Thyroid hormone stimulates de novo growth hormone synthesis in cultured GH1 cells: Evidence for the accumulation of a rate limiting

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 73, No. 1, pp , October 1976 Biochemistry Thyroid hormone stimulates de novo growth hormone synthesis in cultured GH1 cells: Evidence for the accumulation of a rate limiting RNA species in the induction process (nuclear receptor/triiodothyronine) HERBERT H. SAMUELS AND LAWRENCE E. SHAPIRO The Endocrine Division, Department of Medicine, New York University Medical Center, New York, N.Y. 116 Communicated by H. Sherwood Lawrence, July 1, 1976 ABSTRACT We have previously demonstrated with radioimmunoassay techniques that physiologic concentrations of thyroid hormone stimulate an increase in growth hormone production in GH, cells in culture. In this study, with radiolabel techniques and selective immunoprecipitation, we demonstrate that Ltriiodothyronine stimulates growth hormone production solely by inducing an increase in the de novo synthesis of the polypeptide hormone. L-Triiodothyronine stimulated synthetic rates of growth hormone by 1.5-fold in 1.5 hr, -fold in.5 hr, to a maimal of 3- to -fold after 8.5 hr of incubation. The time interval between significant L-triiodothyronine binding to putative nuclear receptors and a detectable increase in growth hormone synthesis was 5-6 min. Studies on the effect of actinomycin D, 3'-deoyadenosine, and cycloheimide support the thesis that L-triiodothyronine induces the accumulation of an RNA species which is rate limiting for growth hormone synthesis and lends further support for a primary action of thyroid hormone at the nuclear level. We have previously documented that GH1 cells, a growth hormone and prolactin producing rat pituitary cell line, is a valid cell culture system which can be utilized to define the molecular action of physiological concentrations of thyroid hormone in mammalian cells (1). In this system, L-triiodothyronine (T3) stimulates an increase in growth hormone production (, 3) and inhibits prolactin secretion (). The physiological relevance of these effects in GH1 cells is underscored by the observations that thyroid hormone plays a similar role in the production and -secretion of growth hormone and prolactin in the adenohypophysis in mvo (-6). Based on biologic dose-response relationships to thyroid hormone and a variety of hormonal analogs, we identified and characterized nuclear receptors for the thyroid hormones which appear to mediate these biologic responses in GH, cells 7). These putative nuclear receptors can be identified with intact cells (7), isolated nuclei in vitro (7, 8) or with the binding protein after isolation from nuclei in a soluble form (9). Similar observations have also been made on thyroid hormone binding in rat liver by Oppenheimer and his coworkers (1-1) and more recently by several other investigators (13-15). All of our previous studies, however, quantitated growth hormone production by radioimmunoassay techniques. Therefore, although the nuclear localization of cellular receptors for thyroid hormone suggests that T3 stimulates growth hormone production by regulating the epression of the growth hormone "genome," it is essential to clarify whether the growth hormone response is related to an increase in the synthetic rate or a modification of the degradation rate of the polypeptide hormone. Abbreviations: T3, L-triiodothyronine; NaDodSO, sodium dodecyl sulfate; TCA, trichloroacetic acid; AMD, actinomycin D In this study, we document that the stimulation of growth hormone production by T3 is solely related to an increase in the synthetic rate and present evidence for the accumulation of an RNA species which is rate limiting for growth hormone synthesis. MATERIALS AND METHODS Hormones and Chemicals. L-[3,,5-3H]Leucine (specific activity of 8 Ci/mmol) and Aquasol liquid scintillation counting solution were obtained from the New England Nuclear Corp., Boston, Mass. [15I]T3 (initial specific activity, 3 