Proinsulin binds to a growth peptide receptor and stimulates DNA synthesis in chick embryo fibroblasts.

Proinsulin Binds to a Growth Peptide Receptor and Stimulates DNA Synthesis in Chick Embryo Fibroblasts1 S. PETER NISSLEY, MATTHEW M. RECHLER, ALAN C. MOSES, PATRICIA A. SHORT, AND JUDY M. PODSKALNY Endocrinology Section, Metabolism Branch, National Cancer Institute and Diabetes Branch, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 ABSTRACT. Proinsulin, the biosynthetic precursor of insulin, has 3-5% the potency of insulin in various in vitro assays for insulin-like metabolicactivity. We now report that proinsulin was approximately 60% as potent as insulin and 20% as potent as the growth polypeptide, multiplication stimulating activity (MSA), in stimulating [3H]thymidine incorporation into DNA in chick embryo fibroblasts (CEFs). The stimulation by maximally effective concentrations of proinsulin plus MSA or insulin was not additive. The [3H]thymidine incorporation data reflected DNA synthesis since proinsulin also stimulated DNA synthesis as measured by flow microfluorometry and promoted the multiplication of CEFs. Proinsulin also competed with [125I]iodoMSA for binding to CEFs, and with 60% the

potency of insulin. Moreover, MSA binding was inhibited by the same concentrations of proinsulin that were required to stimulate [3H]thymidine incorporation into DNA. The activity of proinsulin in the [3H]thymidine incorporation and MSA radioreceptor assays appeared to be an intrinsic property of the proinsulin molecule, since there was no evidence for conversion of proinsulin to insulin or proinsulin intermediates during incubation. In addition, C-peptide, the peptide bridge connecting the insulin A and B chains in the proinsulin molecule, was inactive in stimulating thymidine incorporation into DNA and in competing with labeled MSA for binding to CEFs. (Endocrinology 101: 708, 1977)

"|\/TULTIPLICATION stimulating activity -LVA (MSA), a family of polypeptides synthesized by certain rat liver cell lines, stimulates the multiplication of chick embryo fibroblasts (CEFs) (1,2). MSA is similar to the acid ethanol-soluble portion of non-suppressible insulin-like activity (NSILA-s), and to somatomedins A and C, human plasma polypeptides which may be circulating second messengers for the action of growth hormone on skeletal tissue (3-5). These similarities include physicalchemical properties, weak insulin-like activity, competition for binding of the peptides to the same membrane receptors and stimulation of growth-related processes in cells in culture (1,2,4-8). Using radioiodinated purified MSA we have recently described a specific growth peptide receptor in CEFs (9,10). Only

somatomedin A, NSILA-s, MSA itself, and insulin competed with [125I]iodoMSA for binding to this receptor (8-10). MSA and insulin, which are almost equipotent in displacing [125I]iodoMSA from the receptor, were also equal in their ability to stimulate [3H]thymidine incorporation into DNA (9). In addition, the concentrations of MSA or insulin required for half-maximal stimulation of [3H]thymidine incorporation into DNA corresponded to the concentrations required for half-maximal displacement of [125I]iodoMSA from the CEF MSA receptor. When maximally effective concentrations of MSA and insulin were combined in the [3H]thymidine incorporation assay, the response was not additive. These results suggested that MSA and insulin stimulated DNA synthesis in CEFs via interaction with the growth receptor defined by [125I]iodoMSA binding. Proinsulin, the biosynthetic precursor of insulin in the pancreatic beta cell, has usually been reported to have only 3-5% the

Received November 1, 1976. 1 Presented at the 1976 meeting of the Society of Biological Chemists, San Francisco, Fed Proc 35: 1628, 1976.

