Characterization of Unprocessed Insulin Proreceptors Transfected With cDNA With Arg735 -+ Ser735 Point Mutation

in COS 7 Cells at the Cleavage

Site

Masaaki Sugibayashi, Yukio Shigeta, Hiroshi Teraoka, and Masashi Kobayashi We previously reported on patients with severe insulin resistance due to unprocessed insulin proreceptors. A structural change of the cleavage site from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser due to G + T point mutation appeared to be the cause for failure to process the proreceptors. To determine whether the mutation of insulin proreceptors at the cleavage site was responsible for unprocessed insulin receptors and to elucidate the structural and binding characteristics of the proreceptors, we transfected cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. At 72 hours after transfection, insulin binding increased to the maximum in cells transfected with either normal or mutated cDNA, and insulin binding was 40 and 14 times higher than that of nontransfected cells, respectively. The declining rate of insulin binding after reaching the maximum was delayed in cells transfected with mutated cDNA. Affinity cross-linking and surface-labeling studies showed a 135kilodalton (kD), normal a-subunit in the cells transfected with normal cDNA and a 210-kD proreceptor in the mutant cells. The proreceptors were cleaved by trypsin to yield normal-sized IX- and p-subunits. The sensitivity to trypsin was similar to that demonstrated in patients’ cells, and the most effective concentration for the cleavage was 0.025%. Autophosphorylation resulted in decreased 32P incorporation into proreceptors of cells transfected with mutated cDNA at both basal and insulin-stimulated states, without a change in insulin sensitivity. Competitive binding studies with insulin, proinsulin, and miniproinsulin showed that the proreceptors had a lower relative affinity for proinsulin, but this characteristic disappeared after trypsin treatment. These results suggest that the mutation was the cause of the unprocessed insulin proreceptors in the patients with insulin resistance, and that the expressed proreceptors on the COS 7 cells transfected with mutated cDNA had similar binding specificity and sensitivity to trypsin compared with the patient’s proreceptors. However, the proreceptors expressed on COS 7 cells had impaired intramolecular signal transduction and slightly higher binding ability than those of the patients. Copyright 0 1992 by W.B. Saunders Company

T

HE FIRST STEP in insulin action is binding to insulin receptors on the plasma membrane. Thus, decreased binding causes insulin resistance.‘-‘0 We previously found siblings who had a normal receptor number with decreased receptor affinity to insulin due to a point mutation at the cleavage site. “s’~ The mutation changed the structure of the cleavage site from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser, which was thought to be the cause of the failure to process proreceptors in the cells of the patient.13-15 To determine whether the mutation is responsible for the unprocessed insulin receptors in the patients, it is necessary to examine the expressed insulin receptors after transfection with mutated cDNA. Recently, we constructed mutated cDNA by site-directed mutagenesis and transiently transfected pGEM3 vectors with a mutated cDNA into monkey kidneyderived fibroblasts.lb In this report, we describe the characteristics of the expressed mutant receptors that showed decreased binding affinity and defective coupling in signal transmission, and show that the G + T point mutation was the cause of the failure of proreceptor processing.

From the Third Depurtmenr of Medicine, Skigu University of Medical Science, Ok&u, Skiga; and the Skionogi Research Laboratories, Osaka, Japan. Suppotied in part by a research grant for Intractable Disease from the Ministry of Health and Welfare. and a grant-in-aid from the Ministry of Education, Science, and Culture, Japan. Address reprint requests to Masashi Kobayashi, MD, First Depatiment of Medicine, Toyama Medical and Pharmaceutical University Sugitani, Toyama 930-01, Japan. 0026-0495/9214108-0003$03.00J0