Ci/mmol) was obtained from Abbott Laboratories, North Chicago, Ill. Purified rat growth hormone free of prolactin for immunization and immunoprecipitation, and rat prolactin were obtained through the generosity of Arthur Parlow and the rat pituitary hormone program of the National Institutes of Health. Acrylamide and bisacrylamide were obtained from the Ames Co., Elkhart, Ind. Triton X-1 was obtained from Packard Instrument Co. and sodium dodecyl sulfate (NaDodSO) from BDH Chemicals Ltd., Poole, Enland. All other chemicals were obtained from Sigma, St. Louis, Mo. Preparation of Immune Plasma to Rat Growth Hormone. Antibody to rat growth hormone was raised in a 17-kg female baboon (Papio Cynocephalus). A primary injection of 51g of purified rat growth hormone, dissolved in 5 mm NaOH, was administered with an equal volume of Freund's adjuvant by subcutaneous injection. This procedure was repeated at 3- to -week intervals, and 1-1 days after the previous immunization ml of blood was collected into ml of citrate-detrose solution to prevent clotting as described by Bancroft (16). The antigrowth hormone plasma obtained was stored at -. Cell Culture Conditions. The growth of GH, cells as monolayer cultures in plastic flasks with a surface area of 5 cm and 75 cm (Falcon Plastics, Cockeysville, Md.) was carried out as previously described (1, ). One million cells were inoculated into 5 cm flasks with growth medium (Ham's F-1 medium containing.5% fetal calf serum and 15% horse serum, Grand Island Biological Co., Grand Island, N.Y.) and incubated at 37 in an atmosphere of 95% air and 5% CO for 8-7 hr. The medium was then replaced with ml of Ham's F-1 medium containing 1% hypothyroid calf serum for 8 hr. This insured complete cellular depletion of thyroid hormone which resulted from the growth of cells in medium containing fetal calf and horse serum (1, 9). The medium was then replaced with Ham's F-1 medium containing hypothyroid calf serum (1) and incubated with the final concentrations of T3 in the total medium for the times indicated in the tet.

2 I37 Biochemistry: Samuels and Shapiro For protein synthetic studies, the medium was replaced with 1 ml of "incorporation" media which consisted of two parts of Ham's F-1 medium, seven parts of calciumi free Hanks' basal salt solution which contained.6 mm MgCl, and one part of hypothyroid calf serum. The T3 concentration was maintained at the same level as the prior incubation. The flasks were incubated for min at 37 in an incubator to permit equilibration of intracellular and etracellular amino acid pools. [3H] Leucine (5,uCi) ;was added to each flask, the caps were sealed, and the flasks were incubated by partially submerging in a 37 water bath to allow for precise temperature control. At the times indicated in the tet the incorporation media was removed, and ml of.85% NaCl at was added to each flask. All subsequent studies were carried out at unless noted. Each flask was rinsed two additional times with ml of.85% NaCl and the cells were lysed with a 1 ml solution of 5 mm Tris-acetate at ph 7.6 (5 ), 75mM NaCl, 1.1 mm MgCl.5% Triton X-1, and harvested with a rubber policeman. Trichloroacetic acid (TCA) (final concentration 1%) was added to 5-5 jil of cell suspension. The TCA samples were centrifuged at X g and the resultant pellet and supernatant fractions were used to quantitate total cell protein synthetic rates and the size of the intracellular free [3H]leucine pool. The remaining cell suspensions were agitated with a vorte mier and centrifuged at X g for 15 min. The derived fractions of Triton X-1 lysed cell supernatant were utilized for separation of radiolabeled growth hormone from other radiolabeled cell proteins by immunoprecipitation as described below. Immunoprecipitation of Radiolabeled Growth Hormone and NaDodSO/Polyacrylamide Gel Electrophoresis. Radiolabeled growth hormone for calibration of the immunoprecipitation procedure was prepared by incubating GH, cells with [3H]leucine (5 MCi/ml) for hr. The media, which contained 3H-labeled