©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Degradation of G/G Is Accelerated by Agonist Occupancy of , , and Adrenergic Receptors (*)

(Received for publication, March 29, 1995; and in revised form, May 9, 1995)

Alan Wise , Tae Weon Lee , David J. MacEwan , Graeme Milligan (§)

From the Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Cells of clones of rat 1 fibroblasts transfected to express the molecularly defined , , or adrenoreceptors and prelabeled with myo-[H]inositol were each shown to generate high levels of inositol phosphates when exposed to the adrenoreceptor agonist phenylephrine. Maintained exposure of each of these cells to phenylephrine resulted in a large down-regulation of the receptors and also a marked down-regulation of cellular levels of both of the phosphoinositidase C-linked G-proteins G and G. To examine the mechanism of phenylephrine-induced down-regulation of G and G, pulse-chase S-amino acid labeling experiments were performed with each of the , , and adrenoreceptor-expressing cell lines. The rate of degradation of G/G, which was adequately modeled by a monoexponential with half-life between 33 and 40 h in each of the cell lines in the absence of agonist, was accelerated substantially (some 4-fold) in the presence of phenylephrine. By contrast, the rate of degradation of the G-protein G2, which would not be anticipated to be activated by members of the adrenoreceptor family, was unaltered by the presence of phenylephrine. Levels of mRNA encoding G and G were not substantially altered by exposure of the cells to phenylephrine in any of the cell lines studied.


INTRODUCTION

There are multiple closely related adrenoreceptor subtypes that have been indicated by comparisons of the pharmacological profiles of ligands in different tissues(1) . Three distinct adrenoreceptor cDNA species have currently been isolated(2, 3, 4, 5) , , , and , but there has been considerable debate as to how closely these reflect the pharmacologically defined subtypes (see (1) for review), even if all of these cDNA species show the same signal transduction mechanisms following their heterologous expression in cell lines. This has arisen partially because of the relative pharmacological similarity of the subtypes and partially because the first isolated cDNA species were derived from a number of different species(1, 6) . Current opinion favors the view that the cloned adrenoreceptor corresponds to the pharmacologically defined adrenoreceptor(1) , that the cloned and pharmacologically defined adrenoreceptors are identical(1) , and that the cloned adrenoreceptor may be the equivalent of the pharmacologically defined adrenoreceptor(1) .()Despite these ongoing concerns, it is generally accepted that the primary signaling function of adrenoreceptor subtypes is to stimulate the hydrolysis of inositol-containing phospholipids via interaction with pertussis toxin-insensitive G-proteins of the G/G family (7) with subsequent activation of phospholipase C activity(7) .

Although it has been well established that sustained exposure of many G-protein-coupled receptors to agonist can result in a reduction in cellular levels of the receptor (a process known as down-regulation), it has only been in the recent past that agonist-mediated reduction in cellular levels of G-proteins has also been observed (see (8) for review). Even in such cases, information on the mechanism(s) responsible for these effects is fragmentary(8) . To attempt to address this point directly, in the present report we have used clonal cell lines derived from rat 1 fibroblasts following stable transfection with the cloned rat , the hamster , and the bovine adrenoreceptor cDNA species. We note that in cells expressing each of the three receptor species, sustained exposure to phenylephrine results in a large, selective down-regulation of G and G as well as in down-regulation of the receptors. These are the G-proteins that have been demonstrated to couple adrenoreceptors to phosphoinositidase C activity and the hydrolysis of inositol-containing phospholipids(7) . In each case, we demonstrate that the basal rate of turnover of these G-proteins is described adequately by a monoexponential with a t between 33 and 40 h, while upon exposure to agonist, this rate of degradation is markedly increased, such that a substantial fraction of the cellular content of these two G-proteins now has a t of between 7 and 10 h. In contrast, no marked alterations in amounts of mRNA encoding the G-proteins was observed following agonist treatment.

These data indicate that G-proteins activated by a receptor are degraded considerably more rapidly than those in the inactive state and provide a mechanistic explanation for how receptor agonists can control the cellular content of G-proteins, which interact with that receptor.


EXPERIMENTAL PROCEDURES

Materials

All materials for tissue culture were supplied by Life Technologies, Inc. (Paisley, Strathclyde, Scotland). [H]Prazosin (24 Ci/mmol) and myo-[2-H]inositol (17.6 Ci/mmol) were obtained from Amersham International. TranS-label (1180 Ci/mmol) was purchased from ICN Biomedicals Inc. All other chemicals were from Sigma or Fisons plc and were of the highest purity available.

Cells

Rat 1-fibroblasts transfected to stably express the rat , the hamster , and the bovine adrenoreceptor cDNA species were obtained from Dr. D. E. Clarke (Syntex, Palo Alto, CA) under license to Syntex from Dr. L. F. Allen (Duke University, NC).

Cell Culture

Rat 1 fibroblasts stably expressing adrenoreceptor subtypes were maintained in tissue culture in Dulbecco's modified Eagle's medium (DMEM)()containing 5% (v/v) newborn calf serum, glutamine, penicillin, and streptomycin. For most experiments, cells were grown until close to confluency and then either harvested or subcultured in a 1:10 ratio. In preparation for labeling with TranS-label for the pulse-chase experiments, cells were trypsinized and seeded in 6-well culture plates. At about 70% confluency, 2/3 of the growth medium was replaced with DMEM lacking methionine and cysteine, supplemented with glutamine, antibiotics, and 50 µCi/ml TranS-label (final concentration in well). After the labeling period (16 h), the radioactive medium was removed, and the now close to confluent cell layer was washed once with 1 ml of normal DMEM culture medium. They were subsequently incubated in 1.5 ml/well normal DMEM culture medium in the presence or absence of phenylephrine (100 µM). At appropriate times, the medium was removed and cells were dissolved in 1% (w/v) SDS (200 µl/well). The cell suspension was heated in a screw-cap test tube to 100 °C for 20 min to denature proteins and nucleic acids; then, samples were either stored at -20 °C or processed directly for immunoprecipitation.

Immunoprecipitation of S-Labeled G-proteins

To the 200 µl of SDS-denatured cell suspension was added 800 µl of solubilization buffer (1% (w/v) Triton X-100, 10 mM EDTA, 100 mM NaHPO, 10 mM NaF, 100 µM NaVO, 50 mM HEPES, pH 7.2) and 100 µl of Pansorbin (Calbiochem). Samples were incubated at 4 °C with continuous rotation for 1-2 h for nonspecific preclearing. Following centrifugation of the samples (16,000 g, 2 min, 4 °C), the supernatant was collected and subjected to immunoprecipitation by addition of 100 µl of protein A-agarose along with 10 µl of the specific G-protein antiserum and incubated at 4 °C for 4 h. Immune complexes were then recovered by centrifugation (16000 g, 30 s at 4 °C) and washed by resuspension-centrifugation three times each with 1 ml of wash buffer (1% (w/v) Triton X-100, 100 mM NaCl, 100 mM NaF, 50 mM NaHPO, 50 mM HEPES, pH 7.2, 0.5% (w/v) SDS). The final protein A-agarose pellet was resuspended in Laemmli sample buffer, heated at 100 °C for 5 min, and electrophoresed by SDS-PAGE (10% (w/v) acrylamide). Following resolution of the proteins, the gel was stained with Coomassie Blue and then dried. The dried gels were exposed either to photographic film or to phosphor storage screen autoradiography according to manufacturer's instructions using a Fujix bio-imaging analyzer linked to an Apple Macintosh Quadra 650 personal computer. Autoradiographs were analyzed such that the time-course data were expressed as a percentage of the control-specific G/G protein radiolabeling found at zero time.

Immunological Studies

The generation and specificities of the various antisera used in this study have been fully described previously(9, 10) . Each antiserum was produced in a New Zealand White rabbit using a conjugate of a synthetic peptide with keyhole-limpet hemeocyanin (Calbiochem) as antigen. Membrane samples were resolved by SDS-PAGE (10% (w/v) acrylamide gels) overnight at 60 V. Proteins were subsequently transferred to nitrocellulose (Schleicher and Schuell) and blocked for 3 h in 5% (w/v) gelatin in phosphate-buffered saline (PBS), pH 7.5. Primary antisera were added in 1% gelatin in PBS containing 0.2% (v/v) Nonidet P-40 and incubated overnight. The primary antiserum was removed, and the blot was washed extensively with PBS containing 0.2% Nonidet P-40. Secondary antiserum (donkey anti-rabbit IgG coupled to horseradish peroxidase) in 1% gelatin, PBS, 0.2% Nonidet P-40 was added and left for 3 h. After removal of the secondary antiserum, the blot was washed extensively as above and developed with o-dianisidine hydrochloride (Sigma) as substrate for horseradish peroxidase as described(11) .

Preparation of Membranes

Membrane fractions were prepared from cell pastes that had been stored at -80 °C following harvest. Cell pellets were resuspended in 5 ml of 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5 (buffer A), and rupture of the cells was achieved with 25 strokes of a hand-held Teflon on-glass homogenizer. Unbroken cells and nuclei were removed from the resulting homogenate by centrifugation at 500 g for 10 min in a Beckman L5-50B centrifuge with a Ti-50 rotor. The supernatant fraction was then centrifuged at 48,000 g for 10 min, and the pellet was washed and resuspended in 10 ml of buffer A. Membrane fractions were recovered following a second centrifugation at 48,000 g for 10 min, and pellets were resuspended in buffer A to a final protein concentration of 1-3 mg/ml and stored at -80 °C until required.

[H]Prazosin Binding Experiments

Binding assays were initiated by the addition of 5-15 µg of protein to an assay buffer (50 mM Tris-HCl, 0.5 mM EDTA, pH 7.4) containing [H]prazosin (12, 13) (0.005-1 nM in saturation assays and 0.1 µM for competition assays) in the absence or presence of increasing concentrations of the test drugs (500 µl, final volume). Nonspecific binding was determined in the presence of 10 µM phentolamine. Reactions were incubated for 30 min at 25 °C, and bound ligand was separated from free by vacuum filtration through GF/B filters. The filters were washed with 2 5 ml assay buffer, and bound ligand was estimated by liquid scintillation spectrometry.

Expression of G in Escherichia coli

An expression system utilizing the promoter for bacteriophage T7 RNA polymerase in the prokaryotic expression vector pT7.7 (14) was employed to overexpress cDNA encoding hamster G in E. coli. To ensure maximal expression of G, a PCR-based strategy described in (15) was used to position the cDNA downstream from the T7 promoter at the optimum distance from the ribosome binding site. The expression construct was transformed into E. coli BL21 (DE3) cells, which contain a single copy of the gene for T7 RNA polymerase under control of the isopropyl-1-thio--D-galactopyranoside-inducible lac UV5 promoter. Isopropyl-1-thio--D-galactopyranoside-induced cells were recovered by centrifugation and stored as pastes at -80 °C.

Adrenoreceptor Regulation of Inositol Phosphate Production

Cells were seeded in 24-well plates and labeled to isotopic equilibrium by incubation with 1 µCi/ml myo-[2-H]inositol in 0.5 ml inositol-free DMEM containing 1% (v/v) dialyzed newborn calf serum for 36 h. On the day of experiments, the labeling medium was removed, and cells were washed twice with 0.5 ml of Hank's buffered saline, pH 7.4, containing 1% (w/v) bovine serum albumin and 10 mM glucose (HBG). Cells were then washed twice for 10 min with HBG supplemented with 10 mM LiCl (HBG/LiCl) and subsequently stimulated with agonist in HBG/LiCl for 20 min. All incubations were performed at 37 °C. Reactions were terminated by the addition of 0.5 ml of ice-cold methanol. Cells were then scraped and transferred to vials, and chloroform was added to a ratio of 1:2 (CHCl:MeOH). Total inositol phosphates were extracted for 30 min prior to the addition of chloroform and water to a final ratio of 1:10:9 (CHCl:MeOH:HO). The upper phase was taken, and total inositol phosphates were analyzed by batch chromatography on Dowex-1-formate as previously described(16, 17) .

Reverse Transcriptase-Polymerase Chain Reaction

The reverse transcriptase-PCR procedure was essentially as previously described(18) .

RNA Extractions

Total RNA was extracted using RNAzol B (Biogenesis). Purity and quantification of RNA were assessed by spectrophotometric A/A ratios.

Reverse Transcription

Samples of 5-10 µg of RNA (20 µl) were denatured by incubation at 65 °C for 10 min followed by chilling on ice and reverse transcribed in 33 µl of reaction mixture using first strand cDNA synthesis kit (Pharmacia Biotech Inc.) as detailed by the manufacturer. Incubation was carried out at 37 °C for 1 h. The reactions were terminated by heating samples at 95 °C for 5 min followed by transfer to ice.

Polymerase Chain Reaction

PCR reactions were carried out using the following primers: sense, 5`-TTGGAAGGAGCCAGTGC3`; / antisense, 5`-GAAGGCGCGCTTGAACT-3`; sense, 5`-ACAAGGAATGCGGAGTC-3`; sense, 5`-TTCTCCGTGAGACTGCT-3`; antisense, 5`-CCAGGTCCTTGTGCTGT-3`; G sense, 5`CCACCTGAATTCGAGCATGCC-3`; G antisense, 5`-GCGTGGGTCCTCTCCGGGCTCGGG-3`; G sense, 5`-ATGACTTGGACCGTGTAGCCGACC-3`; G sense, 5`-ACGTGGACCGCATCGCCACAGTAG-3`; G/G antisense, 5`-CCATGCGGTTCTCATTGTCTGACT-3`.

Amplifications were performed in 50 µl of buffer containing 25 pmol of primers and 2.5 units of Taq polymerase (Promega) using a HYBAID Omnigene temperature cycler. Amplifications of adrenoreceptor subtypes were carried out as follows: 95 °C/5 min, 49 °C/30 s, 72 °C/1 min (1 cycle); 95 °C/30 s, 49 °C/30 s, 72 °C/1 min (30 cycles); 95 °C/30 s, 49 °C/30 s, 72 °C/5 min (1 cycle). Conditions used for amplification of G, G, and G were: 95 °C/5 min, 60 °C/30 s, 72 °C/1 min (1 cycle); 95 °C/30 s, 60 °C/30 s, 72 °C/1 min (30 cycles); 95 °C/30 s, 60 °C/30 s, 72 °C/5 min (1 cycle). Reaction products were then separated by 1.5-1.75% agarose gel electrophoresis. In each case, the size of the generated product was that anticipated from the selected primers.

Northern Analysis

Total RNA (20-30 µg) was run on 1% (w/v) formaldehyde gels and transferred to Hybond N nylon membrane (Amersham). G-, G-, and G-specific probes were made by PCR-amplifying reverse transcribed total RNA as described above. Amplified products were electrophoresed on 1% (w/v) agarose gels, excised, and labeled by random priming (Rediprime labeling system, Amersham International). RNA blots were hybridized at 42 °C in 50% (v/v) formamide, 2 Denhardt's solution (0.04% (w/v) Ficoll, 0.04% (w/v) polyvinylpyrrolidone, 0.04% (w/v) bovine serum albumin), 5 SSPE (750 mM NaCl, 5 mM EDTA, 44 mM NaHPO, pH 7.4), 0.1% (w/v) SDS, and 50 µg/ml fragmented salmon sperm DNA and then washed at 55 °C in 2 SSC (300 mM NaCl, 30 mM NaCHO, pH 7.0), 0.1% (w/v) SDS.

Data Analysis

Analysis was performed using the Kaleidagraph (version 2.1) curve fitting package with an Apple Macintosh computer.


RESULTS

Rat 1 fibroblast cells transfected to stably express each of the three cloned adrenoreceptor subtypes were used for the present study. The presence of , , and adrenoreceptor mRNA in appropriately transfected cells only was confirmed by reverse transcriptase-PCR analysis of RNA isolated from untransfected parental rat 1 cells and each of the clonal cell lines examined in this study using oligonucleotide primers specific for each of the three molecularly defined adrenoreceptor subtypes (data not shown).

Membranes derived from all three clonal cell lines were examined for their levels of expression of the adrenoreceptor subtypes by measuring the specific binding of the adrenoreceptor antagonist [H]prazosin. , , and adrenoreceptor subtypes were found to be expressed at levels of 2.1 ± 0.1, 2.8 ± 0.5, and 7.0 ± 0.9 pmol/mg membrane protein, with K values for the binding of [H]prazosin of 110 ± 20, 76 ± 13, and 150 ± 26 pM, respectively (data not shown). Displacement of specific [H]prazosin binding by varying concentrations of the adrenoreceptor agonist phenylephrine was achieved with pIC values (and Hill coefficients) of 5.1 ± 0.1 (n = 0.76 ± 0.09), 4.7 ± 0.2 (n = 1.06 ± 0.07), 5.2 ± 0.1 (n 0.90 ± 0.06), respectively, for the , , and adrenoreceptor subtypes (Fig. 1). Addition of the poorly hydrolyzed analogue of GTP, Gpp(NH)p (100 µM), to such assays produced no significant alterations in the positions of such displacement curves (5.1 ± 0.1 (n = 0.99 ± 0.03), 4.6 ± 0.1 (n = 0.89 ± 0.05), 4.9 ± 0.2 (n 1.12 ± 0.16) for the , , and adrenoreceptor subtypes) (Fig. 1).


Figure 1: The ability of phenylephrine to displace [H]prazosin binding in membranes of adrenoreceptor-expressing cells. The ability of phenylephrine in the absence (filledsymbols) or presence (opensymbols) of Gpp(NH)p (100 µM) to displace the specific binding of [H]prazosin to membranes of (I), (II), and (III) adrenoreceptor-expressing rat 1 fibroblasts was assessed as described under ``Experimental Procedures.'' Typical examples, representative of four independent experiments, are displayed (see ``Results'' for details).



Accumulation of inositol phosphates in LiCl (10 mM)-treated adrenoreceptor subtype-expressing cells, which had been labeled for 36 h with myo-[2-H]inositol, was found to be markedly stimulated by phenylephrine (Fig. 2), however, in a manner that was insensitive to pretreatment of the cells with pertussis toxin (25 ng/ml, 16 h) (data not shown), confirming a functional coupling of these receptors to the cellular G-protein machinery. Half-maximal stimulation of inositol phosphate generation in response to phenylephrine was produced with between 0.3 and 1 µM agonist in a range of experiments with each of the three adrenoreceptor subtype-expressing cell lines (Fig. 2).


Figure 2: The adrenoreceptor subtypes all cause stimulation of inositol phosphate production. Rat 1 fibroblasts transfected to stably express the (I), (II), and adrenoreceptor subtypes (III) were labeled with myo-[2-H]inositol (1 µCi/ml) for 36 h prior to treatment with varying concentrations of phenylephrine for 20 min. Total inositol phosphates were measured as described under ``Experimental Procedures.'' Stimulation of inositol phosphate generation was found to be dose dependent with an EC for phenylephrine between 0.3 and 1.0 µM for all three cell lines in a range of experiments. The data displayed are the mean of triplicate assays from representative experiments; bars represent S.E.



Sustained exposure of the three cell lines to phenylephrine (100 µM, 6 h) led to a marked reduction of detectable adrenoreceptors in all three cases (Table 1). Sustained exposure (16 h) of cells expressing each of the three adrenoreceptor subtypes to varying concentrations of phenylephrine also resulted in a reduction of membrane-associated levels of a combination of the phosphoinositidase C-linked G-proteins G and G by, maximally, some 50-70% as determined by immunoblotting of membranes of agonist-treated and untreated cells with antiserum CQ(9) , which identifies the C-terminal decapeptide, which is entirely conserved between these two closely related G-proteins (Fig. 3). No significant alterations in immunologically detected levels of other G-protein subunits expressed by these cells (G, G) were noted to be associated with phenylephrine treatment (data not shown). Down-regulation of G/G was found to be dose dependent with an EC for phenylephrine close to 600 nM for all three adrenoreceptor subtype-expressing cells (Fig. 3). In cells expressing the and adrenoreceptor subtypes, half-maximal effects in response to a maximally effective concentration of phenylephrine (1 mM) were produced after 8 h, whereas in cells expressing the subtype, this figure was determined to be 4 h (Fig. 4), a difference that might reflect the higher levels of expression of the receptor in these cells (see above). In all cells treated with agonist over a sustained period (16 h), a similar membrane-bound plateau level of G/G was established at some 30-50% of that present in untreated cells.




Figure 3: Phenylephrine - mediated down-regulation of G/G levels in membranes of adrenoreceptor subtype-expressing rat 1 fibroblasts. Membranes (15 µg) derived from (a), (b), and (c) adrenoreceptor-expressing rat 1 fibroblasts, which were either untreated or had been treated with varying concentrations of phenylephrine for 16 h, were resolved by SDS-PAGE (10% (w/v) acrylamide) and then immunoblotted using the anti-G/G antiserum CQ as primary reagent. Quantitative analysis from these studies on the adrenoreceptor-expressing cell line is displayed in d.




Figure 4: Time course of agonist-mediated down-regulation of G/G. Membranes (15 µg) derived from (a), (b), and (c) adrenoreceptor-expressing rat 1 fibroblasts either untreated or treated with 1 mM phenylephrine for the times indicated were resolved by SDS-PAGE (10% (w/v) acrylamide) and subsequently immunoblotted using antiserum CQ as described under ``Experimental Procedures.'' Quantitative analysis from these studies on the adrenoreceptor-expressing cell line is displayed in d.



The amount of expressed recombinant G in whole cell extracts of E. coli BL21 (DE3) cells and of G/G in the adrenoreceptor-expressing cells was estimated by comparison with known levels of purified G/G following immunoblotting with antiserum CQ (Fig. 5). Standard curves for the reaction of recombinant G with this antiserum was obtained using various dilutions of the prokaryotically expressed G-protein subunit. Such standard curves allowed measurement of the amounts of G/G in membranes of all three adrenoreceptor subtype-expressing rat 1 fibroblast clones. The steady-state level of G/G was found to be similar in all three clonal cell lines (46 pmol/mg membrane protein). In a typical example, as displayed in Fig. 5, sustained challenge of the adrenoreceptor-expressing cells with a maximally effective concentration of phenylephrine (1 mM, 16 h) resulted in membrane-associated immunodetectable G/G being reduced to 16 pmol/mg membrane protein.


Figure 5: Quantitation of G/G levels in adrenoreceptor subtype-expressing rat 1 fibroblasts. E. coli BL21 DE3 cells were transformed with hamster G cDNA subcloned into the prokaryotic expression vector pT7.7 (see ``Experimental Procedures''). Cultures of transformants were induced to express recombinant G by the addition of 1 mM isopropyl-1-thio--D-galactopyranoside and grown for a further 4 h prior to recovery by centrifugation. Known quantities of purified G/G from liver (lane1 = 0, lane 2 = 10, lane 3 = 50, lane 4 = 75, lane 5 = 100, lane 6 = 150, and lane 7 = 200 ng) were resolved by SDS-PAGE together with varying amounts of E. coli extract-expressing recombinant G (lane8 = 125, lane9 = 250, and lane10 = 500 ng) and membranes (15 µg) from adrenoreceptor-expressing rat 1 fibroblasts (lanes11 and 12), untreated (lane11) or treated with phenylephrine (1 mM, 16 h) (lane12). In the example displayed, levels of G/G were calculated to be 46 pmol/mg membrane protein in untreated cells, whereas in cells treated with agonist over a sustained period, levels of G/G were reduced to 16 pmol/mg membrane protein.



G and G comigrate in 10% (w/v) acrylamide SDS-PAGE. Therefore, to determine both the relative levels of expression of these two phosphoinositidase C-linked G-proteins in the rat 1-derived clonal cell lines and whether phenylephrine-mediated down-regulation of G/G in any of these cells was selective for either of these highly homologous G-protein subunits, membranes from untreated and phenylephrine (1 mM)-treated cells were resolved by SDS-PAGE using 12.5% (w/v) acrylamide gels containing 6 M urea, conditions that we have previously shown to be effective in resolving these G-proteins(19, 20, 21) . Fig. 6demonstrates that steady-state levels of G were substantially (over 2-fold) greater than G in these cells and that phenylephrine-driven down-regulation of membrane-associated G and G from the clonal cell lines expressing each of the three adrenoreceptor subtypes was non-selective between these G-protein subunits.


Figure 6: Phenylephrine treatment of adrenoreceptor subtype-expressing cells results in down-regulation of both G and G. Membranes (60 µg) from (lanes1 and 2), (lanes3 and 4), and (lanes5 and 6) adrenoreceptor-expressing rat 1 fibroblasts, untreated (lanes2, 4, and 6) or treated with phenylephrine (1 mM, 16 h) (lanes1, 3, and 5), were resolved by SDS-PAGE in a 12.5% (w/v) acrylamide, 0.0625% (w/v) bis-acrylamide gel system containing 6 M urea and subsequently immunoblotted as in Fig. 4.



To address the mechanism of phenylephrine-mediated reduction in membrane-bound levels of G/G, cells of the adrenoreceptor subtype-expressing clones were incubated with TranS-label for 16 h, and subsequently the decay of S with time in immunoprecipitated G/G was monitored in cells that were either untreated or treated with phenylephrine (100 µM) (Fig. 7-9). Analysis of the rate of decay of S-labeled G/G indicated that in the untreated (Fig. 7), (Fig. 8), and (Fig. 9) adrenoreceptor subtype-expressing cells, this process was described adequately by monoexponentials with estimated half-time (t) ranging from 33 to 40 h. However, the decay of S-labeled G/G in each of the cell lines in the presence of phenylephrine was more rapid (Fig. 7-9), and the data were more effectively modeled by a two-component fit than by a single exponential (Fig. 7-9). Addition of phenylephrine was associated with a substantial component of the decay in which t for G/G was markedly accelerated to 10.2, 10.9, and 7.7 h in rat 1 fibroblasts expressing , , and adrenoreceptor subtypes, respectively. There was, however, a second component of the decay rate for G/G that was not enhanced compared to the single phase decay observed in the untreated cells. Because of the lower steady-state level of expression of G relative to G, we did not attempt to examine whether the agonist-induced acceleration of G/G degradation could be observed independently for both of these G-proteins. In contrast to the effect of phenylephrine on the rate of removal of S-labeled G/G, this agonist had no effect in any of the adrenoreceptor subtype-expressing cells on the rate of decay of S-labeled G2, which had been immunoprecipitated with antiserum SG (data not shown). This G-protein has been shown to be involved in receptor-mediated inhibition of adenylyl cyclase(11, 22) .


Figure 7: Time course of adrenoreceptor-stimulated enhancement of G/G protein degradation. Whole cell S-amino acid pulse-chase analysis of G/G from -expressing rat-1 cells isotopically labeled and experimentally processed as described under ``Experimental Procedures'' is shown. In the chase phase, cells were incubated with medium containing non-radiolabeled amino acids in the absence (filledsymbols) or presence (opensymbols) of 100 µM phenylephrine. Cell activity was stopped at the indicated time, and the cell extract was processed for immunoprecipitation and analysis of radiolabeled G/G. Results represent the means from four independent experiments.




Figure 8: Time course of adrenoreceptor-stimulated enhancement of G/G protein degradation. Whole cell S-amino acid pulse-chase analysis of G/G from -expressing rat-1 cells isotopically labeled and experimentally processed as described under ``Experimental Procedures'' is shown. In the chase phase, cells were incubated with medium containing non-radiolabeled amino acids in the absence (filledsymbols) or presence (opensymbols) of 100 µM phenylephrine. Results represent the means from four independent experiments.




Figure 9: Time course of adrenoreceptor-stimulated enhancement of G/G protein degradation. Whole cell S-amino acid pulse-chase analysis of G/G from adrenoreceptor-expressing rat 1 cells isotopically labeled and experimentally processed as described under ``Experimental Procedures'' is shown. In the chase phase, cells were incubated with medium containing non-radiolabeled amino acids in the absence (filledsymbols) or presence (opensymbols) of 100 µM phenylephrine. Results represent the means from four independent experiments.



To investigate whether transcriptional or translational controls were likely to contribute to the process of agonist-driven G-protein down-regulation, Northern blot analysis of G, G, and G mRNA levels in cells expressing the adrenoreceptor subtype was performed in the absence or presence of phenylephrine treatment. Levels of mRNA encoding each of the above subunits were not substantially altered by exposure of the cells to phenylephrine (data not shown).


DISCUSSION

Although the basic observation that maintained agonist activation of a G-protein-linked receptor can result in a marked and selective reduction in cellular levels of the G-protein(s) activated by that receptor is now well established (see (8) for review), far less is known about the mechanisms responsible for such phenomena. Studies from Hadcock et al.(23, 24) havenoted complex regulation of G-proteins following receptor stimulation, including alterations in both protein and mRNA stability of a variety of G-proteins, which is then integrated to result in up-regulation of some G-proteins and down-regulation of others. By contrast, Mitchell et al.(25) noted that muscarinic m1 acetylcholine receptor-mediated down-regulation of the subunits of the phosphoinositidase C-linked G-proteins G/G was accompanied by a selective accelerated rate of degradation of these G-proteins. In the present study, we have sought to further analyze such effects by examining the process of down-regulation of the subunits of G and/or G in rat 1 cells transfected to express individual molecularly defined adrenoreceptor subtypes.

In such cells, expressing one of the rat , the hamster , and the bovine adrenoreceptors and labeled with myo-[H]inositol, exposure to the adrenoreceptor agonist phenylephrine resulted in stimulation of inositol phosphate production in a fashion that was resistant to pretreatment of the cells with pertussis toxin. Such a feature was hardly unexpected but is the pattern anticipated for receptors that couple to phosphoinositidase C-linked G-proteins of the G family. Maintained exposure of these cells to phenylephrine resulted in down-regulation of each of the receptor subtypes (Table 1) and selective down-regulation of some combination of the subunits of G/G. Agonist-induced down-regulation of G-protein-linked receptors is a common regulatory feature. However, in the subfamily of adrenoreceptors, while both the C10 and C2 receptor are readily down-regulated by agonist treatment, the C4 receptor has been reported to be largely resistant to down-regulation (26) . Mutation of the site for palmitoylation in the C-terminal tail of the C10 adrenoreceptor has been reported to render it resistant to agonist-mediated down-regulation without altering its ability to couple to the G-like G-proteins (27, 28) . Although direct information is not currently available, all three adrenoreceptor cDNA species used in this study have cysteine residues in their predicted C-terminal tail in a context that makes them likely sites of palmitoylation. It would be interesting to examine if mutation of these residues results in an agonist-mediated down-regulation resistant form of these receptors and whether this interferes with agonist-mediated down-regulation of G/G. Agonist-mediated down-regulation of the subunits of G/G has now been reported for a variety of receptors, including the muscarinic m1 acetylcholine receptor(19, 25) , the long isoform of the thyrotropin-releasing hormone receptor(29) , and the gonadotropin-releasing hormone receptor (21) . However, only for the first of these has any mechanistic analysis been provided. In Chinese hamster ovary cells transfected to express the rat muscarinic m1 receptor, accelerated degradation of a combination of G/G was recorded without detectable alteration in levels of mRNA of either of these polypeptides (25) . In the present study, we expand those observations to show that in the genetic background of rat 1 fibroblasts, the basal half-life of the subunits of G/G can be adequately modeled as a single monoexponential consistent with t in the region of 33-40 h and that agonist occupancy of any of the adrenoreceptor subtypes leads to a proportion of the cellular G/G population being degraded much more rapidly. Data from each of the systems, however, are not consistent with all of the cellular content of these G-proteins being degraded more rapidly in the presence of agonist. A maximally effective concentration of phenylephrine was able to cause down-regulation of between 50 and 70% of the total G/G population in these cells in a range of experiments. As the immunoprecipitation experiments that were performed in the G-protein turnover studies made use of an antiserum that identifies G and G equally(9) , as it is directed at an epitope that is identical in these two G-proteins, and these two G-proteins are widely co-expressed(30) , then we wished to examine the possibility that each of the adrenoreceptor subtypes was able to cause down-regulation of only one of these two G-proteins. To do so, we took advantage of our previous observations that separation of these two polypeptides can be achieved in SDS-PAGE systems that incorporate high concentrations of urea (19, 20, 21) . Immunoblotting of membranes of the clones used in this study with antiserum CQ following their resolution in such gels demonstrated that the steady-state levels of G expression was over 2-fold higher than that of G. Furthermore, they indicated that sustained phenylephrine occupancy of each of the adrenoreceptor subtypes resulted in a down-regulation of both G and G. Although we have not analyzed the relative cellular distribution of G and G in these clonal cell lines, we have previously been able to show in cellular fractionation studies of Chinese hamster ovary cells on sucrose density gradients that the subcellular distribution of these two G-proteins is identical(20) .

