Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sorin, B.
Right arrow Articles by Vacher, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sorin, B.
Right arrow Articles by Vacher, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 273, Issue 43, 28461-28469, October 23, 1998


Distinct Cytoplasmic Regions of the Prolactin Receptor Are Required for Prolactin-induced Calcium Entry*

Bruno SorinDagger §, Olivier Goupille, Anne M. VacherDagger , Jacqueline Paly, Jean Djiane, and Pierre VacherDagger parallel

From the Dagger  Laboratoire de Neurophysiologie, Centre National de la Recherche Scientifique UMR 5543, Université de Bordeaux 2, 33076 Bordeaux Cédex, France and the  Unité d'Endocrinologie Moléculaire, Institut National de la Recherche Agronomique, 78352 Jouy en Josas Cédex, France

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Two cytoplasmic regions of the prolactin (PRL) receptor are well documented for their participation in PRL signal transduction, the membrane proximal box 1 and the COOH-terminal region. In order to study the role of these regions in PRL-induced Ca2+ increase, we use Chinese hamster ovary (CHO) cells stably transfected with mutated PRL receptor cDNA. These cells express the long form of PRL receptor deleted from box 1 (CHO Delta 1 cells) or the 141 amino acids of the COOH-terminal region (CHO H3 cells). The patch-clamp technique in "whole-cell" configuration and microfluorimetric techniques were used singly or in combination. Data obtained for these cells were compared with those we have recently published using CHO cells expressing the wild-type long form of the PRL receptor (CHO TSE32). In contrast to CHO TSE32 cells, exposure of CHO Delta 1 or H3 cells to PRL (0.05-50 nM) did not modify [Ca2+]i. We have previously shown that the PRL-induced calcium influx via voltage-insensitive, Ca2+ channels was due to the activation of tyrosine kinase-dependent K+ channels that hyperpolarize the CHO TSE32 cell membrane (hyperpolarization-driven Ca2+ influx). Therefore, two events are involved in PRL-induced Ca2+ changes (i) JAK2-activation of K+ channels and (ii) intracellular messenger-opening of Ca2+ channels. In CHO Delta 1 cells, PRL (0.05-50 nM) neither hyperpolarized the membrane potential nor stimulated the JAK2-dependent K+ current, confirming the pivotal role played by box 1/JAK2 in the PRL-induced activation of K+ channels. However, when these cells were voltage-clamped below the resting membrane potential, application of 5 nM PRL resulted in an increase in Ca2+ influx. Therefore, box 1/JAK2 was not involved in the opening of these Ca2+ channels. In CHO H3 cells, 5 nM PRL activated the K+ current and hyperpolarized the membrane potential without any effect on [Ca2+]i. Moreover, PRL was also ineffective on CHO H3 cells voltage-clamped below the resting membrane potential. Therefore, the COOH-terminal region is involved in the production of the intracellular messenger that opens voltage-independent Ca2+ channels.

We conclude from these findings that box 1 and COOH-terminal regions are both needed for PRL-induced Ca2+ changes.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The peptide hormone, prolactin (PRL),1 exerts pleiotropic biological effects in a wide variety of cells and tissues via membrane receptors (1). PRL receptors (PRL-R) belong genetically to the larger family of receptors known as the hematopoietic-cytokine superfamily (2). These receptors present common structural features in the extracellular domain, in particular two cysteine pairs and ws/ws box. In the cytoplasmic domain, a proline-rich motif (box 1) located in the membrane proximal region is common to several receptors in this superfamily (3). The intracellular domain of receptors in this family lacks intrinsic kinase activity and varies in both length and sequence. The short form of PRL-R (57 or 30-50 amino acids) was at first only found in rat and mouse cells. However, recent reports presented some evidence for the expression of short and long forms of PRL-R in other species (4). While the physiological functions of the short form remain controversial, the long form has been found to be capable of activating transcription of genes involved in cell differentiation (5).

Recent studies have been marked by considerable progress in understanding the intracellular signaling mechanisms for the different members of this receptor superfamily. These receptors associate with and activate several cytoplasmic tyrosine kinases in the Janus tyrosine kinase family. It has been shown for PRL-R that box 1 was required for JAK2 association with this receptor and phosphorylation (6) but that it was not sufficient for signal transduction (7). Deletion of the carboxyl-terminal 141 amino acids results in partial loss of transcriptional signaling activity (8). In the same way, a recent report indicated that the carboxyl-terminal portion of the growth hormone receptor is also required to activate transcription (9). Proteins, such as signal transducers and activators of transcription (STAT), are assumed to associate, through their SH2 domains, with COOH-terminal phosphotyrosines of activated receptors, where they become phosphorylated and activated (10).

To study early events in PRL signal transduction, we have developed a CHO cell line (CHO TSE32) stably transfected with the cDNA of the long form of rabbit (rb) mammary PRL-R (11). These CHO-transfected cells respond to PRL by stimulating the co-transfected milk protein gene promoter (12), proving that such cells are fully capable of transmitting the PRL signal and that PRL-R is functional. Using this cell line, we demonstrated that exposure of cells to physiological concentrations of PRL (5 nM) resulted in an increase in the cytosolic free calcium concentration ([Ca2+]i) (13) by stimulating both Ca2+ entry and mobilization from intracellular Ca2+ stores. Electrophysiological techniques were used to improve characterization of the early effects of PRL on membrane ion conductances. We have recently shown that PRL-induced Ca2+ entry was due to JAK2-dependent stimulation of calcium and voltage-activated potassium channels (14, 15). The resulting hyperpolarization stimulates Ca2+ entry through voltage-insensitive Ca2+ channels (14), which require PRL-induced production of a cytosolic messenger to be opened.

In this article, we report on the structure-function relationships of PRL-R: using microfluorimetry and electrophysiology on CHO cells expressing mutated PRL-R, we confirm that box 1 is required for activation of the Ca2+- and voltage-dependent K+ conductance and demonstrate, for the first time, that the COOH-terminal region of PRL-R is necessary for Ca2+ entry.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cell Cultures-- The CHO cells (TSE32, H3, Delta 1) were grown in Ham's F-12 medium (Seromed, Stasbourg, France) containing 10% (v/v) fetal calf serum (Life Technologies, Inc.). The medium was changed every 2-3 days. Cells were maintained at 37 °C in a humidified atmosphere gassed with 95% air, 5% CO2. In order to avoid occupancy of PRL receptors by lactogenic factors contained in the culture medium serum, cells were transferred into a serum-free medium 6-24 h before the experiments. This medium was derived from the GC3 medium described by Gasser et al. (16) and is a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 (seromed) supplemented with nonessential amino acids (Life Technologies, Inc.), insulin (Sigma; 80 milliunits/ml), glutamine (Sigma; 2.5 nM), and transferrin (Life Technologies, Inc.; 10 µg/ml).

oPRL Binding on Cell Membrane and Western Blot Analysis-- "Whole cell" binding and Scatchard analysis were performed essentially as described previously (8). Purification of the rbPRL-R complexes, immunoprecipitation, and Western blot Analysis were previously described (8).

Electrophysiological Recordings-- The cultures were viewed under phase contrast with a "Leitz-Diavert" (Leitz, Germany) inverted microscope. Electrodes were positioned with "Leitz" (Germany) micromanipulators. Grounding was through a silver chloride-coated silver wire inserted into an agar bridge (4% agar in electrode solution). An Axopatch-1D amplifier (Axon Instruments, Inc., Foster City, Ca) was used for whole cell recordings. Stimulus control and data acquisition and processing were carried out with a PC computer AT-80386 (Tandon, Moorpark, Ca), fitted with a Labmaster TL-1 interface, using Pclamp 5.5.1 software (Axon Instruments Inc., interface and software). Electrode offset was balanced before forming a "giga seal." Leakage and capacitive current subtraction protocols were composed of four or five hyperpolarizing pulses, one-fourth or one-fifth pulse, respectively, and were applied from a holding potential before test pulses eliciting active responses. During data analysis, leak data were subtracted from the raw data. Series resistance were compensated and calculated before and after compensation. Recordings where series resistance resulted in a 5 mV or greater error in voltage commands were discarded. Currents were low pass-filtered at 2 KHz with an eight-pole Bessel filter (-3dB) and digitized at 10 KHz for storage and analysis.

