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Volume 271, Number 1, Issue of January 5, 1996 pp. 574-580
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
G RNA Antisense Expression Demonstrates the Exclusive Coupling of Peptide YY Receptors to G Proteins in Renal Proximal Tubule Cells (*)

(Received for publication, June 22, 1995; and in revised form, September 11, 1995)

Thierry Voisin (§),  ,  Anne-Marie Lorinet ,  Jean-José Maoret ,  Alain Couvineau ,  Marc Laburthe (§)

From the Unité de Recherche de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, INSERM U410, Faculté de Médecine Xavier Bichat, BP 416, 75870 Paris, Cedex 18, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A clone PKSV-PCT Cl.10 referred to as Cl.10 was selected from the PKSV-PCT renal proximal tubule cell line which expressed peptide YY (PYY) receptors (Voisin, T., Bens, M., Cluzeaud, F., Vandewalle, A., and Laburthe, M.(1993) J. Biol. Chem. 268, 20547-20554). In order to identify G(i) protein(s) coupled to PYY receptors, antisense Galpha(i) protein RNAs were expressed in Cl.10 cells by transfecting the pcDNA3 vector into which were inserted 39 bases of the 5`-noncoding region of Galpha or Galpha used as specific antisense templates. A Cl.10/alpha clone was selected which displayed a drastic decrease (>90%) of the expression of Galpha without changes of Galpha, Galpha(s), and Gbeta subunits (Galpha is not present in Cl.10 cells) as evidenced by Western blots. When compared to untransfected cells, this clone exhibited: (i) an increase in the dissociation constant of PYY receptors (5.3 versus 0.6 nM) identical to that observed in pertussis toxin-treated untransfected cells; (ii) an absence of inhibition of I-PYY binding by guanosine 5`-O-(thiotriphosphate) (GTPS); and (iii) the failure of PYY to inhibit cAMP levels and to stimulate [methyl-^3H]thymidine incorporation into DNA. A clone was also selected which exhibited a specific decrease (>80%) of Galpha as compared to untransfected cells. The sensitivity to GTPS and the dissociation constant of PYY receptors as well as PYY-mediated inhibition of cAMP were identical to those observed in untransfected cells. These findings support an exclusive coupling of PYY receptors to Galpha.

INTRODUCTION

Following its discovery in rat intestinal epithelial cells(1) , the peptide YY (PYY) (^1)receptor has been characterized in dog adipocytes (2) and the proximal tubule PKSV-PCT cell line derived from kidneys of transgenic mice(3) . This receptor is PYY-preferring since it binds the intestinal hormone PYY (4) with high affinity and the neuropeptide NPY (4) with a 10-fold lower affinity(1, 2, 3, 4) . PYY and NPY triggers several biological effects through interaction with PYY receptors, including inhibition of adenylyl cyclase activity(2, 3, 4, 5) , inhibition of Cl secretion in the small intestine(4, 6, 7) , inhibition of lipolysis in fat cells(2) , and stimulation of epithelial cell growth(3, 8, 9) . The PYY receptor resembles the Y2 subtype of NPY receptor (9, 10, 11) which does not discriminate between PYY and NPY but, like the PYY receptor, binds long COOH-terminal fragments of PYY or NPY (5, 7) . Its pharmacology is clearly different from that of other receptors for the PP-fold family of peptides, including the Y1 and Y3 subtypes of NPY receptors and PP receptors(9, 10, 11) . Like most receptors for this family of peptides(9) , with the exception of the Y1 subtype of NPY receptor(12, 13, 14) , the PYY receptor is not yet cloned. However, it has been characterized as a M(r) 44,000 glycoprotein by cross-linking experiments and hydrodynamic studies (15) .

Recent studies characterized PYY receptors in the PKSV-PCT cell line (3) derived from microdissected proximal convoluted tubules of kidneys from transgenic mice harboring the simian virus 40 (SV40) large T antigen placed under the control of the rat L-type pyruvate kinase 5`-regulatory sequence(16, 17) . PYY receptor-mediated events are triggered through interaction of PYY receptors with pertussis toxin-sensitive G(i) proteins in PKSV-PCT cells. Indeed, preincubation of cells with pertussis toxin completely reverses the PYY-induced inhibition of cAMP production and stimulation of cell growth and converts PYY receptors to a low affinity state(3) . In view of the fact that (i) multiple G(i) proteins including G, G, and G can contribute to heptahelical receptor-mediated inhibition of adenylyl cyclase (18) and (ii) pertussis toxin-sensitive G proteins have been shown to be crucial for the mitogenic action of several agents(19) , the characterization of the pertussis toxin-sensitive G(i) protein(s) coupled to PYY receptors is an important step leading to further understanding of the mechanism of action of this recently discovered receptor.

In the present work, we have developed the stable expression of antisense Galpha(i) RNA in a clone PKSV-PCT Cl.10 (referred to as Cl.10 below) isolated from the parent PKSV-PCT cells in order to identify G(i) proteins coupled to PYY receptors. By studying receptor affinity and regulation of ligand binding by GTPS, inhibition of cAMP production and stimulation of cell growth in Cl.10 cell clones in which endogenous Galpha(i) proteins were permanently down-regulated after transfection with antisense Galpha(i) expression vectors, we provide evidence for the exclusive coupling of the PYY receptor to the G protein.

