Human Growth Hormone-stimulated Growth of Human Cultured Lymphocytes (IM-9) and Its Inhibition by Phorbol Diesters through Down-regulation of the Hormone Receptors POSSIBLE INVOLVEMENT OF PHOSPHORYLATION OF A 55,000 MOLECULAR WEIGHT PROTEIN ASSOCIATED WITH THE RECEPTOR IN THE DOWN-REGULATION*

We report in this paper that human growth hormone (hGH) stimulates the growth of human cultured IM-9 lymphocytes in a low concentration (3%) of serum. The hormone-stimulated growth was inhibited with the phorbol diesters phorbol 12-myristate 13-acetate and phorbol 12,13-dibutylate (PDBu). The binding exper- iments of ‘251-hGH to the phorbol diester-treated cells and to their detergent-solubilized receptors revealed that the phorbol diesters caused internalization of the hGH receptors from the cell surfaces but did not sig-nificantly affect their affinity (K, 8.5 X 10’ M-l).

We report in this paper that human growth hormone (hGH) stimulates the growth of human cultured IM-9 lymphocytes in a low concentration (3%) of serum. The hormone-stimulated growth was inhibited with the phorbol diesters phorbol 12-myristate 13-acetate and phorbol 12,13-dibutylate (PDBu). The binding experiments of '251-hGH to the phorbol diester-treated cells and to their detergent-solubilized receptors revealed that the phorbol diesters caused internalization of the hGH receptors from the cell surfaces but did not significantly affect their affinity (K, = 8.5 X 10' M-l). About half of the receptors (1.4 x 103/cell) were internalized in 30 min at 37 "C, and the half-effective doses of phorbol 12-myristate 13-acetate and PDBu were 5 and 35 nM, respectively.
When culture was continued after washing with the culture medium, the phorbol diester-treated cells recovered their hGH-responsive growth, and the number of the surface hGH receptors was restored.
The down-regulation of the hormone receptor was also induced with another phorbol diester, phorbol 12,13-didecanoate, but not with the phorbol or phorbol monoesters phorbol 12-myristate and phorbol13-acetate.
Staurosporine and sphingosine, inhibitors of protein kinase C, inhibited the phorbol diester-caused down-regulation with a half-inhibitory dose (ICso) of 8 nM and 130 FM, respectively. This suggests that protein kinase C was involved in the reaction. When 32Pi-loaded IM-9 cells were stimulated with PDBu at 37 "C, the phosphorylation of M, 55,000, 88,000, and 114,000 proteins increased rapidly.
The PDBu-stimulated phosphorylation of 55,000 protein was also inhibited by staurosporine at 10 nM, which was a comparable concentration to inhibit the phorbol diester-induced down-regulation of hGH receptors.
Furthermore, among these proteins, the 55,000 protein was specifically coisolated with the 2) immunoisolation using protein A-cellulose columns; and 3) affinity purification by hGH-fixed agarose gel. These results suggest that phorbol diesters reduce the hGH-stimulated growth of cultured IM-9 lymphocytes by the down-regulation of hGH receptors and that the receptor-associated 55,000 protein may be involved in this regulation through phosphorylation by protein kinase C.
Human growth hormone (hGH),' a peptide hormone (molecular weight, 22,000) required for normal growth in man, has been synthesized by biotechnological methods and is used widely as a therapeutic drug for growth-deficient syndromes. However, the mechanisms of the hormone action have not been established mainly because it has different activities, both direct and indirect (via insulin-like growth factor l), and shows various activities in different tissues and cells, including liver, cartilage, lymphocytes, and adipocytes (l-3). The amino acid sequences of the growth hormone receptors of rabbit and human livers have been deduced from sequences of cloned DNAs, and the structures have been found to be distinct from the receptors of other growth factors (4).
Recently, it has been demonstrated that growth hormone has an important role in regulating many immunological phenomena via specific receptors on lymphocytes or phagocytes (for review, see 5). Human IM-9 lymphocytes, which have high affinity receptors for growth hormone (6), have been used for the radioreceptor assay of growth hormones (7) or for biochemical studies of the hormone receptors (8-10). The biological functions and dynamics of the receptors have, however, been obscure, partly because hGH does not promote growth in cultured lymphocytes under common culture conditions.