Spectrofluorimetric Assay of Cytosolic Calcium-- These experiments were performed using the fluorescent probe indo-1, as already described (13). The cells were incubated with 5 µM indopentaacetoxymethyl ester (indo-1/AM) and 0.02% Pluronic F127 (Molecular Probes, Eugene, OR) in Hank's solution for 30 min at 37 ± 1 °C, then washed and maintained at room temperature in the same saline solution before the fluorescence measurements. This procedure resulted in an intracellular indo-1 concentration of between 20 and 60 µM, estimated from comparison with the loading via a patch pipette.

For single cell measurements the dual emission microspectrofluorimeter was constructed from a Nikon Diaphot inverted microscope (Nikon France, Paris, France) fitted with epifluorescence (× 100 oil immersion fluorescence objective; numerical aperture, 1.3). For excitation of indo-1, a collimated light beam from a 100-watt mercury arc lamp (Nikon) was filtered at 355 nm and reflected from a dichroic mirror (380 nm). The emitted fluorescence signal was passed through a pinhole diaphragm slightly larger than the selected cell and directed onto another dichroic mirror (455 nm). Transmitted light was filtered at 480 nm, reflected light was filtered at 405 nm, and the intensities were recorded by separate photometers (P1, Nikon). Single photon currents were converted to voltage signals, divided on-line by a monolithic laser-trimmed two-quadrant divider (AD535, Analog Devices, Norwood, MA). Under these experimental conditions, the r = F405/F480 ratio was recorded on-line as a voltage signal and was expressed as [Ca2+]i using the formula derived by Grinkiewicz et al. (17). Ca2+ calibrations were obtained under simultaneous whole cell clamp and microspectrofluorimetric measurements. The patch pipettes were filled with internal solution containing 10 mM EGTA (solution A), 10 mM CaCl2 (solution B), or 9.2 mM EGTA and 5.4 mM CaCl2 (solution C). Solutions A and B were used to estimate minimum and maximum values, Rmin and Rmax, respectively. Solution C was used to evaluate the product of the apparent dissociation constant (Kd) and the ratio of fluorescence of free indo-1 divided by the fluorescence of Ca2+-bound indo-1 with 355 nm excitation and 480 nm emission (beta ). The latter solution had a free Ca2+ of 300 nM, calculated using the stability constants and computer program of Fabiato and Fabiato (18). Rmin, Rmax, and Kdxbeta averaged 0.039 ± 0.01 (n = 15), 0.65 ± 0.08 (n = 17), and 581 ± 22 nM (n = 15), respectively.

In other experiments [Ca2+]i was measured on cell populations using a Hitachi F2000 spectrofluorometer. The glass coverslide carrying the cells was positioned on a plastic holder in a quartz cuvette. The indo-1 fluorescence response to the intracellular calcium concentration was calibrated from the ratio of 405/480 nm fluorescence values after subtraction of the background fluorescence of the cells at 405 and 480 nm as described by Grinkiewicz et al. (17). The dissociation constant for the indo-1·Ca2+ complex was taken as 405 nM. The Rmax and Rmin values were calculated from measurements using 25 µM digitonine and 5 mM EGTA.

Simultaneous Electrophysiological and Microfluorimetric Recordings-- These experiments were performed using the fluorescent Ca2+ probe indo-1. The cells were loaded with indo-1 pentasodium salt (30 µM) via the patch pipette. Indo-1 salt diffused gradually through the patch pipette to the recorded cell. [Ca2+]i was determined as described above.

Recording Solutions-- For patch-clamp, spectrofluorimetry, and combined studies the standard extracellular solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 0.3 Na2HPO4, 0.4 KH2PO4, 4 NaHCO3, 5 glucose, 10 HEPES. The osmolality of the external salt solution was adjusted to 300-310 mosm/kg with sucrose, and pH adjusted to 7.3 ± 0.01 with NaOH. In some experiments, ion channel inhibitors were added to the bathing solution: (i) tetrodotoxin (TTX, 1-5 µM) to prevent activation of the fast sodium current, or (ii) cobalt (5 mM) or nickel (5 mM) to prevent activation of calcium currents. For whole cell studies the recording pipette was filled with an artificial intracellular saline containing (in mM): 150 potassium gluconate, 2 MgCl2, 1.1 EGTA, 5 HEPES (pH 7.3 ± 0.01 with KOH), osmolality 290 mosm/kg. In all electrophysiological studies 3 µM ATP was added to the internal solution.

In single cell experiments, a "pouring" pipette with a tip opening of 10-20 µm was used for local drug application to the investigated cell. This pipette was filled with the same extracellular saline as that used in the bath and the drug under investigation was added to it in appropriate concentrations. The pipette was brought close to the investigated cell at a distance of 50-100 µm. In cell population experiments the drugs and reagents were added directly to the cuvette under continuous stirring. All experiments were performed at room temperature (20-22 °C).

Chemicals-- PRL (oPRL-19) was kindly provided by Dr. A. F. Parlow (National Hormone and Pituitary Program, University of Maryland School of Medicine, Baltimore, MD). CTX, MgATP, TTX, indo-1-AM were from Sigma, charybdotoxin (CTX), and iberiotoxin were obtained from Latoxan (Rosans, France). Antibody anti-JAK2 and JAK2 immunizing peptide were purchased from Upstate Biotechnology, Inc. Normal rabbit serum was from Sera Lab (London, United Kingdom).

Data and Statistical Analysis-- Peak currents in whole cell recordings were measured using the automatic peak detection function in the clampan section of the pclamp software. Late currents were measured isochronally before the end of the pulse. Results are expressed as mean ± S.D. where appropriate. Each experiment was repeated several times. Student's t test was used for statistical comparison among means and differences with p < 0.05 were considered significant.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Characterization of rbPRL-R Deletion Mutants Expressed in CHO Stable Transfectants-- To analyze the functional domains of the rbPRL-R involved in prolactin-induced calcium entry, we designated two mutated forms of the rbPRL-R (Fig. 1A). One form is a carboxyl-terminal deleted mutant lacking the last 141 residues (H3/T451). The other form is an internal deletion mutant lacking the proline-rich homology region called box 1 (Delta 1/Delta 245-267). The corresponding cDNA were inserted in pECE expression vector (19). CHO cells were stably transfected with wild-type rbPRL-R (WT, CHO TSE32) and mutated forms, H3 and Delta 1. Binding studies (Fig. 1B) indicate that mutant receptor forms (H3 and Delta 1) have higher binding capacities for oPRL than the wild-type receptor. Scatchard analysis indicate that mutant receptor forms have similar binding affinity to wild-type receptor but are more expressed at the cell surface. The size of the different receptor forms and their expression were then determined in CHO stable transfectants. Solubilized proteins were immunoprecipitated by anti-receptor polyclonal antibody developed against recombinant rbPRL-R extracellular domain expressed in Escherichia coli, alpha 102, and the blot was revealed with a second polyclonal antibody (alpha 46). As shown in Fig. 1C, the different CHO stable clones express receptor proteins of expected molecular sizes for the WT (~100 kDa), Delta 1 (~95 kDa), and H3 (~80 kDa). We confirmed that more receptor proteins are expressed for the two receptor mutants than for WT receptor form.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 1.   Characterization of wild-type (WT/TSE32) and deletion mutants (Delta 1/Delta 245-267; H3/T451) expressed in stably transfected CHO cells. A, schematic representation of rabbit WT and cytoplasmic deletion mutants of PRL-R; numbers indicate position of amino acids residues in the sequence; box 1 (first homology domain with growth hormone receptor) are indicated as hatched rectangles; the transmembrane domain as a solid box; tyrosine residues in the cytoplasmic domain are indicated by Y. B, binding properties of rbPRL-R WT and mutants expressed in CHO cells. The affinity constant (Ka) and the number of receptors (binding sites) were calculated from the Scatchard analysis of competition experiments using CHO stable transfectants as described under "Experimental Procedures." C, cell lysates of CHO cells expressing WT and mutated rbPRL-R were immunoprecipitated with the anti-rbPRL-R 102 and analyzed by immunoblotting with the anti-rbPRL-R antibody 46. The migration positions of molecular mass standards (in kDa) are indicated on the left. The arrows indicate the migration of the different rbPRL-Rs.

