Inefficient Secretion of Human H27A-Prolactin, a Mutant That Does Not Bind Zn2+

Zhenyu Sun, Min S. Lee, Harrison K. Rhee, Joanne M. Arrandale and Priscilla S. Dannies

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human PRL binds Zn2+, but the function of the binding is not known. We investigated the effect on PRL production in pituitary cells by obtaining clones of GH4C1 cells stably transfected with human H27A-PRL, a mutant that does not bind Zn2+. Unexpectedly, clones transfected with the mutant human PRL made little rat PRL. Untransfected GH4C1 cells made between 0.5 to 10 µg rat PRL/105 cells in 24 h. Clones transfected with vector alone (four of four), wild type human PRL (six of six), or with human K69A-PRL (two of two) made amounts of rat PRL in the same range. Clones transfected with human H27A-PRL (five of five) made 0.003–0.1 µg rat PRL/105 cells in 24 h, and the production of rat PRL mRNA was reduced. Human H27A-PRL was not efficiently secreted; 20–40% newly synthesized H27A-PRL was degraded by 60 min, and there was usually a delay in release of newly synthesized H27A-PRL. Reduction of rat PRL production is not mediated through the PRL receptor, because no sequences for the receptor in GH4C1 cells were detected by RT-PCR. Proteins involved in folding, such as BiP, were not specifically elevated in the H27A-PRL clones. In transient transfections, in which cells have not undergone selection, we found no evidence for disulfide-bonded aggregates of the mutant protein. The results indicate that Zn2+ binding stabilizes PRL in the secretory pathway; the instablility of the mutant protein may trigger effects that suppress rat PRL production directly or that indirectly result in selection of clones with low rat PRL production.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Zinc ions have been recognized as playing an important structural role in many proteins (1), including the ability to stabilize insulin in a hexamer, the form in which it is stored in secretory granules. Binding Zn2+ is important for insulin storage because a mutant of human insulin that does not bind Zn2+ well is not properly processed (2). Zn2+ binding also may be important for storage of other proteins concentrated in secretory granules. Human GH binds Zn2+ (3). Binding of Zn2+ affects the properties of human GH in solution; Zn2+ causes human GH to form dimers and increases its stability, properties that have been proposed to be important in storing GH in a concentrated form in secretory granules (3). Whether it plays such a role in intact cells has not been shown.

Human PRL, which has a structure like that of GH (4, 5), also binds Zn2+ (6). Histidine 18 in human GH is required to bind Zn2+ (3), and the topologically equivalent histidine 27 is required in PRL. The two hormones differ, however, in the effects of Zn2+ binding. PRL does not require Zn2+ binding for lactogenic activity as human GH does, and the ability to bind Zn2+ and to self-associate in solution are not coupled in PRL as they are in human GH (6). Loss of histidine 18 in human GH reduces Zn2+ binding and dimer formation (3). Loss of histidine 27 in human PRL reduces Zn2+ binding, but not the ability to self-associate in the presence of Zn2+ (6). Although the binding site for Zn2+ that requires histidine is not necessary for PRL to associate in a cell-free system, it may be necessary for proper processing and packaging of PRL in cells, and we have investigated this question by expressing human H27A-PRL in GH4C1 cells, which are a rat pituitary cell line.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the same way in which we had previously isolated clones that expressed human wild type PRL (7), we isolated stably transfected clones of GH4C1 cells that expressed human H27A-PRL. Unexpectedly, clones transfected with human H27A-PRL produced reduced amounts of rat PRL (Table 1Go). The amounts of rat PRL made by untransfected GH4C1 cells varies in different experiments, and in ten experiments with untransfected cells performed during the time the transfected clones were analyzed, the amounts of rat PRL in the cultures ranged from 0.5–10 µg/105 cells in 24 h. All clones transfected with vector alone, human wild type PRL, or human K69A-PRL, which were obtained at the same time as the H27A-PRL clones, produced amounts of rat PRL within the range of the untransfected cells. All clones transfected with human H27A-PRL made less rat PRL, from 0.003–0.1 µg/105 cells in 24 h. These values were obtained by RIA; we also confirmed qualitatively by immunoblots that clones H27A6 and H27A8 contained much less rat PRL than GH4C1 cells (not shown).


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Table 1. Production of Rat and Human PRL by Untransfected GH4C1 Cell and Transfected Clones

 
The decrease in rat PRL production was accompanied by a decrease in rat PRL mRNA. Northern analysis of RNA extracted from GH4C1 cells and from a clone, H27A6, indicated there was less rat PRL mRNA in the transfected clone (Fig. 1Go). The filter was assayed subsequently with cyclophilin to demonstrate that we had recovered RNA from the transfected clone. In Fig. 1Go, the amount of cyclophilin mRNA was higher in the transfected clone, but in a second experiment it was not. In each case, rat PRL mRNA was reduced more than 30-fold in the transfected clone.



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Figure 1. Northern Analysis of mRNA from Equal Numbers of GH4C1 Cells and a Clone Transfected with Human H27A-PRL

Top panel, rat PRL; bottom panel, the same filter anaylzed for cyclophilin.

 
GH4C1 cells in the absence of estradiol, insulin, and epidermal growth factor store little rat PRL relative to the amount they secrete, usually less than 5% of the amount secreted in 24 h; when treated with hormones, cells store 15% or more (7). We attempted measurements of rat PRL stored in clones transfected with human H27A-PRL, but found no consistent results, even in separate experiments using the same clone; intracellular PRL in control or treated cells varied from 5–30% of the amount secreted in 24 h. The variation was in contrast to our previous studies with clones expressing human wild type PRL and other mutants, where the results were consistent (7).

This type of analysis is a measure of storage if the protein is stable. We examined the stability of H27A-PRL to determine whether intracellular degradation might account for the variability we encountered. In clone H27A6, we detected incorporation of 35S-labeled amino acids during a 10-min pulse only into human PRL (Fig. 2Go). In these series of experiments, PRL was precipitated with concentrations of antisera to rat PRL sufficient to precipitate both rat and human PRL completely, and we determined that the band we detected was human PRL by its mobility, and, in separate experiments, by using dilutions of antisera specific to each species. The inability to detect rat PRL synthesis, although it is detected in the medium, reflects the small amounts of rat PRL that this clone usually made as well as the comparatively fewer methionines in rat compared with human PRL.



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Figure 2. Fate of Newly Synthesized Human H27A-PRL

Cells were incubated for 10 min with 35S-labeled amino acids, lysed at indicated intervals after the 10-min pulse, and PRL immunoprecipitated, and the immunoprecipitate was subjected to electrophoresis. Bottom panel, Radioactivity detected in the position corresponding to human PRL in the cells (inside) and in the medium (outside). Top panel, Quantification of [35S]PRL. {circ}, [35S]PRL in the medium; {blacksquare}, [35S]PRL in the cells.

 
We did not detect newly synthesized human H27A-PRL in the medium for times up to 90 min in the experiment shown in Fig. 2Go. The lack of detection of secreted H27A-PRL was not caused by degradation in the medium because we incubated recombinant H27A-PRL with the cells in the conditions that we use for the pulse-chase procedure, and we recovered all the added hormone, measured by RIA. The lack of secretion was not seen in all experiments; in five of six experiments with this clone and two of three experiments with clone H27A8, we detected no secretion during a 90-min chase period, but in one experiment with each clone, there was detectable secretion at 30 min, as we found with rat PRL and human wild type PRL (Fig. 3Go).



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Figure 3. Fate of Newly Synthesized Human Wild Type and Rat PRL

In an independent experiment, rat and human [35S]PRL were also stable, but relatively more rat PRL, 40% of the total, was secreted by 90 min. Bottom panel, Radioactivity detected in the positions corresponding to human PRL and rat PRL. Middle panel, Quantitation of human [35S]PRL; top panel, quantification of rat [35S]PRL. {circ}, [35S]PRL in medium; •, [35S]PRL in cells; {blacksquare}, total [35S]PRL.

 
Intracellular human H27A-PRL was not completely stable. In the experiment shown in Fig. 2Go, there was 20% less [35S]H27A-PRL at the end of the chase period than at the end of the 10-min pulse. The decrease was reproducible; the amount of [35S]H27A-PRL decreased 20–40% by 60 min after the pulse in all six experiments. The time that we first detected the decrease was more variable, ranging from 20–60 min after the pulse. We did not detect such decreases in the clones expressing wild type human PRL (Ref. 7 and Fig. 3Go). The variation in amount and time of degradation and secretion of H27A-PRL is likely to explain the variability we encountered when we attempted to measure storage.

We determined whether human H27A-PRL was present in a regulated pathway by stimulating release using high K+ concentrations to depolarize the cell and stimulate Ca2+ entry (8). Perifusing the cells with high K+ concentrations increased human PRL release 2-fold (Fig. 4Go). The magnitude of the stimulation is similar to the 2- to 4-fold increases that we detected in untransfected GH4C1 cells at this time (7). The mutant therefore has the ability to enter a regulated pathway.



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Figure 4. Stimulation of Human H27A-PRL Release by High Concentrations of Potassium Chloride in Perifused Cells

Four minute fractions were collected, and the flow rate was 1 ml/8 min. The delay time before added components appear in the medium is 10 min.

 
The decrease in rat PRL production caused by expression of H27A-PRL does not appear to be caused by simply interfering with folding or stability of rat PRL, since rat PRL synthesis and mRNA production were reduced, and amounts of rat PRL were decreased as measured by [35]S-amino acid incorporation, RIA, and immunoblots.

The possible mechanisms for reducing rat PRL mRNA include a feedback cycle activated inappropriately by H27A-PRL to reduce rat PRL production or by unwitting selection for a property in addition to G418 resistance. The most obvious way for feedback to occur is through the PRL receptor. We determined whether the PRL receptor was present in GH4C1 cells by RT-PCR using primers that would detect the long and the short form of the receptor. We were easily able to detect appropriate PCR bands using 1 µg total RNA from Nb2 cells but did not detect any in 1 µg RNA from GH4C1 cells in several experiments. Mixing equal amounts of RNA from GH4C1 cells and Nb2 cells did not reduce the ability to form product from Nb2 cells (Fig. 5Go). We also mixed 106 GH4C1 cells with 105 Nb2 cells before extracting the RNA and found no difference in the amount of product compared with 105 Nb2 cells extracted alone (not shown). The GH4C1 cells therefore do not interfere with the extraction of RNA or the RT-PCR assay and do not contain enough mRNA for either form of the receptor to be detectable. It appears unlikely that feedback is occurring through the PRL receptor.



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Figure 5. Products Formed by RT-PCR to Assay for the Presence of the Short Form of the Rat PRL Receptor (Lanes 1 to 6) and the Long Form (Lanes 7 to 11)

In the first lane, an Nb2 PRL receptor plasmid was used as a control. Lanes 2 and 7, 1 µg Nb2 RNA; lanes 3 and 8, 1 µg untreated GH4C1 RNA: lanes 4 and 9, 1 µg hormone-treated GH4C1 RNA; lanes 5 and 10, 1 µg each Nb2 and untreated GH4C1 RNA; lanes 6 and 11, 1 µg each Nb2 and hormone-treated GH4C1 RNA.

 
A second mechanism by which feedback may occur is through communication between the secretory pathway and the nucleus. Misfolded proteins in the endoplasmic reticulum result in a signal that increases transcription of proteins that assist in folding in the secretory pathway, including GRP78 (glucose-regulated protein 78, also known as BiP) (9, 10). We examined proteins known to reside in the endoplasmic reticulum to determine whether they were induced specifically in clones expressing human H27A-PRL. There was no increase in protein disulfide isomerase (Fig. 6Go and Table 2Go). Three other proteins, GRP78, GRP94, and calreticulin, may be somewhat increased compared with GH4C1 cells, but were not reproducibly different from cells transfected with human wild type PRL (Fig. 6Go and Table 2Go). Production of H27A-PRL therefore does not appear to trigger specifically the pathway that activates GRP78.



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Figure 6. Immunoblots of Several Proteins Located in the Endoplasmic Reticulum in GH4C1 Cells, Two Clones That Make Wild Type Human PRL (Wt-hPRL), and Two Clones That Make Human H27A-PRL (H27A-hPRL)

 

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Table 2. Amount of Proteins in the Endoplasmic Reticulum Relative to that in GH4C1 Cells

 
Selection for clones with properties in addition to resistance to G418 may occur if the transfected product interferes with cell growth in a manner that can be bypassed. A simple means for expression of H27A-PRL to cause selection may be that it initially folds so poorly in the cells that it causes a disadvantage during selection. We have therefore used transient transfections of GH4C1 cells to determine how H27A-PRL behaves in unselected cells. We found transient transfections have been useful only for qualitative conclusions because of the low transfection efficiency and the variation among replicates. The procedure that gave the best production of human PRL resulted in expression of ß-galactosidase in 0.5 to 3% of the cells. Conditions that gave higher transfection efficiencies resulted in substantially reduced human PRL production. Variation in the amount of human PRL produced in replicate cultures was as much as 2-fold, and cotransfection with ß-galactosidase for normalization did not improve this variation.

Although the information gained was only qualitative, these experiments showed that there was no extensive misfolding of H27A-PRL. Both wild type and the mutant were synthesized in a 10-min pulse, both were present 90 min after the pulse, and small amounts of each form were secreted (Fig. 7Go). Expression of either form has no effect on production of rat PRL, according to RIA measurement, but the percentage of cells transfected is so small that we would not predict that there would be an effect. We also performed immunoblots on cell extracts run under reducing or nonreducing conditions (Fig. 8Go). We found no evidence for disulfide-bonded aggregates; both human wild type and mutant PRL ran as monomer in the presence or absence of reducing agents.



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Figure 7. Immunoprecipitates of PRL from Cells Transiently Transfected with Human Wild Type or H27A-PRL After a 10-min Pulse with 35S-Labeled Amino Acids or an 80-min Chase

In this experiment, antibody to human PRL was used at a dilution that precipitated all human PRL, but not all the rat PRL in the cells. Because of the low transfection transficiency, most rat PRL is made in cells that do not express human PRL. Two other experiments gave similar results.

 


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Figure 8. Immunoblots of Cells Transiently Transfected with Human Wild Type or H27A-PRL or a Mixture of the Two

The antibody to human PRL was used at a dilution that did not cross-react with rat PRL standard at the amounts used (100 ng/gel), but the amount of rat PRL in the cells was so much greater than human PRL that it is detected, as seen in control cultures, or cultures treated with Lipofectamine alone. The last three lanes are, in order, extracts from cultures treated with 3 µg human wild type PRL DNA, 3 µg H27A-PRL DNA, or 1.5 µg wild type plus 1.5 µg H27A-PRL DNA to keep the total amount of DNA in the transfections constant. The gel in the upper panel was run under reducing conditions, and the one in the lower panel was run under nonreducing condtions. The band at the top of the gel is a nonspecific band detected in cell extracts.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The original purpose of this investigation was to determine whether the ability to bind Zn2+ was necessary for transport and secretion of PRL. Zn2+ has been shown to reduce solubilization of PRL from dense cores of granules (11). Both human GH and human PRL bind Zn2+, and Zn2+ has been detected in granules containing PRL or GH (12). Zn2+ was not detected in all granules; whether the variation actually exists or is a result of the staining technique has not yet been resolved. Human GH is more stable in the presence of Zn2+ (3), and Cunningham and co-workers therefore predicted Zn2+ binding would enhance stability of the stored form of these hormones, which is exactly what we found.

Human H27A-PRL, which has a greatly reduced ability to bind Zn2+ (6), folds correctly, since it is transported through the secretory pathway and is recognized in a RIA. It is also transported in a regulated pathway where release is stimulated by depolarization. The major effect of the H27A mutation appears to be on the stability of PRL; in the pulse chase experiments, some of the newly synthesized H27A-PRL was degraded, and there was usually a delay in the secretion of newly synthesized hormone. The conditions of the pulse chase experiments with medium changes and the resulting fluctuations in factors, such as pH and temperature, may affect stability of the mutant protein more markedly than wild type. The instability of the mutant protein, induced by the experimental conditions, may interfere with transport through the secretory pathway.

Although the instability of the H27A mutant in cells was predicted, we did not expect the expression of endogenous rat PRL would be reduced in the clones. Such a finding may be analogous to certain cases of GH deficiency in which the presence of a mutant GH gene suppresses secretion of the product of the normal gene (13, 14, 15). A mutation in vasopressin that causes it to be dominant negative for secretion has also been described (16). The mechanisms by which secretion of a normal protein is reduced by a mutant protein are not known, although direct interactions between normal and mutant proteins during folding so that they are not correctly transported have been suggested. Results with H27A-PRL demonstrate that, at least in this case, the cause is more complex because the production of PRL mRNA is reduced as well. In addition, when we examined the production of human wild type and H27A-PRL in cells that had not undergone selection procedures, we found no marked differences between the two; therefore, gross misfolding and aggregation of H27A-PRL is unlikely to be a problem.

We found no evidence for the PRL receptor in GH4C1 cells by PCR techniques, which indicates there is not a possibility for feedback mediated through the receptor. We had previously proposed that the PRL receptor might play a direct role in causing PRL concentration into dense core granules, as an explanation for the effects of mutations in human PRL on rat PRL storage (7); our inability to detect the PRL receptor eliminates this mechanism and raises the possibilities of indirect mechanisms, such as selection or feedback, for the effects on storage as well.

Communication from the secretory pathway to the nucleus is known to occur through several different mechanisms. Unfolded proteins in the endoplasmic reticulum activate a transmembrane kinase that results in increased transcription of chaperones, such as GRP78; this pathway has been extensively investigated in yeast (10, 17, 18, 19, 20). Unfolded proteins in the endoplasmic reticulum also activate an interferon-inducible, double-stranded RNA-dependent protein kinase and the transcription factor NF-{kappa}B, processes that appear to involve Ca2+ (21, 22, 23). The response to unfolded proteins is complex and involves several pathways; although we did not find specific induction of GRP78, other responses may have occurred. We found no evidence for massive misfolding of H27A-PRL, such as disulfide-bonded aggregates, but the instability that we detected may cause stress for the cells. Because the mutant is not completely stable in cells, it may require more use of chaperones and consumption of ATP to fold and maintain correct conformations as it traverses the secretory pathway. Such increased activity may result in a signal to the nucleus that directly regulates synthesis of endogenous PRL or that indirectly results in selection of clones that produce less rat PRL.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of Stable Transfectants
The cDNA for human PRL was in pcDNA-NEO (7). Histidine 27 in human PRL was mutated to alanine by PCR, using the primer extension technique of Ho et al. (24), and for primers, SP6, T7, 5'-TCCGCCTACATCCATAACCTCTCC-3', and 5'-GTTATGGATGTAGGCGGACAGGAC-3'. Lysine 69 was mutated in the same way, using SP6, T7, 5'-CCCGAAGACGCGGAGCAAGCC-3' and 5'-TTGGGCTTGCTCCGCGTG-3'. Synthesis of primers and complete sequencing of all constructions to confirm they were correct was done by the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University.