In this paper, we present lines of evidence that hGH is involved in controlling the growth of IM-9 lymphocytes. We also describe the mechanisms by which phorbol diesters regulate the hGH receptors in these cells. of the phorbol diester-treated cells (5-8 X 106/tube), in 0.5 ml of TBS containing 4-7 ng of '*'I-hGH (55-105 &i/pg) and 0.1% bovine serum albumin was incubated at 4 "C for 2 h with occasional mixing.
The "51-hGH-bound cells were washed with TBS containing 0.1% bovine serum albumin either by centrifugation (100 x g, 10 min) at 4 "C or bv a cell harvester (Brandel Co., Gaithersburg, MD), and the radioactivity of the bound ?-hGH was determined with a y-ray counter.
The hGH receptors of the phorbol diester-treated cells (1 X lo'), suspended in 2 ml of HBSS containing 5 mM diisopropyl fluorophosphate, were solubilized with 2% Nonidet P-40 under a single pulse of ultrasound (10 s) from a sonicator (Heat Systems model W-220F equipped with a cup horn) at 80% power and 0 "C. The sonicate was centrifuged (25,000 X g, 15 min) at 4 "C, and the supernatant was subjected to "'I-hGH-binding assay according to Cuatrecasas (11). The release of the '*"I-hGH-binding activity from intact cells to the extract was about 84%. In both the intact cells and the solubilized receptors, nonspecific binding was defined as the binding in the presence of a 200.fold excess-of unlabeled hGH.
All of the '251-hGH-binding experiments were carried out at O-4 "C with standard deviations of less than 6.4%. Protein Phosphorylation of ZM-9 Cells-The IM-9 cells (2 X 10") washed with phosphate-free minimum essential medium were divided (4 x lo7 cells/tube) and preincubated in 4 ml of the same medium containing 5 mM HEPES (pH 7.2) and "P, (carrier-free, 200 &i/ tube) at 37 "C for 60 min. After incubation with inhibitors or vehicle (dimethyl sulfoxide) at 37 "C for 10 min, the "Pi-loaded cells were treated with phorbol diester for 3 min at 37 "C and washed with icecold TBS containing 2 mM sodium phosphate and 2 mM potassium pyrophosphate as described above. The washed cells were then solubilized in 1 ml of TBS containing 2% Nonidet P-40 by sonication as above in the presence of the following enzyme inhibitors: 50 mM NaF, 5 mM EDTA, 2 mM potassium pyrophosphate, 5 mM diisopropyl fluorophosphate, 1 mM benzamidine, 20 pM leupeptin, and 18 FM pepstatin.
The sonicates were centrifuged (25,000 X g, 20 min) at 4 "C, and the supernatants were used as the cell extract for the isolation of hGH receptors.
To 40 ~1 of the extract was added 100 ~1 of sample buffer (12) containing 2% SDS and 10 mM dithiothreitol, and heat treatment was performed at 80 "C for 1 min. A sample (15 ul) was subiected to SDS-PAGE according to Laemmli (12) and autoradiographed (13). Zmmunoorecioitation of hGH Recenters-Two hundred microliters of the cell extract prepared as above was diluted with 600 ~1 of TBS containing 0.1% Nonidet P-40,0.3 mM phenylmethylsulfonyl fluoride, 2 mM potassium pyrophophate, and 1 mM MgCl,. Two microliters of hGH or bovine GH solution (1 mg/ml phosphate-buffered saline) was added. After the mixture was incubated at 4 "C for 15 min, 20 ~1 of rabbit anti-hGH antibodv or normal rabbit IgG (10 mg/ml of TBS) was added and incubated at 4 "C for 60 min. Goat a&i-rabbit IgG serum (45 ~1,Cappel) was then added, and immunoprecipitates were obtained bv incubation at O-4 "C for 17 h followed by centrifugation at 2000 X 2 for 20 min. The precipitates were suspended in 10 ml of the same buffer used for the dilution and collected by centrifugation (2000 x a!. for 20 min) at 4 "C. The precipitates were solubilized in iO0 ~1 ofSDS-PAGE sample buffer (12)  The materials bound to the column by ionic interaction were removed by washing with 1 ml of the buffer containing 0.5 M NaCl, and specifically bound hGH receptors were eluted with 2.8 ml of the buffer containing 6 M urea. More than 99% of i*'I-hGH-binding activity was absorbed to the column, and no activity was detected in the NaCl-eluted fraction.
The recovery of the activity in the urea-eluted fraction was calculated to be 21% after the removal of urea by dialysis.