An epitope-tagged constitutively activated mutant of G has been demonstrated to have a substantially reduced half-life compared with the epitope-tagged wild type protein when expressed in S49 lymphoma cyc cells(31) , and agonist activation of an IP prostanoid receptor can result in down-regulation of this epitope-tagged variant of G when this G-protein is expressed in neuroblastoma NG108-15 cells(32) . Thus, although it has not been formally demonstrated for any G-protein other than G, it is reasonable to surmise that activation of the G-protein might be the key feature that controls its rate of degradation. The palmitoylation status of both the activated mutant of G and the wild type protein following activation of a G-linked receptor is altered compared with the basal state of the wild type protein(33, 34, 35) . Kinetic evidence indicates that this is likely to reflect accelerated depalmitoylation(35) . How relevant this is to agonist-mediated down-regulation of G remains to be explored, but it is certainly true that in a number of systems, agonist occupation of G-linked receptors has been noted to result in a large selective down-regulation of this G-protein(36, 37, 38) . It will thus now be of considerable interest to examine whether agonist activation of the adrenoreceptor subtypes in these cells results in an alteration in the palmitoylation status of the subunits of G/G. As with the previous studies on muscarinic m1 receptor regulation of G/G levels(25) , Northern blot analysis of mRNAs corresponding to these G-proteins in untreated and phenylephrine-treated cells did not provide evidence for regulation at the mRNA level.

The data provided herein demonstrate that agonist occupation of adrenoreceptor subtypes can selectively regulate the cellular levels of both G and G. The mechanism of this effect is a selective acceleration of the rate of degradation of these G-proteins.


FOOTNOTES

*
These studies were supported by a project grant (to G. M.) from the Biotechnology and Bioscience Research Council Intracellular Signaling Initiative. 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 all correspondence should be addressed: Fax: 44-141-330-4620; GBCA32{at}UDCF.GLA.AC.UK

We define the adrenoreceptor subtypes using the nomenclature originally assigned to the cDNA species that were used in the study, except that the cDNA originally named the adrenoreceptor is referred to as the adrenoreceptor, as this is now widely accepted to be equivalent to the pharmacologically defined adrenoreceptor.

The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; Gpp(NH)p, guanyl-5`-yl imidodiphosphate.