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PROINSULIN AND CHICK EMBRYO FIBROBLASTS activity of insulin in in vitro assays for insulin (11-13). We now report the potency of proinsulin (relative to insulin) in the [ 3 H]thymidine incorporation and MSA radioreceptor assays in CEFs. Materials and Methods Materials Carrier free NaI25I was obtained from Amersham-Searle and [3H-methyl]thymidine was purchased from Schwarz/Mann. Crystalline Zn porcine insulin (Lot #615 C63-10), porcine proinsulin (Lot #615-112B-85-C), and porcine C-peptide (Lot #615-D63-64) were gifts from R. C. Chance, Eli Lilly Laboratories. Fetal calf serum was obtained from GIBCO. Crystalline bovine serum albumin (A4378) and fatty acidfree crystalline bovine serum albumin (A7511) was purchased from Sigma Chemical Company. Colchicine was purchased from Calbiochem. Purification of MSA MSA was purified from the serum-free conditioned media of a line of Buffalo rat liver cells (BRL 3A) originated by H. Coon and obtained from H. M. Temin. MSA was identified by the ability to stimulate [3H]thymidine incorporation into DNA in CEFs (1). The purification scheme was modified from previously described methods (1,14) and will be reported in detail in a manuscript in preparation. Briefly, Dowex MSA was prepared as described by Dulak and Temin (1), and then chromatographed on Sephadex G-75 in 1M acetic acid. MSA appeared in three peaks. Fractions from the center peak were combined on the basis of the protein pattern seen on analytical disc acrylamide electrophoresis (pH 2.7, 9M urea). The pooled MSA fractions from Sephadex G-75 were further purified by preparative disc acrylamide electrophoresis (pH 2.7, 9M urea). Fractions showing a single protein band on analytical disc acrylamide electrophoresis were used for subsequent experiments. The specific activity of this MSA relative to porcine insulin in the CEF [3H]thymidine incorporation assay, and the 50-fold purification over conditioned medium are the same as reported by Smith and Temin for their MSA preparation (15).

Measurement of [3H]thymidine into DNA in CEFs

709

incorporation

Tertiary cultures of CEFs (1 x 106 cells/60 mm dish) derived from carcasses of 12 day old embryos were plated in ET medium (Temin's modified Eagle's medium with 20% by volume tryptose phosphate broth) containing 0.4% fetal calf serum. After 3-5 days at 37 C in a 5% CO2 atmosphere, these serum-starved cells are growth arrested and synchronized in G, of the cell cycle. The medium was removed and replaced with 3 ml of E medium (Temin's modified Eagle's medium) containing MSA, insulin or proinsulin. After 10-13 h the cells were exposed to a 1 h pulse of [3H]thymidine (0.4 /u.Ci/2 ml minimal essential Eagle's medium) and the radioactivity incorporated into acid-precipitable material (DNA) determined as previously described (16). Preparation of [125I]iodoMSA Purified MSA was iodinated with Na125I to a specific activity of 60-235 Ci/g by a modification of the chloramine-T procedure (17). [l25I]IodoMSA gave a single sharp peak on Sephadex G-50 gel filtration in 1M acetic acid containing 1 mg/ml bovine serum albumin and co-migrated with MSA on analytical disc acrylamide electrophoresis (pH 2.7, 9M urea). [nH]lodoMSA binding to CEFs Tertiary cultures of CEFs were prepared exactly as described above for the [3H]thymidine incorporation assay. The binding of [125I]iodoMSA to CEFs was performed as described previously (9). Briefly, CEFs were detached from the dishes by gentle trypsinization (0.05% trypsin, 0.5 mM EDTA, 37 C, 5 min). The binding assay mixture included: [125I]iodoMSA, ^250 pg; 1-2 x 106 CEFs; unlabelled peptides as indicated; in a total volume of 0.5 ml HEPES binding buffer (HEPES, O.lM; NaCl, 0.12M; KC1, 5 mM; MgSO4, 1.2 mM; glucose 8 mM, bovine serum albumin 10 mg/ml) at pH 8.0. Binding experiments were performed in plastic tubes at 22 C for 3 h, conditions previously shown to give a steady state (10). Cells were then sedimented by centrifugation for 1 min in a Beckman Microfuge, the pellets excised and bound radioactivity determined in a gamma spectrophotometer at 85% efficiency. Non-