growth hormone, was then dialyzed against three echanges of 1 volumes of 5 mm Tris acetate -at ph 7.6 (5), and.15 M NaCl. Complete immunoprecipitation of 3H-labeled growth hormone with the lot of immune plasma used in this study occurred with. ml of plasma and 5,ug of purified growth hormone. This was checked by Na- DodSO/polyacrylamide gel electrophoresis as described below. Rat prolactin, Triton X-1, or the addition of cell lysate had no influence on the immunoprecipitation reaction. The immunoprecipitation reaction contained 1,u of growth hormone standard (5,ug/ml),.17 ml of Triton X-1 cell lysate, and. ml of immune plasma which contained 5 mm Tris-acetate at ph 7.6 (5). The growth hormone standard was omitted from parallel samples which served as "blanks" which were used to correct for the small degree of nonspecific precipitation of radiolabeled cell protein in the precipitin reaction. All studies reflect the results from triplicate flasks, and each flask was assayed by immunoprecipitation in triplicate. The samples were incubated for -8 hr, then centrifuged at X g, and washed twice in buffer solution which contained 5 mm Tris-acetate at ph 7.6 (5),.1 M NaCl, 1% (vol/vol) Triton X-1, and 1% sodium deoycholate. The resultant immune precipitates were then heated at 1 in 1% NaDodSO, 1 mm NaPO at ph 7., and 1% -mercaptoethanol (vol/vol) for min, and were analyzed by NaDodSO/ polyacrylamide gel electrophoresis by the method of Laemmli (17) using 15% acrylamide and.% bisacrylamide in disk gels 1 cm long. The gels which contained radiolabeled material were sliced into 1.5 mm sections and were analyzed in a liquid scintillation counter as previously described (18). The position of growth hormone on the gel was determined by electrophoresing 5,ug of purified growth hormone in a parallel gel. 3 E 5 ~ 1 CE' 15 (5i Proc. Natl. Acad. Sci. USA 73 (1976) z lw OZ 8 WX 6 1 Xk Ov 3t B t S '. cr ds 6 8 / M I NUTES CELLS TIME (MINUTES) 1, b rn 6 ior Q : Ir 6 In FIG. 1. Estimation of the intracellular half-life and the rate of secretion of formed radiolabeled growth hormone in GH1 cell cultures. The eperiment was carried out as described in Materials and Methods with 5 nm T3 (@-*), and without T3 (o--- -). The inset illustrates the kinetics of loss of intracellular growth hormone with () and without T3 (). The results reflect the total cell and media growth hormone per 5 cm flask. Gel electrophoretic studies documented that 3H-labeled growth hormone was identified only when the growth hormone standard was present in the immune precipitation reaction and that the amount of 3H-labeled growth hormone in the immune precipitate could be routinely quantitated by subtraction of the small amount of radioactivity in the "blank" samples from those which were immunoprecipitated with the growth hormone standard. Kinetics of [15I]T3 Nuclear Binding in Intact GH1 Cells. The nuclear binding of [15I]T3 (5 nm) with intact suspended GH1 cells was carried out as previously described (7) ecept the media contained 1% serum. DNA was determined by the method of Burton (19) and the magnitude of "nonspecific" binding, determined by simultaneously incubating cells with a -fold molar ecess of nonradioactive T3, was less than 8% of the total [15I]T3 bound. RESULTS AND DISCUSSION Kinetics of [3H]Leucine Incorporation into Growth Hormone and Total Cell Protein and Estimation of the Eit Rate of Growth Hormone from the Intracellular Pool. To eamine the effect of T3 on influencing growth hormone synthetic rates, we needed documentation that T3 does not influence the eit rate of growth hormone from the intracellular pool which would reflect secretion and/or intracellular degradation (). In addition, in estimating synthetic rates the time of GH1 cell incubation with [3H]leucine must be sufficiently short such that the incorporation rate is linear and is not significantly influenced by the half-life of intracellular growth hormone (). Fig. 1 illustrates an eperiment to show whether T3 influences the intracellular half-life of radiolabeled growth hormone. The cell cultures were first incubated with 5 nm T3 for hr and then with [3H]leucine for 5 min. The media was echanged twice, and the cell cultures were then incubated with incubation media which contained 1 mm L-leucine for the times indicated in Fig. 1. At each time point, both intracellular and etracellular growth hormone were quantitated by immunoprecipitation. Although T3 had no effect on the total [3H]leucine incorporation during the 5 min incubation (6 X CE- (5

3 Biochemistry: Samuels and Shapiro Proc. Nati. Acad. Sci. USA 73 (1976) 3371 M 3 mo a_ o,,, U '-~ 1 H~ I - bmweessammamb- I.- LLJ w = &-. *I co I', 1 o 8 X< 6 < C= a C Z _~ bj 5 lo TIME (MINUTES) FIG.. Kinetics of [3H]leucine incorporation into growth hormone (-*), total cell protein (O--- -o), and the free intracellular [3H]leucine pool (^-.-M. The results reflect the total incorporation of isotope per 5 cm flask. 16 dpm ± 5% per flask), incorporation into radiolabeled growth hormone was 3fold greater in the T3-treated cultures. The decay rate for intracellular growth hormone was essentially identical in T3 and control cell cultures and, as illustrated in the inset in Fig. 1, the half-life of intracellular growth hormone was estimated to be 7 min. Concomitant with the loss of intracellular growth hormone is an increase in etracellular growth hormone and the total radiolabeled growth hormone per culture (cells plus media) remained unchanged during the course of incubation. This indicates that the eit rate of growth hormone from the intracellular pool reflects hormone secretion and growth hormone does not appear to be degraded by the cell. Fig. illustrates the labeling kinetics of intracellular free [3H]leucine and the growth hormone and total cell protein pools between 8 and 35 min of incubation. Within 8 min, as measured by the TCA-soluble radioactivity, [3H]leucine is rapidly transported into the cell and totally equilibrates with the intracellular leucine pool. The incorporation rate of [3H]leucine into growth hormone and total cell protein pools was linear for the initial 15 min but diminished at longer incubation times which reflects the rate of loss of protein from the radiolabeled pool. Growth hormone synthetic rates were therefore estimated with [3H]leucine incorporation times of 1-15 min. Based on an intracellular growth hormone half-life of 7 min, these incubation times might slightly underestimate the absolute synthetic rate. Because the intracellular half-life was not altered by T3, these incubation times give a valid comparison of the effect of T3 on growth hormone synthesis in relation to control cell cultures. NaDodSO/Polyacrylamide Gel Electrophoresis of Growth Hormone Immune Precipitates and Labeled Cell Protein. The effect of 5 nm T3 (free T3 was.55 nm) on growth hormone and total protein synthetic rates were eamined after a -hr incubation with hormone followed by a 15 min incubation with [3H]leucine. Fig. 3 compares the gel electrophoretic pattern of total radiolabeled cell protein (Fig. 3A) and immunoprecipitated growth hormone (Fig. 3B) from T3-treated and control cell cultures. T3 had no effect on the incorporation rate of [3H]leucine into total cell protein, although the gel electrophoretic patterns did resolve a few differences between the T3 and control cell cultures. Gel electrophoresis of the immune precipitates (Fig. 3B) identified a single sharp radioactive peak which migrated identically as the growth hormone standard. This documents the specificity of the imon M J I LJ Ir- _o LA r-i C) I X cl, 3ga (D 3 1 I? : 1 I FRACTlON NO. FIG. 3. NaDodSO/polyacrylamide gel electrophoresis of total radiolabeled cell protein (Fig. 3A), and growth hormone immune precipitates (Fig. 3B), in cultures incubated without (-) and with 5 nm T3 (O... ). The migration was from right to left and the arrow indicates the position of a purified growth hormone standard which was electrophoresed in a parallel gel. The results reflect the total incorporation of isotope per 5 cm flask. mune precipitation reaction for separating growth hormone from other radiolabeled proteins. In contrast to total radiolabeled cell protein, a comparison of the immunoprecipitated growth hormone peaks indicates that T3 induced a -fold increase in the rate of synthesis of growth hormone. Kinetics of Nuclear Binding-and the Induction of Growth Hormone Synthesis by T3. Fig. compares the kinetics of the induction of growth hormone synthesis to the time course of binding of 5 nm T3 to nuclear receptors in intact cells. No increase in total cell protein [3Hjleucine incorporation or in [3H]leucine transport was observed during the course of the eperiment. T3 increased the rate of growth hormone synthesis by 1.5-fold in 1.5 hr, -fold in.5 hr, and to a maimal.9-fold by 8.5 hr of incubation. The kinetics-of T3 nuclear binding, carried out in serum containing media, is essentially identical to our previous studies using serum free media (7).- An eamination of these curves indicates a time interval of 5-6 min from the point of significant occupancy of nuclear sites by T3 and a detectable increase in growth hormone synthesis. This effect is the earliest inducible biologic response reported for physiologic concentrations of thyroid hormone and is similar to the time interval reported for the induction of the estrogen dependent "induced protein" in rat uterine tissue (1). Role of RNA and Proteins Synthesis in the Induction of Growth Hormone by T3. The major evidence that the thyroid hormone nuclear binding protein -functions as a cellular receptor is based primarily on the agreement between affinity characteristics and biologic response (1, 7, 1). The effect of T3 in stimulating growth hormone synthesis is not immediate even though T3 enters the cell very rapidly (Fig. ). This suggests that T3 stimulates the accumulation of a cellular factor which is rate limiting with regard to growth

4 337 Biochemistry: Samuels and Shapiro I cm To z o:: H z C-j C-) H 3 1 r3 N UCLEAR BIN/ DING GR-GOWrH/ HORMOW E 5 ~~SYNTESIS I I if I TIME (HOURS) 1 D O.c 8 m m co a 6 < CD Ad L) C zm oo ELL to FIG.. Kinetics of induction of growth hormone synthesis (-), and relationship to the kinetics of [15I]T3 nuclear receptor binding (,-- -A&). [3H]Leucine incorporation into total cell protein was.6 X 16 dpm ± 5% per 5 cm flask. hormone synthetic rates. In addition, the results of Figs. 1 and indicate that T3 induces an increase in growth hormone synthetic rates without altering the total rate of GH, cell translation. These observations are consistent with a nuclear action of T3 which is reflected by an increase in the accumulation of growth hormone messenger RNA. The kinetics of the response do not support a direct and immediate selective effect of T3 on growth hormone synthesis at the level of translation. The induction, however, may result from a direct translational effect of T3 on stimulating the synthesis of protein factors which accumulate and then, in turn, selectively accelerate the rate of growth hormone synthesis on preformed growth hormone messenger RNA. Therefore, the T3 induction of growth hormone synthesis independent of an effect on total cell protein synthetic rates may be mediated by an etranuclear mechanism. We attempted to differentiate between the above possibilities. If the assumption is made that T3 acts at the nuclear level by regulating a transcriptional or post-transcriptional event which increases growth hormone messenger RNA concentrations, then by inhibiting RNA synthesis with actinomycin D (AMD), prior to cellular incubation with T3, the induction of growth hormone should be inhibited. Furthermore, if growth hormone synthesis is first maimally induced by T3 prior to incubation with AMD, then the rate of growth hormone synthesis might be epected to continue at the same rate if the growth hormone messenger RNA has a long half-life. Similar results might be epected with 3'-deoyadenosine (3'-dA) which has been reported to inhibit the nuclear polyadenylation of heterogeneous nuclear RNA and the emergence of messenger RNA into cytoplasmic polysomes (). Fig. 5 illustrates the results of an eperiment where this was eamined for AMD (.8 Aug/ml) and 3'-dA (3 Atg/ml). After a.5-hr incubation, AMD and 3'-dA inhibited the rate of incorporation of [3H]leucine into total cell protein by 5% and 6%, respectively. Therefore, to eamine the influence of these compounds on the induction of growth hormone, we epressed the rate of growth hormone synthesis relative to total cell protein synthesis. Both AMD and 3'-dA inhibited the growth hormone response by 9% when added to GH, cell cultures min prior to the addition of T3. In contrast, when these inhibitors were added to cells which were first incubated with T3 for 6 hr, the relative rate of growth hormone synthesis was unaltered and remained identical to that determined after 6 hr of T3 incubation. These results are consistent with the concept that T3 stimulates the accumulation of a relatively stable RNA IL- LL z o 8 I- IJ Hr Proc. Natl. Acad. Sci. USA 73 (1976) 3~ l 3-dA AMD --da E-AMD TI ME (HOURS) FIG. 5. Influence of AMD and 3'-dA on the induction of growth hormone synthesis (-) by T3. AMD,.8,g/ml, (o-o) and 3'-dA, 3 Ag/ml, (O--- -) were added to cells min prior to T3 (5 nm) and 6 hr after T3 incubation. The total incubation time with the inhibitors in each case was.5 hr. [3H]Leucine incorporation into total cell protein without the inhibitors was.6 X 16 dpm + 5% per 5 cm flask. species which is involved in the T3-induced increase in the rate of synthesis of growth hormone. The results of Fig. 5, however, may also be consistent with an etra-nuclear action of thyroid hormone. As mentioned above, T3 might directly stimulate the translational synthesis of factors which accumulate and selectively stimulate the rate of growth hormone synthesis at the translation level on preformed growth hormone messenger RNA. We eamined, therefore, whether protein synthesis is necessary for the induction of the growth hormone response by T3. GH1 cells were incubated in groups with and without S nm T3 and cells in each group were also incubated from the beginning of the eperiment with 5,gM cycloheimide. At the times indicated in Fig. 6, the media of the cell cultures were washed twice with ml of Ham's F-1 media to remove cycloheimide, and the rate of growth hormone and total cell protein synthesis was then quantitated. This concentration of cycloheimide inhibited cell protein synthesis and growth hormone synthesis by 95% and, after the media was echanged twice with fresh media, the rate of protein synthesis was rapidly restored to that of control cell cultures. Similar observations on total cell protein synthesis have been previously reported by Peterkofsky and Tomkins for HTC cells (3). Fig. 6 illustrates that T3 induced a gradual increase in the rate of synthesis of growth hormone to -fold after 3.5 hr of incubation. When the cell cultures with T3 were washed free of cycloheimide (wash ) after 3 hr of incubation the rate of synthesis determined 35 min later was essentially identical to that determined for the cultures which were incubated with only T3 from the beginning of the eperiment. This was not observed for cell cultures incubated for 3 hr with cycloheimide without T3 (wash ). The rapid attainment of the rate of growth hormone synthesis is not eplained by an effect of T3 occurring during the 35-min cycloheimide-free period between 3 hr and 3 hr and 35 min. No effect of T3 is observed during the same 35-min period in the cycloheimide free cultures at the beginning of the eperiment, or if cycloheimide is removed from T3-treated cultures at the early part of the incubation (wash 1). The rapidity at which the growth hormone synthetic rate is attained after removal of cycloheimide compared to the kinetics of the induced response suggests that a nonprotein factor accumulates during the 3 hr incubation which is rate limiting for growth hormone synthesis. These observations taken along with the studies using AMD and 3'-dA suggest that the rate /?

5 Biochemistry: cqr ZZW wdz bo Q:a IJ 8 -i HO ~ 3-1 WASH I Samuels and Shapiro I~~- WASH 1 3 TIME (HOURS) FIG. 6. Kinetics of the growth hormone response with cycloheimide present during the incubation period. GH1 cell cultures were incubated with 5 nm T3 (-), with T3 plus 5 gm cycloheimide ( ), without T3 (-), and without T3 plus cycloheimide (O--- -). At the two indicated times the growth hormone synthetic rate was determined after the flasks were washed free of cycloheimide by two echanges of fresh Ham's F-1 media. [3H]Leucine incorporation into total cell protein was. X 16 dpm 8% per 5 cm flask. limiting factor is an RNA molecule presumably growth hormone messenger RNA. It should be pointed out that this study does not differentiate between a T3 effect on increasing the accumulation of growth hormone messenger RNA or a messenger RNA which codes for the synthesis of a specific translational factor which can then selectively stimulate the synthesis of growth hormone even though total cell translation rates remain unchanged. An eamination of the effect of thyroid hormone on regulating growth hormone messenger RNA levels and its relation to the rate of growth hormone synthesis may help to resolve this question. By either mechanism, the results are consistent with the thesis that T3 stimulates the accumulation of an RNA species which is rate limiting for growth hormone synthesis and provides supportive biologic evidence in addition to receptor localition studies for a very early primary cellular action of thyroid hormone at the nuclear level. This research was supported by an Ella Maude Lander Brady Me- Proc. Natl. Acad. Sci. USA 73 (1976) 3373 morial Grant from the American Cancer Society (BC-13D) and National Institutes of Health Grant AM Samuels, H. H., Tsai, J. S. & Cintron, R. (1973) Science 181, Tsai, J. S. & Samuels, H. H. (197) Biochem. Blophys. Res. Commun. 59, Samuels, H. H. & Tsai, J. S. (1975) Seventh International Thyroid Conference (Ecerpta Medica International Congress Series no. 361), Solomon, J. & Greep, R.. (1959) Endocrinology 65, Snyder, P. J., Jacobs, L. S., Utiger, R. D. & Daughaday, W. H. (1973) J. Clin. Invest. 5, Hervas, F., Morreale de Escobar, G. & Escobar Del Rey, F. (1975) Endocrinology 97, Samuels, H. H. & Tsai, J. S. (1973) Proc. Natl. Acad. Sci. USA 7, Samuels, H. H. & Tsai, J. S. (197) J. Clin. Invest. 53, Samuels, H. H., Tsai, J.-S., Casanova, J. & Stanley, F. (197) J. Clin. Invest. 5, Surks, M. I., Koerner, D., Dillman, W. & Oppenheimer, J. H. (1973) J. Biol. Chem. 8, Oppenheimer, J. H., Schwartz, H. L., Koerner, D. & Surks, M. I. (197) J. CGn. Invest. 53, Koerner, D., Schwartz, H. L., Surks, M. L., Oppenheimer, J. H. & Jorgensen, E. C. (1975) J. Blol. Chem. 5, DeGroot, L. J. & Strausser, J. L. (197) Endocrinology 95, Spindler, B. J., MacLeod, K. M., Ring, J. & Bater, J. D. (1975) J. Biol. Chem. 5, Thomopoulos, P., Dastugue, B. & Defer, N. (197) Biochem. Biophys. Res. Commun. 58, Bancroft, F. C. (1973) Endocrinology 9, 11-11; 17. Laemmli, U. K. (197) Nature 7, Ikeda, H. (1971) Nature New Biol. 3, Burton, K. (1956) Biochem. J. 6, Schimke, R. T. (1975) in Methods in Enzymology, eds. O'Malley, B. W. & Hardman, J. G. (Academic Press, New York), Vol., pp Katzenellenbogen, B. S. & Gorski, J., (197) J. Biol. Chem. 7,, Adesnik, M., Salditt, M., Thomas, W. & Darnell, J. E. (197) J. Mol. Biol. 71, Peterkofsky, B. & Tomkins, G. M. (1968) Proc. Natl. Acad. Sci. USA 6, -8.