EXPERIMENTAL PROCEDURES

Materials

Synthetic porcine PYY, porcine NPY, rat PP, and Tyr^0-PTH(1-34) were purchased from Peninsula Laboratories (Belmont, CA). I-Na (IMS300) and [methyl-^3H]thymidine were from Amersham Corp. (Les Ulis, France). Geneticin (G418) and culture media DMEM and Ham's F-12 were purchased from Life Technologies, Inc. (Cergy Pontoise, France). Transferrin, sodium selenate, dexamethasone, triiodothyronine, insulin, glutamine, forskolin, epidermal growth factor, HEPES, PMSF, TLCK, bacitracin, pertussis toxin, and the other highly purified chemicals used were purchased from Sigma. BSA (Pentex, fraction V) was obtained from Miles Laboratories (Elkart, NJ). Anti-alpha/alpha (AS7), anti-alpha/alpha(0) (EC2), anti-alpha(s) (A572), and anti-beta (U49) antibodies were from DuPont NEN. I-Tyr-monoiodo PYY (referred to as I-PYY below) was prepared and purified as described elsewhere(3) .

Cultured Cells

The PKSV-PCT cell line was derived from microdissected proximal convoluted tubules from the kidney of a transgenic mouse (L-PK/Tag1) carrying the large T and small t antigens of SV40 placed under the control of the rat L-type pyruvate kinase promoter gene(16, 17) . PKSV-PCT cells were cloned by limiting dilution. Briefly, a monodispersed cell suspension was distributed to microtest plates at a mean ratio of 0.25 cell/well. Those wells containing only one cell, as ascertained by microscopic inspection by two independent observers, were identified with their coordinates on the plates. Cells grown in wells observed to initially contain one cell were subsequently transferred to increasingly larger culture vessels. Among the 12 clones obtained, the clone Cl.10 was selected on the basis of its high binding capacity for PYY. Cl.10 cells were cultured in a standard culture medium (DMEM:Ham's F-12, 1:1 (v/v); 60 nM sodium selenate; 5 µg/ml transferrin; 2 mM glutamine; 50 nM dexamethasone; 1 nM triiodothyronine; 10 nM epidermal growth factor; 2% fetal calf serum; 20 mM HEPES, pH 7.4), supplemented with 5 µg/ml insulin and 20 mMD-glucose at 37 °C in 5% CO(2), 95% air atmosphere as previously described(3, 16, 17) . As previously shown for the parent cell line PKSV-PCT(3, 16, 17) , such culture conditions with D-glucose-enriched medium favors the activation of Tag transcripts and cell growth. All studies on the Cl.10 cell line were performed between the 4th and 12th passages on sets of cells seeded on plastic culture flasks (25- or 75-cm^2 surface). The cells were routinely passaged every 7 days.

Construction of the Antisense Galpha(i) Subunit Expression Vectors

The pcDNA3 expression vector (Invitrogen, San Diego, CA) was used to construct antisense Galpha(i) subunit expression vectors. It contains enhancer/promoter sequences of the human cytomegalovirus intermediate early gene and a polyadenylation signal from the bovine growth hormone gene, an ampicillin resistance gene and a Col E1 origin of replication for selection and maintenance in Escherichia coli, a neomycin-resistant gene expressed from the SV40 early promoter for selection of stable transformants in the presence of G418, and T7 and Sp6 promoters flanking the multiple cloning site. The 39 bases of the 5`-noncoding region, immediately upstream of end including the ATG translation initiation codon of Galpha (5`-GCGTGTGGGGGCCAGGCCGGGCCGGCGGACGGCAGGATG-3`) and Galpha (5`-GCGAGCCAGGGCCCGGTCCCCTCTCCGGCCGCCGTCATG-3`) were selected for use as antisense probes (20, 21) to take advantage of the diversity of the nucleotide sequence in this region and to provide specificity (48% identity in noncoding region versus >85% identity in coding region). Oligodeoxynucleotides of this sequence and the complementary strand were synthesized commercially (Eurogentec, Eraing, Belgium). The construction of vectors outlined below was performed with the use of standard techniques. Complementary oligodeoxynucleotides were hybridized together, and the double-stranded DNA was inserted into the EcoRV cloning site of the polylinker of pcDNA3. After transformation into competent E. coli XL-1, ampicillin-resistant clones were selected for the presence of inserts by restriction mapping and analyzed for the orientation of the inserts by DNA sequencing.

Transfection with Antisense Galpha(i) Subunit Expression Vector

Cl.10 cells were transfected by electroporation using a gene pulser (Electroporator II, Invitrogen). 5 times 10^6 exponentially growing cells were preincubated on ice for 5 min with 20 µg of pcDNA3 plasmid encoding Galpha or Galpha antisenses or without a cDNA insert and 20 µg of salmon sperm DNA carrier in cold DMEM/Ham's F-12 medium with 100 IU/ml penicillin and 100 µg/ml streptomycin. Electroporation was performed at 330 V and 500 µF. After electroporation, cells were kept on ice for 5 min, added to 5 ml of culture medium, and transferred in a 25-cm^2 plastic culture flask. 48 h after electroporation, transfected Cl.10 cells were selected by addition of geneticin to a final concentration of 400 µg/ml for 3 weeks. Cl.10 cells which were resistant to geneticin were subsequently cloned by limiting dilution as described above and characterized for their Galpha content by Western blotting (see below).

Preparation of Particulate Fraction of Cultured Cells

Control and transfected Cl.10 cells were grown in 75-cm^2 plastic culture flasks for 6-13 days (see legends to figures) as described above. Cells were washed three times with 0.13 M PBS (pH 7.4), harvested using a rubber policeman, and centrifuged at 2,000 times g for 5 min at 4 °C. The cell pellet was then exposed for 30 min to hypoosmotic 5 mM HEPES buffer (pH 7.4) as described elsewhere (3) . Thereafter, aliquots of cell suspensions were centrifuged at 20,000 times g for 15 min, washed with 20 mM HEPES buffer (pH 7.4), pelleted, and stored at -80 °C until used. This particulate fraction from cell homogenates will be referred to as a membrane preparation.

