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Endocrinology Vol. 139, No. 6 2727-2733
Copyright © 1998 by The Endocrine Society

ARTICLES

Stabilization of the Receptor Protein Tyrosine Phosphatase-Like Protein ICA512 in GH4C1 Cells upon Treatment with Estradiol, Insulin, and Epidermal Growth Factor1

Min S. Lee, Ronald Dirkx, Jr., Michele Solimena and Priscilla S. Dannies

Departments of Pharmacology and Internal Medicine, Section of Endocrinology, Yale University School of Medicine, New Haven, Connecticut 06510

Address all correspondence and requests for reprints to: Dr. Priscilla S. Dannies, Department of Pharmacology, 333 Cedar Street, Yale University School of Medicine, New Haven, Connecticut 06510.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment with 1 nM estradiol, 300 nM insulin, and 5 nM epidermal growth factor induces secretory granule accumulation and prolactin storage in GH4C1 rat pituitary tumor cells. The same triple treatment induced more than 6-fold accumulation of both the precursor (100 kDa, pro-ICA512) and the mature forms (60–70 kDa, ICA512 transmembrane fragment) of ICA512, a receptor protein tyrosine phosphatase-like protein that is preferentially localized in secretory granule membranes. Accumulation of ICA512 resembles that of prolactin storage, for the combination of all three, estradiol, insulin, and epidermal growth factor, gave the greatest induction, which was maximal at 4 days. This effect was specific, as the levels of the small GTP-binding protein Rab3, which is also associated with secretory granule membranes, were unaffected by the triple hormone/growth factor treatment. Increased transcription and translation of ICA512 could only partially account for its 6-fold accumulation, as ICA512 messenger RNA and ICA512 synthesis levels were 1.8 ± 0.35- and 1.6 ± 0.17-fold in triple treated GH4C1 cells compared with those in untreated cells, respectively. Pulse-chase procedures showed that pro-ICA512 was more stable in treated cells. These results indicate that the enlargement of the secretory granule compartment results in the stabilization of ICA512 and raise the possibility that trafficking of secretory granules may affect ICA512’s function and vice versa.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HORMONES and neuropeptides of neuroendocrine cells are stored in and released from dense core vesicles, which are commonly referred to as secretory granules. Intrinsic membrane proteins in these vesicles have been identified (1), some of which are found in both secretory granules and smaller vesicles (2, 3, 4). The smaller vesicles are synaptic vesicles and synaptic-like microvesicles, the other regulated secretory vesicles of neuroendocrine cells (5). Some of these proteins are thought to play a role in the exocytosis of secretory vesicles; examples include synaptotagmin, a Ca2+ sensor in neurotransmitter release (6, 7, 8), and synaptobrevin (also referred to as VAMP), a component of the exocytotic fusion complex (9). Information has been recently obtained about proteins involved in the biogenesis of secretory granules from the trans-Golgi network using permeabilized cells (1, 10). Despite this progress, much remains to be understood about the molecular mechanisms involved in the formation and trafficking of these organelles.

GH4C1 cells are rat pituitary tumor cells that serve as a model to investigate the formation of secretory granules. In the absence of estradiol, the cells contain few secretory granules, but in its presence, especially upon addition of insulin and epidermal growth factor (EGF), the number of secretory granules increases about 50-fold (11). This combined hormone/growth factor treatment also causes the accumulation of PRL stores in the cells (11). Somewhat unexpectedly, the increase in PRL storage is specific, as the storage of rat GH and secretogranin II, two other secreted proteins made by GH4C1 cells, is not enhanced upon hormone/growth factor treatment (12, 13). Estradiol and EGF treatment of GH4C1 cells transfected with insulin does not cause increased storage of insulin (12). Furthermore, expression in GH4C1 cells of human PRL, which is 61% identical to rat PRL, prevents the storage of the latter, whereas a single amino acid mutation in the former ablates its ability to block rat PRL storage (14). These findings suggest that although the number of secretory granules per cell rises dramatically, the amount of cargo within the granules is specifically regulated. In light of these observations, we have been interested in determining whether the expression of other proteins of secretory granules is regulated by the hormone/growth factor treatment.

Recently, two proteins with homology to receptor-type protein tyrosine phosphatases that are enriched in neuroendocrine cells and associated with neurosecretory granules have been identified (15, 16). The first of the proteins that was identified is ICA512 (also know as IA-2{alpha}), an islet autoantigen of insulin-dependent diabetes mellitus (17, 18) that has been cloned in several species, including human (17, 18), rat (19), and mouse (20). The human complementary DNA (cDNA) of ICA512 encodes for a type I transmembrane protein of 979 amino acids with a signal peptide (amino acids 1–26), an ectodomain (amino acids 27–575), a single transmembrane domain (amino acids 576–600), and a cytoplasmic domain (amino acids 601–979). Although the cytoplasmic domain includes a region (amino acids 696–979) with homology to protein tyrosine phosphatases, ICA512 does not appear to have this activity (15). ICA512 is processed within its ectodomain, as are several receptor-type protein tyrosine phosphatases. Cleavage of the 106-kDa pro-ICA512 generates a transmembrane fragment of about 65 kDa (amino acids 449–979) and a putative N-terminal fragment (amino acids 27–448), whose fate remains to be established. The other member of this family, phogrin or IA-2 (16, 21), is also widely expressed in mammalian neuroendocrine tissues and is found in secretory granule membranes (16). Although the functions of ICA512 and phogrin are not known, their association with secretory granules suggests their participation in some aspect of granule formation or function. In this study we have examined the pattern of expression of ICA512 in GH4C1 cells grown in the absence or presence of estradiol, insulin, and EGF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
GH4C1 cells were grown in stock cultures in a 1:1 mixture of DMEM and Ham’s F-10 nutrient mixture supplemented with 15% horse serum. For induction with hormones, cells were plated in the same medium supplemented with 15% gelding serum alone or plus 1 nM estradiol, 300 nM insulin, and 5 nM EGF at a density of 2.5 x 105 cells/100-mm plate; the low density enhances the storage effect (22). The medium was replaced on the third day, and the cells were used on the fourth day unless otherwise indicated.

Antibodies
Two rabbit anti-ICA512 polyclonal antisera were generated against either the ectodomain (amino acids 389–575, anti-ICA512ecto) or the cytoplasmic domain (amino acids 643–979, anti-ICA512cyt) of human ICA512 using the respective recombinant GST-ICA512 fusion proteins as immunogens (15). Generation and characterization of the mouse monoclonal antibody directed against the cytoplasmic domain of ICA512 (amino acids 601–979) will be described elsewhere. The monoclonal antibody against PRL, 6F11, was a gift from Dr. Jonathan Scammell (23). Monoclonal antibodies against Rab3a and Rab3a/b were provided by Dr. Reinhard Jahn (24).

Immunoblots
After counting, cells were dissolved in loading buffer [50 mM Tris (pH 6.8), 2% SDS, 0.01% bromophenol blue, 10% glycerol, 10% ß-mercaptoethanol, and 200 mM dithiothreitol] and heated at 100 C for 5 min followed by 60 C for 15 min. Aliquots equal to 50,000 cells were subjected to electrophoresis on 12.5% acrylamide. Proteins were transferred to Immobilon membranes (Millipore, Bedford, MA) at 300 mA for 1.5 h, and membranes were incubated with the primary antibody overnight, followed by incubation with rabbit antimouse Ig for the blots of Rab3a and Rab3a/b and then incubation with 10 µCi [125I]protein A (DuPont-New England Nuclear, MA) for all blots for 1 h. Anti-ICA512ecto was used at a dilution of 1:1200, and anti-ICA512cyt and anti-Rabs were used at a dilution of 1:1000. The Molecular Imager system (Bio-Rad, Hercules, CA) was used to detect and quantitate the bound radioactivity.

Confocal immunomicroscopy
GH4C1 cells were plated on glass coverslips and cultured with or without hormone/growth factor treatment for 4 days, then fixed with 4% paraformaldehyde and 120 mM sodium phosphate buffer, pH 7.4. Immunocytochemistry and confocal microscopy were performed as previously described (15). Rabbit anti-ICA512ecto and anti-PRL antisera were used at 1:250 and 1:200 dilutions, respectively. Mouse anti-ICA512cyt and anti-PRL ascites were used at 1:100 and 1:250 dilutions, respectively. Lyssamine-rhodamine-conjugated goat antirabbit IgG and fluorescein-conjugated goat antimouse IgGs (Sigma, S. Louis, MO) were used at 1:50 and 1:30 dilutions, respectively.

Determination of cell volume
Cells were dispersed with pancreatin treatment and resuspended in medium. Just before sizing, 0.1 ml was diluted into 10 ml 0.9% NaCl. Cell size was determined with a Coulter counter (Coulter Electronics, Hialeah, FL) with a Channelyzer accessory, which was calibrated with 10.03-µm microspheres.

Pulse-chase labeling and immunoprecipitations
After 4 days of culture with or without hormone/growth factor treatment, cultures were labeled in DMEM with no methionine or cysteine and with 0.1% horse serum, 1 mM NaHCO3, and 20 mM HEPES, pH 7.0. After rinsing, cells were incubated with 400 µCi Express 35S Protein Labeling Mix (DuPont-New England Nuclear, Boston, MA) for 20 min and then in Ham’s F-10 nutrient mixture with 5% horse serum, 1 mM NaHCO3, and 20 mM HEPES (pH 7.5), and 1.5 mM methionine and cysteine. Cells were lysed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 100 µg/ml phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 2% Triton X-100, 0.5% sodium deoxycholate, 2.5 µg/ml leupeptin, and 2.5 µg/ml pepstatin. Lysates were incubated with preimmune serum and protein A-Sepharose beads (5 mg/sample; Pharmacia, Piscataway, NJ) overnight, and then with both polyclonal antiserum and protein A-Sepharose beads for 18 h. The beads were washed once with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.2% Triton X-100, 1 mM EDTA (pH 8.0), 0.02% sodium azide, 0.25% BSA, 2.5 µg/ml leupeptin, and 2.5 µg/ml pepstatin for 30 min, and then twice with 10 mM Tris-HCl (pH 7.0), 0.2% Triton X-100, 2.5 µg/ml leupeptin, and 2.5 µg/ml pepstatin for 30 min/wash. Proteins were eluted from the beads by heating at 100 C for 5 min followed by incubation at 60 C for 15 min in 125 mM Tris (pH 6.8), 4% SDS, 0.02% bromophenol blue, 10% glycerol, 10% ß-mercaptoethanol, and 200 mM dithithreitol. After SDS-gel electrophoresis was performed, gels were dried at 75 C for 2 h under vacuum. Results were corrected for cell number using values obtained from replicate plates. The variation in cell numbers on replicate plates is less than 10%.

Northern blotting
An aliquot of cells was removed for counting, and the rest was used for RNA extraction using RNeasy (Qiagen, Chatsworth, CA). Aliquots equivalent to equal numbers of cells (usually about 5 x 105) were used for electrophoresis and hybridization, carried out as previously described (12). 32P probes were generated by random priming. The probe for ICA512 include the 1134-bp clone of human ICA512 cDNA (17) and a 660-bp cDNA probe for rat Rab3a, provided by Dr. Pietro De Camilli (Yale University, New Haven, CT). To normalize the levels of ICA512 signals in relation to the amount of total messenger RNAs (mRNAs) loaded on the gels, blots were stripped and rehybridized with probes for Rab3a or cyclophilin A, and then the ratio of the amount of ICA512 in treated cells to that in untreated cells was calculated. RNA markers were obtained from Ambion (Austin, TX).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The expression of ICA512 in GH4C1 cells with or without hormone/growth factor treatment was investigated by immunoblotting cell extracts using antisera directed against the ectodomain (anti-ICA512ecto) or the cytoplasmic domain of the protein (anti-ICA512cyt; Fig. 1Go). Both antisera recognized the same set of proteins with apparent mobilities of about 100 and about 60–70 kDa. The predicted molecular mass of ICA512 is 106 kDa, including the signal peptide, so it is likely that the 100-kDa form corresponds to pro-ICA512. It has been previously shown that the 60- to 70-kDa protein corresponds to the ICA512 transmembrane fragment (ICA512 TMF) generated by the cleavage of the protein within its ectodomain at a conserved processing site for convertases (15). In rat ICA512, this cleavage site is located between lysine at position 452 and serine at position 453. The processed products are larger than predicted from its amino acid composition, most likely because of its putative N-glycosylation at the asparagines in positions 510 and 528.



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  		Figure 1. Immunoblots of two independent experiments using rabbit antisera against the ectodomain (ICA512ecto) or the cytoplasmic domain (ICA512cyt) of ICA512. Cells were incubated for 4 days with or without hormone/growth factors, as indicated. Amounts of lysates equivalent to an equal number of cells were applied to each lane and separated by SDS-PAGE before immunoblotting. No protein were detected with preimmune sera. Hormones/growth factors were used at the following concentrations: estradiol (E2), 1 nM; EGF, 5 nM; and insulin, 300 nM.

 
Levels of both pro-ICA512 and ICA512 TMF were increased after treatment with estradiol, EGF, and insulin combined (Figs. 1Go and 2Go). Although there was variation in the amount of increase with hormone/growth factor treatment, the induction was reproducible; treatment increased ICA512 levels in each of 12 experiments in which the combined treatment was compared with controls. The combination of estradiol and EGF was also effective (Figs. 1Go and 2Go). This induction pattern closely resembles that of PRL storage, in which these two combinations are most effective (22). The mean increase in ICA512 after the triple hormone/growth factor treatment was about 6-fold in the five experiments in which we examined all combinations, with pro-ICA512 being induced to about the same extent as ICA512 TMF (Fig. 2Go). The accumulation of ICA512 was a slow process, reaching a maximum 4 days after the start of hormone/growth factor treatment (Fig. 3Go) as was also the case for the increase in PRL storage (25).



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  		Figure 2. Quantitative measurement of ICA512 in untreated (control) and hormone/growth factor-treated GH4C1 cells. Data are expressed as the ratio of hormone/growth factor-treated samples to the untreated within the same experiment and are the mean ± SE of five independent experiments in which all combinations were tested. Equal numbers of cells were incubated as described in Fig. 1Go. Although ICA512 induction levels varied from one experiment to another, treatment with all three hormones/growth factors caused an accumulation of ICA512 in all experiments. Open bars, pro-ICA512; filled bars, ICA512 TMF.

 


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  		Figure 3. Time course of ICA512 induction by estradiol, EGF, and insulin. Data are the mean of two or three experiments except for day 6, which is a single experiment. The error bars show the SE; on day 1, the error bars are within the symbols. Open circles, pro-ICA512; filled squares, ICA512 TMF.

 
The increased levels of ICA512 in hormone/growth factor-treated cells did not simply reflect an overall increase in protein content. The total amount of protein in these cells was only 1.8-fold higher in treated than in control cells (0.45 mg protein/106 cells in treated cultures and 0.25 mg protein/106 cells in untreated cultures). In addition, not all proteins associated with neurosecretory granules accumulated in response to the hormone/growth factor treatment. Rab3a and Rab3b are two guanosine triphosphatases associated with neurosecretory granule membranes; Rab3b is the predominant isoform expressed in pituitary cells (26, 27). Immunoblotting with an antibody that recognizes these Rab3 isoforms showed no changes in their levels between control and treated cells (Fig. 4Go). In three independent experiments, the ratio of Rab3a/b in treated cells compared with that in untreated cells was 1.03 ± 0.13 (mean ± SE). Similarly, no changes were detected when immunoblots were performed using an antibody specific for Rab3a (data not shown).



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  		Figure 4. Immunoblot of Rab3a/b in GH4C1 cells. Cells were incubated for 4 days with or without the addition of 1 nM estradiol, 5 nM EGF, and 200 nM insulin to the culture medium. Amounts of lysates equivalent to an equal number of cells were applied to each lane and separated by SDS-PAGE before immunoblotting. C, Untreated control cells; T, hormone/growth factor-treated cells.

 
The pattern of expression of ICA512 in GH4C1 cells was further investigated by confocal immunomicroscopy in control and treated cells. ICA512 immunoreactivity was low in untreated cells, but very prominent upon hormone/growth factor treatment, results consistent with immunoblot analyses. Hormone/growth factor treatment of GH4C1 cells resulted in flattening on the coverslips and the extension of neurite-like processes. Independent measurements of cell volume indicated that the average cell volume increased 1.35-fold upon hormone/growth factor treatment (774 ± 39 µm3 in untreated cells vs. 1047 ± 52 µm3 in treated cells). In most ICA512-immunoreactive GH4C1 cells, the protein was concentrated at the tip of neurite-like processes (Fig. 5FGo), where PRL immunoreactivity may also occur (Fig. 5EGo); this staining is likely to be secretory granules. Note, however, that some cells contained appreciable amounts of ICA512, but not PRL, and vice versa, indicating that expression of these two proteins may be dissociated.



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  		Figure 5. Confocal immunomicroscopy for PRL (A, C, E, and G) and ICA512 (B, D, F, and H) in control (top panels) and hormone/growth factor-treated cells (bottom panels). Double immunolocalization was performed with mouse anti-PRL (A and E) and rabbit anti-ICA512ecto (B and F) antibodies or with rabbit anti-PRL (C and G) and mouse anti-ICA512cyt (D and H). Low levels of ICA512 and PRL were detectable in untreated cells, primarily in the perinuclear region (presumably corresponding to the Golgi complex), whereas both PRL and ICA512 immunoreactivities increased upon hormone/growth factor treatment. ICA512 immunoreactivity was consistently more pronounced in specimens immunostained with the monoclonal anti-ICA512cyt than in those immunostained with the affinity-purified rabbit antibodies against the ICA512 ectodomain. Colocalization of ICA512 and PRL at the tip of the neurite-like processes in treated cells (E and F) is consistent with the association of both proteins with secretory granules. The ratio between PRL and ICA512 varies from one cell to another. Hormone/growth factor treatment not only causes an increase in the average volume of GH4C1 cells (see text), but induces their flattening on the substrate, a phenomena that accounts for their considerably larger appearance compared with untreated cells.

 
Higher levels of ICA512 after hormone/growth factor treatment could result from several different mechanisms, either alone or in combination, including increased transcription of the gene, increased translation of the mRNA, and stabilization of the protein. To determine whether treatment with estradiol, EGF, and insulin would induce the expression of ICA512 mRNA, we performed Northern blotting on RNA extracted from control or treated GH4C1 cells. The ICA512 probe hybridized primarily with a 4-kilobase transcript, consistent with previous results (28). In a total of four independent experiments, ICA512 mRNA was increased 1.8 ± 0.35-fold (mean ± SE) in treated compared with untreated cells. These data indicate that the higher amounts of ICA512 recovered from treated cells can only be partially accounted for by induction of ICA512 transcription.

To measure the rate of synthesis of ICA512 in control and treated cells, we performed pulse-chase labeling experiments, followed by immunoprecipitation with anti-ICA512 antibodies. After a 20-min pulse, newly synthesized pro-ICA512, but not ICA512 TMF, was detectable in both control and treated cells (Fig. 6Go). In four separate experiments, the increase in newly synthesized pro-ICA512 in treated vs. control cells was 1.6 ± 0.17-fold (mean ± SE); this relatively small increase in ICA512 synthesis is consistent with the similar increase in ICA512 mRNA levels. These results indicate that increased synthesis is insufficient to account for the increase in ICA512 and suggested that hormone/growth factor treatment may result in ICA512 accumulation by stabilizing the protein. After a 60-min chase period, more pro-ICA512 was detectable in treated cells than in untreated cells (Fig. 6Go). In three independent experiments, the amount of pro-ICA512 present after 60 min of chase was 128 ± 7.3% of the amount in the initial pulse; in untreated cells, it was 51.7 ± 12.9% (mean ± SE), indicating that pro-ICA512 was more stable in treated cells. The increase in counts in pro-ICA512 during the chase period in treated cells is likely to reflect the inability to prevent reincorporation of radioactive amino acids, which is also found in pulse-chase measurements of PRL (29). The slower mobility of pro-ICA512 after the chase than after the pulse is likely to result from modification of its carbohydrate chain as the protein proceeds along the secretory pathway. At times longer than 60 min, it became increasingly difficult to resolve pro-ICA512 from background, which precluded obtaining precise half-lives. At no time in any experiment was ICA512 TMF clearly resolved from background radioactivity (Fig. 6Go and data not shown), although the gel area where ICA512 TMF migrates was slightly more radioactive in the case of the treated cell samples. Several factors may have contributed to the lack of detection of the ICA512 TMF. First, the methionine and cysteine content of ICA512 TMF is two thirds that of pro-ICA512, so that ICA512 TMF is expected to contain less radiolabeling even if all pro-ICA512 converts to it. Second, the diffuse migration of ICA512 TMF (Fig. 1Go) will reduce the number of counts per unit of surface area that are detected by phosphorimaging. Third, processing of pro-ICA512 may not be a synchronous event, limiting the amount of ICA512 TMF that is generated at any given time. In conclusion, these data indicate that stabilization of ICA512 significantly contributes to the increased amounts of the protein after hormone/growth factor-treatment of GH4C1 cells.



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  		Figure 6. Pulse-chase analysis of newly synthesized ICA512. Untreated (C) or estradiol-, insulin-, and EGF-treated GH4C1 cells (T) were incubated with 35S-labeled amino acids and collected immediately or subjected to a 60-min chase period before lysis and immunoprecipitation. Newly synthesized pro-ICA512 was more stable in treated cells, but newly generated ICA512 TMF was virtually undetectable in both set of cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The transmembrane fragment of ICA512 (ICA512 TMF) is the predominant form in bovine posterior pituitary (15). It is also the more abundant form of the protein in other neuroendocrine tissues (Hermel, J. M., R. Dirkx, and M. Solimena, unpublished observations), although the ratio between pro-ICA512 and ICA512 TMF varies from case to case. In GH4C1 cells, pro-ICA512 is readily detectable, usually in amounts that are about half of the processed form. The most likely explanation for the relative abundance of pro-ICA512 is that GH4C1 cells are deficient in convertases. This possibility is supported by the findings that GH4C1 cells do not efficiently process a variety of secretory granule proteins, including somatostatin, proinsulin, or POMC (12, 30, 31), but are capable of cleaving POMC upon cotransfection of the appropriate convertase (31).

We have shown here that the hormone/growth factor treatment that induces PRL storage stabilizes ICA512. This conclusion is based on the evidence that ICA512 mRNA and synthesis increase less than 2-fold, whereas the protein levels increased 6-fold. Since precursor proteins usually have a short half-life compared with their processed forms, it is usually expected that an increase in the half-life of a protein would result in the accumulation of its mature form. Our results, however, indicate that after hormone/growth factor treatment of GH4C1 cells, both pro-ICA512 and the ICA512 TMF increase to an approximately equal extent. Such a finding implies that both ICA512 forms are stabilized, which means that the half-life of pro-ICA512 will depend on both its degradation as well as its conversion to a mature form. Our inability to detect the appearance of ICA512 TMF during pulse-chase procedures did not allow us to resolve the contributions of these two components, but we demonstrated that pro-ICA512 was more stable in the treated cells.

ICA512 has some properties that are similar to those of RESP18, which is a protein that is also specifically expressed in neuroendocrine cells (32, 33, 34). It is also in the secretory pathway, as a luminal protein of the endoplasmic reticulum that is rapidly degraded further along the secretory pathway, but which does appear in secretory granules when expressed in large amounts (33). Levels of RESP18 are also regulated by steroids in a fashion similar to those of ICA512; glucocorticoid treatment stabilizes RESP18 in AtT-20 cells (32). Both of these proteins may be important in ways yet to be determined in the secretory functions of neuroendocrine cells.

The amount of ICA512 among GH4C1-treated cells, as revealed by confocal immunomicroscopy, is quite heterogeneous. Some cells contain much more protein than others, and the increase in ICA512 content in a given cell does not always correlate with a parallel increment in the amount of PRL and vice versa. These data suggest that the accumulation of these two secretory granule polypeptides can occur independently. Heterogeneity in the amount of cargo proteins in secretory granules of GH4C1 cells has been previously described. For instance, the ratio between PRL and secretogranin II has been shown to vary from one cell to another (11, 35, 36). Furthermore, the hormone/growth factor treatment does not affect PRL and secretogranin II in the same way. Although the triple treatment induces an increase in the synthesis of both proteins, the amount of intracellular secretogranin II increases proportionally to its synthesis, whereas intracellular PRL accumulation is well above its increased production (11, 12, 13). Similarly, the 50-fold increased mean number of secretory granules per section after treatment (from 1 to 50) is accompanied by a large variability in the number of granules present in each individual cell, with 10% of the treated cells having less than 5 granules/section (11).

The stabilization of ICA512 in hormone/growth factor-treated GH4C1 cells parallels the accumulation of secretory granules, as the characteristics of induction, time course, and effective hormone/growth factor combinations for the two processes are similar. Thus, the data presented here provide additional evidence for the specific association of ICA512 with secretory granules of neuroendocrine cells. It is tempting to speculate that the half-life of ICA512, like that of other receptors, depends upon its trafficking, such as its exposure at the cell surface and internalization. According to this hypothesis, it is conceivable that stabilization of ICA512 results from its accumulation on secretory granules, a compartment that has a slow turnover compared with that of other secretory vesicles and which is significantly enlarged in treated GH4C1 cells. In addition, the increased number of secretory granules in these cells may reduce the probability of each individual granule undergoing exocytosis, a situation that may decrease the overall turnover of ICA512.

In conclusion, these data further support the association of ICA512 with secretory granules. Future studies will be required to determine whether and how the trafficking of secretory granules affects the function of ICA512 and vice versa.


   Acknowledgments
 
We thank Dr. J.-H. Hermel for advice, Dr. P. De Camilli for providing us with the Rab3a cDNA and for critical reading of the manuscript, Drs. R. Jahn and J. Scammell for the gift of antibodies, and Drs. D. Michaels and D. U. Rabin of Bayer Corp. for their generous support.


   Footnotes
 
1 This work was supported in part by grants from the American Diabetes Association and the Donaghue Foundation (to M.S.), and NIH Grant DK-46807 and a grant from the American Diabetes Association (to P.S.D.). Confocal immunomicroscopy was supported by the DERC Cell Biology Core Foundation. Back

Received October 23, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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