Comparison of Passive Membrane Properties, Voltage-dependent Conductances, and [Ca2+]i in CHO H3 and Delta 1 Cells-- The mean input resistance measured with constant current hyperpolarizing pulses was 1.22 ± 0.41 GOmega (n = 28) in CHO TSE32 cells, 1.17 ± 0.39 GOmega (n = 35) in CHO Delta 1 cells, and 1.16 ± 0.36 GOmega (n = 31) in CHO H3 cells. Immediately after establishment of whole cell recording, the mean-resting membrane potential of CHO TSE32, Delta 1 and H3 cells was -31.2 ± 7.2 mV (n = 28), -33.4 ± 7.5 mV (n = 35), and -32.8 ± 9.0 mV (n = 28), respectively. These values did not differ significantly from one cell model to another. We have shown that three types of voltage-activated ion conductances are present in most CHO K1 native cells and CHO TSE32 cells (14, 20): (i) a large Ca2+- and voltage-activated K+ conductance ("maxi-K+" channel), (ii) a TTX-sensitive Na+ conductance, and (iii) a Ca2+ conductance, similar to the L-type conductance of excitable cells. The expression of these conductances varied from cell to cell in CHO K1, CHO TSE32, CHO Delta 1, and CHO H3 cells, but, qualitatively, the voltage-dependent ion conductances were the same. The values of intracellular calcium in CHO TSE32, CHO Delta 1, and CHO H3 cells measured by spectrofluorimetry using the fluorescent Ca2+ probe indo-1 were also very close and exhibited stable resting values (CHO TSE32: 147 ± 15 nM, n = 48; CHO Delta 1: 149 ± 19 nM, n = 47; CHO H3: 150 ± 18 nM, n = 97). Therefore transfection of mutated PRL-receptor cDNA did not affect passive membrane properties, voltage-dependent membrane ion conductances, or intracellular Ca2+ levels.

Effects of PRL on [Ca2+]i in CHO TSE32, CHO Delta 1, and CHO H3 Cells-- We have recently used "single cell" microfluorimetry to show that physiological concentrations of PRL stimulate Ca2+ entry and/or induce a mobilization of calcium ions stored in intracellular compartments in CHO TSE32 cells (13). In this paper, we confirm this result on cell populations. Fig. 2A shows the PRL-induced Ca2+ increase in CHO TSE32. Continuous perfusion of 5 nM PRL resulted in an increase in [Ca2+]i (amplitude: 263 ± 14 nM, n = 36). Under this experimental procedure, Ca2+ returned very slowly to basal value. When PRL was removed from the cuvette, Ca2+ returned more rapidly to basal value (about 200 s).


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 2.   Time course of changes in [Ca2+]i in CHO cells in response to stimulation with 5 nM PRL. [Ca2+]i was recorded from cell populations loaded with indo-1. Experiments were conducted on CHO cells expressing the native rabbit long form of the PRL receptor (A, CHO TSE32), and two mutants: the long form where amino acids 245-267 had been deleted (B, CHO Delta 1), and the long form truncated to remove the last 141 amino acids (C, CHO H3). The examples shown are representative for 36, 34, and 32 recordings under identical conditions.

By contrast, application of the same concentration of PRL (5 nM) did not affect [Ca2+]i in CHO Delta 1 (Fig. 2B) or in CHO H3 cells (Fig. 2C). On the contrary, PRL slightly decreased the [Ca2+]i in both cell lines (CHO Delta 1: -19 ± 5 nM, n = 34; CHO H3: -22 ± 6 nM, n = 32). This effect persisted as long as PRL was present in the bath. On termination of the PRL application, [Ca2+]i slowly returned to baseline levels (within 200 s).

As we have observed several types of response to PRL (see Ref. 13 and Fig. 3A) in individual CHO TSE32 cells, we also studied the effects of PRL on [Ca2+]i in CHO Delta 1 and H3 cells using microspectrofluorimetry on single cells. As shown in Fig. 3, B and C, 5 nM PRL induced a slight decrease in [Ca2+]i in about 50% of the cells from both cell lines. The amplitude of this PRL-induced [Ca2+]i decrease averaged 20 nM, ranging from 5 to 35 nM in both cell lines. In the other 50% of the cells 5 nM PRL had no significant effect on [Ca2+]i. Other concentrations of PRL were tested. Fifty nanomolar ovine PRL decreased [Ca2+]i in 2 out of 9 CHO Delta 1 cells (-20 ± 7 nM) and 2 out of 10 CHO H3 cells (-17 ± 3 nM). A 100-fold lower concentration of PRL (0.5 nM) was as efficient as 5 nM (20 out of 31 CHO Delta 1 cells, -22 ± 7 nM; 12 out of 20 CHO H3 cells, -24 ± 6 nM). A lower concentration (0.05 nM) had more attenuated effects (2 out of 15 CHO Delta 1 cells, -17 ± 5 nM; 3 out of 10 CHO H3 cells, -20 ± 5 nM). 0.005 nM PRL was ineffective on [Ca2+]i in both cell lines (n = 7). Therefore, although a systematic dose-response study was not carried out, PRL did not increase [Ca2+]i in CHO Delta 1 and CHO H3, whatever the concentration used.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 3.   Effects of brief applications of PRL on [Ca2+]i in mutated CHO cells. [Ca2+]i was measured in individual CHO TSE32 (A), CHO Delta 1 (B), and H3 (C) cells loaded with indo-1. PRL (5 nM) was applied continuously during the period covered by bars. Examples shown are representative of the response to PRL (A, 69 out of 103 cells; B, 17 out of 33 cells tested; C, 24 out of 47 cells tested). In CHO TSE32 cells (A) PRL responses differed in their kinetics (slow response, a; fast response, b).

Effects of PRL on Voltage-activated, Ca2+-dependent K+ Conductance of CHO Delta 1 and H3 Cells-- To investigate the physiological action of PRL on K+ conductance, CHO cells were voltage-clamped at -40 mV, close to the mean resting membrane potential. Contamination of K+ current recordings with Na+ was avoided by the use of TTX (2 µM) containing external solution.

Application of PRL (5 nM) to CHO Delta 1 cells had no effect on the amplitude or kinetics of the K+ current (I-V relationships and time course) in the majority of recorded cells (9 of 15 cells). In the other cells, PRL depressed the amplitude of the steady-state K+ outward current (for a voltage step from -40 to +60 mV, control: +72.5 ± 5.7 pA, t0: +45.6 ± 4.8 pA, t0 + 200: 52.5 ± 5.1 pA, n = 6). Fig. 4A presents characteristic I-V relationships before (control), during (t0), and 200 s after (t0 + 200) 5 nM PRL application, Fig. 4B is a representative time course of the response. PRL-induced decrease was very slow. Maximal effect was obtained 200-300 s after the end of ejection. This effect was partially (~50%) reversible within 6-12 min after termination of the drug application (n = 6).


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 4.   Effects of PRL on K+ current in CHO Delta 1 (A and B) and H3 (C and D) cells. A and C, current-potential relationships for both cell types: top, example of an effect of 5 nM PRL on K+ current recorded before (control, bullet ), during (t0, black-triangle), and 200 s after the end of hormone application (t0 + 200, ). K+ current was evoked by 100-ms pulses from holding potential of -40 mV to +20 mV test potential. Lower panel, I-V relationships for control (bullet ), t0 (black-triangle), t0 + 200 (). Symbols represent K+ current amplitudes measured at the end of step depolarization. The examples shown are representative for 6 (CHO Delta 1) and 7 (CHO H3) cells. In the other cells (CHO Delta 1, 9; CHO H3, 5) PRL had no significant effect. B and D, time courses of K+ carried stimulation by PRL in CHO Delta 1 (B) and H3 (D) cells. Top, illustrates an example of the effects of PRL on K+ current. Lower panel, plot of K+ current versus time in the presence or absence of 5 nM PRL and 50 nM iberiotoxin (B) or 50 nM CTX (D), two calcium-dependent K+ channel inhibitors. Symbols represent K+ current amplitudes measured by 100 ms pulses at the end of a step depolarization to +20 mV from Vh of -40 mV.

On the other hand, PRL (5 nM) considerably increased the amplitude of the steady-state K+ current in 7 of 12 CHO H3 cells. In these cells, the mean amplitude of the K+ current (for a step from -40 to +60 mV) was not significantly modified during hormone application (t0), whereas it was markedly enhanced from 28 ± 4.6 pA under control conditions to 115 ± 23 pA 200 s after the end of the hormone application (t0 + 200) (Fig. 4C). This PRL-induced increase was very slow (time to peak 169 ± 56 s) with an incomplete return to basal level after 8-12 min (Fig. 4D). These results for CHO H3 cells are similar to those previously reported for CHO TSE32 cells (14). In both cases the outward current was characterized by application of calcium-dependent K+ channel inhibitors (50 nM charybdotoxin or 50 nM iberiotoxin) at the end of the recording (Fig. 4, B and D).

Effects of PRL on Membrane Potential of CHO Delta 1 and H3 Cells-- Local application of PRL (5 nM) to 6 of 10 CHO Delta 1 cells current clamped at resting potential caused a prolonged depolarization (15-25 mV) associated with a 150% increase in input resistance, as can be seen in Fig. 5A, a. These responses to PRL were reversible within 3-5 min after the peptide application was terminated. A similar response was obtained when we applied 50 nM CTX to CHO cells, instead of PRL (data not shown) or during PRL-induced depolarization (Fig. 5A, b).


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 5.   Effects of PRL on the membrane potential of CHO Delta 1 (A) and CHO H3 (B) cells. Recording obtained under zero current conditions (current clamp). Hyperpolarizing current traces were periodically injected to monitor membrane resistance. Upper and lower traces display membrane potential and current, respectively. Dotted lines show the zero membrane potential. 5 nM PRL was applied within a few minutes after the establishment of whole cell recording. 50 nM Charybdotoxin was added to block K+ channels (b).

When the same concentration of PRL was applied to CHO H3 cells, 5 out of 9 cells that hyperpolarized (10 to 20 mV) in response to the peptide remained so for a prolonged period during which the membrane conductance was markedly increased (Fig. 5B, a). These effects took considerably longer to reverse in these cells. Application of CTX (50 nM) blocked PRL-induced membrane hyperpolarization (Fig. 5B, b).

Simultaneous Monitoring of [Ca2+]i, Membrane Potential, and Current in CHO Delta 1 and H3 Cells Stimulated with 5 nM PRL-- As demonstrated by our previous studies (13, 14) PRL stimulates Ca2+ entry through voltage-insensitive nonspecific channels by hyperpolarizing the membrane potential of CHO cells expressing the long form of PRL receptor. To further investigate the action mechanism of PRL in CHO cells expressing the mutated receptors, the relation between intracellular calcium concentration changes and calcium influx via voltage-independent Ca2+ channels was systematically investigated, using the patch-clamp technique combined with dual emission [Ca2+]i recordings obtained with indo-1. These experiments were conducted under voltage-clamp conditions, including inhibitors of both Na+ (tetrodotoxin, extracellularly applied) and K+ channels (NMG gluconate in the recording pipette) to isolate Ca2+ currents for monitoring. Two approaches were used (i) PRL was applied to the recorded cell after the patch of membrane was disrupted and control recordings were made (Figs. 6A and 7A) and (ii) PRL was applied a few minutes before the establishment of whole cell recordings (Figs. 6B and 7B), therefore without control recordings on the same cell. These experiments made it possible to vary the holding potential in one cell and follow the subsequent changes in [Ca2+]i.


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 6.   Simultaneous recording of membrane potential, ionic currents, and cytosolic Ca2+ in single CHO Delta 1 cells. Combined recordings of membrane potential, ionic currents, and [Ca2+]i were obtained with a combination of microfluorimetry and patch-clamp recording techniques in the whole cell configuration under conditions that block Na+ and K+ currents as described under "Experimental Procedures." Dotted lines indicate the minimal and maximal [Ca2+]i values. A, under voltage-clamp conditions (Vh of -40 mV), 5 nM PRL-induced increase in [Ca2+]i was associated with activation of an inward current and an increase in membrane conductance. Following hormone application, membrane potential was stepped down from -40, -60, and -80 mV, until [Ca2+]i reached a plateau. Finally, membrane potential was depolarized to 0 mV. B, cells were pretreated with 5 nM PRL before the establishment of whole cell recording and variations in the holding potential were performed shortly after this in order to avoid or to minimize the effects of prolonged dialysis of whole cell recording. Under these conditions, hyperpolarization-driven calcium entry was greater than in A, suggesting the involvement of an intracellular second messenger in this mechanism. m.p., membrane potential; Im, whole cell membrane current. Traces are representative of 20 experiments.


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 7.   Simultaneous recording of membrane potential, ionic currents, and cytosolic Ca2+ in single CHO H3 cells. Data were obtained as in Fig. 6. A, under voltage clamp conditions (Vh = -80 mV) 5 nM PRL induced a slight decrease in [Ca2+]i associated with a small outward current and a slight decrease in membrane conductance. B, pretreatment of the cells with 5 nM PRL before the establishment of whole cell recording and variations in the holding potential did not improve the PRL response. m.p., membrane potential; Im, whole cell membrane current. Traces are representative of 17 experiments.

In unstimulated CHO Delta 1 cells, maintenance of the holding potential of -40 mV resulted in a stable current base line and equally a stable [Ca2+]i level of about 150 nM (range: 120-180 nM) (Fig. 6A). Upon shifting the holding potential to -60 or -80 mV, no current or [Ca2+]i changes were triggered in the absence of PRL (data not shown). On the other hand, application of 5 nM PRL to 7 out of 12 CHO Delta 1 cells voltage-clamped at -40 mV induced a long-lasting inward current of modest amplitude (<10 pA, Fig. 6A, middle trace). This current displayed slow activation and very slow, if any, inactivation. It was associated with a slight increase in the conductance amplitude (about 25%) and a distinguishable increase in background noise in the current trace. Concomitantly to the onset of the inward current, a slowly developing [Ca2+]i rise became detectable in the indo-1 recording (Fig. 6A, lower trace). Within 100-120 s after hormone application, a new steady state [Ca2+]i of 210 nM (range, 170-245 nM) was reached (plateau). Successive 20-mV step decreases in holding voltage below -40 mV resulted in gradual stepwise increases in [Ca2+]i (Fig. 6A, lower trace). At -80 mV [Ca2+]i reached a stable maximal value (385 ± 53 nM, n = 5) within 20 s. Upon depolarization to 0 mV, [Ca2+]i gradually returned to the original level within 1 to 2 min.

In PRL-pretreated CHO Delta 1 cells, exploration of membrane potential from 0 to -80 mV stimulated Ca2+ influx in 8 out of 8 cells tested. Hyperpolarization to -40, -60, and then -80 mV led to subsequent Ca2+ rises, increasing with the amplitude of the hyperpolarizing potential, reaching a maximal value (683 ± 89 nM) at -80 mV (Fig. 6B). Depolarization to 0 mV again decreased [Ca2+]i to basal level.

The percentage of responding cells and the response amplitudes were lower in the first experimental approach. This discrepancy may be due to a longer delay between the rupture of the patch membrane and the examination of the PRL effects in this case. Moreover, in the second experimental approach, when the exploration of the membrane potential was performed more than 20 min after establishing the whole cell recording, the hyperpolarization-driven calcium entry was drastically reduced or absent. Taken together, these data confirm the involvement of a cytosolic, diffusible second messenger in the PRL-induced Ca2+ entry (14), and show that this messenger can be produced in response to PRL in CHO Delta 1 cells.

A similar enhancement of steady state [Ca2+]i levels upon PRL application was observed under conditions where neither Na+ nor K+ were blocked (in 4 out of 9 cells tested, data not shown). Under these conditions, obviously no inward current was detectable. There was, however, a small outward current due, at least in part, to the activation of a Ca2+-dependent K+ current as shown in CHO TSE32 cells (14) in parallel with the calcium increase.

Application of 5 nM PRL to CHO H3 cells voltage clamped at 0, -20, -40, -60, or -80 mV (Fig. 7A) never increased [Ca2+]i, nor induced inward current. In some cells (3 out of 8 cells tested) PRL provoked a slight, long-lasting decrease in [Ca2+]i (10-30 nM, 8-10 min) associated with a discrete diminution of the membrane conductance (10-15%) and a more discrete outward current (4.3 ± 1.7 pA).

In PRL-pretreated CHO H3 cells, continuous variation of holding potential from 0 to -80 mV had very little effect on [Ca2+]i at the beginning of the recording (Fig. 7B), comparable to the recordings in unstimulated CHO cells. Higher (50 nM) or lower (0.5 and 0.05 nM) concentrations of PRL had no greater effect on the [Ca2+]i of these cells (data not shown). From these data, it appears that, in CHO H3 cells, PRL did not produced the intracellular messenger which opened the voltage-insensitive Ca2+ channels.

Effects of PRL on Voltage-dependent Inward Currents in CHO Delta 1 and H3 Cells-- We have previously shown that CHO cells possess two voltage-dependent inward currents: (i) a TTX-sensitive Na+ channel and (ii) an L-type Ca2+ channel (20). PRL (5 nM) had no effect on the Na+ channel but slightly inhibited the voltage-dependent Ca2+ current in CHO TSE32 cells (14). When the outward current was completely blocked by replacing intracellular K+ with N-methyl-D-glucamine, and the L-type Ca2+ current by adding nifedipine (0.5 µM), PRL (0.5 to 50 nM) caused no change in the Na+ current in CHO Delta 1 cells or in CHO H3 cells (data not shown), whatever the amplitude of step depolarizations (current-voltage relationships) or the delay between hormone application and current eliciting (time course).

In order to record voltage-activated Ca2+ current, nifedipine was replaced by TTX (2 µM). Repetitive (every 20 s) voltage steps (time course) from -40 to +20 mV elicited Ca2+ currents whose amplitudes were not affected by PRL (5 nM) in the majority of CHO Delta 1 (7 of 12 cells) and CHO H3 (9 of 15 cells) cells (Fig. 8B). In the responding CHO Delta 1 (Fig. 8A) and CHO H3 cells, PRL induced a slight increase in Ca2+ current amplitude (CHO Delta 1, control: -28.8 ± 4.5 pA, PRL, -36 ± 5.7 pA, n = 5; CHO H3, control: -30.8 ± 5.3 pA, PRL, -37.2 ± 4.5 pA). These effects of PRL were observed in both cell types at all potentials where the current was activated (data not shown).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 8.   Effects of PRL on voltage-dependent Ca2+ current in CHO Delta 1 (A) and H3 (B) cells. Top, Ca2+ currents were evoked by 100-ms pulses from holding potential of -40 mV to +20 mV test potential. Lower panel, time course of the voltage-dependent Ca2+ channels in the control and in the presence of 5 nM PRL and 0.5 µM nifedipine, a specific L-type Ca2+ channel inhibitor. The examples shown are representative for 5 CHO Delta 1 and 9 CHO H3 cells.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In our previous research we established that, in CHO cells, transfected with cDNA of the long form of rbPRL-R (CHO TSE32), the primary ionic events in PRL-R signal transduction were the activation of Ca2+-dependent K+ channels and Ca2+ entry (13, 14). More recently, our data have suggested that at least one of the kinases involved in channel stimulation by PRL may be JAK2 tyrosine kinase (15). The mutagenesis of cloned receptors is a powerful tool that can help elucidate the mechanism whereby the ligand binding signal is tranduced and interpreted by a responding cell. So, in these experiments, we used CHO cells expressing wild type and two deletion mutants of the rbPRL-R, to elucidate the structure-function relationships of PRL receptors. Stably transfected CHO cell lines were characterized by PRL binding analysis and the receptor size was confirmed by Western blot analysis.

The major findings in this study are that (i) if the membrane proximal, proline-rich region called box 1 is deleted (CHO Delta 1), PRL stimulates neither K+ channels nor the subsequent Ca2+ entry through voltage-independent, hyperpolarization-driven Ca2+ channels and (ii) in the absence of the COOH-terminal region (CHO H3), PRL does not induce Ca2+ influx though it activates K+ channels. We conclude from these findings that box 1 and the COOH-terminal region of rbPRL-R are both required for PRL-induced calcium entry.

Box B1 Region of PRL-R Is Necessary for K+ Channel Activation but Not Sufficient for Ca2+ Entry Stimulation-- Previous works have shown that box 1 is essential for PRL signal transduction (19). For example, the box 1 region and, particularly, the last proline was shown to be critical for JAK2 phosphorylation and association with PRL-R (21). Treatment of CHO TSE32 cells with a series of tyrosine kinase inhibitors or intracellular dialysis of an anti-JAK2 tyrosine kinase antibody via a patch pipette, completely inhibits the basal and PRL stimulated activity of Ca2+-dependent K+ channels (15). From these findings it was concluded that JAK2 tyrosine kinase, constitutively associated with PRL-R, was implicated in basal activity and PRL stimulation of Ca2+-dependent K+ channels. However, it could not be excluded that these effects could be due to direct effects of JAK2 on K+ channels that prevented further activation by PRL. In unstimulated CHO cells expressing box 1-deleted PRL-R (Delta 1), we show that the amplitude of the K+ current was not significantly modified as compared with CHO TSE32 or native CHO K1 cells. This observation suggests that all K+ channels are controlled by JAK2 tyrosine kinase, but only a small part of them are coupled to PRL-R. We have recently shown that PRL-induced K+ channel activation resulted in a transient, slow hyperpolarization in CHO TSE32 (14). Conversely, in CHO Delta 1 cells PRL decreased the same K+ current and depolarized the membrane potential. These effects may be due to the activation of a protein phosphatase that dephosphorylates K+ channels. This could explain the PRL-induced decrease in [Ca2+]i we have observed in CHO Delta 1 cells. Protein phosphatases actively participate in transduction pathways (22, 23). Furthermore, receptor or protein kinase down-regulation and ion channel activity involve protein phosphatase activities (24, 25). We have recently shown that, like PRL, orthovanadate, a tyrosine phosphatase inhibitor, was capable of increasing K+ channel activity in CHO TSE32 cells (15). From this study we have concluded that the functioning of PRL-stimulated K+ channels is modulated by protein tyrosine kinases and protein tyrosine phosphatases and thus regulated by constitutive tyrosine phosphorylation/dephosphorylation. What protein tyrosine phosphatase could be involved in this effect? A complex of PRL-R·JAK2·PTP1D appears to be necessary for initiating PRL-R signaling (26). PTP1D acts as a positive regulator of PRL-R-dependent induction of beta -casein gene transcription. Their involvement is unlikely in our case since, in CHO Delta 1 cells, PRL inhibited K+ current, although JAK2 can neither associate with PRL-R nor form the complex in these cells. Other phosphatases appear to be implicated in cytokine receptor signaling (27). Their participation in the PRL-induced modulation of ion channels is under investigation in our laboratory.

In CHO H3 cells (lacking the last 141 amino acids of the COOH-terminal region) PRL was always able to activate the K+ channels and hyperpolarize the membrane potential to the same extent as in CHO TS32 cells (Figs. 4, C and D, 5B). In addition, intracellular dialysis of these cells with anti-JAK2 tyrosine kinase antibody by the patch pipette decreased K+ conductance and prevented PRL-stimulation of the K+ channels (data not shown), as we have previously shown in CHO cells expressing the wild-type long form rbPRL-R (15). These results are not surprising, as we have previously shown (8) that PRL was still able to induce JAK2 tyrosine phosphorylation in CHO cells expressing the carboxyl-terminal truncated PRL-R (named T451 in this paper).

Data shown in this study further implicates the box 1-JAK2 couple in PRL-induced activation of K+ currents and demonstrate that the COOH-terminal region does not participate to the cascade of events leading to the activation of K+ channels.

At least in some cell types, proliferative signals seem only to require box 1 (6, 28). A number of studies have shown that the mechanisms promoting proliferation and tumorization processes in various cell models (melanoma, breast cancer, prostate cancer, lymphocytes, neuroblastoma, brown fat cells, etc.) often involve the activation of voltage- and/or calcium-activated K+ channels (29-33). Most of these studies were performed on T-lymphocytes. Indeed, the proliferation of these cells induced by various mitogenic stimuli, such as PRL, is modulated by potassium current blockers (34-36). Because malignant (Nb2) lymphocytes proliferate independently of calcium influx and extracellular calcium concentrations have no influence on PRL-induced Nb2 proliferation (36-38), Wang et al. (39) have proposed that potassium current per se rather than potassium current modulation of calcium influx mediates prolactin-induced proliferation of Nb2 cells (39). The predominant form of PRLR expressed in this cell line is a natural deletion mutant of the long form lacking a 198-amino acid segment of the cytoplasmic domain. However, this mutant receptor retains box 1 and box 2 and tyrosine phosphorylation participates in the anti-apoptotic effect of PRL in Nb2 cells (36). These studies are in agreement with the present work, showing that PRL-induced K+ channel activation requires box 1 but not the COOH-terminal region. To our knowledge, no other studies have shown the effect of PRL on K+ conductance in another cell model.

To our knowledge, no previous studies have shown the participation of K+ channels in the signal transduction of the other members of the cytokine receptor superfamily. However, it has been reported that growth hormone (GH) can cause an increase in [Ca2+]i, independently of JAK2 (40) or other tyrosine kinases (41). This Ca2+ increase was attributed to a stimulation of voltage-dependent Ca2+ channels (40). We have shown (14, 42, 43) that CHO cells possess L-type voltage-dependent Ca2+ channels, but they can be only recorded with extracellular medium containing 60 mM Ca2+ and, even under these particular conditions, the maximal amplitude of the voltage-dependent Ca2+ current remains very low (10-20 pA). According to our findings, it was not possible to detect any [Ca2+]i increase in response to an electrical depolarization in CHO cells by combining electrophysiology and microspectrofluorimetry on the same cell.2 Furthermore, more recently, we have shown that (i) GH inhibited L-type voltage-dependent Ca2+ channels, and (ii) the GH-induced Ca2+ increase was not affected by dihydropyridines in CHO cells expressing rabbit growth hormone receptor (43). Using these cells, we completely blocked Ca2+ response to GH by using pretreatment with tyrosine kinase inhibitors.3 Tyrosine kinase inhibitors blocked the erythropoietin-induced increase in [Ca2+]i (44), suggesting a role for tyrosine phosphorylation but not for serine-threonine kinases in the erythropoietin modulation of intracellular calcium. The tyrosine kinase-ion channel coupling remains unclear in the cytokine receptor superfamily and requires further investigation.

COOH-terminal Region of the PRL-R but Not Box B1 Is Necessary for PRL Activation of the Voltage-independent Ca2+ Channels-- We have previously shown that PRL-induced Ca2+ entry resulted from the activation of hyperpolarization-driven channels (14). Since the effect of PRL on these channels diminished during the electrophysiological recordings (patch-clamp configuration) because the saline patch pipette solution diluted the intracellular medium, we have postulated that one or more diffusible cytosolic second messengers and/or enzymatic activities were required. Earlier studies have shown the presence of a variety of second messenger-operated voltage-insensitive Ca2+ conductances in many cell types (45, 46). Inositol 1,3,4,5-tetrakisphosphate (IP4) is known to operate voltage-independent Ca2+ conductance and to induce Ca2+ entry (45). We have recently shown in CHOTSE32 cells that: (i) PRL induces rapid increases in two inositol phospholipids, phosphoinositol 4,5-P2 and phosphoinositol 3,4,5-P3 (47), (ii) PRL also increases the production of IP4 without any significant increase in inositol 1,4,5-trisphosphate (48), (iii) IP4 causes an increase in the open probability of a voltage-independent Ca2+ channel (48), and (iv) PRL activates the same channel in "cell attached" configuration of the patch-clamp technique (48). PRL has no effect on voltage-independent Ca2+ channels in cell-free configurations, confirming the involvement of a cytosolic messenger. Taken together, all these results demonstrate that PRL stimulates Ca2+ entry through voltage-independent Ca2+ channels by stimulating IP4 production. The mechanism by which PRL stimulates IP4 production remains unclear. To our knowledge, a metabolic pathway from phosphoinositol 3,4,5-P3 to inositol 1,3,4,5-P4 has not yet been described.

Exploration of the membrane potential during simultaneous measurements of [Ca+]i and ionic currents shows that, in PRL-R, the last 141 amino acids in the COOH-terminal regions necessary for the activation of the voltage-independent Ca2+ channels, but box 1 is not (Figs. 6 and 7). The effects of PRL on the phosphoinositide metabolism and inositol phosphate production in CHO H3 cells are currently under investigation in our laboratory. It can be postulated that the three tyrosine residues in the 141 amino acids play a pivotal role. In order to confirm the role of these residues and specify which tyrosine is involved in channel activation, we will work on CHO cells expressing point mutated PRL-R. PRL-R intracytoplasmic domain can be used as a bait to clone potential PRL-R interacting proteins by the yeast two-hybrid interacting cloning strategy (23, 49). This strategy may help us to specify how IP4 is produced in response to PRL.

To our knowledge, none of the studies have shown a direct effect of PRL on Ca2+ influx in cells expressing natural form and we have found only two studies of Nb2 cells, where the role of Ca2+ in PRL-induced events was inferred from biochemical and pharmacological experiments (37, 38). Furthermore, in our experiments, PRL failed to induce any [Ca2+]i increase in Nb2 cells.4

We have developed an in vitro functional test consisting of the co-transfection in CHO cells of PRL-R cDNAs with a chimeric gene encompassing a milk protein gene promoter (beta -lactoglobulin) fused to the coding sequence of the chloramphenicol acetyltransferase gene (5). Using this test, we have recently shown that PRL was still able to activate the transcription of the target gene in CHO cells expressing the rbPRL-R, where the last 141 amino acids of the COOH-terminal region have been deleted (T451 mutant) (8). However, the T451 mutant was less active (2.0-fold induction) than the wild-type receptor (4.5-fold induction). Thus, the tyrosine phosphorylation of the carboxyl-terminal part of the rbPRL-R appears to be a positive modulator in signal transduction to milk protein genes. On the other hand, the T451 mutant is also about half as active in activating STAT5. In addition, recent findings indicate that serine/threonine phosphorylation of STAT proteins increases DNA binding (50, 51). MAPK is a candidate for this action. As its activity is dependent on Ca2+ influx (52), the lower activation of STAT5 and the consecutive lower transcriptional activity of the T451 mutant may be explained by the absence of an increase in [Ca2+]i in response to PRL in CHO cells expressing this receptor.

The COOH-terminal region (amino acids 454-506) of the growth hormone receptor is also required for Ca2+ signaling (40). Calcium is an ubiquitous intracellular messenger and regulator of cellular activities, including proliferation, mitosis, muscle contraction, energy metabolism, secretion, etc. For example, increases in intracellular Ca2+ has been associated with the induction of proliferation-associated immediate early genes, including c-fos and c-jun (53). Calcium influx through store-operated Ca2+ channels also seems to play a role in the activation of MAPK pathways (52). Billestrup et al. (40) have reported that a GH-induced rise in [Ca2+]i is required for GH-stimulated insulin gene transcription in RIN 5AH cells.

In conclusion, the data presented in this article confirm the involvement of box 1/JAK2 tyrosine kinase in PRL-induced K+ conductance activation. For the first time we show that the COOH-terminal part of the long form rbPRL-R is involved in the opening of the voltage-independent Ca2+ channels expressed in CHO cells. Thus, both box 1 and the last 141 amino acids of the COOH-terminal region of the PRL-R are required to produce the PRL-induced rise in [Ca2+]i. Mutated PRL receptors may help us to further investigate the early effects of PRL on ion conductance and [Ca2+]i and to determine if these effects converge upon MAPK and STAT5 activation and milk gene transcription.

    ACKNOWLEDGEMENTS

We are grateful to D. Varoqueaux for excellent technical assistance. We are indebted to Dr. A. F. Parlow, National Pituitary Agency, Pituitary Hormone Distribution Program, National Institute of Diabetes and Digestive and Kidney Diseases for the gift of ovine PRL.

    FOOTNOTES

* This work was supported in part by grants from Centre National de la Recherche Scientifique and Université de Bordeaux 2 (UMR 5543).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by Ministère de l'Enseignement Supérieur et de la Recherche Grant 97-5-23436.

parallel To whom correspondence should be addressed: CNRS UMR 5543, Université de Bordeaux 2, BP 22, 146 rue Léo Saignat, 33076 Bordeaux cédex, France.

The abbreviations used are: PRL, prolactin; STAT, signal transducers and activators of transcription; CHO, Chinese hamster ovary; rb, rabbit; TTX, tetrodotoxin; CTX, charybdotoxin; GH, growth hormone; IP4, inositol (1,3,4,5)-tetrakisphosphate.

2 B. Sorin, O. Goupille, A. M. Vacher, J. Paly, J. Djiane, and P. Vacher, unpublished data.

3 G. Boquet, J. Paly, J. Djiane, and B. Dufy, submitted for publication.

4 B. Sorin, O. Goupille, A. M. Vacher, J. Paly, J. Djiane, and P. Vacher, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Kelly, P. A., Djiane, J., Postel-Vinay, M. C., and Edery, M. (1991) Endocr. Rev. 12, 235-251[Abstract/Free Full Text]
  2. Cosman, D. (1993) Cytokine 5, 95-106[CrossRef][Medline] [Order article via Infotrieve]
  3. O'Neal, K. D., and Yu-Lee, L. Y. (1994) J. Biol. Chem. 269, 26076-26082[Abstract/Free Full Text]
  4. Bignon, C., Binart, N., Ormandy, C., Schuler, L. A., Kelly, P. A., and Djiane, J. (1997) J. Mol. Endocrinol. 19, 109-120[Abstract/Free Full Text]
  5. Lesueur, L., Edery, M., Ali, S., Paly, J., Kelly, P. A., and Djiane, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 824-828[Abstract/Free Full Text]
  6. DaSilva, L., Howard, O. M. Z., Rui, H., Kirken, R. A., and Farrar, W. L. (1994) J. Biol. Chem. 269, 18267-18270[Abstract/Free Full Text]
  7. Lebrun, J.-J., Ali, S., Ullrich, A., and Kelly, P. A. (1995) J. Biol. Chem. 270, 10664-10670[Abstract/Free Full Text]
  8. Goupille, O., Daniel, N., Bignon, C., Jolivet, G., and Djiane, J. (1997) Mol. Cell. Endocrinol. 127, 155-169[CrossRef][Medline] [Order article via Infotrieve]
  9. Sotiropoulos, A., Perrot-Applanat, M., Dinerstein, H., Pallier, A., Postel-Vinay, M. C., Finidori, J., and Kelly, P. A. (1994) Endocrinology 135, 1292-1298[Abstract]
  10. Goffin, V., and Kelly, P. A. (1996) Clin. Endocrinol. 45, 247-255[CrossRef][Medline] [Order article via Infotrieve]
  11. Edery, M., Jolicoeur, C., Levy-Meyrueis, C., Dusanter-Fourt, I., Pétridou, B., Boutin, J. M., Lesueur, L., Kelly, P. A., and Djiane, J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2112-2116[Abstract/Free Full Text]
  12. Bignon, C., Daniel, N., and Djiane, J. (1993) Biotechniques 15, 243-245[Medline] [Order article via Infotrieve]
  13. Vacher, P., Chuoï, M. T. V., Paly, J., Djiane, J., and Dufy, B. (1994) Endocrinology 134, 1213-1218[Abstract/Free Full Text]
  14. Prevarskaya, N., Skryma, R., Vacher, P., Daniel, N., Bignon, C., Djiane, J., and Dufy, B. (1994) Am. J. Physiol. 267, C554-C562[Abstract/Free Full Text]
  15. Prevarskaya, N. B., Skryma, R. N., Vacher, P., Daniel, N., Djiane, J., and Dufy, B. (1995) J. Biol. Chem. 270, 24292-24299[Abstract/Free Full Text]
  16. Gasser, F., Mulsant, P., and Gillois, M. (1985) Dev. Biol. 21, 588-592
  17. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract/Free Full Text]
  18. Fabiato, A., and Fabiato, F. (1979) J. Physiol. 75, 463-505
  19. Edery, M., Levi-Meyrueis, C., Paly, J., Kelly, P. A., and Djiane, J. (1994) Mol. Cell. Endocrinol. 102, 39-44[CrossRef][Medline] [Order article via Infotrieve]
  20. Skryma, R., Prevarskaya, N., Vacher, P., and Dufy, B. (1994) Am. J. Physiol. 267, C544-C553[Abstract/Free Full Text]
  21. Pezet, A., Buteau, H., Kelly, P., and Edery, M. (1997) Mol. Cell. Endocrinol. 129, 199-208[CrossRef][Medline] [Order article via Infotrieve]
  22. Pallen, C. J., Tan, Y. H., and Guy, G. R. (1992) Curr. Opin. Cell Biol. 4, 1000-1007[CrossRef][Medline] [Order article via Infotrieve]
  23. Yu-Lee, L.-Y. (1997) Proc. Soc. Exp. Biol. Med. 215, 35-52[CrossRef][Medline] [Order article via Infotrieve]
  24. Daniel, N., Waters, M. J., Bignon, C., and Djiane, J. (1996) Mol. Cell. Endocrinol. 118, 25-35[CrossRef][Medline] [Order article via Infotrieve]
  25. Cataldi, M., Taglialatela, M., Guerriero, S., Amoroso, S., Lombardi, G., di Renzo, G., and Annunziato, L. (1996) J. Biol. Chem. 271, 9441-9446[Abstract/Free Full Text]
  26. Ali, S., Chen, Z., Lebrun, J. J., Vogel, W., Kharitonenkov, A., Kelly, P. A., and Ullrich, A. (1996) EMBO J. 15, 135-142[Medline] [Order article via Infotrieve]
  27. Yi, T., Mui, A. L.-F., Krystal, G., and Ihle, J. N. (1993) Mol. Cell. Biol. 13, 7577-7586[Abstract/Free Full Text]
  28. Das, R., and Vonderhaar, B. K. (1995) Mol. Endocrinol. 9, 1750-1759[Abstract/Free Full Text]
  29. Nilius, B., and Wohlrab, W. (1992) J. Physiol. 445, 537-548[Abstract/Free Full Text]
  30. Wegman, E. A., Young, J. A., and Cook, D. I. (1991) Pflügers Arch. 417, 562-570[CrossRef][Medline] [Order article via Infotrieve]
  31. Skryma, R., Prevarskaya, N., Dufy-Barbe, L., Odessa, M. F., Audin, J., and Dufy, B. (1997) Prostate 33, 112-122[CrossRef][Medline] [Order article via Infotrieve]
  32. Rouzaire-Dubois, B., and Dubois, J. M. (1991) Cell Signal. 3, 333-339[CrossRef][Medline] [Order article via Infotrieve]
  33. Paponne, P. A., and Ortiz-Miranda, S. J. (1993) Am. J. Physiol. 264, 1014-1019
  34. Lee, S. C., Sabath, D. E., Deutsch, C., and Prystowski, M. B. (1986) J. Cell Biol. 102, 1200-1208[Abstract/Free Full Text]
  35. Lewis, R. S., and Cahalan, M. D. (1990) Annu. Rev. Physiol. 52, 415-430[CrossRef][Medline] [Order article via Infotrieve]
  36. LaVoie, H. A., and Witorsch, R. J. (1995) Proc. Soc. Exp. Biol. Med. 209, 257-269[CrossRef][Medline] [Order article via Infotrieve]
  37. Buckley, A. R., Montgomery, D. W., Kibler, R., Putnam, C. W., Zukoski, C. F., Gout, P. W., Beer, C. T., and Russell, D. H. (1986) Immunopharmacology 12, 37-51[CrossRef][Medline] [Order article via Infotrieve]
  38. Murphy, P. R., DiMattia, G. E., and Friesen, H. G. (1988) Endocrinology 122, 2476-2485[Abstract/Free Full Text]
  39. Wang, Y.-F., Jia, H., Walker, A. M., and Cukierman, S. (1992) J. Cell. Physiol. 152, 185-189[CrossRef][Medline] [Order article via Infotrieve]
  40. Billestrup, N., Bouchelouche, P., Allevato, G., Ilondo, M., and Nielsen, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2725-2729[Abstract/Free Full Text]
  41. Ilondo, M. M., Meyts, P. D., and Bouchelouche, P. (1994) Biochem. Biophys. Res. Commun. 202, 391-397[CrossRef][Medline] [Order article via Infotrieve]
  42. Skryma, R., Prevarskaya, N., Vacher, P., and Dufy, B. (1994) FEBS Lett. 349, 289-294[CrossRef][Medline] [Order article via Infotrieve]
  43. Boquet, G., Barakat, L., Paly, J., Djiane, J., and Dufy, B. (1997) Mol. Cel. Endocrinol. 131, 109-120[CrossRef][Medline] [Order article via Infotrieve]
  44. Miller, B. A., Bell, L. L., Lynch, C. J., and Cheung, J. Y. (1994) Cell Calcium 18, 481-490
  45. Penner, R., Matthews, G., and Neher, E. (1988) Nature 334, 499-504[CrossRef][Medline] [Order article via Infotrieve]
  46. Tscharner, V. V., Prod'hom, B., Baggiolini, M., and Reuter, H. (1986) Nature 324, 369-372[CrossRef][Medline] [Order article via Infotrieve]
  47. Ratovondrahona, D., Fournier, B., Odessa, M. F., and Dufy, B. (1998) Biochem. Biophys. Res. Commun. 243, 127-130[CrossRef][Medline] [Order article via Infotrieve]
  48. Ratovondrahona, D., Fahmi, M., Fournier, B., Djiane, J., and Dufy, B. (1998) J. Mol. Endocrinol. 21, 85-95[Abstract]
  49. Fields, S., and Song, O.-Y. (1989) Nature 340, 245-246[CrossRef][Medline] [Order article via Infotrieve]
  50. Wen, Z., Zhang, Z., and Darnell, J. E. (1995) Cell 82, 241-250[CrossRef][Medline] [Order article via Infotrieve]
  51. Zhang, X., Blenis, J., Li, H. C., Schindler, C., and Chgen-Kiang, S. (1995) Science 267, 1991-1994
  52. Rodland, K. D., Wersto, R. P., Hobson, S., and Kohn, E. C. (1997) Mol. Endocrinol. 11, 281-291[Abstract/Free Full Text]
  53. Pribnow, D., Muldoon, L., Fajardo, M., Theodor, L., Chen, L.-Y. S., and Magun, B. E. (1992) Mol. Endocrinol. 6, 1003-1012[Abstract/Free Full Text]