Cl.10 Cell Treatment with Pertussis Toxin

In some experiments, confluent cells grown in 25-cm^2 plastic culture flasks were treated overnight with pertussis toxin (0.4 µg/ml). A membrane fraction was then prepared as described above and used immediately for binding experiments. This procedure was also applied to confluent cells grown in 12-well trays before cellular cAMP assay.

Binding of I-PYY to Membrane-bound Receptors

Binding of I-PYY to membrane preparations was conducted as described previously(3, 8, 15) . Briefly, membranes (200 µg of protein/ml) were incubated for 90 min at 30 °C in 250 µl of incubation buffer (20 mM HEPES buffer (pH 7.4), 2% (w/v) BSA, 17 mg/liter PMSF, 10 mg/liter TLCK, 10 mg/liter pepstatin, 10 mg/liter leupeptin, and 100 mg/liter bacitracin) containing 0.05 nMI-PYY (2,200 Ci/mmol) with or without unlabeled PYY or other competing peptides. At the end of the incubation, 150-µl aliquots of membranes were mixed with 150 µl of ice-cold incubation buffer. Bound and free peptides were separated by centrifugation at 20,000 times g for 10 min, and membrane pellets were washed twice with 10% (w/v) sucrose in 20 mM HEPES buffer (pH 7.4). The radioactivity was then counted with a counter. The nonspecific binding represented about 20% of total binding. All binding data were analyzed using the LIGAND computer program developed by Munson and Rodbard(22) .

Binding of I-Tyr^0-PTH(1-34) to Membrane-bound Receptors

I-Tyr^0-PTH(1-34) (1,300 Ci/mmol) was prepared by the chloramine T method and purified on a column of Sephadex G-50. Membranes (200 µg of protein/ml) were incubated for 60 min at 30 °C with 0.03 nM tracer as described above for PYY, and bound and free peptides were separated by centrifugation (see above).

Cyclic AMP Measurement

Cellular cAMP content was assayed as previously described(3) . Cells in 12-well trays were incubated in the presence or absence of 10 µM forskolin in 1 ml of DMEM/Ham's F-12 containing 2% (w/v) BSA, 0.1% (w/v) bacitracin, and 0.2 mM 3-isobutyl-1-methylxanthine without or with 0.1 µM PYY for 40 min at 37 °C. At the end of the incubation, the medium was rapidly removed, cells were washed in 1 ml of PBS (pH 7.4), and 1 ml of 1 M perchloric acid was added. After centrifugation for 10 min at 4,000 times g, the cAMP present in the supernatant was succinylated, and its concentration was measured by radioimmunoassay as described elsewhere(8) . Data are reported as picomoles of cAMP per mg of protein. Cell protein determinations were made in parallel wells.

Immunoblotting of Galpha(i), Galpha(s), and Gbeta Subunits of G Proteins

Membranes (50 µg of protein) were solubilized as previously described(23) . Then, samples were alkylated prior to electrophoresis in order to enhance the clarity and hence resolution of polypeptide bands(3) . Samples were heated at 100 °C for 5 min, and proteins were separated in a 10% polyacrylamide gel. Proteins were transferred to nitrocellulose as described elsewhere(23) . The nitrocellulose sheets were incubated with anti-alpha(s) (A572, dilution 1/500), anti-alpha/alpha (AS7, dilution 1/1,000), anti-alpha/alpha(0) (EC2, dilution 1/1,000) or anti-beta (U49, 1/5,000) followed by washing and incubation with I-labeled goat antibodies to rabbit IgG in 50 mM Tris-HCl, 500 mM NaCl, and 0.02% NaN(3) for 2 h. After extensive washing, nitrocellulose sheets were dried prior to autoradiography(23) . Gels were calibrated with molecular weight marker proteins: myosin (200,000), phosphorylase b (97,000), BSA (68,000), ovalbumin (43,000), and carbonic anhydrase (29,000). Autoradiograms of the dried immunoblots were scanned with a Macintosh Onescanner densitometer in order to estimate the relative amount of G protein subunits.

[methyl-^3H]Thymidine Incorporation

Cell growth kinetics were estimated by [methyl-^3H]thymidine (25 Ci/mmol) incorporation. Cells, detached with 2.7 mM EDTA, were seeded (15,000 cells/dish) in 12-well trays and grown for 3 days in standard culture medium (see above). Cells were then incubated in the absence or presence of 0.1 µM PYY for 24 h in a serum- and growth factor-free culture medium. Cells were then incubated for 6 h with [methyl-^3H]thymidine (0.5 µCi/well) and rinsed three times with 1 ml of ice-cold PBS. 1 ml of 5% trichloroacetic acid was added for 30 min at 4 °C. The trichloroacetic acid was removed and discarded after which the cells were further incubated for 30 min at 37 °C with 1 ml of 0.3 N NaOH. After neutralization with acetic acid, the radioactivity in the cell extracts was measured by scintillation counting, and the results were expressed as counts/min/10^6 cells. Cells were counted before addition of trichloroacetic acid.

Confocal Laser Scanning Microscopy

Cells grown on 12-mm glass coverslips were preincubated in 80 mM PIPES buffer (pH 6.8), containing 1 mM MgCl(2), 5 mM EGTA, and 0.075% saponin, for 5 min and then fixed at room temperature with 2% paraformaldehyde in PBS for 15 min, washed with PBS, quenched in 50 mM NH(4)Cl in PBS, and then blocked and permeabilized in 0.075% saponin in PBS for 20 min. The coverslips were then incubated with anti-alpha (AS7 diluted 1/50) or anti-alpha (EC2 diluted 1/50) for 45 min. Antibodies were diluted in gelatin-saponin-PBS. After washing, the coverslips were incubated for 45 min with a fluorescein isothiocyanate goat anti-rabbit antibody (diluted 1/500). The coverslips were mounted in Glycergel, and selected fields were scanned using a True Confocal Scanner Leica TCS 4D comprising a Leica Diaplan inverted microscope equipped with an argon-krypton ion laser (488 nm) with an output power of 2-50 mW and a VME bus MC 68020/68881 computer system coupled to an optical disk for image storage (Leica Lasertchnik GmbH). The emitted light was collected through a long-pass filter on the target of the photo multiplier. Each sample was treated with a kalman filter to increase the ratio signal versus background. All image-generating and processing operations were carried out using the Leica CLSM software package. Screen images were taken on Kodak Ektachrome using a 35-mm camera.

Morphology and Measurement of Transepithelial Resistance of Filter-grown Cells

Cells were plated (500,000 cells/filter) on nitrocellulose filters (Millipore HAHY; porosity, 0.45 µm; diameter, 1.2 cm). Seven days later the transepithelial resistance was measured using a Millicell electrical resistance system (ERS, Millipore Corporation) as described elsewhere(16) , and thereafter the filters were fixed in Bouin's fluid and embedded in paraffin. Cross sections (4 µm) were stained with hematein eosin and observed by light microscopy.

Protein Determination and Measurement of Enzyme Activities

Proteins were measured using a protein assay kit (Bio-Rad) based on the method of Bradford (24) with BSA as a standard. The activities of aminopeptidase N and dipeptidyl peptidase IV were determined as described elsewhere(25) .

Statistical Analysis

Results are expressed as means ± S.E. from (n) separate experiments. Statistical significance between groups was calculated by the Student's t test.

RESULTS

Characterization of the Cl.10 Clone

Among the 12 clones obtained by limiting dilution from the parent PKSV-PCT cell line, four exhibited the ability to bind I-PYY specifically. The highest binding was observed with clone 10, and the properties of PYY receptors in this clone were further investigated. Cl.10 cells express a typical PYY receptor as described in the parent PKSV-PCT cell line (3) and rat intestinal epithelial crypt cells(8) . PYY receptors in Cl.10 cells discriminate between PYY and the naturally occurring related peptides NPY and PP. The peptide concentrations that induced half-maximal inhibition of I-PYY binding (IC) were as follows (Fig. 1A): PYY (IC = 0.6 ± 0.1 nM) < NPY (IC = 6.3 ± 0.6 nM) PP (IC > 1 µM). Scatchard analysis of binding data indicated the presence of one class of binding site with a dissociation constant of 0.63 ± 0.20 nM and a binding capacity of 145 ± 22 fmol/mg protein (three experiments). As shown in Fig. 1B, PYY, in the concentration range between 10 and 10M, inhibited forskolin-induced cAMP production in cultured Cl.10 cells. A 70% inhibition was observed at high PYY concentration (1 µM) and half-maximal inhibition was obtained for 5.2 ± 0.5 nM PYY. The inhibitory effect of PYY on cAMP production was completely abolished when cultured Cl.10 cells were preincubated with pertussis toxin (Fig. 1B), indicating a pertussis toxin-sensitive G(i) protein-mediated event. Further experiments were conducted to determine the G protein profile in Cl.10 cells. Among the putative G protein subunits involved in the coupling of PYY receptors to biological events in Cl.10 cells, Western blot analysis indicated the presence of the M(r) 39,000 Galpha subunit and the M(r) 42,000 Galpha subunit but not Galpha subunits (Fig. 2). We also demonstrated the presence of M(r) 42,000 Galpha(s) subunits and M(r) 35,000 Gbeta subunits by immunoblotting (Fig. 2). The subcellular localization of Galpha and Galpha subunits in Cl.10 cells was studied by immunofluorescence using laser confocal microscopy (Fig. 3). The Galpha protein was preferentially localized to the plasma membranes but a faint staining was also detectable in the cytosol at the endoplasmic reticulum level. In contrast, the Galpha protein was mainly present on the perinuclear Golgi complex of Cl.10 cells although a significant staining was also seen at the plasma membrane (Fig. 3).

Figure 1: Peptide specificity of PYY receptors and PYY-induced inhibition of cAMP in Cl.10 cells. A, peptide specificity of PYY receptors was investigated with membranes from Cl.10 cells. Membranes were incubated with 0.05 nMI-PYY and increasing concentrations of unlabeled PYY (bullet), NPY (circle), or PP () as described under ``Experimental Procedures.'' Nonspecific binding was determined in the presence of 1 µM unlabeled PYY. Results are the means ± S.E. from three experiments. B, inhibition of forskolin-stimulated cAMP production in Cl.10 cells. Cellular cAMP content was determined on cells pretreated (circle) or not (bullet) with 0.4 µg/ml pertussis toxin for 18 h. Thereafter, cells were incubated in the presence of 10M forskolin and increasing amounts of PYY for 40 min at 37 °C. The cAMP content was determined as described under ``Experimental Procedures.'' Each value is the mean ± S.E. of three determinations.


Figure 2: Western blot analysis of Galpha and Gbeta subunits of G(i) and G(s) proteins in Cl.10 cells and Cl.10/alpha cells. Cell membrane proteins (50 µg/lane) were subjected to 10% acrylamide slab gel electrophoresis. After transfer onto nitrocellulose sheets, bands were revealed using antisera against the alpha/alpha, alpha/alpha(0), alpha(s), or beta subunits of G proteins. Gels were calibrated with several molecular weight marker proteins as described under ``Experimental Procedures.'' For the sake of clarity, only two protein markers are shown, i.e. ovalbumin (43 kDa) and carbonic anhydrase (29 kDa). The Galpha protein which would migrate above the Galpha protein in these electrophoresis conditions (23) was not detected in Cl.10 cells. The same holds true for the Galpha(o) protein which is not expressed in epithelial cells. For details, see ``Experimental Procedures.''


Figure 3: Localization of Galpha and Galpha subunits in Cl.10 cells by indirect immunofluorescence using confocal laser scanning microscopy. Cells grown on 12-mm glass coverslips were permeabilized with saponin and then incubated with anti-alpha (left) or anti-alpha (right) antibodies. After subsequent incubation with fluorescein isothiocyanate goat anti-rabbit antibody, cells were analyzed by confocal microscopy as described under ``Experimental Procedures.''


Transfection of Cl.10 Cells with the pcDNA3 Antisense Galpha Expression Vector

In view of the preferential localization of the Galpha protein to the plasma membranes of Cl.10 cells, this cell clone was first transfected with the antisense Galpha subunit expression vector pcDNA3/Galpha (see ``Experimental Procedures''). After transfection, selection, and cloning, 20 clones were isolated, and their level of expression of Galpha was characterized by Western blot. Among three clones with a marked decrease in Galpha content as compared to the parent Cl.10 cells or Cl.10 cells transfected with vector alone, one clone was selected because it displayed an important down-regulation in the expression of Galpha protein (>90%) as compared to Cl.10 cells transfected with vector alone (Fig. 2). This clone named Cl.10/alpha was further studied for the specificity of the Galpha protein quenching. It appeared that the Galpha protein content of Cl.10/alpha cells was not modified as assessed by Western blotting (Fig. 2). Nor was there any modification of Galpha(s) or the Gbeta-subunit protein contents in the Cl.10/alpha clone as compared to Cl.10 cells transfected with vector alone (Fig. 2). It is therefore likely that expression of antisense Galpha RNA in the Cl.10/alpha clone resulted in the selective loss of the Galpha protein. Since the Galpha protein was previously shown to be involved in cell differentiation(20, 21) , Cl.10/alpha cells were further investigated for markers of epithelial cell differentiation and compared to parent Cl.10 cells. The two cell populations exhibited similar morphology being organized as monolayers with epithelioid shapes (Fig. 4, A and B) and forming domes (Fig. 4, C and D) which are indicators of fluid transport(26) . Neither were there significant differences at the biochemical level since the aminopeptidase N activities (22.3 ± 1.2 and 20.0 ± 1.7 milliunits/mg of protein in Cl.10 cells and Cl.10/alpha cells, respectively) and the dipeptidylpeptidase IV activities (6.1 ± 0.1 and 6.0 ± 0.1 milliunit/mg of protein in Cl.10 cells and Cl.10/alpha cells, respectively) were similar in the two clones (3 experiments). Finally, the transepithelial resistance of filter-grown Cl.10 cells and Cl.10/alpha cells was also very similar i.e. 57.2 ± 4.3 and 53.4 ± 5.2 ohmsbulletcm^2, respectively (three experiments). Therefore, there was no morphological, biochemical, or electrophysiological evidence for a major change in the differentiation of Cl.10 cells upon transfection of the pcDNA3/Galpha expression vector.

Figure 4: Light microscopic appearance of Cl.10 cells and Cl.10/alpha cells. Top, light micrograph of Cl.10 cells (A) and Cl.10/alpha cells (B) grown on porous filters. Filters were fixed in Bouin's fluid and embedded in paraffin, and cross sections were stained with hematein eosin and examined. Bars, 10 µm. Bottom, phase-contrast micrograph of confluent Cl.10 cells (C) and Cl.10/alpha cells (D) grown on plastic Petri dishes. Note the presence of numerous domes, indicating the fluid transport capacities of both cell clones. Bars, 50 µm.


PYY Receptors and PYY Receptor-mediated Events in the Cl.10/alphaCell Clone

Competitive inhibition of I-PYY binding by unlabeled PYY was performed on membranes prepared from Cl.10/alpha cells and control Cl.10 cells. These experiments revealed a marked decrease in I-PYY binding in Cl.10/alpha cells. Scatchard analysis gave a straight line in both cell clones (Fig. 5A) with a marked increase of the dissociation constant in Cl.10/alpha cells (5.33 ± 1.59 nMversus 0.63 ± 0.20 nM; three experiments, p < 0.01) without a change in the binding capacity, i.e. 155 ± 20 and 145 ± 22 fmol/mg of protein (three experiments) in Cl.10/alpha cells and Cl.10 cells, respectively. These data demonstrated that the drastic loss of Galpha in Cl.10/alpha cells resulted in the conversion of all PYY receptors to a low affinity state. In this context, it is worth pointing out that the binding parameters of PYY in Cl.10/alpha cells were identical to those observed in Cl.10 cells which had been preincubated overnight with pertussis toxin (Fig. 5A) for which Scatchard analysis revealed an 8-fold increase of the dissociation constant without a change in the binding capacity. Therefore, inhibition of Galpha(i) function by pertussis toxin and decrease of Galpha content by expression of Galpha antisense RNA resulted in the same shift in the dissociation constant of PYY, suggesting that the G protein plays a pivotal role in the effector-receptor coupling of PYY receptors in Cl.10 cells. As a control, we also investigated G(s) protein-coupled PTH receptors (27) in Cl.10/alpha cells and Cl.10 cells. The specific binding of I-Tyr^0-PTH was identical in the two clones (not shown), demonstrating no alteration in PTH receptors in Cl.10/alpha cells consistent with the absence of changes in Galpha(s) protein expression after transfection with Galpha antisense RNA (Fig. 2). To further implicate the coupling of PYY receptors with the G protein in Cl.10 cells, we investigated the effect of GTPS in inhibiting I-PYY binding in Cl.10/alpha cells and control Cl.10 cells. Fig. 5B shows that GTPS was active in Cl.10 cells, whereas in the same concentration range GTPS had no effect in Cl.10/alpha cells.

Figure 5: PYY binding to Cl.10 cells and after expression of antisense Galpha RNA in the Cl.10/alpha clone: Scatchard analysis and effect of GTPS. A, saturation analysis was conducted as described under ``Experimental Procedures'' in the presence of a fixed concentration of I-PYY (0.05 nM) and increasing concentrations of unlabeled PYY. Nonspecific binding was determined in the presence of 1 µM unlabeled PYY. Binding experiments were performed on membranes prepared from Cl.10 cells (bullet), Cl.10 cells pretreated overnight with 0.4 µg/ml of pertussis toxin (circle) or Cl.10/alpha cells (). Scatchard plots were analyzed using the LIGAND computor program(22) . Results shown are from a typical experiment. Two other experiments gave similar results. B, effect of GTPS on PYY binding to membranes from Cl.10 cells (bullet) and Cl.10/alpha cells (circle). Experiments were carried out in the presence of a fixed concentration of I-PYY (0.05 nM) and increasing concentrations of GTPS. Nonspecific binding was determined in the presence of 1 µM unlabeled PYY and substracted from total binding.


As PYY inhibited cAMP production and stimulated cell growth in the mouse proximal tubule cell line PKSV-PCT(3) , we further investigated the influence of expression of Galpha antisense RNA on both processes in Cl.10/alpha and control Cl.10 cells. As shown in Fig. 6, PYY inhibited both basal and forskolin-stimulated cAMP production in Cl.10 cells. In contrast, PYY failed to alter basal and forskolin-stimulated cAMP levels in Cl.10/alpha cells (Fig. 6). Therefore, it appeared that the down-regulation of Galpha expression in Cl.10/alpha cells completely reversed the PYY receptor-mediated inhibition of cAMP production. As previously observed in F9 teratocarcinoma cells(20) , the suppression of Galpha did not change basal or forskolin-stimulated cAMP levels which were identical in Cl.10/alpha and control Cl.10 cells. This suggested that Galpha itself does not play a major role in the control of cAMP production unless it is activated by receptors such as the PYY receptor. As shown on Fig. 7, PYY stimulated the incorporation of [methyl-^3H]thymidine into DNA of Cl.10 cells, whereas it had no effect in Cl.10/alpha cells. This is in line with the fact that cell growth in Cl.10 cells is a forskolin sensitive cAMP-dependent process (data not shown). Altogether these data support the notion that Galpha is responsible for the coupling of PYY receptors to adenylyl cyclase and the subsequent stimulatory effect on cAMP-dependent incorporation of [methyl-^3H]thymidine into DNA. However, the decrease in [methyl-^3H]thymidine incorporation into DNA in Cl.10/alpha cells (Fig. 7), where there is no change in basal cAMP level (Fig. 6) suggested that G might also participate in cAMP-independent pathway(s) for the control of DNA synthesis in Cl.10 cells. The microtubule-associated protein kinase cascade which can be activated by G(28, 29, 30) is a good candidate.

Figure 6: Effect of PYY on basal and forskolin-stimulated cAMP levels in Cl.10 cells or after expression of antisense Galpha RNA in Cl.10/alpha cells or antisense Galpha RNA in Cl.10/alpha cells. PYY effect on cAMP production was investigated in Cl.10, Cl.10/alpha cells or Cl.10/alpha cells. Cells were incubated with (right) or without (left) 10M forskolin in the absence (hatched bars) or in the presence (solid bars) of 1 µM PYY for 30 min at 37 °C. The cellular cAMP content was then determined as described under ``Experimental Procedures.'' Each value is the mean ± S.E. of three determinations. *p < 0.001 versus control without PYY; NS, nonsignificant.


Figure 7: Effect of PYY on [methyl-^3H]thymidine incorporation into DNA of Cl.10 cells or after expression of antisense Galpha RNA in Cl.10/alpha cells. Two days after seeding, cells were cultured in fetal calf serum-deprived medium in the presence (solid bars) or absence (hatched bars) of 0.1 µM PYY for 18 h. Cells were then pulsed for the last 6 h of incubation with 0.5 µCi/ml [methyl-^3H]thymidine as described under ``Experimental Procedures.'' Values are the means ± S.E. from 12 experiments. *p < 0.005 versus control without PYY; NS, nonsignificant.


Expression of Galpha Antisense RNA in Cl.10 Cells

Although Galpha appeared to be necessary and sufficient to account for the PYY receptor-mediated effects in Cl.10 cells, we decided to confirm this by down-regulating the expression of the other Galpha(i) protein in Cl.10 cells, i.e. Galpha (see Fig. 2). The antisense Galpha subunit expression vector pcDNA3/Galpha (see ``Experimental Procedures'') was therefore transfected into Cl.10 cells, and after selection we isolated a clone (Cl.10/alpha) that displayed a large reduction in the expression of the Galpha protein (>80%) with no alteration in the expression of the Galpha protein (Fig. 8A). Nor was there any change in the expression Galpha(s) or Gbeta subunits (not shown). As observed in the parent Cl.10 cells, the binding of I-PYY was inhibited by GTPS in Cl.10/alpha cells [55% inhibition for 0.1 mM GTPS] in contrast to that which had been observed in Cl.10/alpha (Fig. 5). As expected, Scatchard analysis of PYY binding in Cl.10/alpha cells and control Cl.10 cells (Fig. 8B) gave similar results, indicating that the dissociation constant of PYY receptors was not modified after specific down-regulation of the expression of the Galpha protein. Likewise, PYY inhibited basal and forskolin-stimulated cAMP production in Cl.10/alpha cells in the same manner as in Cl.10 cells (Fig. 6).

Figure 8: Western blot analysis of Galpha and Galpha proteins and Scatchard plot of PYY binding after expression of antisense Galpha RNA in the Cl.10/alpha clone. A, Western blot analysis of Galpha and Galpha subunits of G(i) proteins in membranes prepared from Cl.10/alpha and control Cl.10 cells. Cell membrane proteins (50 µg/lane) were subjected to 10% acrylamide slab gel electrophoresis. After transfer on nitrocellulose sheets, bands were revealed using antisera against the alpha/alpha or alpha/alpha(0) subunits of G proteins. Gels were calibrated with several molecular weight marker proteins as described under ``Experimental Procedures.'' For the sake of clarity, only two protein markers are shown on the figure, i.e. ovalbumin (43,000) and carbonic anhydrase (29,000). For details, see ``Experimental Procedures.'' B, PYY binding to membranes from Cl.10/alpha cells (circle) and control Cl.10 cells (bullet). Membranes were incubated with I-PYY (0.05 nM) and increasing concentrations of unlabeled PYY. Nonspecific binding was determined in the presence of 1 µM unlabeled PYY. Results are from a typical experiment. Another experiment gave similar data. See ``Experimental Procedures'' for details.


DISCUSSION

The present investigation which takes advantage of the powerful antisense RNA technology is the first to demonstrate the coupling of PYY receptors to the G protein. This was possible because selection of 39 bases of the 5`-noncoding region of Galpha or Galpha(20) for use as antisense templates provided the necessary nucleotide sequence specificity, e.g. only 48% identity in this region(31, 32) , whereas selection of templates in the coding region with >85% identity (31, 32) would have probably failed to ensure such specificity. In fact, the isolation of clones after transfection of Cl.10 cells with antisense Galpha or Galpha expression vectors resulted in stable cell lines which showed a 90% decrease of Galpha (Cl.10/alpha cells) and 80% decrease of Galpha (Cl.10/alpha cells), respectively. The mechanism whereby the production of antisense RNA in those cells blocks the expression of targeted proteins is not known but hybridization of antisense RNA with the corresponding mRNA was shown to prevent translation and/or to enhance mRNA degradation (reviewed in (33) ).

A great deal of evidence argues for an exclusive role of G among other candidate G(i) proteins for mediating PYY receptor signal transduction in the mouse kidney proximal tubule cell clone Cl.10 isolated from PKSV-PCT cells. Thus, the Cl.10/alpha clone in which the synthesis of the Galpha protein was down-regulated by expression of antisense Galpha RNA exhibits the following properties: (i) an increase of the dissociation constant of PYY receptors which was identical to that observed when the untransfected Cl.10 cells had been pretreated with pertussis toxin. Since Galpha and Galpha are substrates for pertussis toxin and Galpha is not present in Cl.10 cells, these data alone strongly suggest that Galpha does not participate significantly in the direct coupling of PYY receptors with G proteins. (ii) The inhibition of PYY binding by GTPS could not be observed, again ruling out a major role of Galpha in controlling the dissociation of PYY from PYY receptors. (iii) Basal and forskolin-stimulated cAMP levels as well as incorporation of [methyl-^3H]thymidine into DNA were totally unaffected by PYY, suggesting that Galpha was also crucial for PYY receptor-mediated events. Finally, the fact that the Cl.10/alpha clone, in which the synthesis of the Galpha protein was specifically down-regulated by expression of antisense Galpha RNA, did not exhibit any modification in the dissociation constant of PYY receptor or in the sensitivity to GTPS further confirmed that Galpha was not coupled to PYY receptors in Cl.10 cells. Furthermore, the subcellular distribution of Galpha and Galpha in Cl.10 cells, as determined by confocal laser microscopy, is consistent with the coupling of PYY receptors to Galpha rather than to Galpha. Indeed, Galpha is found associated mainly with plasma membranes where PYY receptors (3) and adenylyl cyclase (18) are located; in contrast Galpha is preferentially localized on the perinuclear Golgi complex. The localization of Galpha is in line with recent observations indicating that Galpha is involved in intracellular processes in epithelial cells such as autophagic sequestration (34) and Golgi trafficking(35, 36, 37) . Finally, the absence of Galpha not only in Cl.10 cells but also in other PYY receptor-containing epithelia, such as the rat intestinal epithelium(1, 23) , lends support to the fact that Galpha is not coupled to PYY receptors, at least in epithelial cells. By using antibodies to Galpha(i) subunits, the PYY-mediated inhibition of adenylyl cyclase, which occurs through the Y2 subtype of NPY receptor in a neuronal cell line, was shown to involve both G and G, with G possibly playing the more important role(38) . This contrasts with the exclusive coupling of PYY receptors with G in renal proximal tubule cells. Whether the difference is related to receptors, i.e. NPY Y2 receptors (38) versus PYY-preferring receptors (this study) and/or tissues, i.e. neuronal cells (38) versus epithelial cells (this study), is not known. What is known is that both G and G participate in the inhibition of adenylyl cyclase (39, 40, 41, 42) and that a specific receptor may signal through distinct Galpha(i) proteins to inhibit adenylyl cyclase(42) .

In view of the fact that G protein subunits are generally considered to be expressed in large excess over individual G protein-coupled receptors(43) , it was intriguing to observe that a 90% decrease of the expression of Galpha in Cl.10/alpha cells totally abolished the regulation of PYY binding by GTPS as well as PYY receptor-mediated inhibition of cAMP production and stimulation of [methyl-^3H]thymidine incorporation into DNA. We have no definitive answer to this issue. It can be hypothesized that: (i) a threshold amount of Galpha protein is necessary to interact significantly with PYY receptors. After transfection of Cl.10 cells with the pcDNA3/Galpha expression vector, we have isolated two other cell clones which exhibited a 60% decrease in Galpha content as compared to the parent Cl.10 cells. We have examined PYY receptors and PYY-mediated inhibition of cAMP production in one of these clones. We obtained essentially the same data as in Cl.10/alpha cells with a 90% decrease in Galpha content, i.e. an increase in the dissociation constant of PYY receptors and the failure of PYY to inhibit basal and forskolin-stimulated cAMP levels. (^2)Although Western blotting cannot be considered as a quantitative method for measuring protein levels, these data show that partial inhibition of Galpha expression is sufficient for uncoupling PYY receptors and suggest that a critical amount of Galpha is necessary to maintain the functional response and high affinity ligand binding. (ii) The remaining low amount of Galpha protein in Cl.10/alpha cells is not localized to the plasma membrane where PYY receptors are present and functionally coupled to adenylyl cyclase(3) . Confocal laser microscopy of the remaining Galpha protein in Cl.10/alpha cells (not shown) did not favor this hypothesis. However, in view of the importance of membrane organization in G protein mechanisms(44) , we cannot exclude the possibility that the nonhomogeneous localization of Galpha(i) to patches within the plasma membrane (45) was modified in Cl.10/alpha cells.

Types V and VI appear to be the dominant forms of adenylyl cyclase in peripheral tissues(18) . The three isoforms of Galpha(i) have been shown to be equally potent and efficacious in inhibiting Galpha(s)- and forskolin-stimulated type V and type VI adenylyl cyclase(18, 46) . Therefore, it is not surprising that down-regulation of the expression of Galpha in Cl.10/alpha cells abolished PYY receptor-mediated inhibition of adenylyl cyclase and subsequent cAMP-dependent effects. The reason why basal as well as forskolin-stimulated levels of cAMP are identical in Cl.10 and Cl.10/alpha cells is less clear. This phenomenon has been previously observed in F9 teratocarcinoma cells expressing Galpha antisense RNA (20) and could be due to the fact that multiple G protein subunits, including Galpha, Galpha, and also beta(18) , participate in the inhibiting tonus of adenylyl cyclase in Cl.10 cells and/or that G-mediated inhibition of adenylyl cyclase is strictly dependent on the activation of inhibitory receptors, such as PYY receptors, by agonists.

In conclusion, our antisense RNA technology studies indicate that PYY receptors are coupled with a strict specificity to Galpha in the proximal tubule Cl.10 cell clone and that Galpha is responsible for PYY receptor-mediated inhibition of adenylyl cyclase and stimulation of cell growth. These findings further document the mechanism of PYY receptor-mediated responses in epithelial cells.

FOOTNOTES

*
This work was supported in part by NATO, Association Française de Lutte contre la Mucoviscidose Grant AFLM (to M. L.), Association pour la Recherche sur le Cancer Grant ARC 6404 (to M. L.), Faculté de Médecine X. Bichat, Université Paris VII, and Centre National de la Recherche Scientifique (CNRS). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: INSERM U410, Faculté de Médecine X. Bichat, BP 416, 75870 Paris Cedex 18, France. Fax: 33-1-42-28-87-65 (laboratory) or 33-1-44-85-61-24 (M. L.).

(^1)
The abbreviations used are: PYY, peptide YY; I-PYY, I-Tyr-monoiodo-PYY; NPY, neuropeptide Y; PP, pancreatic polypeptide; GTPS, guanosine 5`-O-(thiotriphosphate); G(i), inhibitory regulatory GTP-binding protein of adenylyl cyclase; G(s), stimulatory regulatory GTP-binding protein of adenylyl cyclase; BSA, bovine serum albumin; SV40, simian virus 40; Tag, large T antigen; PMSF, phenylmethylsulfonyl fluoride; TLCK, Nalpha-p-tosyl-L-lysine chloromethyl ketone; PIPES, piperazine-N,N`-bis-(2-ethanesulfonic acid); PTH, parathyroid hormone; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium.

(^2)
T. Voisin, A. M. Lorinet, and M. Laburthe, unpublished results.

ACKNOWLEDGEMENTS

We thank Dr. Alain Vandewalle for his kind gift of PKSV-PCT cells and Dr. Jean-François Fléjou for his efforts in some of the morphological studies. We also thank the IFR Cellules épithéliales for confocal microscopy facilities.

Note Added in Proof-While this paper was under review, the cloning of a cDNA encoding a human Y2 subtype of NPY receptor was reported (J. Biol. Chem.270, 22661-22664, 1995).

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