All transfectants were isolated in the same way. Cells were transfected by electroporation, and stable clones were obtained by growth in 0.5 mg/ml G418. Visible colonies appeared between 2 and 3 weeks. The medium from colonies was screened for human PRL, and those that had accumulated the most hormone were selected and expanded. In the transfection with the empty vector, the colonies were randomly selected. Assays for PRL production, expressed as amount per cell number, were begun soon after the clones were expanded. Cells were subcultured once a week, and assays for rat and human PRL were performed within early passages for all clones.

For induction of PRL storage, cells were incubated in medium containing 15% gelding serum alone or plus 1 nM estradiol, 300 nM insulin, 5 nM epidermal growth factor, as previously described (25).

Pulse Chase Procedure
Cells were treated with 1 nM estradiol, 300 nM insulin, and 5 nM epidermal growth factor for 7 days. Labeling was performed in Ham’s F10 nutrient mixture with 0.1% horse serum, 1 mM NaHC03, and 20 mM HEPES, pH 7.5. After rinsing, cells were incubated with 400 µC Expre35S35S Protein Labeling Mix, (Dupont New England Nuclear, Boston, MA) for 4–10 min and then in medium with 1.5 mM methionine and cysteine for additional time. Cells were lysed in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EDTA plus 2.5 µg/ml leupeptin, 2.5 µg/ml pepstatin, 1 µg/ml aprotinin, 1 mM benzamidine, 0.4 mM phenylmethylsulfonylfluoride (PMSF), 0.5% SDS, and 0.25% BSA. Medium and lysate were incubated with antiserum for 18 h at 4 C. For most experiments, we used rabbit anti-rat PRL from Arnel (New York, NY) at a concentration (1:100) that was sufficient to precipitate both rat and human PRL. Where indicated, we used anti-human PRL from NIH (PRL-IC-5) at a concentration, 1:600, that precipitated all the human PRL in the culture, but not all the rat PRL. At these concentrations, the antisera recognize denatured PRL. The immunoprecipitates were collected with Protein A-Sepharose beads (1.875 mg/sample; Pharmacia, Piscataway, NJ) and washed three times with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP40, 0.25% gelatin, 1 mM EDTA and three times with 10 mM Tris-HCl, pH 7.5, and 0.1% NP40 for 30 min per wash. PRL was eluted from the beads by boiling for 5 min in 50 mM Tris, pH 7.0, 4% glycerol, 2% SDS, and 10% ß-mercaptoethanol, followed by 60 C for 15 min. PRLs were resolved by SDS gel electrophoresis (7). Human PRL migrates more slowly than rat PRL (7). Rat and human PRL produced by the clones were also distinguished in preliminary experiments by the ability to be precipitated by diluted concentrations of their respective antisera, as well as the RF values (the distance migrated relative to the front).

Immunoblots
Cells were treated as described above, the cells collected, an aliquot removed for counting, and the rest centrifuged and dissolved in loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.02% bromophenol blue, 20% glycerol, 10% ß-mercaptoethanol, and 200 mM dithiothreitol), and heated at 100 C for 5 min, followed by 60 C for 15 min. Samples from equal numbers of cells were run on 12.5% polyacrylamide gels at 120 V. Proteins were transferred from gels to Immobilon Membranes (Millipore, Bedford, MA) at 300 mA for 1.5 h. Membranes were incubated with antibody overnight, followed by incubation with rabbit antimouse immunoglobulin when monoclonal antibodies were used, and finally with 10 µCi [125I]Protein A (specific activity, 8.5 µCi/µg, Dupont New England Nuclear) for 1 h. Antisera were from Affinity Bioreagents (Neshanic Station, NJ) for proteins other than PRL. Antisera for human PRL was that for immunocytochemistry from NIH. A Molecular Imager system (Bio-Rad, Hercules, CA) was used to detect and quantitate the bound radioactivity.

Perifusion
Cells cultured on Cytodex II beads (Pharmacia) were perifused as previously described (25).

RIA
Rat and human PRL were detected by RIA. Reference preparations NIDDK-pPRL-RP.1 and NIDDK-rPRL RP-3 were used for standards and were provided by the National Hormone and Pituitary Program, the National Institutes of Diabetes and Digestive and Kidney Diseases, the National Institutes of Child Health and Human Development, and the US Department of Agriculture. Rat and human [125I]PRL were from Dupont NEN. Human H27A-PRL does not completely displace human [125I]PRL in the assay (6); the samples were measured in a range where displacement occurs, and the standard curves were parallel. Each assay was specific for rat or human PRL and did not recognize the other species.

Northern Analysis
Total RNA was prepared by an acid-guanidinium thiocyanate-phenol-chloroform technique (26), subjected to electrophoresis, and transferred to nitrocellulose. RNA was hybridized to a rat PRL probe from Dr. Richard Maurer (University of Oregon), labeled with 32P by random priming.

RT-PCR
Cells were disrupted in 6 ml 4 M guanidinium isothiocyanate, 25 mM sodium citrate, pH 6.5, 0.5% sarcosyl, 1.1 M ß-mercaptoethanol and passed three times through a 25-gauge needle. The homogenate was then layered onto 4 ml 5.7 M CsCl, 25 mM sodium acetate, pH 6.0. Solutions were centrifuged at 175,000 x g for 21 h at 22 C. The supernatants were aspirated and the RNA pellets were washed once with 50% ethanol and then resuspended in H2O. Total RNA was added to 1.25 µg oligo(dT)12-18primer (GIBCO, Grand Island, NY), 0.5 mM deoxynucleotide triphosphates, 10 U of rRNasin (Promega, Madison, WI), 500 U of SuperScript II RNAse H- RT (GIBCO), 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT in a total reaction volume of 50 µl and incubated at 42 C for 15 min. The RNA template was digested with 2.7 U of ribonuclease H (GIBCO) at 37 C for 15 min. Five microliters of reverse transcription reaction were added to a total volume of 100 µl containing 20 mM Tris-HCl, pH 8.4, 50 mM KCL, 1.5 mM MgCl2, 0.5 µM of each primer, and 2.5 U of Taq DNA polyerase (GIBCO). PCR was performed for 30 cycles with a denaturing temperature of 94 C for 1 min, an annealing temperature of 65 C for 2 min, and an elongation temperature of 72 C for 2 min. The forward primer for both the long and short form of the receptor was 5'-CATGGATACTGGACTAGATGGAGC-3'. The reverse primer for the long form was 5'-CTCAGCAGCTATTCAGACTTG-3', to give a 250-bp fragment. The reverse primer for the short form was 5'-TCCTATTTGAGTCTGCAGCTTCAGTAGTCA-3', to give a 335-bp fragment. The plasmid containing sequences for the PRL receptor used as a control was a gift from Lee Yuan Yu-Lee (27).

Transient Transfections
Hormone-treated cells were transfected with Lipofectamine (GIBCO), using 2.5 x 105 cells, 3.2 µg DNA, 9.2 µl Lipofectamine for 6 h per 60-mm plate, according to the instructions supplied. PRL in the vector used in stable transfectants, pcDNAneo, was not produced well in transient transfections, so for these experiments, PRL was used in pcDNA3 (Invitrogen, San Diego, CA).


    FOOTNOTES
 
Address requests for reprints to: Priscilla S. Dannies, Yale University School of Medicine, 333 Cedar Street, Department of Pharmacology, New Haven, Connecticut 06520-8066.

This work was supported by NIH Grant DK-46807.

Received for publication June 2, 1997. Accepted for publication June 24, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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