The samples were concentrated by Molcut II and analyzed on SDS-PAGE by autoradiography.

RESULTS
Human Growth Hormone-stimulated Growth of IM-9 Lymphocytes and Inhibitory Effect of Phorbol Diesters on the Growth-The growth of IM-9 lymphocyte is dependent on the presence of fetal bovine serum in the culture medium, and usually 10% of the serum is required for normal growth. When the concentration of the serum was lowered to 3%, cell growth was reduced to less than 30% 48 h after the culture had begun (data not shown). However, cultivating IM-9 lymphocytes in RPM1 1640 containing 3% fetal bovine serum for 24 h caused the cells to respond dose dependently to hGH in the culture medium ( Fig. 1). In the presence of 10 nM hGH, growth was restored to more than 80% of that in 10% fetal bovine serum in RPM1 1640, as measured by [3H]thymidine incorporation into DNA. Since other human cell lines such as K-562, HL-60, and CCRF-CEM, which do not have detectable hGH receptors on their surfaces, showed no response to hGH under the same culture conditions (data not shown), the effect of hGH on the growth stimulation of IM-9 cells under the fetal bovine serum-lowered conditions appears to be a receptormediated phenomenon. Serum concentration in the culture medium seemed to be critical to elicit the hGH dependence of the growth. IM-9 cells kept their ability to grow in RPM1 1640 containing 5% fetal bovine serum and were able to respond only weakly to hGH. When the cells were cultured in RPM1 1640 containing 1% of fetal bovine serum, more than half of the cells died within 24 h.
It has been shown that phorbol diesters can modulate cell growth positively (15-17)  of IM-9 cells in RPM1 1640 containing 3% fetal bovine serum. As shown in Fig. 1, the addition of PMA in the culture medium (100 nM) completely abolished the responsiveness of the cells to hGH, although PMA did not show any cytotoxicity at this concentration (the viability of the cells tested by the dye exclusion test was 98%). Washing the ceils to remove unbound PMA restored the cell responsiveness to hGH under the same culture conditions.
Phorbol Diester-induced Decrease of lz51-hGH Binding-To elucidate the mechanisms of PMA-caused cell unresponsiveness to hGH, we studied the effects of phorbol diesters on lz51-hGH binding to IM-9 cells. The cells were preincubated with a phorbol diester in HBSS at 37 "C for 30 min and harvested. lz51-hGH was bound at 4 "C for 2 h in 10 mM Tris-HCl (pH 7.2)-buffered saline. Fig. 2 shows that PMA and PDBu dose dependently decreased the specific binding of iz51-hGH to the receptors on the cell surfaces. Half-effective doses of PMA and PDBu were 5 and 35 nM, respectively. These effects of phorbol diesters did not occur when the cells were treated at 4 "C, although the specific binding of the lz51-hGH to the IM-9 cells was not affected at this temperature (data not shown). It is possible that the mobility of the hGH receptors on the cell surfaces is involved in the phorbol diester-induced decrease of "'1-hGH binding.

Time Course of Phorbol Diester-induced Change of hGH
Receptors on IM-9 Cell Surfaces-To investigate the biochemical effects on the hGH receptors which occur after the phorbol diester treatment, we studied the time course of the effect of PDBu (1 PM) on the iz51-hGH binding to the IM-9 cells. Fig. 3A shows that the specific binding of '251-hGH to IM-9 cells decreased rapidly depending on the time of pretreat- In another experiment, the cells were treated with PDBu for 30 min to reduce the cell surface binding sites, washed with culture medium to remove the unbound drug, and cultured for 20 h in RPM1 1640 containing 10% fetal bovine serum to study the change in the number of receptors on the cell surfaces and cellular morphology. A typical result is shown in Fig. 3B. '251-hGH-binding capacity remained at a low level (about 50% of the original capacity) for about 10 h after the PDBu treatment and then gradually recovered to the same level as the phorbol diester-untreated cells 20 h after the removal of the drug from the medium.
These kinetic profiles of the change of the number of hGH receptors after the phorbol diester treatment (Fig. 3, A and B), together with the result shown in Fig. 2 appear to explain the drug-induced change in the responsiveness to the hormone (Fig. 1).
The phorbol diester treatment caused the IM-9 cell membranes to become ruffled or damaged, and washing followed by incubation for 20 h allowed the membranes to return to sharply outlined forms similar to those in untreated cells.