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
R. Shao, M. Nutu, B. Weijdegard, E. Egecioglu, J. Fernandez-Rodriguez, E. Tallet, V. Goffin, C. Ling, and H. Billig
Differences in Prolactin Receptor (PRLR) in Mouse and Human Fallopian Tubes: Evidence for Multiple Regulatory Mechanisms Controlling PRLR Isoform Expression in Mice
Biol Reprod, October 1, 2008; 79(4): 748 - 757.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
D. R. Grattan, C. L. Jasoni, X. Liu, G. M. Anderson, and A. E. Herbison
Prolactin Regulation of Gonadotropin-Releasing Hormone Neurons to Suppress Luteinizing Hormone Secretion in Mice
Endocrinology, September 1, 2007; 148(9): 4344 - 4351.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
I. C. Kokay, P. M. Bull, R. L. Davis, M. Ludwig, and D. R. Grattan
Expression of the long form of the prolactin receptor in magnocellular oxytocin neurons is associated with specific prolactin regulation of oxytocin neurons
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2006; 290(5): R1216 - R1225.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Lajus, P. Vacher, D. Huber, M. Dubois, M.-N. Benassy, Y. Ushkaryov, and J. Lang
{alpha}-Latrotoxin Induces Exocytosis by Inhibition of Voltage-dependent K+ Channels and by Stimulation of L-type Ca2+ Channels via Latrophilin in beta-Cells
J. Biol. Chem., March 3, 2006; 281(9): 5522 - 5531.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
Q. Zhang, M. Kohler, S.-N. Yang, F. Zhang, O. Larsson, and P.-O. Berggren
Growth Hormone Promotes Ca2+-Induced Ca2+ Release in Insulin-Secreting Cells by Ryanodine Receptor Tyrosine Phosphorylation
Mol. Endocrinol., July 1, 2004; 18(7): 1658 - 1669.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
T. Ducret, A.-M. Vacher, and P. Vacher
Effects of Prolactin on Ionic Membrane Conductances in the Human Malignant Astrocytoma Cell Line U87-MG
J Neurophysiol, March 1, 2004; 91(3): 1203 - 1216.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. P. Tam, P. Lau, J. Djiane, D. J. Hilton, and M. J. Waters
Tissue-Specific Induction of SOCS Gene Expression by PRL
Endocrinology, November 1, 2001; 142(11): 5015 - 5026.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
M. E. Freeman, B. Kanyicska, A. Lerant, and G. Nagy
Prolactin: Structure, Function, and Regulation of Secretion
Physiol Rev, October 1, 2000; 80(4): 1523 - 1631.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
J. M. Bowen and P. L. Keyes
Repeated Exposure to Prolactin Is Required to Induce Luteal Regression in the Hypophysectomized Rat
Biol Reprod, April 1, 2000; 63(4): 1179 - 1184.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
V. Kansra, C. Groves, J. C. Gutierrez-Ramos, and R. D. Polakiewicz
Phosphatidylinositol 3-Kinase-dependent Extracellular Calcium Influx Is Essential for CX3CR1-mediated Activation of the Mitogen-activated Protein Kinase Cascade
J. Biol. Chem., August 17, 2001; 276(34): 31831 - 31838.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sorin, B.
Right arrow Articles by Vacher, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sorin, B.
Right arrow Articles by Vacher, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1998 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement