al. (39) have shown that a Val(80) 3 Alamutation was not as effective in disrupting dimeriza-tion as was predicted from the detergent studies.

Why does introduction of the dimerizing TMdomain of GpA in the insulin receptor lead to aninhibition of insulin signaling instead of activation,as was seen with the neuVal3Glu TM domain? Similarresults were observed when the GpA TM domain wasintroduced into the neu/erbB2 protein, wherestrong dimerization was observed but without trans-forming activity, clearly demonstrating that dimeriza-tion of a tyrosine-kinase receptor is not by itselfsufficient for its activation (40). It thus seems thatnot all dimerizing TM domains are equivalent intheir ability to activate receptor tyrosine-kinases, andthis may be related to the known differences instructure of the TM domains themselves. It has beenestablished that the GpA TM domain promotes aright-handed interaction of a-helices (37), whereasthe neu/erbB2 TM domain adopts a left-handedcoiled-coil structure (41). Thus, we suggest thatthese two different dimerizing TM domain se-quences may impose very different geometries ofinteraction between intracytoplasmic kinase do-

1355TM DOMAIN INACTIVATION OF INSULIN RECEPTORS

mains, thereby favoring (erbB2 TM domain) orimpeding (GpA TM domain) the mechanism oftransphosphorylation between dimerized partners.In the case of the insulin receptor-GpA TM domainchimeras studied here, it is also possible that such amodified orientation of the kinase domains leads tointermolecular transphosphorylation between a2b2

holoreceptors instead of the normal intramoleculartransphosphorylation between the two ab receptorhalves of the receptor. It has been shown thatintermolecular phosphorylation between insulin ho-loreceptors is unable to stimulate substrate kinaseactivity (42). Such a mechanism may be operative inour chimeric receptors, which do not signal whileexhibiting detectable autophosphorylation after in-sulin stimulation in vitro. Another possibility is thatthe abnormal geometry imposed by the GpA TMdomain may impair either the phosphorylation ofkey tyrosine residues or the normal interaction ofphosphotyrosines with SH2/PTB domains proteins,or both. To test our geometrical hypothesis, we haveundertaken the construction and characterization ofother modifications of the GpA TM domain insertedin the insulin receptor. For example, addition orsubstraction of one amino acid of the GpA TMdomain may reorient its dimerization interface, andtherefore the tyrosine kinase monomers, modifyingtheir interactions during activation and oligomeriza-tion.

CONCLUSION

Introduction of the GpA TM sequence in the insulinreceptor led to changes in the receptor structure.The chimeric receptors displayed altered tyrosinekinase activation by insulin, with a decreased auto-phosphorylation and substrate phosphorylation.This, in turn, led to an almost complete abolition ofinsulin responsiveness in cells transfected with thechimeric IR-GpA receptors. Studies of the sites ofphosphorylation of the chimeric receptors and oftheir interactions with SH2/PTB signaling proteins,together with the characterization of other muta-tions, should provide further insight into the mech-anism of transphosphorylation of insulin receptorsand into the role of their TM domain.

We wish to thank Ms. Caroline Waltzinger (Illkirch,France) for her help with the fluorescence-activated cellsorter, and Drs. Bentley Cheatham (Boston, Mass.) andKenneth Siddle (Cambridge, U.K.) for the gift of anti-insulinreceptor antibodies. We also thank the Eli Lilly Co. (India-napolis, Ind.) for the gift of recombinant human insulin. Thiswork was supported by the Institut National de la Sante et dela Recherche Medicale (INSERM) and by grants from theAssociation pour la Recherche sur le Cancer and the LigueNationale contre le Cancer.

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Received for publication October 5, 1998.Revised for publication February 24, 1999.

1357TM DOMAIN INACTIVATION OF INSULIN RECEPTORS