Internalization of hGH Receptors Caused by Phorbol Diesters-Phorbol diesters have been shown to lead to the enhanced internalization of a variety of cell surface receptors (19-24). Therefore, we investigated whether the phorbol diester-induced change of hGH binding was due to a decrease in the number of the hGH receptors, a decrease in their affinity, or both. Scatchard plot analysis (25) of PDBu-treated and -untreated IM-9 cells revealed that the number of the receptors of hGH was decreased from 2.7 X lo3 to 1.3 X lo3 sites/ cell by PDBu, but there was no significant change in their affinity (& = 8.5 X 10' M-') (Fig. 4). These results are fully consistent with the observation shown in Fig. 3A which indicates that the binding of lZ51-hGH was decreased by PDBu treatment to about 50% of that of untreated cells. When all the receptors were extracted from the whole cells by sonication in the presence of Nonidet P-40, which solubilized the active receptors with the highest recovery (about 84%) among the detergents tested, almost the same hGH-binding activity appeared in the extracts from either PDBu-treated or -untreated cells (Table I).
These results indicate the involvement of protein kinase C in the down-regulation of the hGH receptors on IM-9 cells. Effects of Staurosporine and Sphingosine on the Downregulation of hGH Receptor-If protein kinase C is involved in the down-regulation of hGH receptors on IM-9 cells, an inhibitor of this enzyme should prevent the down-regulation. We therefore investigated the effect of staurosporine, a strong inhibitor of protein kinase C (29), on the PDBu-induced decrease of hGH receptors on IM-9 lymphocyte surfaces. A typical result is shown in Fig. 6A. Staurosporine strongly inhibited the phobol diester-induced down-regulation of the hGH receptors at low concentrations (I& = 7 nM). It is reported that the I& of staurosporine against isolated protein kinase C was 2.7 nM (29). Therefore it appears that the inhibition of the down-regulation by staurosporine was caused by the suppression of protein kinase C activity. Sphingosine, which was used as an inhibitor of protein kinase C (30, 31), also showed an inhibitory activity against the down-regulation (Fig. 6B). These results suggest that protein phosphorylation catalyzed by protein kinase C is essential to the down-regulation of the receptors.
Phorbol Diester-stimulated Protein Phosphorylation and Its Inhibition by Staurosporine-We studied the effect of phorbol diester on protein phosphorylation of IM-9 lymphocytes preincubated with 32Pi. As shown in Fig. 7, PDBu stimulated the phosphorylation of M, 55,000, 88,000, and 114,000 proteins. The reaction was rapid and reached a maximum within 2 min. This clearly preceded the down-regulation of the hGH receptors. The PDBu-stimulated phosphorylation was inhibited by staurosporine, dose dependently. The effective concentration of staurosporine for the phosphorylation of the 55,000 protein was comparable with that in the inhibition of PDBu-induced down-regulation (Fig. 6A). To inhibit the phosphorylation of M, 88,000 and 114,000 proteins, higher concentrations of staurosporine were required. Immunoprecipitation of hGH Receptors-It has been reported that some receptors for peptide ligands are phosphorylated by phorbol diester-activated protein kinase C (19-22, 32). We investigated whether the PDBu-stimulated phosphoproteins were hGH receptors or not. After PDBu-stimulated phosphorylation was performed as in Fig. 7, hGH receptors were solubilized with Nonidet P-40, and ligand-receptor complexes were made by addition of hGH. The complexes were then precipitated by anti-hGH antibody. The recovery of hGH receptors in the precipitate was estimated to be more than 88% from a similar cold experiment.
As shown in Fig. 8, the PDBu-stimulated 55,000 phosphoprotein was specifically precipitated with hGH and anti-hGH antibody.
Several other phosphoproteins were also observed in the precipitates; however, the 55,000 phosphoprotein seemed to be a constituent of hGH receptors or a receptorassociated protein. To clarify this point, we tried to purify the hGH receptors by the following two different techniques.

Immunoisolation of hGH Receptor by Protein A-Cellulose-
We cross-linked hGH with its receptors on the "'P-loaded cells using ultraviolet irradiation (14). The hGH-receptor conjugates were isolated from the solubilized cells as an anti-hGH antibody-hGH-receptor ternary complex by affinity chromatography on a protein A-cellulose column. The direct affinity labeling by UV irradiation was mild and easy to control with much less overcross-linking.