820

MATERIALS

AND METHODS

Materials Purified porcine insulin and proinsulin were gifts from Shimizu Pharmaceutical (Shizuoka, Japan), and miniproinsulin was a gift from Novo Industry (Bagsvaerd, Denmark). Na[lZSI] and [@?P] adenosine triphosphate (ATP) were purchased from New England Nuclear (Boston. MA): Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), and trypsin were from Gibco (Grand Island, NY,); lactoperoxidase and chloroquine were from Sigma (St Louis, MO); disuccinimidyl suberate (DSS) was from Pier-Chemical (Rockford, IL); protein A (Pansorbin) was from Calbiochem-Behring (La Jolla, CA); and wheat germ agglutinin (WGA) agarose and diethylaminoethyl (DEAE)-dextran were from Pharmacia (Uppsala, Sweden). Insulin receptor cDNA was kindly supplied by Dr G.I. Bell of Chicago University, and COS 7 cells and monkey kidney-derived fibroblasts, by Dr Sokawa of Kyoto University, Japan. All restriction enzymes were purchased from Takara Shuzo (Kyoto, Japan). Anti-insulin receptor antibody was obtained from a patient with type B insulin resistance, as previously described.17 Construction of Plasmid for Transfection A eukaryotic expression vector, designated pGEM3SV. was constructed from pGEM3 and pKSV-IO by standard procedures.‘s A full~length cDNA of human insulin receptor (HIR) carrying 130 base pairs (bp) and 940 bp of the 5’. and 3’-noncoding region. respectively, was prepared. ly The cDNA had an additional 36 nucleotides just before the cleavage site, as Ebina et al described.‘” This DNA fragment containing the entire coding sequence of HIR cDNA was blunt-ended with the Klenow enzyme and inserted into the expression vector, pGEM3, after the Bg1II site had been blunt-ended. The resulting plasmid was named pGEM3SV-HIR and contained full-length HIR cDNA behind the SV40 early

Metabolism,

Vol41, No 8 (August), 1992: pp 820-826

SIGNAL TRANSDUCTION

pJOmOtOJ, splicing, origin of replication.

and

IN INSULIN PRORECEPTORS

polyadenylation

signals,

and

821

the

SV40

control mutant intact

Site-Directed In Vitro Mutagenesis BumHI-PstI containing 586 bp fragments encoding a normal connecting peptide between a- and p-subunits was subcloned into M13mplO; single-stranded DNA from this recombinant phage was used as the template for oligonucleotide-directed mutagenesis.” The oligonucleotide used was 5’GGAAACGCAGTTCCC’ITGGCGS’ to create the Arg --f d Ser mutation as detected in the patient, as previously reported.” A mutant Ml3 plaque was identified and the double-stranded replicative form was isolated. The BatnHI-PstI fragment was then cloned into BarnHI-Pstlcleaved pGEM3SVHIR, and the resulting plasmid, pGEM3SVHIR, was used for transfection. The structure of the mutant plasmid was verified by restriction enzyme analysis and nucleotide sequencing.

Q-O7.0-

4.4-

Cells and Transfection with 10% FCS. COS 7 cells were grown in DMEM supplemented Cells were transfected with 5 +g plasmid DNA per lo5 cells, using the DEAE-dextran method.‘2

Northern Blot Analysis

l.Q-

Total RNA was extracted by the acid guanidnium phenolchloroform method23 and electrophoresed after denaturation with 0.5 mol/L glyoxal. The RNA was blotted onto nylon and hybridized by standard methods. The probes were a 3.8-kb Sac1 fragment of HIR cDNA and an 0.9-kb &al1 fragment of p-actin cDNA.

Insulin Binding Insulin binding was studied in a monolayer in 6-well dishes at 15°C for 3.5 hours and normalized to percent bound per lo5 cells.

(kb)

Affinity ‘Cross-Linking The cells were incubated with 1251-labe1ed insulin for I6 hours at 4°C and cross-linked with 1 mmol/L DSS. After solubilization and immunoprecipitation with anti-insulin receptor antibody plus Pansorbin,Z4 the sample was treated with Laemmli buffer containing 100 mmol/L dithiothreitol (DIT), and was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE).” ‘?

P-actin-

Surface Labeling The 106 cells were iodinated with 50 to 100 $Zi/mL NalZSI by 25 pg/mL lactoperoxidase. The cells were then solubilized and precipitated with anti-insulin receptor antibody, and the subsequent steps were similar to those of the affinity cross-linking.”

Preparation of Partially Purified Insulin Receptor The cells were washed with phosphate-buffered saline (PBS) and solubilized in a solution of 50 mmol/L HEPES buffer containing 1% Triton X-100, 2 mmol/L phenylmethyl sulphonyl fluoride. and 1 pg/mL aprotinin, pH 7.6. The cell extract was then stirred at 4°C for 60 minutes. After removing insoluble material by centrifugation at 200,000 xg for 60 minutes, supernatants were applied to a WGA-agarose column. After extensive washing with buffer containing 50 mmol/L HEPES, 150 mmol/L NaCI, and 0.1% Triton X-100, pH 7.4, glycoprotein-enriched preparations were eluted by a buffer containing 0.3 mol/L N-acety-o-glucosamine in 50 mmol/L HEPES. 150 mmol/LNaCI, and 0.1% Triton X-100, pH 7.6.’

Fia 1. Northern blot analvsis of COS 7 cells. At 72 hours after transfection, total RNA was &acted. RNA, 20 pg. was applied to a 1% agarose gel and blotted onto a nylon membrane. Hybridization with insulin receptor cDNA or @actin cDNA, and autoradiography were performed following standard procedures. Control. wild-type COS 7 cells; mutant, COS 7 cells transfected with mutated HIR cDNA; intact, CO.5 7 cells transfected with normal HIR cDNA.

Autophosphorylation Assays The lectin-purified extracts were preincubated with various concentrations of insulin at 4°C for 16 hours. The phosphorylation reaction was initiated by adding a solution composed of 20 mmol/L Mn acetate, 5 mmol/L cytidine triphosphate, 20 bmol/L ATP. and 30 FCi [Y-~~P]ATP. After incubation at 4°C for 10 minutes, the reaction was terminated by adding stop solution containing 0.2% Triton X-100. 10 mmol/L EDTA, 100 mmol/L NaF. 20 mmol/L

SUGIBAYASHI ET AL

822

sodium pyrophosphate. 20 mmol/L ATP. and 20 mmol/L HEPES, pH 7.6.” Trypsin Treatment After transfection with mutant cDNA, COS 7 cells were washed twice with PBS and incubated with tlypsin at various concentrations at 25°C for 5 minutes. RESULTS

Northern Blot Analysis After transfection with normal or mutant cDNA. we examined mRNA levels of insulin receptors by Northern blot analysis. In both groups of cells transfected with normal HIR-cDNA or mutated HIR cDNA, there were markedly increased mRNA signals in contrast to those of control, nontransfected cells (Fig 1). The mRNA expression was comparable in two transfected cell lines when they were normalized with p-actin mRNA. Insulin Binding Insulin binding in cells reached the maximum at 72 hours after transfection with normal or mutant cDNA, and then decreased gradually (Table 1). Cells that were not transfected showed very low insulin binding, ie, 0.5% per IO5 cells, indicating that COS 7 cells had a negligible amount of insulin receptors in the plasma membranes. Insulin binding to the cells transfected with normal HIR cDNA (intact cells) increased to 40 times that of the wild-type cells, ie, 20.5% per lo5 cells. In contrast, insulin binding to cells transfected with mutated cDNA was only 35% that of the cells transfected with normal HIR cDNA, as shown in Table 1. Scatchard analysis of the binding curves in cells transfected with normal or mutated HIR cDNA at 72 hours after transfection is shown in Fig 2. In cells transfected with mutated cDNA, insulin binding was decreased due to decreased affinity, which was similar to that of the patients’ cells. Both high- and low-affinity sites had significantly different K, between the two groups. Since maximal binding for the two cell groups occurred 72 hours after transfection, we calculated the declining rate of insulin binding, ie, the time required to reach 50% of maximal binding (Fig 3). It was 105 hours for cells transfected with normal cDNA and 135 hours for those with mutated cDNA. Thus, the rate of increasing binding was comparable for the two groups, but the declining rate was decreased in the cells transfected with mutant cDNA. These results indicated that relatively more receptors expressed by mutant cDNA remained at the plasma membrane for a longer period after transfection.

1000

2000 Bound

4000

3000

insulin

(pg/l

O5 cells)

Fig 2. Scatchard analysis of insulin binding to COS 7 calls transfected with normal or mutated HIR cDNA at 72 hours after transfection. (0-O) Mutant transfected with mutated HIR cDNA; (O-O) intact transfected with normal HIR cDNA. Data represent mean of four experiments. High-affinity site: (intact) K.1 = (1.5 k 0.3) x 1Oqal/M, RO, = (1.1 * 0.3) x 10 sites/cell; [mutant) K.2 = (0.4 _+0.2) x 10’4 l/M, b2 = 10.7 k 0.2) x 10s sites/cell; Kal versus K.2, P < .05; R,, versus RO*,NS. Low-affinity site: (intact)K,3 = (1.0 + 0.3) x 10” 1 IM, Ro3 = (7.1 + 1.9) x 10 sites/cell; (mutant) K.4 = (0.3 + 0.1) x 1Oq4 l/M, & = IS.4 2 1.4) x 1C sites/cell; K.3 versus K.4, P < .05; Rot versus RW, NS. Data are means f SEM of four experiments.

Afinity Cross-Linking To examine the molecular mass of the expressed insulin receptors in these cells, affinity cross-linking was performed. As shown in Fig 4, the molecular mass of insulin receptors expressed in cells transfected with normal HIR cDNA was 135 kD. namely. the a-subunit. However, transfection with mutated cDNA produced only 210-kD unprocessed proreceptors, and 0.025% trypsin treatment changed them into the 135-kD a-subunit. Furthermore, decreased binding affinity to the 210-kD proreceptor was apparent, since the intensity of the autoradiography at 210 kD was markedly lower compared with that of the 135 kD produced by trypsin treatment. Surface Labeling We examined the amount of insulin receptors on the cell surface that could be precipitated with anti-receptor antibody by surface labeling with Na[1?51]. Transfection with mutant cDNA produced 210-kD proreceptors, and trypsin treatment converted them into 135-kD a-subunits (Fig 5). Furthermore, the intensity of autoradiography at 210 kD measured by a densitometer was similar to that of the

Table 1. Time-Course of Insulin Binding After Transfection

0

24

48

72

20.5 k 4.8 7.2 i 0.2* 0.7 + 0.4

Intact (%)

0.5 + 0.2

0.6 + 0.1

10.3 * 1.4

Mutant 1%)

0.5 _f 0.2

0.5 2 0.1

2.5 2 0.2t

Control (%)

0.5 k 0.2

0.5 + 0.2

0.5 k 0.4

96 11.0 +

120

144

7.6 + 0.1

4.5 t 0.1

5.0 _f 1.5*

4.0 _t 0.2t

3.2 t 0.2t

0.5 2 0.2

0.6 k 0.3

0.5 + 0.2

1.9

NOTE. Values are mean 2 SEM of six experiments. Intact, transfection with normal HIR cDNA; mutant, transfection with mutant HIR cDNA; control, no transfection.

lP < .05 intact versus mutant. tP < .Ol intact versus mutant.

SIGNAL TRANSDUCTION

IN INSULIN PRORECEPTORS

823

control

mutant

mutant

Intact

100 90 I c" 5 80. 8 .", 70. .j 60.-(fi50E .5 40E 5 30x

2010

(kOa)

I

__--0 24

I

48

72

96

120

144

hours

Fig 3. Time-course of insulin binding to COS 7 cells after transfection with intact (O-O) or mutated (0-O) HIR cDNA. Percent binding at 72 hours was defined as 100% for intact and mutated HIR cDNA-transfected cells.

If+Ab Trywin

control

mutant

mutant

(-)

(+)

(-)

(+)

(-)

(+)

(-)

(+)

(-)

(-)

(-)

(-)

(+)

(+)

(-)

(-)

Fig 5. Surface labeling of COS 7 cells with [9]. At 72 hours after transfection, COS 7 cells were treated with or without 0.025% trypsin and then surface-labeled with Na[rnll. The subsequent procedure was similar to that of affinity cross-linking. Results were normalized to lo6 cells in each group. Control, wild-type COS 7 cells; mutant, COS 7 cells transfected with mutated HIR cDNA; intact, COS 7 cells transfected with normal HIR cDNA.

Intact

135-kD a-subunit of cells transfected with normal cDNA, indicating that the amount of receptors on the cell surface was equal in the two cell groups. Thus, mutated, unprocessed proreceptors were normally synthesized at the transcription levels as shown by Northern blot analysis. They were then normally glycosylated and transported to the cell surface.

210 135 ~

Autophosphorylation (kDa)

IR-Ab

(-1

(+I

(-1

(+I

(-1

(+I

(-1

(+I

Trypsin

(-1

(-)

(-)

(-1

(+I

(+)

(-1

(-1

Fig 4. Affinity cross-linking of COS 7 ceils. At 72 hours after transfection, COS 7 cells were treated with or without 0.025% trypsin, and insulin receptors were cross-linked with [Y]-insulin. After immunoprecipitation with (+) or without (-) anti-insulin receptor antibody (IR-Ab) plus Pansorbin, the sample was mixed with Laemmli buffer containing 199 mmol/L DlT and applied to SDS-PAGE. Control, nontransfected wild type; mutant, transfection with mutated HIR cDNA; intact, transfection with normal HIR cDNA.

Autophosphorylation of insulin receptors in these cells is shown in Fig 6. The insulin-stimulated incorporation of 32P into proreceptors was decreased in cells transfected with mutated cDNA compared with that of the a-subunit of normal insulin receptors. It was still decreased even after being normalized by the amount of bound insulin (Fig 6B). Thus, autophosphorylation produced by the same amount of insulin bound to the mutant proreceptor was much lower compared with that bound to normal receptors. Therefore, intramolecular signal transduction was impaired in proreceptors expressed in COS 7 cells. Ttypsin Treatment The effect of trypsin digestion at various concentrations on insulin binding to cells transfected with mutant or

SUGIBAYASHI ET AL

824

(Fig 8B and C). Thus, these results showed no change in EDss for miniproinsulin, but decreased the relative affinity for proinsulin in binding to proreceptors (P < .05). Therefore, the proreceptors had relatively low affinity to proinsulin, but had normal affinity to miniproinsulin.

Intact

mutant

mutant

210 -

DISCUSSION

95 -

(kDa)

Insulin Trypsin

B

0

101000

?3---l _

0 53-

10 1000 +

0

ng/ml

10 1000

0

7c

-

(a) ”

Fig 6. Autophosphorylation of COS 7 cells treated with or without 0.025% trypsin 72 hours after transfection. After solubilized, lectinpurified preparations were preincubated with various concentrations of ins&n, they were phosphorylated at 4°C for 10 minutes, as described in the Methods. (A) Autoradiography of autophosphorylated COS 7 cells. (B) Analysis of data of 3zP incorporation into 6-subunits in each group. (0-O) Mutant transfected with mutated HIR cDNA; (A----A) mutant treated with 0.025% trypsin; (O-O) intact transfectad with normal HIR cDNA.