REFERENCES
  1. Ford, A. P. D. W., Williams, T. J., Blue, D. R., and Clarke, D. E.(1994) Trends Pharmacol. Sci. 15, 167-170 [CrossRef][Medline] [Order article via Infotrieve]
  2. Cotecchia, S., Schwinn, D. A., Randall, R. R., Lefkowitz, R. J., Caron, M. G., and Kobilka, B. K.(1988)Proc. Natl. Acad. Sci. U. S. A. 85, 7159-7163 [Abstract]
  3. Schwinn, D. A., Lomasney, J. W., Lorenz, W., Szklut, P. J., Fremeau, R. T., Jr., Yang-Feng, T. L., Caron, M. G., Lefkowitz, R. J., and Cotecchia, S.(1990) J. Biol. Chem. 265, 8183-8189 [Abstract/Free Full Text]
  4. Lomasney, J. W., Cotecchia, S., Lorenz, W., Leung, W.-Y., Schwinn, D. A., Yang-Feng, T. L., Brownstein, M., Lefkowitz, R. J., and Caron, M. G.(1991) J. Biol. Chem. 266, 6365-6369 [Abstract/Free Full Text]
  5. Perez, D. M., Piascik, M. T., and Graham, R. M.(1991)Mol. Pharmacol. 40, 876-883 [Abstract]
  6. Milligan, G., Svoboda, P., and Brown, C. M.(1994)Biochem. Pharmacol. 48, 1059-1071 [Medline] [Order article via Infotrieve]
  7. Wu, D., Katz, A., Lee, C. H., and Simon, M. I.(1992)J. Biol. Chem. 267, 25798-25802 [Abstract/Free Full Text]
  8. Milligan, G.(1993) Trends Pharmacol. Sci. 14, 413-418 [CrossRef][Medline] [Order article via Infotrieve]
  9. Mitchell, F. M., Mullaney, I., Godfrey, P. P., Arkinstall, S. J., Wakelam, M. J. O., and Milligan, G.(1991)FEBS Lett. 287, 171-174 [CrossRef][Medline] [Order article via Infotrieve]
  10. Green, A., Johnson, J. L., and Milligan, G.(1990)J. Biol. Chem. 265, 5206-5210 [Abstract/Free Full Text]
  11. McKenzie, F. R., and Milligan, G.(1990)Biochem. J. 267, 391-398 [Medline] [Order article via Infotrieve]
  12. Morrow, A. L., and Creese, I.(1986)Mol. Pharmacol. 29, 321-330 [Abstract]
  13. Morrow, A. L., Battaglia, S., Norman, A. B., and Creese, I.(1985) Eur. J. Pharmacol. 109, 285-287 [CrossRef][Medline] [Order article via Infotrieve]
  14. Tabor, S., and Richardson, C. C.(1985)Proc. Natl. Acad. Sci. U. S. A. 82, 1074-1078 [Abstract]
  15. Finan, P. M., White, I. R., Redpath, S. H., Findlay, J. B. C., and Millner, P. A.(1994) Plant. Mol. Biol. 25, 59-67 [Medline] [Order article via Infotrieve]
  16. Plevin, R., Palmer, S., Gardner, S. D., and Wakelam, M. J. O.(1990)Biochem. J. 268, 605-610 [Medline] [Order article via Infotrieve]
  17. MacNulty, E. E., McClue, S. J., Carr, I. C., Jess, T., Wakelam, M. J. O., and Milligan, G. (1992)J. Biol. Chem. 267, 2149-2156 [Abstract/Free Full Text]
  18. Steel, M. C., and Buckley, N. J.(1993)Mol. Pharmacol. 43, 694-701 [Abstract]
  19. Mullaney, I., Mitchell, F. M., McCallum, J. F., Buckley, N. J., and Milligan, G.(1993) FEBS Lett. 324, 241-245 [CrossRef][Medline] [Order article via Infotrieve]
  20. Svoboda, P., and Milligan, G.(1994)Eur. J. Biochem. 224, 455-462 [Abstract]
  21. Shah, B. H., and Milligan, G.(1994)Mol. Pharmacol. 46, 1-7 [Abstract]
  22. Simonds, W. F., Goldsmith, P. K., Codina, J., Unson, C. G., and Spiegel, A. M. (1989)Proc. Natl. Acad. Sci. U. S. A. 86, 7809-7813 [Abstract]
  23. Hadcock, J. R., Ros, M., Watkins, D. C., and Malbon, C. C.(1990)J. Biol. Chem. 265, 14784-14790 [Abstract/Free Full Text]
  24. Hadcock, J. R., Port, J. D., and Malbon, C. C.(1991)J. Biol. Chem. 266, 11915-11922 [Abstract/Free Full Text]
  25. Mitchell, F. M., Buckley, N. J., and Milligan, G.(1993)Biochem. J. 293, 495-499 [Medline] [Order article via Infotrieve]
  26. Eason, M. G., and Liggett, S. B.(1992)J. Biol. Chem. 267, 25473-25479 [Abstract/Free Full Text]
  27. Eason, M. G., Jacinto, M. T., Theiss, C. T., and Liggett, S. B.(1991)Proc. Natl. Acad. Sci. U. S. A. 91, 11178-11182 [Abstract/Free Full Text]
  28. Kennedy, M. E., and Limbird, L. E.(1993)J. Biol. Chem. 268, 8003-8011 [Abstract/Free Full Text]
  29. Kim, G.-D., Carr, I. C., Anderson, L. A., Zabavnik, J., Eidne, K. A., and Milligan, G. (1994)J. Biol. Chem. 269, 19933-19940 [Abstract/Free Full Text]
  30. Strathmann, M., and Simon, M. I.(1990)Proc. Natl. Acad. Sci. U. S. A. 87, 9113-9117 [Abstract]
  31. Levis, M. J., and Bourne, H. R.(1992)J. Cell Biol. 119, 1297-1307 [Abstract]
  32. Mullaney, I., and Milligan, G.(1994)FEBS Lett. 353, 231-234 [CrossRef][Medline] [Order article via Infotrieve]
  33. Mumby, S. M., Kleuss, C., and Gilman, A. G.(1994)Proc. Natl. Acad. Sci. U. S. A. 91, 2800-2804 [Abstract]
  34. Degtyarev, M. Y., Spiegel, A. M., and Jones, T. L. Z.(1993)J. Biol. Chem. 268, 23769-23772 [Abstract/Free Full Text]
  35. Wedegaertner, P. B., and Bourne, H. R.(1994)Cell 77, 1063-1070 [Medline] [Order article via Infotrieve]
  36. McKenzie, F. R., and Milligan, G.(1990)J. Biol. Chem. 265, 17084-17093 [Abstract/Free Full Text]
  37. Adie, E. J., Mullaney, I., McKenzie, F. R., and Milligan, G.(1992)Biochem. J. 285, 529-536 [Medline] [Order article via Infotrieve]
  38. Adie, E. J., and Milligan, G.(1994)Biochem. J. 300, 709-715 [Medline] [Order article via Infotrieve]

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