To determine the molecular weight of the hGH-receptor conjugates, we first conducted a similar experiment with '"'I-hGH and "'P-unloaded cells. The hGH-receptor conjugates appeared as a rather broad band with a molecular weight of 135,000 (Fig.  9A, lane I). Since this band disappeared in the presence of 100 times excess of unlabeled hGH (Fig. 9A, lane 2), this 135,000 protein band seems to be the cross-linked hGHreceptor conjugates. These observations are consistent with the results reported previously (8,9). In a similar experiment, when the cells were prelabeled with 32Pi, treated with PDBu for 3 min, and the hGH and receptors were cross-linked by UV irradiation, no radioactive protein band was detected around 135,000 (Fig. 9B). In this case, two radioactive bands with lower molecular weight (50,000 and 55,000) were observed. They were not observed when normal rabbit IgG was used in place of the anti-hGH antibody. This 55,000 band appeared only with the cells treated with PDBu and seems to be identical to the 55,000 band shown in Figs. 7 and 8. Identity   FIG. 8. Immunoprecipitates of hGH receptors from phosphorylated IM-9 cell extracts. IM-9 cells preincubated with "P, were stimulated by PDBu as in Fig. 7 and extracted with Nonidet P-40. hGH or bovine GH and anti-hGH antibody or normal rabbit IgG were added to the extract.
Finally, goat anti-rabbit IgG antibody was added, and the resultant precipitates were analyzed by SDS-PAGE and autoradiography.
of the M, 50,000 protein is unclear at present. Since the phosphoprotein was observed only when protein A column was used, it may not be related with hGH receptors. However, we cannot rule out the possibility that it is a receptor-associated protein.
Isolation of hGH Receptor by hGH-Agarose Gel-We isolated the hGH receptors directly using hGH-fixed agarose gel without anti-hGH antibody.
More than 99% of hGH-binding activity was absorbed to the affinity gel, and after extensive washing, a purified hGH receptor fraction was obtained by elution with 6 M urea. Although the PDBu treatment caused no significant change in the amount of 55,000 protein, in the purified receptor fraction, the autoradiograms show clearly that the 55,000 protein phosphorylated with PDBu was copurified with the hGH receptor (Fig. 10B). As "P was not detected at the position of hGH receptors (llO,OOO), the receptor itself does not seem to be phosphorylated.
The 55,000 protein, a portion of which seemed to associate with the hGH receptors, may play an important role in the phorbol diester-induced down-regulation of the receptor through its phosphorylation.

DISCUSSION
In this paper we present simple culture conditions for observing hGH-stimulated growth of IM-9 lymphocytes. When IM-9 cells were cultured in a medium with low serum content (3%) for 24 h, the cells acquired the hGH dependence for growth. However, with a serum content less than l%, the 1 2 were the same as those in A (above) except unlabeled hGH and '"P,-loaded cells were used instead of '""I-labeled hGH and cold cells. The autoradiograms show the "P-incorporated proteins in the ternary complexes of antibody-hGH-receptor isolated by protein Acellulose.
cells did not proliferate even in the presence of hGH. Above 5% serum, the cells readily proliferated, but the dependence was much lower than it was in 3% serum. When the serum concentration in the medium was lowered from 10 to 3% and the cells were immediately stimulated by adding hGH, no clear growth response to the hormone was observed (data not shown). Therefore, it seems necessary to culture the cells in a medium containing 3% serum for more than several hours to obtain substantial responsiveness to the hormone.
The hGH-stimulated growth of IM-9 cells appears to be a receptor-mediated event for the following reasons. 1) Other human cell lines such as K-562, HL-60, CCRF-CEM (32) and WI-38, which have no detectable hGH receptors on their surfaces, did not respond to hGH in a medium with low serum content (3%) after the same treatment of the cells. 2) When the cells were treated with a phorbol diester, the number of hGH receptors expressed on the cell surfaces was decreased to half of those on the untreated cells, and the hormone responsiveness was completely abolished (Figs. 1 and 3). 3) After washing and incubation, both the hGH responsiveness ( Fig. 1) and the number of receptors were restored in the phorbol diester-treated IM-9 cells (Fig. 3). Since the number of the hGH receptors and their affinity for the ligand remained unchanged during the cultivation of the cells in medium containing 3% serum, the acquired dependence of the growth on hGH was not able to be explained simply by the changes in the receptor number and/or affinity for the ligand.
We examined the changes of the hGH receptors and the effects of the phorbol diesters, activators, and inhibitors of protein kinase C on the receptor internalization.
The results shown in this paper indicate that hGH receptors of IM-9 cells are linked to a system that modulates the growth of the cells. However, whether or not the hGH receptors are functioning in a medium with high serum concentration (10%) remains undetermined.
One possibility is that the hGH receptors are always working, and when the serum concentration is high, endogenous growth hormone is utilized. By lowering the concentration of endogenous growth hormone by decreasing the serum concentration in the medium, the cell may become responsive to the exogenous growth hormone. However, this possibility is unlikely since the cells in low serum concentration must be cultivated for more than several hours to acquire responsiveness.
An alternative explanation is that when the cells are exposed to the low serum condition, which is apparently unfavorable to growth, the cells may use a system linked to the hGH receptors for growth, and more than several hours is necessary for the cells to switch to this system.
The present studies demonstrated that tumor-promoting phorbol diesters down-regulated without ligand binding both the hGH-stimulated growth and the hGH receptors of IM-9 lymphocytes through an internalization of hGH receptors without any change of their affinity to the hormone. It has been reported that hGH receptors were down-regulated by a ligand-induced process (9,33,34 (19)(20)(21)(22)(23)(24). The fact that activators of protein kinase C decrease the number of specific binding sites of hGH on IM-9 cells (Fig. 4) and that staurosporine and sphingosine, inhibitors of protein kinase C, inhibit the PDBu-induced decrease of the number of hGH receptors (Fig. 6) strongly suggest that protein kinase C is involved in the phorbol diester-induced downregulation of the hGH receptors. In general, phosphorylation machineries on cell membranes have been shown to he involved in signal transduction and in the dynamic movement or interaction of proteins (35-37). Recently, it has been reported that phosphorylation of certain receptors for peptide ligands induced down-regulation either through internalization (such as transferrin receptors (19,20), T-cell receptors (21,22), and insulin receptors (38,39)) or by decreasing their affinity (such as epidermal growth factor receptors (18) and tumor necrosis factor receptors (24)).
As with other receptor systems (19-24), the internalization of hGH receptors reached a plateau at 50% reduction of surface receptors. Two different explanations are possible for this. 1) As in the case of transferrin receptors (19), all the receptors are recycled under a rate constant such that half of the receptors are exposed to the cell surfaces. 2) There are two distinct sets of hGH receptor systems on IM-9 cell surfaces, only one of which responds to growth control, and its expression is modulated on the cell surface by phorbol diesters. The other set of hGH receptors is not coupled to growth control and always remains on the cell surfaces.
When IM-9 cells were treated with PDBu, the M, 55,000 protein and some other proteins were rapidly phosphorylated. Although the 55,000 phosphoprotein was partly copurified with hGH receptors, it does not seem to be a hGH-binding molecule itself, since the molecular weight of the hGH receptor of IM-9 cells has been estimated to be 110,000 (8,9). We confirmed this by the ultraviolet-irradiated cross-linking of hGH to its receptors (Fig. 9). By this technique, covalently bound hGH-receptor conjugates migrated by SDS-PAGE as a rather broad band with a molecular weight of 135,000. When the cross-linking was carried out using the cells preincubated with 32Pi according to the conditions in the experiment shown in Fig. 9A, 32P was not incorporated into the 135,000 protein (Fig. 9B). We therefore suggest that the hGH receptors of IM-9 cells are not phosphorylated in response to PDBu. Although the function of the 55,000 protein remains to be determined, we assume that at least a portion of it is associated with hGH receptors to form a functional complex, since it is copurified with hGH receptors by two immunoisolation techniques (Figs. 8 and 9). Further evidence for this observation comes from the fact that the phosphorylated 55,000 protein was purified from the IM-9 cells stimulated with PDBu by affinity chromatography using an hGH-agarose column (Fig. 10). Its function may be controlled through the phosphorylation by protein kinase C. Since the 55,000 protein seems to be more abundant than hGH receptors in the cells, the protein may interact with various other receptors or transmembrane proteins and modulate their dynamics like submembranous cytoskeieton proteins (40).
It may be possible to speculate that the internalization is essential to express the function of hGH, as in the case of interleukin 2 (41), transferrin (19,20), interferons (42), and tumor necrosis factor (24).
During the preparation of this manuscript, Palmer and Wallis (43) reported that in serum-free medium, IM-9 cells produced insulin-like growth factor 1 in response to hGH but with no growth and low viability (50%). Carter-Su et al. (44)  ;: