Degradation of G11α/Gqα Is Accelerated by Agonist Occupancy of α1A/D, α1B, and α1C Adrenergic Receptors
1995; Elsevier BV; Volume: 270; Issue: 29 Linguagem: Inglês
10.1074/jbc.270.29.17196
ISSN1083-351X
AutoresAlan Wise, Tae Weon Lee, David J. MacEwan, Graeme Milligan,
Tópico(s)Caveolin-1 and cellular processes
ResumoCells of clones of rat 1 fibroblasts transfected to express the molecularly defined α1A/D, α1B, or α1C adrenoreceptors and prelabeled with myo-[3H]inositol were each shown to generate high levels of inositol phosphates when exposed to the α1 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 Gqα and G11α. To examine the mechanism of phenylephrine-induced down-regulation of Gqα and G11α, pulse-chase 35S-amino acid labeling experiments were performed with each of the α1A/D, α1B, and α1C adrenoreceptor-expressing cell lines. The rate of degradation of G11α/Gqα, 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 Gi2α, which would not be anticipated to be activated by members of the α1 adrenoreceptor family, was unaltered by the presence of phenylephrine. Levels of mRNA encoding Gqα and G11α were not substantially altered by exposure of the cells to phenylephrine in any of the cell lines studied. Cells of clones of rat 1 fibroblasts transfected to express the molecularly defined α1A/D, α1B, or α1C adrenoreceptors and prelabeled with myo-[3H]inositol were each shown to generate high levels of inositol phosphates when exposed to the α1 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 Gqα and G11α. To examine the mechanism of phenylephrine-induced down-regulation of Gqα and G11α, pulse-chase 35S-amino acid labeling experiments were performed with each of the α1A/D, α1B, and α1C adrenoreceptor-expressing cell lines. The rate of degradation of G11α/Gqα, 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 Gi2α, which would not be anticipated to be activated by members of the α1 adrenoreceptor family, was unaltered by the presence of phenylephrine. Levels of mRNA encoding Gqα and G11α were not substantially altered by exposure of the cells to phenylephrine in any of the cell lines studied. INTRODUCTIONThere are multiple closely related α1 adrenoreceptor subtypes that have been indicated by comparisons of the pharmacological profiles of ligands in different tissues(1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar). Three distinct α1 adrenoreceptor cDNA species have currently been isolated(2Cotecchia S. Schwinn D.A. Randall R.R. Lefkowitz R.J. Caron M.G. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7159-7163Crossref PubMed Scopus (481) Google Scholar, 3Schwinn D.A. Lomasney J.W. Lorenz W. Szklut P.J. Fremeau Jr., R.T. Yang-Feng T.L. Caron M.G. Lefkowitz R.J. Cotecchia S. J. Biol. Chem. 1990; 265: 8183-8189Abstract Full Text PDF PubMed Google Scholar, 4Lomasney J.W. Cotecchia S. Lorenz W. Leung W.-Y. Schwinn D.A. Yang-Feng T.L. Brownstein M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1991; 266: 6365-6369Abstract Full Text PDF PubMed Google Scholar, 5Perez D.M. Piascik M.T. Graham R.M. Mol. Pharmacol. 1991; 40: 876-883PubMed Google Scholar), α1A/D, α1B, and α1C, but there has been considerable debate as to how closely these reflect the pharmacologically defined subtypes (see (1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar) 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(1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 6Milligan G. Svoboda P. Brown C.M. Biochem. Pharmacol. 1994; 48: 1059-1071Crossref PubMed Scopus (48) Google Scholar). Current opinion favors the view that the cloned α1A adrenoreceptor corresponds to the pharmacologically defined α1D adrenoreceptor(1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar), that the cloned and pharmacologically defined α1B adrenoreceptors are identical(1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar), and that the cloned α1C adrenoreceptor may be the equivalent of the pharmacologically defined α1A adrenoreceptor(1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar).1 1We define the α1 adrenoreceptor subtypes using the nomenclature originally assigned to the cDNA species that were used in the study, except that the cDNA originally named the α1A adrenoreceptor is referred to as the α1A/D adrenoreceptor, as this is now widely accepted to be equivalent to the pharmacologically defined α1D adrenoreceptor. Despite these ongoing concerns, it is generally accepted that the primary signaling function of α1 adrenoreceptor subtypes is to stimulate the hydrolysis of inositol-containing phospholipids via interaction with pertussis toxin-insensitive G-proteins of the Gq/G11 family (7Wu D. Katz A. Lee C.H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar) with subsequent activation of phospholipase Cβ activity(7Wu D. Katz A. Lee C.H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar).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 (8Milligan G. Trends Pharmacol. Sci. 1993; 14: 413-418Abstract Full Text PDF PubMed Scopus (110) Google Scholar) for review). Even in such cases, information on the mechanism(s) responsible for these effects is fragmentary(8Milligan G. Trends Pharmacol. Sci. 1993; 14: 413-418Abstract Full Text PDF PubMed Scopus (110) Google Scholar). 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 α1A/D, the hamster α1B, and the bovine α1C 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 G11α and Gqα as well as in down-regulation of the receptors. These are the G-proteins that have been demonstrated to couple α1 adrenoreceptors to phosphoinositidase C activity and the hydrolysis of inositol-containing phospholipids(7Wu D. Katz A. Lee C.H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar). In each case, we demonstrate that the basal rate of turnover of these G-proteins is described adequately by a monoexponential with a t0.5 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 t0.5 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.RESULTSRat 1 fibroblast cells transfected to stably express each of the three cloned α1 adrenoreceptor subtypes were used for the present study. The presence of α1A/D, α1B, and α1C 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 α1 adrenoreceptor subtypes (data not shown).Membranes derived from all three clonal cell lines were examined for their levels of expression of the α1 adrenoreceptor subtypes by measuring the specific binding of the α1 adrenoreceptor antagonist [3H]prazosin. α1A/D, α1B, and α1C 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 Kd values for the binding of [3H]prazosin of 110 ± 20, 76 ± 13, and 150 ± 26 pM, respectively (data not shown). Displacement of specific [3H]prazosin binding by varying concentrations of the α1 adrenoreceptor agonist phenylephrine was achieved with pIC50 values (and Hill coefficients) of 5.1 ± 0.1 (nH = 0.76 ± 0.09), 4.7 ± 0.2 (nH = 1.06 ± 0.07), 5.2 ± 0.1 (nH 0.90 ± 0.06), respectively, for the α1A/D, α1B, and α1C 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 (nH = 0.99 ± 0.03), 4.6 ± 0.1 (nH = 0.89 ± 0.05), 4.9 ± 0.2 (nH 1.12 ± 0.16) for the α1A/D, α1B, and α1C adrenoreceptor subtypes) (Fig. 1).Accumulation of inositol phosphates in LiCl (10 mM)-treated α1 adrenoreceptor subtype-expressing cells, which had been labeled for 36 h with myo-[2-3H]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 α1 adrenoreceptor subtype-expressing cell lines (Fig. 2).Figure 2:The α1 adrenoreceptor subtypes all cause stimulation of inositol phosphate production. Rat 1 fibroblasts transfected to stably express the α1A/D (I), α1B (II), and α1C adrenoreceptor subtypes (III) were labeled with myo-[2-3H]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 EC50 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Sustained exposure of the three cell lines to phenylephrine (100 μM, 6 h) led to a marked reduction of detectable α1 adrenoreceptors in all three cases (Table 1). Sustained exposure (16 h) of cells expressing each of the three α1 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 Gqα and G11α by, maximally, some 50-70% as determined by immunoblotting of membranes of agonist-treated and untreated cells with antiserum CQ(9Mitchell F.M. Mullaney I. Godfrey P.P. Arkinstall S.J. Wakelam M.J.O. Milligan G. FEBS Lett. 1991; 287: 171-174Crossref PubMed Scopus (47) Google Scholar), 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 α subunits expressed by these cells (Gsα, Gi2α) were noted to be associated with phenylephrine treatment (data not shown). Down-regulation of Gqα/G11α was found to be dose dependent with an EC50 for phenylephrine close to 600 nM for all three α1 adrenoreceptor subtype-expressing cells (Fig. 3). In cells expressing the α1A/D and α1B 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 α1C 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 Gqα/G11α was established at some 30-50% of that present in untreated cells.Tabled 1 Open table in a new tab Figure 3:Phenylephrine - mediated down-regulation of Gqα/G11α levels in membranes of α1 adrenoreceptor subtype-expressing rat 1 fibroblasts. Membranes (15 μg) derived from α1A/D (a), α1B (b), and α1C (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-Gqα/G11α antiserum CQ as primary reagent. Quantitative analysis from these studies on the α1C adrenoreceptor-expressing cell line is displayed in d.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4:Time course of agonist-mediated down-regulation of Gqα/G11α. Membranes (15 μg) derived from α1A/D (a), α1B (b), and α1C (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 α1C adrenoreceptor-expressing cell line is displayed in d.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The amount of expressed recombinant Gqα in whole cell extracts of E. coli BL21 (DE3) cells and of Gqα/G11α in the α1 adrenoreceptor-expressing cells was estimated by comparison with known levels of purified Gqα/G11α following immunoblotting with antiserum CQ (Fig. 5). Standard curves for the reaction of recombinant Gqα with this antiserum was obtained using various dilutions of the prokaryotically expressed G-protein α subunit. Such standard curves allowed measurement of the amounts of Gqα/G11α in membranes of all three α1 adrenoreceptor subtype-expressing rat 1 fibroblast clones. The steady-state level of Gqα/G11α 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 α1A/D adrenoreceptor-expressing cells with a maximally effective concentration of phenylephrine (1 mM, 16 h) resulted in membrane-associated immunodetectable Gqα/G11α being reduced to 16 pmol/mg membrane protein.Figure 5:Quantitation of Gqα/G11α levels in α1 adrenoreceptor subtype-expressing rat 1 fibroblasts. E. coli BL21 DE3 cells were transformed with hamster Gqα cDNA subcloned into the prokaryotic expression vector pT7.7 (see "Experimental Procedures"). Cultures of transformants were induced to express recombinant Gqα by the addition of 1 mM isopropyl-1-thio-β-D-galactopyranoside and grown for a further 4 h prior to recovery by centrifugation. Known quantities of purified Gq/G11α 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 Gqα (lane8 = 125, lane9 = 250, and lane10 = 500 ng) and membranes (15 μg) from α1A/D 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 Gqα/G11α were calculated to be 46 pmol/mg membrane protein in untreated cells, whereas in cells treated with agonist over a sustained period, levels of Gqα/G11α were reduced to 16 pmol/mg membrane protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Gqα and G11α 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 Gqα/G11α in any of these cells was selective for either of these highly homologous G-protein α 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(19Mullaney I. Mitchell F.M. McCallum J.F. Buckley N.J. Milligan G. FEBS Lett. 1993; 324: 241-245Crossref PubMed Scopus (44) Google Scholar, 20Svoboda P. Milligan G. Eur. J. Biochem. 1994; 224: 455-462Crossref PubMed Scopus (38) Google Scholar, 21Shah B.H. Milligan G. Mol. Pharmacol. 1994; 46: 1-7PubMed Google Scholar). Fig. 6 demonstrates that steady-state levels of G11α were substantially (over 2-fold) greater than Gqα in these cells and that phenylephrine-driven down-regulation of membrane-associated Gqα and G11α from the clonal cell lines expressing each of the three α1 adrenoreceptor subtypes was non-selective between these G-protein α subunits.Figure 6:Phenylephrine treatment of α1 adrenoreceptor subtype-expressing cells results in down-regulation of both Gqα and G11α. Membranes (60 μg) from α1A/D (lanes1 and 2), α1B (lanes3 and 4), and α1C (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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To address the mechanism of phenylephrine-mediated reduction in membrane-bound levels of Gqα/G11α, cells of the α1 adrenoreceptor subtype-expressing clones were incubated with Tran35S-label for 16 h, and subsequently the decay of 35S with time in immunoprecipitated Gqα/G11α was monitored in cells that were either untreated or treated with phenylephrine (100 μM) (Fig. 7-9). Analysis of the rate of decay of 35S-labeled Gqα/G11α indicated that in the untreated α1A/D (Fig. 7), α1B (Fig. 8), and α1C (Fig. 9) adrenoreceptor subtype-expressing cells, this process was described adequately by monoexponentials with estimated half-time (t0.5) ranging from 33 to 40 h. However, the decay of 35S-labeled Gqα/G11α 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 t0.5 for Gqα/G11α was markedly accelerated to 10.2, 10.9, and 7.7 h in rat 1 fibroblasts expressing α1A/D, α1B, and α1C adrenoreceptor subtypes, respectively. There was, however, a second component of the decay rate for Gqα/G11α 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 Gqα relative to G11α, we did not attempt to examine whether the agonist-induced acceleration of Gqα/G11α degradation could be observed independently for both of these G-proteins. In contrast to the effect of phenylephrine on the rate of removal of 35S-labeled Gqα/G11α, this agonist had no effect in any of the α1 adrenoreceptor subtype-expressing cells on the rate of decay of 35S-labeled Gi2α, 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(11McKenzie F.R. Milligan G. Biochem. J. 1990; 267: 391-398Crossref PubMed Scopus (199) Google Scholar, 22Simonds W.F. Goldsmith P.K. Codina J. Unson C.G. Spiegel A.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7809-7813Crossref PubMed Scopus (309) Google Scholar).Figure 7:Time course of α1A/D adrenoreceptor-stimulated enhancement of Gqα/G11α protein degradation. Whole cell 35S-amino acid pulse-chase analysis of Gqα/G11α from α1A/D-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 Gqα/G11α. Results represent the means from four independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8:Time course of α1B adrenoreceptor-stimulated enhancement of Gqα/G11α protein degradation. Whole cell 35S-amino acid pulse-chase analysis of Gqα/G11α from α1B-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9:Time course of α1C adrenoreceptor-stimulated enhancement of Gqα/G11α protein degradation. Whole cell 35S-amino acid pulse-chase analysis of Gqα/G11α from α1C 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 Gqα, G11α, and Gsα mRNA levels in cells expressing the α1B adrenoreceptor subtype was performed in the absence or presence of phenylephrine treatment. Levels of mRNA encoding each of the above α subunits were not substantially altered by exposure of the cells to phenylephrine (data not shown).DISCUSSIONAlthough 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 (8Milligan G. Trends Pharmacol. Sci. 1993; 14: 413-418Abstract Full Text PDF PubMed Scopus (110) Google Scholar) for review), far less is known about the mechanisms responsible for such phenomena. Studies from Hadcock et al.(23Hadcock J.R. Ros M. Watkins D.C. Malbon C.C. J. Biol. Chem. 1990; 265: 14784-14790Abstract Full Text PDF PubMed Google Scholar, 24Hadcock J.R. Port J.D. Malbon C.C. J. Biol. Chem. 1991; 266: 11915-11922Abstract Full Text PDF PubMed Google Scholar) 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.(25Mitchell F.M. Buckley N.J. Milligan G. Biochem. J. 1993; 293: 495-499Crossref PubMed Scopus (55) Google Scholar) noted that muscarinic m1 acetylcholine receptor-mediated down-regulation of the α subunits of the phosphoinositidase C-linked G-proteins Gq/G11 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 α subunits of Gq and/or G11 in rat 1 cells transfected to express individual molecularly defined α1 adrenoreceptor subtypes.In such cells, expressing one of the rat α1A/D, the hamster α1B, and the bovine α1C adrenoreceptors and labeled with myo-[3H]inositol, exposure to the α1 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 Gq 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 α subunits of Gq/G11. Agonist-induced down-regulation of G-protein-linked receptors is a common regulatory feature. However, in the subfamily of α2 adrenoreceptors, while both the α2C10 and α2C2 receptor are readily down-regulated by agonist treatment, the α2C4 receptor has been reported to be largely resistant to down-regulation (26Eason M.G. Liggett S.B. J. Biol. Chem. 1992; 267: 25473-25479Abstract Full Text PDF PubMed Google Scholar). Mutation of the site for palmitoylation in the C-terminal tail of the α2C10 adrenoreceptor has been reported to render it resistant to agonist-mediated down-regulation without altering its ability to couple to the Gi-like G-proteins (27Eason M.G. Jacinto M.T. Theiss C.T. Liggett S.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 91: 11178-11182Crossref Scopus (96) Google Scholar, 28Kennedy M.E. Limbird L.E. J. Biol. Chem. 1993; 268: 8003-8011Abstract Full Text PDF PubMed Google Scholar). Although direct information is not currently available, all three α1 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 Gq/G11. Agonist-mediated down-regulation of the α subunits of Gq/G11 has now been reported for a variety of receptors, including the muscarinic m1 acetylcholine receptor(19Mullaney I. Mitchell F.M. McCallum J.F. Buckley N.J. Milligan G. FEBS Lett. 1993; 324: 241-245Crossref PubMed Scopus (44) Google Scholar, 25Mitchell F.M. Buckley N.J. Milligan G. Biochem. J. 1993; 293: 495-499Crossref PubMed Scopus (55) Google Scholar), the long isoform of the thyrotropin-releasing hormone receptor(29Kim G.-D. Carr I.C. Anderson L.A. Zabavnik J. Eidne K.A. Milligan G. J. Biol. Chem. 1994; 269: 19933-19940Abstract Full Text PDF PubMed Google Scholar), and the gonadotropin-releasing hormone receptor (21Shah B.H. Milligan G. Mol. Pharmacol. 1994; 46: 1-7PubMed Google Scholar). 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 Gq/G11 was recorded without detectable alteration in levels of mRNA of either of these polypeptides (25Mitchell F.M. Buckley N.J. Milligan G. Biochem. J. 1993; 293: 495-499Crossref PubMed Scopus (55) Google Scholar). 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 α subunits of Gq/G11 can be adequately modeled as a single monoexponential consistent with t0.5 in the region of 33-40 h and that agonist occupancy of any of the α1 adrenoreceptor subtypes leads to a proportion of the cellular Gq/G11 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 Gqα/G11α 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 Gqα and G11α equally(9Mitchell F.M. Mullaney I. Godfrey P.P. Arkinstall S.J. Wakelam M.J.O. Milligan G. FEBS Lett. 1991; 287: 171-174Crossref PubMed Scopus (47) Google Scholar), as it is directed at an epitope that is identical in these two G-proteins, and these two G-proteins are widely co-expressed(30Strathmann M. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9113-9117Crossref PubMed Scopus (385) Google Scholar), then we wished to examine the possibility that each of the α1 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 (19Mullaney I. Mitchell F.M. McCallum J.F. Buckley N.J. Milligan G. FEBS Lett. 1993; 324: 241-245Crossref PubMed Scopus (44) Google Scholar, 20Svoboda P. Milligan G. Eur. J. Biochem. 1994; 224: 455-462Crossref PubMed Scopus (38) Google Scholar, 21Shah B.H. Milligan G. Mol. Pharmacol. 1994; 46: 1-7PubMed Google Scholar). 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 G11α expression was over 2-fold higher than that of Gqα. Furthermore, they indicated that sustained phenylephrine occupancy of each of the α1 adrenoreceptor subtypes resulted in a down-regulation of both G11α and Gqα. Although we have not analyzed the relative cellular distribution of Gqα and G11α 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(20Svoboda P. Milligan G. Eur. J. Biochem. 1994; 224: 455-462Crossref PubMed Scopus (38) Google Scholar).An epitope-tagged constitutively activated mutant of Gsα 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(31Levis M.J. Bourne H.R. J. Cell Biol. 1992; 119: 1297-1307Crossref PubMed Scopus (179) Google Scholar), and agonist activation of an IP prostanoid receptor can result in down-regulation of this epitope-tagged variant of Gsα when this G-protein is expressed in neuroblastoma NG108-15 cells(32Mullaney I. Milligan G. FEBS Lett. 1994; 353: 231-234Crossref PubMed Scopus (11) Google Scholar). Thus, although it has not been formally demonstrated for any G-protein other than Gsα, 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 Gsα and the wild type protein following activation of a Gsα-linked receptor is altered compared with the basal state of the wild type protein(33Mumby S.M. Kleuss C. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2800-2804Crossref PubMed Scopus (219) Google Scholar, 34Degtyarev M.Y. Spiegel A.M. Jones T.L.Z. J. Biol. Chem. 1993; 268: 23769-23772Abstract Full Text PDF PubMed Google Scholar, 35Wedegaertner P.B. Bourne H.R. Cell. 1994; 77: 1063-1070Abstract Full Text PDF PubMed Scopus (304) Google Scholar). Kinetic evidence indicates that this is likely to reflect accelerated depalmitoylation(35Wedegaertner P.B. Bourne H.R. Cell. 1994; 77: 1063-1070Abstract Full Text PDF PubMed Scopus (304) Google Scholar). How relevant this is to agonist-mediated down-regulation of Gsα remains to be explored, but it is certainly true that in a number of systems, agonist occupation of Gsα-linked receptors has been noted to result in a large selective down-regulation of this G-protein(36McKenzie F.R. Milligan G. J. Biol. Chem. 1990; 265: 17084-17093Abstract Full Text PDF PubMed Google Scholar, 37Adie E.J. Mullaney I. McKenzie F.R. Milligan G. Biochem. J. 1992; 285: 529-536Crossref PubMed Scopus (47) Google Scholar, 38Adie E.J. Milligan G. Biochem. J. 1994; 300: 709-715Crossref PubMed Scopus (34) Google Scholar). It will thus now be of considerable interest to examine whether agonist activation of the α1 adrenoreceptor subtypes in these cells results in an alteration in the palmitoylation status of the α subunits of Gq/G11. As with the previous studies on muscarinic m1 receptor regulation of Gqα/G11α levels(25Mitchell F.M. Buckley N.J. Milligan G. Biochem. J. 1993; 293: 495-499Crossref PubMed Scopus (55) Google Scholar), 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 α1 adrenoreceptor subtypes can selectively regulate the cellular levels of both Gqα and G11α. The mechanism of this effect is a selective acceleration of the rate of degradation of these G-proteins. INTRODUCTIONThere are multiple closely related α1 adrenoreceptor subtypes that have been indicated by comparisons of the pharmacological profiles of ligands in different tissues(1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar). Three distinct α1 adrenoreceptor cDNA species have currently been isolated(2Cotecchia S. Schwinn D.A. Randall R.R. Lefkowitz R.J. Caron M.G. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7159-7163Crossref PubMed Scopus (481) Google Scholar, 3Schwinn D.A. Lomasney J.W. Lorenz W. Szklut P.J. Fremeau Jr., R.T. Yang-Feng T.L. Caron M.G. Lefkowitz R.J. Cotecchia S. J. Biol. Chem. 1990; 265: 8183-8189Abstract Full Text PDF PubMed Google Scholar, 4Lomasney J.W. Cotecchia S. Lorenz W. Leung W.-Y. Schwinn D.A. Yang-Feng T.L. Brownstein M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1991; 266: 6365-6369Abstract Full Text PDF PubMed Google Scholar, 5Perez D.M. Piascik M.T. Graham R.M. Mol. Pharmacol. 1991; 40: 876-883PubMed Google Scholar), α1A/D, α1B, and α1C, but there has been considerable debate as to how closely these reflect the pharmacologically defined subtypes (see (1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar) 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(1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 6Milligan G. Svoboda P. Brown C.M. Biochem. Pharmacol. 1994; 48: 1059-1071Crossref PubMed Scopus (48) Google Scholar). Current opinion favors the view that the cloned α1A adrenoreceptor corresponds to the pharmacologically defined α1D adrenoreceptor(1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar), that the cloned and pharmacologically defined α1B adrenoreceptors are identical(1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar), and that the cloned α1C adrenoreceptor may be the equivalent of the pharmacologically defined α1A adrenoreceptor(1Ford A.P.D.W. Williams T.J. Blue D.R. Clarke D.E. Trends Pharmacol. Sci. 1994; 15: 167-170Abstract Full Text PDF PubMed Scopus (259) Google Scholar).1 1We define the α1 adrenoreceptor subtypes using the nomenclature originally assigned to the cDNA species that were used in the study, except that the cDNA originally named the α1A adrenoreceptor is referred to as the α1A/D adrenoreceptor, as this is now widely accepted to be equivalent to the pharmacologically defined α1D adrenoreceptor. Despite these ongoing concerns, it is generally accepted that the primary signaling function of α1 adrenoreceptor subtypes is to stimulate the hydrolysis of inositol-containing phospholipids via interaction with pertussis toxin-insensitive G-proteins of the Gq/G11 family (7Wu D. Katz A. Lee C.H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar) with subsequent activation of phospholipase Cβ activity(7Wu D. Katz A. Lee C.H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar).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 (8Milligan G. Trends Pharmacol. Sci. 1993; 14: 413-418Abstract Full Text PDF PubMed Scopus (110) Google Scholar) for review). Even in such cases, information on the mechanism(s) responsible for these effects is fragmentary(8Milligan G. Trends Pharmacol. Sci. 1993; 14: 413-418Abstract Full Text PDF PubMed Scopus (110) Google Scholar). 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 α1A/D, the hamster α1B, and the bovine α1C 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 G11α and Gqα as well as in down-regulation of the receptors. These are the G-proteins that have been demonstrated to couple α1 adrenoreceptors to phosphoinositidase C activity and the hydrolysis of inositol-containing phospholipids(7Wu D. Katz A. Lee C.H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar). In each case, we demonstrate that the basal rate of turnover of these G-proteins is described adequately by a monoexponential with a t0.5 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 t0.5 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.
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