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Determination of relative DNA content by flow microfluorometry CEFs were prepared as described for the [3H]thymidine incorporation assay. One /xg/ml of MSA, insulin or proinsulin in 2 ml E medium containing 5 x 10~8M colchicine (to block mitosis) was added to the cells and after 36 h the cells were removed from the dishes with 0.12% trypsin, washed once in serum-containing medium, and then with Dulbecco's phosphatebuffered saline. The cells were finally suspended in cold 50% ethanol and stored at 4 C. After staining with mithramycin the relative DNA content per cell was measured with the flow microfluorometer (18). 6

-

4

-

2

-

Results

Proinsulin stimulated DNA synthesis and cell multiplication in CEFs

200

400

600

800

1000

CONCENTRATION (ng/ml)

FIG. 1. Stimulation of [3H]thymidine incorporation into DNA (per 60 mm dish) by different concentrations of MSA, insulin and proinsulin. The values plotted are averages of determinations on duplicate dishes ofCEFs. specific binding, defined by binding in the presence of 1 /ng/ml MSA, was usually 30% or less of total binding. [l25I]Iodoinsulin binding to cultured lymphocytes (1M-9)

human

Competitive binding experiments for [125I]iodoinsulin binding to IM-9 lymphocytes were performed as described previously (13). Cell multiplication

experiments

Tertiary cultures of CEFs (0.5 x 106 cells/60 mm dish) were plated in ET containing 0.4% fetal calf serum as described above for the [3H]thymidine incorporation assay. After one day, the medium was replaced in 1 /xg/ml MSA, insulin or proinsulin in ET medium containing 0.25% bovine serum albumin. Cells were trypsinized and counted in a Particle Data instrument.

Proinsulin was approximately 60% as potent as insulin (based on molar concentration) in stimulating [3H]thymidine incorporation into DNA in serum-starved CEFS (Fig. 1 and Table 1). Proinsulin was approximately 20% as potent as MSA in the [3H]thymidine incorporation assay. When the [3H]thymidine incorporation data were plotted versus the log of the concentration of MSA, insulin or proinsulin over 3-4 consecutive serial dilutions in the middosage range shown in Fig. 1, straight lines resulted. Application of the Tukey multiple comparison test to each of the assays recorded in Table 1 shows that the slopes for MSA, insulin and proinsulin within the same assay are not significantly different from 3ach other (P > 0.25). In the proinsulin molecule, the A and B chains of insulin are joined by the connecting peptide or C-peptide. In order to determine whether the unexpected potency of proinsulin in the [3H]thymidine incorporation assay could be attributed directly to the C-peptide, porcine C-peptide was tested in the [3H]thymidine incorporation assay (Table 2) and was found not to stimulate [3H]thymidine incorporation into DNA over control levels.


PROINSULIN AND CHICK EMBRYO FIBROBLASTS

711

TABLE 1. Slope (b) and index of precision (X) values for log dose-response curves of [3H]thymidine incorporation into DNA in CEFs Potency of proinsulin

(b)

Experiment no.

MSA

Insulin

Proinsulin

versus MSA

versus insulin

1. 2. 3. 4.

7.8 (5.9-9.7) 10.5(7.2-13.7) 4.9 (3.2-6.5) 1.9(0.8-3.1)

7.7(3.8-11.6) 10.0(8.0-11.9) 4.4 (1.3-7.6) 2.2(1.0-3.3)

6.2 (2.8-9.5) 8.4(4.9-11.8) 5.8 (4.2-7.5) 1.9(1.2-2.5)

0.14 0.42 0.18 0.13

0.58 0.69 0.77 0.48

1. 2. 3. 4.

0.09 0.12 0.13 0.22

0.20 0.06 0.15 0.18

0.12 0.09 0.11 0.20

MSA, insulin, and proinsulin were assayed in the [3H]thymidine incorporation assay in CEFs using serial dilutions from 1 /ig/ml to 31 ng/ml as shown in Fig. 1. The amount of [3H]thymidine incorporated was plotted versus the log of the dose using data corresponding to 3 or 4 concentrations over the mid-dosage range. The 95% confidence limits for the slope are given in parentheses. The molecular weights used for calculating relative potency on a molar basis are: MSA = 9,000, insulin = 6,000 and proinsulin = 10,000. The mean relative potency estimates with 95% confidence limits are 0.63 (0.43-0.83) and 0.22 (0.00-0.44) for proinsulin versus insulin, and proinsulin versus MSA, respectively.

