Co-expression of mutant and normal human insulin receptors in COS 7 cells

425

Biochimica et Biophysica Acta, 1216 (1993) 425-430 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 92561

Co-expression of mutant and normal human insulin receptors in COS 7 cells Hiroshi Maegawa a , , Atsunori Kashiwagi a, Tetsuro Haruta a Katsuya Egawa a Satoshi Ugi a, Rie Tachikawa-Ide a, Masaaki Hasegawa a, Masashi Kobayashi b and Yukio Shigeta a a Third Department of Medicine, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-21 (Japan)and b First Department of Medicine, Toyama Medical & Pharmaceutical University, Sugidani, Toyama, 930-01 (Japan) (Received 15 March 1993) (Revised manuscript received 28 June 1993)

Key words: Insulin receptor; Tyrosine kinase; Co-transfection; Insulin resistance; COS 7 cell; (Human)

In order to assess the interference of the mutant insulin proreceptor on normal receptor function and formation of proreceptor-receptor heterotrimers (a/3-proreceptor), COS 7 cells were transfected with the same amount of expression plasmid (pGEM3SV) containing wild-type, a mutant proreceptor cDNA and both, using the DEAE-dextran method. Scatchard analysis of insulin binding data revealed that there was an approx. 50-fold higher receptor concentration in the transfected cells than in untransfected cells. After 0.025% trypsin treatment, insulin binding to the cells expressed with wild-type, proreceptor and both increased by 1-fold, 2.9-fold and 1.5-fold of the untreated cells, respectively. In the presence of 167 nM insulin, the amounts of phosphate incorporated into the 95 kDa protein /3-subunits and 210 kDa proreceptors from co-transfected cells, were identical to those of an in vitro mixture of the wild-type and the mutant receptors. At 10 nM insulin, the proreceptors from co-transfected cells normally autophosphorylated by insulin stimulation, whereas those mixed in vitro did not (73.3 + 9.3 vs. 29.6 + 2.6% of the maximal effect, n = 4, P < 0.01). However, at a similar concentration of insulin, the phosphate incorporation into Glu-80/Tyr-20 polymers by receptors from co-transfected cells was decreased when compared with a in vitro mixture (9.0 + 2.6 vs. 22.5 + 6.7% of the maximal effect at 4 nM, n = 6, P < 0.01), although the basal and maximally stimulated phosphate incorporation were comparable among these groups. These results suggested that similar amounts of both receptor types were expressed and heterotrimers might be formed resulting that a partially activated proreceptor could interfere with normal receptor kinase activity in the co-transfected cells.

Introduction The first step of insulin action is binding to its receptors in the plasma membrane of target cells. The receptor is a heterotetrameric protein consisting of two extracellular a-subunits and two membrane spanning /3-subunits assembled in an a2/32 structure. Insulin binds to the a-subunits stimulating autophosphorylation and tyrosine kinase activity of the /3-subunits [1]. Thus, a primary defect in insulin receptors leads to the

* Corresponding author. Abbreviations: DMEM, Dulbecco's modified Eagle's minimal essential medium; FCS, fetal calf serum; IGF-1, insulin-like growth factor 1; WGA, wheat germ agglutinin; PEG, poly(ethylene glycol); SDSPAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; aCT, insulin receptor antibody which recognizes the carboxylterminus of receptor/3-subunits.

extreme insulin resistance seen in type A insulin resistance, leprechaunism, and Rabson-Mendenhall syndrome [2]. Studies of patients with severe acquired or familial forms of insulin resistance have led to advances in our understanding of insulin receptor function. Several of these patients have mutations in the coding region of the insulin receptor gene that are either homozygotic or heterozygotic. Some heterozygotic mutations have been reported in the kinase region of the insulin receptor and these presumably interfere with transmembrane signaling by compromising the normal receptor tyrosine kinase function on the another allele (dominant negative effect) [3]. Furthermore, we previously determined by means of in vitro mutagenesis [4,5] that a kinase-negative human insulin receptor (A/K1018) expressed in Rat 1 fibroblasts interfered with normal rat receptor function. Recently, we reported siblings with insulin resistance caused by the decreased ligand affinity of unprocessed insulin

426 proreceptors due to a point mutation at the cleavage site [6-9]. In this study, we determined whether the mutant insulin proreceptor interfered with normal receptor function and whether proreceptor-receptor heterotrimers were formed when both normal and mutant proreceptors were simultaneously expressed in COS 7 ceils at the same levels.

Materials and Methods Materials Purified porcine insulin was a gift from NovoNordisk Pharmar (Copenhagen, Denmark). Na125I, [3'32P]ATP, [125I]insulin-like growth factor 1 (IGF-1) were purchased from New England Nuclear (Boston, USA) and Amersham International (Buckinghamshire, UK). Dulbecco's modified Eagle's minimal essential medium (DMEM), and fetal calf serum (FCS) were from Gibco (Grand Island,USA). Protein A(Pansorbin) was from Calbiochem Behring (La Jolla, USA). Wheat germ agglutinin (WGA) agarose was from Pharmacia (Uppsala, Sweden). The eukaryotic expression vector including normal human insulin receptor (pGEM3SVHIR) and mutant proreceptor (pGEM3SVHIR-Ser-735) were supplied by H. Teraoka of Shionogi Research Laboratories (Osaka, Japan) as previously described [10,11]. Anti-insulin receptor antiserum was obtained from a Type B insulin resistant patient with Sj6gren's syndrome [12] and aCT antibody which recognizes the carboxyl-terminus of insulin receptors /3-subunits was kindly gifted by J.M. Olefsky (University of California, San Diego). All other reagents were of analytical grade from Nakarai Chemical (Kyoto, Japan). COS 7 cells expressing wild-type and mutant insulin proreceptors Wild-type insulin receptors and mutant proreceptors were expressed by means of normal and mutant human insulin receptor cDNA vectors, respectively. COS 7 cells were incubated with normal or mutant cDNA vectors (5 lzg) in DMEM/50 mM Tris-HCl (pH 7.3) containing 0.5 m g / m l DEAE-dextran in 6-well 35 mm dishes at 37°C for 4 h. The ceils were washed, and incubated in D M E M / 2 % FCS/100 ~M chloroquine at 37°C for 3 h. After the incubation with complete DMEM supplemented with 10% FCS for 72 h, the insulin receptor was transiently expressed as previously reported [10,11]. Insulin was labeled with 125I using chloramine T and insulin binding was monitored in a monolayer in the 6-well dishes.

eluate was calculated by Scatchard plots of insulin binding data.

Autophosphorylation and tyrosine kinase assays The lectin-purified extracts (400 fmol insulin binding capacity) were preincubated with insulin (0-167 nM) at 4°C for 16 h. The phosphorylation reaction was initiated by adding a mixture of 20 mM Mn acetate, 5 mM CTP, 50 /xM ATP, and 30 ~Ci of [7-32p]ATP. After incubation at 4°C for 15 min, the reaction was terminated by adding 0.2% Triton X-100 containing, 10 mM EDTA, 100 mM NaF, 20 mM sodium pyrophosphate, 20 mM ATP, and 20 mM Hepes (pH 7.6). After immunoprecipitation with anti-receptor antibody, which recognized a-subunit of insulin receptor, the pellet was dissolved in the Laemmli buffer with 100 mM dithiothreitol and resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE) as previously described [4]. Tyrosine kinase activity of insulin receptors toward the Glu-80/Tyr-20 polymer was assayed by the filter method [13]. Results The cells transfected with the expression vectors containing either the wild-type or the mutant proreceptor cDNA efficiently expressed the respective insulin receptors. Insulin binding to untransfected cells, those transfected with the wild-type cDNA, the mutant cDNA or those co-transfected with both cDNAs were 0.5, 18.0, 7.0 and 11.1% at 0.03 nM insulin, respectively, and Scatchard analysis of these data revealed

0.2

0.15 O~

'~C 0.1

0.05

0

Preparation of partially purified insulin receptors Insulin receptors were purified with WGA column and titrated by insulin binding assay using a poly(ethylene glycol) (PEG) as previously reported [13]. Insulin binding capacity (receptor concentration) of each WGA

100

200

300

t 400

500

600

Bound (fmol) Fig. 1. [~25I]Insulin binding to COS-7 cells transfected with wild-type (©), mutant proreceptor cDNA (e), both; co-transfected (D) and untransfected ( • ) . Binding data were analyzed by Scatchard plots.

427 TABLE I

Effect o f trypsin treatment on insulin binding to transfected COS 7 cells Cell were treated with 0.025% trypsin at 4°C for 20 min. [1251]Insulin binding to the cells was measured using 0.03 nM insulin at 4°C for 16 h. Values are expressed as means_+ S.E. of five separate experiments.

"~" 20

.$ u

0 0

15

Wild type Mutant Co-transfected

2

o')

t-

•-

10

c

'Mutant

Trypsin ( - )

Trypsin ( + )

fold-increase

18.0_+0.9% 7.0 _+0.7% 11.I +0.7%

18.0_+2.2% 20.3 + 1.6% * 16.7+0.6% *

1.0 2.9 1.5

• P < 0.01 vs. trypsin (-).

ig e-

"~ ¢/)

5

e-

Trypsin (-)

Trypsin (+)

Fig. 2. Effect of trypsin treatment on insulin binding to co-transfected cells. After the trypsin treatment, there was a 1.8-fold higher insulin binding to the co-transfected cells than that to the untreated counterparts. Since we found that after the trypsin treatment, insulin binding to the cells that expressed proreceptors increased 2.9-fold, but the binding to the cells expressed with the wild-type receptors was unchanged (Table I), each percentage of two receptor types in the co-transfected cells was calculated based on these findings. Wild-type (normal) receptor (dark-shaded), mutant proreceptor (light-shaded).

that the cells expressing the wild-type, the mutant and both receptors had an approx. 50-fold higher insulin receptor concentration than untransfected cells, and

that the mutant proreceptor had decreased affinity for insulin. Insulin binding to the co-transfected cells was mid-way between those of the cells expressed with wild-type and mutant proreceptors, suggesting that expression of proreceptors had no effects on the ligand binding ability of normal receptors in the co-transfected (Fig. 1). After the treatment of cells with 0.025% trypsin, insulin binding to the cells that expressed proreceptors increased 2.9-fold, whereas that to cells with the wildtype receptors was unchanged. Furthermore, after the trypsin treatment, there was 1.5-fold higher insulin binding to cells co-transfected with both cDNAs than that of the untreated counterparts, suggesting that a similar amount of both receptor types was expressed in co-transfected cells (Table I, Fig. 2). Autophosphorylation activities were determined using partially purified insulin receptors in the cells transfected with two types of cDNA. Furthermore,

210K - ~

95K - ~

Fig. 3. Autophosphorylation o f / 3 - s u b u n i t s of insulin receptors and 210 kDa proreceptors from wild-type (400 fmol insulin binding capacity), mutant (400 fmol), co-transfected cells (800 fmol), in vitro mixing of wild-type and mutant receptors (400 fmol for each). At the treatment of a high concentration of insulin (167 nM), the identical levels of phosphate were incorporated into the 95 kDa protein /3-subunits and 210 kDa proreceptors from the co-transfected cells compared with those of in vitro mixtures of the wild-type and mutant receptors in terms of having the identical receptor number (400 fmol insulin binding capacity).

428

In vitro Mixing

Co-transfected

210K 0 10 167

0 10 167

Insulin (nM) Fig. 4. Dose response of autophosphorylation of proreceptors from both co-transfected cells and in vitro mixture. At high concentration of insulin (167 nM), the phosphorylation levels were comparable among co-transfected cells and in vitro mixture. However, at 10 nM insulin, the proreceptors from co-transfected cells were more phosphorylated than those mixed in vitro as shown in Table II.

T A B L E II

these activities were compared with the activities of the in vitro mixing of equivalent amounts of wild-type and proreceptors. At the maximal insulin stimulation (167 nM), identical levels of phosphate were incorporated into the 95 kDa protein /3-subunits of insulin receptors and 210 kDa proreceptors from co-transfected cells when compared with those of in vitro mixtures of the wild-type and the proreceptors (Fig. 3). It is possible that mutant proreceptors may be less immunoprecip±rated by insulin receptor antibody, which recognizes the a-subunit of insulin receptor, due to some conformational changes. Thus, we performed the immunoprecipitation of the insulin receptors with a different antibody (aCT), which recognizes the carboxylterminus of receptor /3-subunits. However, the max±-

Comparison of autophosphorylation of proreceptor between co-transfected cell and in vitro mixing of wild-type and mutant insulin receptors" Phosphate incorporation into 210 kDa proreceptors from the cotransfected cells and in vitro mixing was assayed by the m e a s u r e m e n t of radioactivity (dpm). Values are expressed as m e a n s + S.E. of four separate experiments. Insulin (nM) basal (A)

Co-transfected 185.5±38.4 In vitro mixing 158.4±31.4 Significance ns

% of the 10 (B)

167 (C)

maximum at 10 nM (B-A)/ (C-A)

612.1±31.9 369.8±24.7 P < 0.01

797.6±54.8 870.0+50.9 ns

73.3+9.3 29.6_+2.6 P < 0.01

T A B L E III

Tyrosine kinase activities of insulin receptors from transfected cells toward the Glu-80 / Tyr-20 polymers Tyrosine kinase activities of insulin receptors containing 200 fmol of insulin binding capacity were assayed by the filter method. Values are expressed as means_+ S.E. of six separate experiments. Insulin (nM) basal (A) Wild type Mutant In vitro mixture Co-transfected Untransfected

4 (B)

167 (C)

1.70 ± 0.22 2.25 _+0.13 3.91 _+0.67 1.71 _+0.22 1.95 _+0.14 3.59 + 0.09 1.94 ± 0.02 2.36 ± 0.15 3.80 _+0.15 1.93 ± 0.11 2.08 ± 0.06 3.65 _+0.12 0.21 ± 0.07 0.21 _+0.20 0.27 _+0.03 (pmol p h o s p h a t e / 2 0 0 fmol insulin receptor)

* P < 0.01 vs. in vitro mixture; # P < 0.01 vs. wild-type receptor.

Foldstimulation (C/A)

% of the maximum at 4 nM (B-A)/(C-A)

2.63 2.11 1.96 1.89 1.29

30.2 ± 0.7% 13.1 _+6.0% # 22.5 _+6.7% 9.0 ± 2.6% *

429 mally stimulated autophosphorylation assessed by a C T antibody immunoprecipitation was comparable between normal and mutant proreceptors. The proreceptors from co-transfected cells were more phosphorylated than those mixed in vitro which was measured in the presence of 10 nM insulin (73.3 _+ 9.26% vs. 29.6 + 2.6% of the maximal effect, n = 4, P < 0.01) as shown in Table II and Fig. 4, although the /3-subunits were comparably phosphorylated between receptors from co-transfected cells and in vitro mixture. On the other hand, the phosphate incorporation into Glu-80/Tyr-20 polymers by receptors from cotransfected cells was significantly decreased compared with those of normal and mutant proreceptors mixed in vitro, at the submaximal concentration (4 nM) of insulin (9.0 _+ 2.6 vs. 22.5 + 6.7% of the maximal effect, n = 6, P < 0.01), although both the basal and the maximally stimulated phosphate incorporation were comparable among these groups (Table III). [~25I]IGF-1 binding to each cell monolayer was comparable ( 5 - 6 % at 0.07 nM IGF-1), and W G A purified receptors had similar binding capacities (10-14 f m o l / ~ l ) among these four types of cell. Thus, there were no significant differences in IGF-I binding between untransfected and transfected COS 7 cells.

Discussion We demonstrated that normal and unprocessed proreceptors were comparably expressed in cotransfected cells in terms of protein content, when the same amount of expression vectors was used. The mutant proreceptors expressed in co-transfected cells were more autophosphorylated than those mixed with normal receptors in vitro. However, tyrosine kinase activities of receptors from co-transfected cells were impaired at the submaximal concentration of insulin when compared with those measured by mixing of the identical receptor concentration in vitro. Treadway et al. [15] reported that the kinase-negative A / K 1 0 1 8 receptors are normally autophosphoryfated. However, they interfere with the normal receptor kinase activity when the heterotetramer (aNormal OCAKflNormalflAK)was generated in vitro. Furthermore, it has been reported that this kinase-deficient A / K 1 0 1 8 and the wild-type receptors are expressed as the heterotetrameric structure ( a N. . . . IOLAK~NormaI~AK) when both cDNAs are transfected simultaneously [16]. In that study, the A / K 1 0 1 8 receptors are intramolecularly trans-phosphorylated by the wild-type receptors, but the opposite is not the case. Therefore, tyrosine kinase activities of these heterotetramers are impaired since both activated tyrosine kinases of each form may be necessary to phosphorylate substrates. These heterotetramers can intramolecularly inhibit the wild-type

receptor tyrosine kinase activity (dominant negative effect) [16]. With regard to the unprocessed mutant proreceptor in the co-expressed cells, heterotrimers (proreceptora/3-subunits complex) might be formed, which may be responsible for inhibiting normal receptor tyrosine kinase. That is, the kinase deficient proreceptor may be trans-phosphorylated by wild-type receptors in an intramolecular manner. However, proreceptors expressed in the co-transfected cells might interfere with normal receptor function (dominant negative) in terms of tyrosine kinase activities at a lower insulin concentration, because both intact activated receptor kinases in one heterotetramer might be necessary for intramolecular substrate phosphorylation. Furthermore, we found that a point mutation at 1048 Asp-Pro at the tyrosine kinase domain of insulin receptor /3-subunit resulted in the impaired tyrosine kinase activities. Using the current experimental protocol, these mutated kinase-deficient receptors impaired the tyrosine kinase activities of the wild-type receptor when expressed simultaneously in the co-transfected cells [17]. Finally, it has been reported that IGF-1 receptor and insulin receptor hybrids may be formed in the transfected cells although they may not interfere with insulin receptor signaling [18,19]. We found that the IGF-1 binding to COS 7 cells and W G A purified receptors was comparable, suggesting that expression of the insulin receptors in COS 7 cells did not any affect IGF-1 binding. In conclusion, among heterozygotic patients with insulin receptor diseases, either a defect in the insulin binding or kinase domains, as well as the formation of heterotetramer may be responsible for insulin resistance, although with varying severity.

Acknowledgements We thank Dr. H. Heraoka for the gift of expression plasmids and COS 7 cells, Dr. J.M. Olefsky for the gift of a C T antibody and Dr. Y. Takata (Toyama Medical and Pharmaceutical University) for useful discussions.

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