We previously reported on siblings with severe insulin resistance due to the presence of 210-kD insulin proreceptors on the cell surface of their transformed lymphocytesii and fibroblasts.14 Gene analysis by polymerase chain reaction demonstrated a G + T point mutation at the cleavage site, ie, from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser.IZ The structural change of the cleavage site appeared to be the most likely cause for unprocessed proreceptors. However, an abnormal processing enzyme that remained unidentified could be the cause for the unprocessed proreceptor. Therefore, transfection assay was necessary to confirm that the mutation of the insulin receptor gene was the cause for extreme insulin resistance. Transfection in COS 7 cells described here confirmed that an Arg + Ser structural change of the cleavage region was responsible for the unprocessed proreceptors. Northern blot analysis showed that the transfection efficiency of both normal and mutated cDNA was similar, and the surface labeling study confirmed that the amount of receptors on the cell surface was comparable between the two groups of cells. These findings indicated that the mutation did not disturb transcription of insulin receptor mRNA and transport of proreceptors to the cell surface after glycosylation in COS 7 cells. Furthermore, normal-sized insulin receptors, which were produced by trypsin cleavage, functioned normally in insulin binding and signal transmission, ie, insulin-stimulated autophosphorylation. These results are generally in agreement with those found in the patients’ cells. We reported that the insulin-binding ability of unprocessed proreceptors from the patients was 20% of normal, which was slightly lower than that of proreceptors expressed in the cells transfected with mutated cDNA.‘i It

22 20 -

normal cDNA was further examined (Fig 7). In cells transfected with mutant cDNA, 0.025% trypsin was the most effective concentration for increasing insulin binding, and binding decreased at higher trypsin concentrations.

B

Binding Specificity

E ;

To characterize insulin binding to transfected cells, two insulin analogues were tested. Fig 8 shows competitive binding of proinsulin and miniproinsulin, which has only two amino acids for the C-peptide.25 In cells transfected with normal cDNA, the 50% effective dose (ED5a) of proinsulin and miniproinsulin was 97 ? 12 and 617 -e 108 nmol/L, respectively (Fig 8A). However, when the cells were transfected with mutant cDNA, the (ED,,) of proinsulin and miniproinsulin was 175 ? 22 and 583 t 67 nmol/L (mean * SEM, n = 4) respectively, and trypsin treatment changed these to 83 f 16 and 492 r 84 nmol/L, respectively

a"

m :

L 3 E

1816141210 a6-

2.5 Trvps~n c~ncentrat~,n

25 (x lo-? %

w/w)

Fig 7. Effect of trypsin treatment on insulin binding to transfected COS 7 cells. At 72 hours after transfection, COS 7 cells ware incubated with indicated concentrations of trypsin at 26°C for 5 minutes, and then insulin binding was performed as described in the Methods. Results are means k SEM. IO-O) Mutant transfected with mutated HIR cDNA; (0-O) intact transfected with normal HIR cDNA.

SIGNAL TRANSDUCTION

0.167 lnsul~n

IN INSULIN PRORECEPTORS

I 67 analogue

16.7 concentration

825

167

1670

hmde)

01

0.167 Insulin

167 analogue

4

I6 7 concentration

167

1670

hde)

BI r$ IOOBP 90. f

m-

i

70. 2

60.

jj

50.

c

40.

E

30.

b R

20.

‘\

IO -

OL

0 167 insulin

1.67 anaiogue

16.7 concentration

167

‘A\ 1670

(nmol/P)

may be due to the difference in the cell types, ie, COS 7 cells, which are monkey-derived fibroblasts, versus transformed lymphocytes or fibroblasts of human origin. Alternatively, the additional 12 amino acids just before the cleavage site in the transfected ceils may cause higher binding ability, since the cDNA used for transfection contained 36 additional nucleotides, as Ebina et al described,20 and the mRNA found in the patients’ cultured lymphocytes did not have these nucleotides. 26However, the difference between the two receptors, ie, the expressed proreceptors by transfection versus the proreceptors on the patients’ cultured lymphocytes, was small, and this indicated that exon 11 does not play a crucial role in receptor binding. Transfection of mutated cDNA in monkey-derived fibroblasts reproduced the phenomena largely demonstrated in the patients’ cells. Thus, no species specificity was seen in the processing or transport of the insulin receptors, at least in human and monkey cells. Unexpectedly, several species of insulin receptor mRNA were detected by Northern blot analysis. Since we transfected a plasmid containing a single species of HIR cDNA, Northern blot analysis should show one band. The reason for this discrepancy is not clear at present. However, no signals were detected with the HIR probe in control cells; therefore, these signals in both groups must be from the transfected plasmids. The doseresponse curve of trypsin digestion in COS 7 cells was similar to that of patients’ cells. Thus, those four basic amino acids were exposed to trypsin attack in cells expressing mutated, abnormal proreceptors with or without the exon 1I region. We also found a similar binding specificity of COS 7 cells compared with that in the patients’ transformed lymphocytes.

Fig 8. Competitive binding ability of proinsulin and miniproinsulin in COS 7 calls transfectad with normal (A) or mutated (6, C) HIR cDNA and incubated with [1Z51]-insulin(0.2 ng/mL) and various concantrations of insulin (0-O). proinsulin (A-----A), or miniproinsulin (O-O). COS 7 calls were treated with (C) or without (A, 6) 0.025% trypsin 72 hours after transfaction, and binding studies were performed as described in the Methods. Results are means T SEM (n = 4).

Regarding intramolecular signal transmission of the receptor, insulin proreceptors in COS 7 cells behaved differently compared with those in patients’ cells.13 The proreceptors expressed on COS 7 cells showed decreased insulin-stimulated autophosphorylation, in contrast to that of the proreceptors on the patients’ lymphocytes. In agreement with the findings shown in this report, transfection with cDNA that had a deleted cleavage site produced mutant proreceptors, which decreased the ability of insulin to stimulate autophosphorylation.27 The discrepancy in the signal transmission study between proreceptors in COS 7 cells and those in the patients’ cells is not clear. It may be due to the difference in the microenvironment or in glycosylation of proreceptors, or to a difference in the tertiary structure of the proreceptors because of the involvement of amino acids encoded by exon 11. Another interesting feature of the unprocessed insulin proreceptors was decreased internalization once they were expressed at the plasma membrane, which allowed these receptors to remain longer at the cell surface. These findings indicated that the abnormal tertiary structure of the C-terminal region of the a-subunit not only decreased binding affinity, but also inhibited internalization. In summary, transfection of cDNA with the G -+ T point mutation at the cleavage site of insulin proreceptors in COS 7 cells generally reproduced the phenomenon found in the previously reported patients with insulin resistance. However, the proreceptor expressed in COS 7 cells showed decreased signal transmission and delayed internalization. The transient transfection system using COS 7 cells was useful in examining the genetic defect of insulin receptors found in patients with severe insulin resistance.

826

SUGIBAYASHI

ET AL

REFERENCES 1. Grunberger G, Comi RJ, Taylor SI, et al: Tyrosine kinase activity of insulin receptor of patients with type A extreme insulin resistance. J Clin Endocrinol Metab 60:381-386, 1984 2. Grigorescue F, Flier JS, Kahn CR: Characterization of binding and phosphorylation defects of erythrocyte insulin receptors in type A syndrome of insulin resistance. Diabetes 35127-138, 1986 3. Hedo JA, Moncada W, Taylor SI: Insulin receptor biosynthesis in cultured lymphocytes from insulin-resistant patients. J Clin Invest 76:2355-2361,1985 4. Cama A, Taylor SI: Tyrosine kinase activity of insulin receptors from an insulin-resistant patient with leprechaunism. Diabetologia 30:631-637, 1987 5. Kobayashi M, Takata Y, Sasaoka T, et al: Fluctuation of insulin resistance in a leprechaun with a primary defect in insulin binding. .I Clin Endocrinol Metab 66:1084-1088,1988 6. Kadowaki T, Bevins CL, Cama A, et al: Two mutant alleles of the insulin gene in a patient with extreme insulin resistance. Science 240:787-790. 1988 7. Moncada VY, Hedo JA, Serrano-Rios M, et al: Insulinreceptor biosynthesis in cultured lymphocytes from an insulinresistant patient (Rabson-Mendenhall syndrome): evidence for defect before insertion of receptor into plasma membrane. Diabetes 35:802-807,1986 8. Takata Y, Kobayashi M, Maegawa H, et al: A primary defect in insulin receptor in a young male with insulin resistance. Metabolism 35950-955, 1986 9. Accili D, Frapier C, Mosthaf L, et al: A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin resistant diabetes. EMBO J 8:2509-2517, 1989 10. Klinkhamer MP, Groen NA, van der Zon GCM, et al: A leucine-to-proline mutation in the insulin receptor in a family with insulin resistance. EMBO J 8:2503-2507,1989 11. Kobayashi M, Sasaoka T. Takata Y, et al: Insulin resistance by uncleaved insulin proreceptor: Emergence of binding site by trypsin. Diabetes 37:653-656,1988 12. Kobayashi M, Sasaoka T, Takata Y, et al: Insulin resistance by unprocessed insulin proreceptors: Point mutation at the cleavage site. Biochem Biophys Res Commun 153:657-663,1988 13. Sasaoka T, Shigeta Y, Takata Y, et al: Binding specificity and intramolecular signal transmission of uncleaved insulin proreceptor in transformed lymphocytes from a patient with extreme insulin resistance. Diabetologia 32:371-377, 1989 14. Sasaoka T, Shigeta Y, Takata Y, et al: Unprocessed insulin

proreceptor in cultured fibroblasts from a patient with extreme insulin resistance. Metabolism 38:990-996, 1989 15. Yoshimasa Y, Seino S, Whittaker J, et al: Insulin-resistant diabetes due to a point mutation that prevents insulin proreceptor processing. Science 240:784-787,1988 16. Kobayashi M, Sugibayashi M, Sasaoka T, et al: Transfection of cDNA with G-T point mutation at the cleavage site of insulin receptors to COS 7 ceils. Biochem Biophys Res Commun 167:10731078,199O 17. Sakata S, Kobayashi M, Miura K, et al: Molecular recognition of human insulin receptor by autoantibodies in a human serum. Immunol Invest 17:237-242,1988 18. Maniatis T, Fritsch E, Sambrook Laboratory Manual. Cold Spring Harbor Harbor, NY, 1982

J: Molecular cloning: A Laboratory, Cold Spring

19. Whittaker J, Okamoto AK, Thys R, et al: High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells. Proc Nat1 Acad Sci USA 84:5237-5241,1987 20. Ebina Y. Ellis L, Jarnargin K. et al: The human receptor cDNA: The structural basis for hormone-activated membrane signaling. Cell 40:747-758, 1985

insulin trans-

21. Nakamaye KL, Eckstein F: Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Res 14:9679-9689,1986 22. Stafford J. Queen transfected immunoglobulin

C: Cell-type specific expression gene. Nature 306:77-79, 1983

23. Chomczynsky P. Sacci N: Single-step method isolation by acid guanidium thiocyanate-phenol-chloroform tion. Anal Biochem 162:156-159, 1987

of a

of RNA extrac-

24. Grunberger G, Comi RJ, Taylor SI, et al: Tyrosine kinase activity of the insulin receptor of patients with type A extreme insulin resistance: studies with circulating mononuclear cells and cultured lymphocytes. J Clin Endocrinol Metab 59:1152-1158, 1984 25. Kobayashi M, Sasaoka T, Sugibayashi M, et al: Receptor binding and biologic activity of biosynthetic human insulin and mini-proinsulin produced by recombinant gene technology. Diabetes Res Clin Pratt 7:25-28, 1989 26. Ullich A, Bell JR, Chen EY, et al: Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature 313:756-761, 1985 27. Williams JF, McClain DA, Dull TJ, et al: Characterization of an insulin receptor mutant lacking the subunit processing site. J Biol Chem 265:8463-8469,199O

Characterization of unprocessed insulin proreceptors in COS 7 cells transfected with cDNA with Arg735----Ser735 point mutation at the cleavage site.

We previously reported on patients with severe insulin resistance due to unprocessed insulin proreceptors. A structural change of the cleavage site fr...
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