In addition to stimulating DNA synthesis in CEFs, proinsulin also stimulated cell multiplication. In a four day growth experiment, proinsulin, like insulin and MSA increased cell number approximately two-fold over control (Fig. 3). In the experiment shown in Fig. 3, the media contained 0.25% bovine serum albumin. Although MSA, insulin and proinsulin stimulated cell multiplication without albumin, the increase in individual responses together with parallel cell number was smaller and less consistent. dose-response curves suggests that pro- In order to determine whether this eninsulin may share a common mechanism of hancing effect of albumin was due to albumin itself or a component bound to action with insulin and MSA. In order to show that the stimulation of albumin, we compared untreated crystalline [3H]thymidine incorporation into DNA re- bovine serum albumin with fatty acid-free flected net DNA synthesis and was not albumin (charcoal-treated). When fatty acid simply attributable to changes in specific free albumin was used, there was no enradioactivity of the thymidine pool or DNA repair, we performed experiments with the TABLE 2. Test of C-peptide in the [3H]thymidine incorporation assay in CEFs flow microfluorometer (18). MSA, insulin and proinsulin were added to serum-starved Additions cpm/dish (xlO~3)* CEFs in the presence of 5 x 10~8M colchicine. Cells were harvested at 36 h and 4.3 Control C-peptide (1 fig/ml) 4.1 analyzed (Table 3). The percentage of cells Proinsulin (1 /Ag/ml) 9.5 in G2 + M in the presence of MSA, insulin Insulin (1 /u.g/ml) 15.0 or proinsulin was 150% of that observed for MSA (1 /ig/ml) 16.9 Serum (10%) 21.8 control cells and was about 50% that observed with media containing 10% fetal calf * The mean of duplicate determinations is shown serum. (duplicate determinations varied from 1.7 to 6.9%).

When various paired combinations of proinsulin, MSA and insulin were tested for their ability to stimulate [3H]thymidine incorporation into DNA, the stimulation observed was less than the sum of the individual stimulations (Fig. 2). Stimulation by calf serum indicates that the cells were capable of a greater degree of response than was achieved with the pairs of polypeptides. Consequently, the non-additivity of the


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712 30

-20

I

5 o 10

MSA (pg/ml)

-

0.5

-

-

0.5 0.5

INSULIN (fig/ml)

-

-

0.5

-

0.5

PROINSULIN l^g/ml)

-

-

-

0.5

-

-

0.5

0.5 0.5 10

SERUM (%)

FiG. 2. Stimulation of [3H]thymidine incorporation (per 60 mm dish) by pairs of growth polypeptides. The final concentration of each polypeptide in the addition experiments was 0.5 /ug/ml. Measurements were made on duplicate dishes of CEFs.

hancement of the growth response to insulin (Fig. 4), suggesting that the enhancing effect of albumin is due to a bound component that is removed by charcoal treatment. Proinsulin competes with [125!]iodoMSA for binding to the CEF groivth peptide receptor

(Fig. 5). Indeed proinsulin competed with [125I]iodoMSA for binding to CEFs over the same concentration range required for stimulation of [ 3 H]thymidine incorporated into DNA. Relative potencies were calculated based on concentrations of MSA, insulin, and proinsulin required for 50% displacement of [125I]iodoMSA. Based on molar concentrations, proinsulin was approximately 60% as potent as insulin in competing for binding to the [125I]iodoMSA receptor. In five experiments the potency of proinsulin relative to insulin was 59, 74, 87, 44 and 29%; mean = 58%. C-peptide did not compete with [125I]iodoMSA for binding.

Proinsulin is not converted to insulin or proinsulin intermediates during incubation The potency of proinsulin (relative to insulin) in the [3H]thymidine incorporation and MSA radioreceptor assays was approxi3

i-

2

-

We have previously proposed that the growth stimulatory effects of insulin and MSA in CEFs were mediated via a specific cell surface receptor that was identified by [125I]iodoMSA binding (9). Accordingly, we next examined whether proinsulin, which exhibited similar growth stimulatory properties, also interacted with the MSA receptor TABLE 3. CEF cell cycle analysis by flow microfluorometry % of Cell population Additions

G,

S

G2 + M

Control MSA (1 /xg/ml) Insulin (1 /xg/ml) Proinsulin (1 /xg/ml) Serum (10%)

75.6 65.8 66.9 68.9 44.8

7.9 6.7 7.0 6.1 7.6

16.6 27.5 26.1 25.0 47.6

FiG. 3. Cell multiplication experiment. MSA, insulin, or proinsulin, 1 /xg/ml was added at zero time in ET medium containing 0.25% bovine serum albumin. Control cultures received ET medium containing 0.25% bovine serum albumin.


PROINSULIN AND CHICK EMBRYO FIBROBLASTS mately 10 times the previously reported potency of proinsulin in in vitro assays for insulin-like activity (11-13). One explanation for the unexpected potency of proinsulin in the former assays is that proinsulin had been partially converted to insulin during the incubation. [131I]Iodoproinsulin, therefore, was incubated with CEFs under the experimental conditions of the two assays and the incubated [131I]iodoproinsulin chromatographed on Sephadex G-50 in 1M acetic acid (Fig. 6). [131I]Iodoproinsulin and [125I]iodoinsulin markers were clearly resolved on this column. There was no change in the elution volume of the [131I]iodoproinsulin during incubation nor was radioactivity detected in the region corresponding to the elution volume of insulin or smaller peptides. We conclude that proinsulin was not converted to insulin during the incubations.

8

BSA (F.A. Free)

< 1

0.05

0.10

0.15

0.20

0.25

0.30

PERCENT CONCENTRATION (wt/vol)

FIG. 4. Comparison of untreated bovine serum albumin with fatty acid-free bovine serum albumin (charcoal-treated) in enhancing the multiplication of CEFs by insulin over three days. Tertiary cultures of chick embryo fibroblasts were plated as described in Materials and Methods. After one day the media were changed to ET medium containing from zero to 0.25% bovine serum albumin or fatty acid-free bovine serum albumin, each alone or together with 10 £ig/ml insulin. The experimental points are the average of duplicate dishes. The cell count at zero time was 4.7 x lOVdish.

713

in

5.0

V — - ° C

-

4.0

Q

PEPTIDE

a

\

\ \\

\

A 3.0 \ s

? \ \

N * PROINSULIN >

\\

V

O INSULIN »MSA

1.0

III

10

100

i i i mi

i

i i i i in

1000


FIG. 5. Competition with [125I]iodoMSA for binding to CEFs by C-peptdde, proinsulin, insulin and MSA. Each assay tube contained 34,100 cpm of [l25I]iodoMSA. Maximal binding was 1,680 cpm and nonspecific binding (binding in the presence of 1 ^g/ml MSA) was 460 cpm.

Desdipeptide proinsulin and desnonapeptide proinsulin, intermediates produced by limited proleolytic cleavage of proinsulin, have been shown to have 4 - 5 fold greater insulin-like activity than proinsulin in in vitro assays for insulin-like metabolic activity (13). Therefore, a second explanation for the unexpected potency of proinsulin (relative to insulin) in the two assays is that proinsulin had been converted to desdipeptide and/or desnonapeptide proinsulin (or other intermediates) during the incubations. Sephadex G-50 chromatography would not exclude conversion to proinsulin to desdipeptide or desnonapeptide proinsulin. In order to determine whether or not proinsulin was converted to these intermediate forms having increased insulin-like activity, proinsulin was incubated with CEFs under experimental conditions of the [3H]thymidine incorporation and MSA radioreceptor assays and the incubated proinsulin assayed for insulin-like activity utilizing the IM-9 lymphocyte insulin


Endo . 1977 Vol 101 . No 3

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radioreceptor assay (Fig. 7). When proinsulin was incubated with CEFs under conditions of the MSA radioreceptor assay and then assayed for insulin-like activity in the lymphocyte insulin radioreceptor

Void

131

20,000 -

Salt l-Proinsulin " 8 l-lnsulin

A 10,000

assay there was little or no increase in insulin-like activity. In agreement with previous reports, the proinsulin preparation utilized in the present studies was only 3% as active as insulin on a molar basis in displacing [125I]iodinsulin from the IM-9 lymphocytes. When proinsulin which had been incubated with CEFs under conditions of the [ 3 H]thymidine incorporation assay was similarly tested in the lymphocyte insulin radioreceptor assay, there was also no conversion to forms with increased insulinlike activity (data not shown). We conclude that the unexpected potency of proinsulin in stimulating [ 3 H]thymidine incorporation into DNA and interacting with the MSA receptor is not due to conversion of proinsulin to insulin or proinsulin intermediates. Discussion

B

Proinsulin has 3-5% of the activity of insulin in stimulating glucose oxidation in isolated fat cells and in insulin radioreceptor assays in a variety of cells and membranes (11-13). We have now shown that proinsulin has considerably greater relative potency in stimulating DNA synthesis in CEFs. Proinsulin was approximately 60% as active as insulin on a molar basis in stimulating [3H]thymidine incorporation into DNA in CEFs; that is 5-10-fold higher than expected on the basis of its insulin-like activity. This unexpected potency appears

20,000

4,000

2,000

30

40

50

FRACTION NUMBER

60

FIG. 6. G-50 Sephadex chromatography of [13lI]iodoproinsulin that had been incubated under conditions of the [3H]thymidine incorporation and MSA radioreceptor assays. (A) Unincubated [131I]iodoproinsulin and [ I25 I]iodoinsulin were chromatographed on Sephadex G-50 in 1M acetic acid containing 1 mg/ml bovine serum albumin on a 50 X 1 cm column. Approximately 0.6 ml fractions were collected. (B) [131I]iodoproinsulin was incubated with CEFs under conditions of the MSA radioreceptor assay and then chromatographed on Sephadex G-50 as in panel A. (C) [13lI]iodoproinsulin was incubated under conditions of the [3H]thymidine incorporation assay and then chromatographed on Sephadex G-50 as in panel A.


715

PROINSULIN AND CHICK EMBRYO FIBROBLASTS to be an intrinsic property of the proinsulin molecule since proinsulin was not converted to insulin or proinsulin intermediates during incubation. Using the radiolabeled growth polypeptide, MSA, we previously have identified a specific receptor on CEFs (9,10) and have proposed that MSA and insulin stimulate DNA synthesis via interaction with this receptor. The data in this report suggest that proinsulin also stimulates DNA synthesis in CEFs via interaction with this same receptor defined by [125I]iodoMSA binding. Proinsulin was 60% as potent as insulin in displacing [125I]iodoMSA from receptors in CEFs and stimulating [3H]thymidine incorporation into DNA. Proinsulin inhibited [125I]iodoMSA binding to CEFs over the same concentration range required for stimulation of [ 3 H]thymidine incorporation into DNA. In addition, when a saturating concentration of proinsulin was added to MSA or insulin, the response in the [ 3 H]thymidine incorporation assay was not additive. The enhanced reactivity of proinsulin in the MSA radioreceptor assay further distinguishes the CEF MSA receptor from the insulin receptor in CEFs (9,10), human skin fibroblasts (19) and various tissues from different species (20,21). In the case of CEFs, [125I]iodoinsulin was shown to bind specifically to cells that had been grown under conditions identical to those described in this paper (10). The [125I]iodoinsulin binding was inhibited by relatively low concentrations of unlabeled insulin (half-maximal inhibition of binding with 1-3 ng/ml insulin). In contrast to the potency of proinsulin relative to insulin in inhibiting [125I]iodoMSA binding to CEFs, proinsulin was only approximately 10% as potent as insulin in competing for [125I]insulin binding to CEFs. Furthermore, MSA was only 5% as potent as insulin in competing with [125I]iodoinsulin for binding to CEFs, whereas insulin was slightly less potent than MSA in competing with [125I]iodoMSA for binding to CEFs. The CEF

-r// i i UNINCUBATED PROINSULIN

100

1000


FIG. 7. Effect of preincubation on proinsulin competition with [125I]iodoinsulin for binding to cultured human lymphocytes (IM-9). Two /u,g/ml of proinsulin was incubated with CEFs under conditions of the MSA radioreceptor assay. Aliquots of incubated proinsulin and unincubated proinsulin were examined for their ability to inhibit [125I]iodoinsulin binding to the insulin receptor in IM-9 lymphocytes.

insulin and MSA receptors are further distinguished from each other by a curved Scatchard plot in the case of insulin binding and a linear Scatchard plot in the case of MSA binding.2 Human skin fibroblasts in culture have an MSA receptor similar to the CEF MSA receptor and the potency of proinsulin (relative to insulin) in inhibiting [125I]iodoMSA binding to the two MSA receptors is similar (23). X-ray crystallographic data suggest that the weak insulin-like activity of proinsulin may be due to steric hinderance by the C-peptide of the insulin receptor binding domain on the insulin molecule (24). Since the C-peptide by itself is not active, the fact that proinsulin is almost as potent as insulin 2

We have not ruled out the possibility that a small fraction of the [125I]iodoMSA binds to the insulin receptor or that, conversely, some [125I]iodoinsulin binds to the MSA receptor. Preliminary data indicate that an antibody directed against the insulin receptor blocks [125I]iodoinsulin binding but does not block [125I]iodoMSA binding in CEFs, providing further evidence that [12ijI]iodoMSA is not binding to the insulin receptor (22).


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NISSLEY ETAL.

in binding to the CEF MSA receptors suggests that the portion of the proinsulin (and insulin) molecules involved in binding to the growth receptor in CEFs may be different than the region of the insulin molecule that interacts with the insulin receptor.

Cytology, Institute, measurestatistical

References 1. Dulak, N. A., and H. M. Temin, J Cell Physiol

2. 3.

4. 5.

6.

7.

8.

10. 11. 12. 13.

Acknowledgments We thank B. J. Fowlkes, Quantitative Pathology Laboratory, National Cancer for performing the flow microfluorometer ments and Dr. David Ailing for help in analysis.

9.

81: 153, 1973. Dulak, N. C , and H. M. Temin, J Cell Physiol 81: 161, 1973. Froesch, E. R., U. Schlumpf, R. Heiman, J. Zapf, R. E. Humbel, and W. J. Ritschard, In Luft, R., and K. Hall (eds.), Advances in Metabolic Disorders, vol. 8, Academic Press, New York, 1975, p. 203. Hall, K., K. Takano, L. Fryklund, and H. Sievertsson, Adv Metab Disord 8: 19, 1975. Van Wyk, J. J., L. E. Underwood, R. L. Hintz, S. J. Voina, R. P. Weaver, and D. R. demons, Recent Prog Horm Res 36: 259, 1974. Froesch, E. R., J. Zapf, C. Meuli, M. Mader, M. Waldvogel, U. Kaufman, and B. Morell, Adv Metab Disord 8: 211, 1975. Megyesi, K., C. R. Kahn, J. Roth, D. M. Neville, Jr., S. P. Nissley, E. R. Froesch, and R. A. Humbel, J Biol Chem 250: 8990, 1975. Rechler, M. M., L. Fryklund, S. P. Nissley, K. Hall, J. M. Podskalny, A. Skottner, and A. C. Moses,

14. 15. 16. 17. 18. 19. 20. 21. 22.

23.

24.

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Prog. 58th Endocrine Society Meeting, San Francisco, 1976 (Abstract #332). Rechler, M. M., J. M. Podskalny, and S. P. Nissley, Nature 259: 134, 1976. Rechler, M. M., J. M. Podskalny, and S. P. Nissley, J Biol Chem 252: 3898, 1977. Kitabchi, A. E., W. C. Duckworth, F. V. Stentz, and S. Yu, CRC Cut Rev Biochem 1: 59, 1972. Freychet, P., J Clin Invest 54: 1020, 1974. Gavin, J. R., D. R. Kahn, P. Gorden, J. Roth, and D. M. Neville, J Clin Endocrinol Metab 41: 438, 1975. Nissley, S. P., J. Passamani, and P. Short, J Cell Physiol 89: 393, 1976. Smith, G. L., and H. M. Temin, J Cell Physiol 84: 181, 1974. Pierson, R. W., and H. M. Temin, J Cell Physiol 79: 319, 1972. Megyesi, K., C. R. Kahn, J. Roth, E. R. Froesch, R. E. Humbel, J. Zapf, and D. M. Neville, Jr., Biochem Biophys Res Commun 57: 307, 1974. Crissman, H. A., and R. A. Tobey, Science 184: 1297, 1974. Rechler, M. M., and J. M. Podskalny, Diabetes 25: 250, 1976. Ginsberg, B. H., In Litwack, G. (ed.), Biochemical Actions of the Hormones, vol. 4, Academic Press, New York (In press). Kahn, C. R., In Korn, E. D. (eds.), Methods in Membrane Biology, vol. 3, Plenum Press, New York, 1975, p. 81. Rechler, M. M., J. M. Podskalny, D. B. Jarrett, S. P. Nissley, A. C. Moses, J. S. Flier, and C. R. Kahn, Prog. 59th Endocrine Society Meeting, Chicago 1977 (Abstract #159). Rechler, M. M., S. P. Nissley, J. M. Podskalny, A. C. Moses, and L. Fryklund,/ Clin Endocrinol Metab 44: 820, 1977. Blundell, T., G. Dodson, D. Hodgkin, and D. Mercola, Adv Prot Chem 26: 279, 1972.


Actin microheterogeneity in chick embryo fibroblasts.

Epidermal growth factor stimulates the phosphorylation of a specific nuclear protein in chick embryo epidermis.

Thrombin receptor 14-amino acid peptide binds to endothelial cells and stimulates calcium transients.

Contribution of human somatomedin activity to the serum growth requirement of human skin fibroblasts and chick embryo fibroblasts in culture.

Specific changes in the synthesis of mitochondrial DNA in chick embryo fibroblasts transformed by Rous sarcoma viruses.

Nonspecific nature of the stimulus to DNA synthesis in cultures of chick embryo cells.

Inhibition of spermidine and spermine synthesis leads to growth arrest of rat embryo fibroblasts in G1.

beta-Hexosaminidase expression in chick embryo fibroblasts in vitro.

Endothelin-1 stimulates DNA synthesis and anchorage-independent growth of Rat-1 fibroblasts through a protein kinase C-dependent mechanism.

Hyaluronidase activity in cultured chick embryo skin fibroblasts.

Growth of the chick embryo in vitro.

Effects of spermidine synthase inhibition in cultured chick embryo fibroblasts.

In vitro evaluation of plasticizer activity on the growth and metabolism of chick embryo fibroblasts and on the development of chick embryo lungs.

Growth hormone (GH) stimulates protein synthesis in cells transfected with GH receptor complementary DNA.

Coordinate control of collagen synthesis and cell growth in chick embryo fibroblasts and the effect of viral transformation on collagen synthesis.

Effect of hyperlipidemic rat serum on the synthesis of collagen by chick embryo fibroblasts.

Differential effects of glucocorticoids on DNA synthesis in normal and virus-transformed chick embryo cells.

Identification of insulin in chick embryo retina during development and its inhibitory effect on DNA synthesis.

A factor derived from chick embryo retina which inhibits DNA synthesis of retina itself.

Reversible inhibition by methioninyl adenylate of protein synthesis and growth in chick enbyo fibroblasts.

Human milk stimulates DNA synthesis and cellular proliferation in cultured fibroblasts.

Correlated effects of external magnesium on cation content and DNA synthesis in culture chicken embryo fibroblasts.

Effect of medium composition on protein degradation and DNA synthesis in rat embryo fibroblasts.

A chimera between platelet-derived growth factor beta-receptor and fibroblast growth factor receptor-1 stimulates pancreatic beta-cell DNA synthesis in the presence of PDGF-BB.