Introduction

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This study aimed to investigate the ability of phospholipase C (PLC)-coupled m3-muscarinic receptors to regulate ₂-adrenergic receptor function. This question has physiological relevance because many cell types, including smooth muscle such as airway smooth muscle (Eglen et al., 1994), coexpress these two G protein-coupled receptors. Our study focuses on two potential regulatory mechanisms. The first of which is based on recent in vitro data suggesting that changes in intracellular calcium (Chuang et al., 1996; Pronin et al., 1997) and membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) (Pitcher et al., 1996; DebBurman et al., 1996) levels may regulate the activity of G protein-coupled receptor kinases (GRKs) known to phosphorylate and desensitize ₂-adrenergic receptors. Because activation of the PLC-pathway will both increase intracellular free calcium and decrease membrane PIP₂ levels, this may be a potential mechanism by which m3-muscarinic receptors could influence homologous GRK-mediated ₂-adrenergic receptor phosphorylation. The second mechanism investigated addresses the possibility that m3-muscarinic receptors may mediate heterologous phosphorylation of the ₂-adrenergic receptor in a GRK-independent manner.

It is now well established that agonist-mediated phosphorylation of the ₂-adrenergic receptor results in receptor desensitization (for reviews see; Hausdorff et al., 1990; Lohse, 1993; Tobin, 1997; Carman and Benovic, 1998; Pitcher et al., 1998). -adrenergic receptor kinase 1 and 2 (GRK-2 and GRK-3) are the principal kinases involved, phosphorylating the receptor on sites in the C-terminal tail (Hausdorff et al., 1990). Recent cloning studies have demonstrated that GRK-2 and GRK-3 are part of the GRK family that currently has six members (GRK-1 through GRK-6; Tobin, 1997; Pitcher et al., 1998). With the exception of GRK-1 (rhodopsin kinase) all of the GRKs bind PIP₂, thereby participating in the anchoring of the kinase to the plasma membrane in a manner that is either cooperative with (i.e., GRK-2 and GRK-3; Touhara et al., 1995; Premont et al., 1996), or independent of (i.e., GRK-4, GRK-5, and GRK-6; Stoffel et al., 1994; Pitcher et al., 1996, Premont et al., 1996) G protein -subunit binding. Furthermore, in reconstitution studies GRK activity can be maintained by a number of phospholipids, including PIP₂ (Onorato et al., 1995; DebBurman et al., 1996; Pitcher et al., 1996). In vitro GRK activity is affected by PIP₂ in a concentration-dependent manner (DebBurman et al., 1996), suggesting that in intact cells changes in membrane levels of PIP₂ may regulate GRK activity.

Recent studies have also implicated Ca²⁺/calmodulin in the regulation of GRK membrane localization. Ca²⁺/calmodulin has been found to inhibit membrane localization of GRK-2 and GRK-3 by competing with -subunit binding (Chuang et al., 1996). Ca²⁺/calmodulin was also able to inhibit GRK-5 activity (Chuang et al., 1996) via a mechanism that may involve direct binding of Ca²⁺/calmodulin to the conserved polybasic N terminus of the kinase common to GRK-4, GRK-5, and GRK-6 (Chuang et al., 1996). This either prevents membrane association directly or stimulates autophosphorylation that results in membrane dissociation (Chuang et al., 1996; Pronin et al., 1997).

These in vitro studies predict that changes in intracellular free calcium and PIP₂ membrane concentrations could affect GRK activity, although this proposal has never been tested in intact cells. If such a mechanism does exist in vivo then stimulation of coexpressed PLC-coupled receptors (for example the m3-muscarinic receptor) with associated changes in intracellular free calcium and PIP₂ concentrations would diminish the ability of GRKs to phosphorylate ₂-adrenergic receptors. In the present study we test whether endogenous receptor kinases, possibly GRKs, responsible for the non-protein kinase A (PKA)-mediated phosphorylation of ₂-adrenergic receptors are regulated by changes in intracellular calcium and PIP₂ in a Chinese hamster ovary (CHO) cell line that is cotransfected with the human ₂-adrenergic and m3-muscarinic receptors.

The second mechanism of receptor cross talk investigated in this study is the possibility that the m3-muscarinic receptor may mediate phosphorylation of the ₂-adrenergic receptor directly. A potential mechanism involving protein kinase C (PKC) has recently been suggested, where PKC stimulation was shown to phosphorylate the ₂-adrenergic receptor at sites in the third intracellular loop resulting in receptor desensitization (Johnson et al., 1990; Yuan et al., 1994). These studies, however, employed phorbol ester stimulation of PKC and as such only imply the potential for a PLC-coupled receptor-mediated regulatory process. In this study we investigated whether m3-muscarinic receptor stimulation can mediate GRK-independent phosphorylation/regulation of the ₂-adrenergic receptor.

	Materials and Methods

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Antibodies and Reagents. Antiserum to the ₂-adrenergic receptor was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-m3-muscarinic antibody (Ab-332) was raised against a glutathione-S-transferase fusion protein containing a region of the third intracellular loop of the muscarinic m3 receptor as previously described (Tobin and Nahorski, 1993). The pCEP plasmid was purchased from Invitrogen. [³²P]Orthophosphate (10 mCi/ml), [³H]cAMP (49Ci/mmol), and [³H]inositol (68Ci/mmol) were purchased from NEN (Boston, MA). [³H]inositol 1,4,5-trisphosphate ]Ins(1,4,5)P₃; 21Ci/mmol] and [³H]CGP-12177 (44Ci/mmol) was obtained from New England Nuclear-DuPont Ltd. (Stevenage, Hertfordshire, UK). Protein A Sepharose was purchased from Pharmacia (Uppsala, Sweden).  Minimal essential medium, fetal calf serum, penicillin/streptomycin, fungizone, and tissue culture flasks were purchased from Life Technologies (Paisley, Renfrewshire, Scotland). Hygromycin B and phorbol-12,13-dibutyrate were purchased from Calbiochem (Nottingham, UK). Myristoylated protein kinase A inhibitor (14-22) amide (N-Myr-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-NH₂) was purchased from Calbiochem. All other reagents were purchased from Sigma Chemical Co. (Poole, Dorset, UK).

Cell Culture. CHO-K1 cells, transfected with cDNA encoding the m1- or m3-muscarinic receptors were kind gifts from Dr. N. J. Buckley (Dept. of Pharmacology, University College, London, England). The CHO-₂/m3 cells were generated by subcloning the cDNA encoding the ₂-adrenergic receptor into the pCEP plasmid, which contains the constituitively expressed hygromycin B resistance gene. The resultant vector was transfected into a CHO-K1 cell line previously transfected with cDNA encoding for the m3-muscarinic receptor (Tobin et al., 1992) using the calcium phosphate precipitation method. ₂-Adrenergic receptor-expressing clones were selected using 200 µg/ml hygromycin B and subsequent clones were tested for ₂-adrenergic receptor and m3-muscarinic receptor expression using [³H]CGP-12177 and [³H]N-methylscopolamine binding. The work described in this article was performed using CHO-₂/m3 clone 27, which expressed 224 ± 33 fmol/mg protein ₂-adrenergic receptor and 1456 ± 240 fmol/mg protein m3-muscarinic receptor. Transfected CHO cells were grown in medium consisting of  minimal essential medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml fungizone, and 225 IU/ml hygromycin B. Cells were incubated in a 5% CO₂, humidified incubator at 37°C.

Membrane Sample Preparation. Membranes were prepared from confluent monolayers of CHO cells by harvesting the cells from 175-cm² flasks in ice-cold PBS/0.5 mM EDTA pH 7.4 solution. The cells were centrifuged at 1000g and the resultant pellet resuspended in ice-cold TE (10 mM Tris-HCl/2.5 mM EDTA, pH 7.4) including 1 mM sodium orthovanadate, 100 µg/ml benzamidine, 1 mM phenylmethylsulfonyl fluoride. The cells were left in ice-cold TE for 10 min to promote cell swelling and subsequently homogenized by a 10-s pulse using a Polytron tissue homogenizer. The resulting cell lysate was centrifuged at 500g and the pellet discarded. The supernatant was diluted with 15 ml of ice-cold TE and further centrifuged at (15000g for 15 min, 4°C). The resultant crude membrane pellet was resuspended in the appropriate buffer ready for use.

Immunoblotting. The protein concentration of the membrane samples prepared from CHO cells expressing recombinant G protein-coupled receptors was adjusted to 1 mg/ml. Fifty micrograms of membrane sample/lane was resolved on a 10% SDS/polyacrylamide gel electrophoresis gel and resolved proteins transferred to nitrocellulose sheets. Nitrocellulose sheets were subsequently blocked with TBS-T [5% (w/v) dried milk, 10 mM Tris (pH 7.4), 0.15 M NaCl, 0.05% (v/v) Tween-20] overnight at 4°C. Nitrocellulose sheets were then probed with the anti-₂-adrenergic receptor antiserum at 1:500 dilution for 90 min. The blots were then washes with TBS-T at room temperature. Detection of immunoreactivity was achieved by horseradish peroxidase-conjugated anti-rabbit antibody and a commercially available enhanced chemiluminescence detection kit (Amersham, UK).

Immunoprecipitation of Phosphorylated Receptors. Intact CHO cells grown in 6-well dishes were washed with 1 ml of phosphate-free Krebs/HEPES buffer (10 mM HEPES, 118 mM NaCl, 4.3 mM, 1.17 mM MgSO₄.7, 1.3 mM CaCl₂, 25.0 mM NaHCO₃, and 11.7 mM glucose (pH 7.4), and the cells were incubated in phosphate-free Krebs/HEPES supplemented with [³²P]orthophosphate (50 µCi/ml) for 1 h at 37°C. Drugs or vehicle were added for varying times according to the experiment and stimulations were terminated by rapid aspiration of the drug-containing media and application of 2 ml of ice-cold solubilisation buffer (10 mM Tris, 10 mM EDTA, 500 mM NaCl, 1% NP-40 (Nonidet P40), 0.1% SDS, 0.5% deoxycholate pH 7.4). Samples were left on ice for 15 min and then cleared by microcentrifugation. Antiserum (0.2 µg) was added and the samples left on ice for 60 to 90 min. Immunocomplexes were isolated on protein-A Sepharose beads and the beads were washed three times with 10× ice-cold TBS-T and 1× ice-cold TE. Isolated immunocomplexes were resolved on 10% SDS/PAGE gels. The gels were dried and subjected to autoradiography and the level of receptor phosphorylation assessed with a Bio-Rad model GS 670 densitometer (Bio-Rad, Hercules, CA).

Isoproterenol Displacement of [³H]CGP 12177 Binding to CHO-₂/m3 Cell Membranes. Isoproterenol displacement of [³H]CGP 12177 binding was performed in membrane preparations from CHO-₂/m3 cells in the presence and absence of the guanine nucleotide GppNHp (100 µM) to assess the proportion of ₂-adrenergic receptor in the high- and low-affinity states. Cell membranes were prepared as described above and resuspended in binding buffer (20 mM Tris, 10 mM MgCl₂, and 1 mM EDTA, pH 7.4). Membranes were freshly prepared before each experiment. Reaction tubes containing a range of concentrations of isoproterenol (10¹⁰ 10⁴ M), ~0.5 nM [³H]CGP 12177 ± 100 µM Gpp[NH]p, and CHO-₂/m3 cell membranes (30-50 µg of protein) in a final volume of 200 µl. Reactions were initiated by the addition of membranes and allowed to proceed for 45 to 60 min at room temperature. The reactions were terminated by rapid vacuum filtration through Whatman GF/B filters followed by 2 × 5 ml washes with ice-cold binding buffer. Filters were removed and membrane samples allowed to extract overnight in 5 ml of scintillant before being placed on the scintillation counter. Results were analyzed using the GraphPad Prism program (GraphPad Software Inc. San Diego, CA).

Isoproterenol Displacement of [³H]CGP 12177 Binding to Intact CHO-₂/m3 Cells. CHO-₂/m3 cells grown on 6-well dishes were washed once with Krebs/HEPES buffer and left to equilibrate for 10 min. Medium was then replaced with Krebs/HEPES buffer containing isoproterenol (10¹⁰ 10⁴ M), ~0.5 nM [³H]CGP 12177 plus either one of vehicle, PKA amide inhibitor [myristoylated protein kinase A inhibitor (14-22) amide (N-Myr-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-NH₂; 20 µM] or the PKA inhibitor H-89 (25 µM). The incubation was allowed to reach equilibrium at 4°C overnight. Cells were then rapidly washed three times with ice-cold Krebs/HEPES buffer and then solubilized in solubilization buffer. The radioactivity in the solubilized extract was then determined by scintillation counting.

Mass Ins(1,4,5)P₃ Determination. Cells grown in 24-well dishes were washed with Krebs/HEPES buffer and challenged with agonists for the appropriate times. Incubations were terminated by rapid aspiration, and addition of ice-cold 0.5 M trichloroacetic acid and transfer to an ice-bath. After 15 min, the supernatant was removed and neutralized by addition of EDTA and freon/tri-n-octylamine as described previously (Tobin et al., 1992). Extracts were brought to pH 7 by addition of NaHCO₃ and stored at 4°C until analysis. Ins(1,4,5)P₃ mass measurements were performed according to a standard method (Challiss et al., 1988).

Mass PIP₂ Determination. Cells grown in 24- well dishes and labeled with [³H]inositol (2.5 µCi/ml) for 48 h, were washed with Krebs/HEPES buffer and challenged with agonists for the appropriate times. Incubations were terminated by rapid aspiration, and addition of 0.5 ml of acidified chloroform/methanol (CHCl₃/CH₃OH/concentrated HCl, 40:80:1). Each well was scraped thoroughly and the lipid extract from duplicates collected and pooled. The combined lipid extract was then resolved by addition of CHCl₃ (0.31 ml) and 0.1 M HCl (0.56 ml), thorough vortex mixing, and centrifugation (1000g, 10 min). A known volume of the CHCl₃ phase was recovered, dried under N₂, deacylated, and the [³H]glycerophosphoinositol (phosphates) resolved exactly as previously described (Challiss et al., 1993).

Single Cell Calcium Measurements. Cells grown for 16 to 24 h on coverslips were incubated in Krebs/HEPES buffer supplemented with 2 µM fura-2 acetoxymethyl ester and 1 mg/ml BSA for 1 h. The coverslips were then washed in Krebs/HEPES buffer and incubated for a further 30 min to allow for complete de-esterification of the dye before being mounted on the stage of a Nikon Diaphot inverted epifluorescence microscope. Krebs/HEPES was continuously perfused over the cells at the rate of 4 ml/min and agonists were applied, where indicated, via the perfusion buffer. Using an intensified charged-couple device camera (Photonic Science) contained in a quanticell 700 system (Applied Imaging), images at wavelengths above 510 nm were collected after excitation at 340 and 380 nm (40 ms at each wavelength). Ratiometric values were converted to approximate [Ca²⁺]using the Grynkiewicz equation (Grynkiewicz et al., 1985).

Measurement of Intracellular cAMP in Permeabilized Cells. cAMP accumulation in permeabilized CHO-₂/m3 cells was assessed using a cAMP binding protein purified from calf adrenal glands (Brown et al., 1971). One flask of confluent CHO-₂/m3 cells was treated with either 1 mM carbachol or vehicle for 10 min and the drug-containing medium rapidly removed. Cells were harvested using ice-cold PBS/0.5 mM EDTA solution and cells were centrifuged at 210g for 2 min. The pellet was washed twice in Ca²⁺-free Krebs/HEPES buffer and the cells centrifuged at 210g for 2 min. The cell pellet was resuspended in 3.2 ml of cytosol-like buffer [CLB: 120 mM KCl, 2 mM KH₂PO₄, 5 mM Na-succinate, 5 mM MgCl₂, and 20 mM HEPES, pH to 7.2 (using KOH)]. -Escin was added to the cell suspension to give a final concentration of 50 µg/ml, and the cell suspension was left on ice for 2 min. The permeabilized cells were subsequently centrifuged at 210g for 2 min and the cell pellet resuspended in CLB (1 mg protein/ml). This procedure results in permeabilization of ~100% of the cells as assessed using the dye Azur-A. Reaction tubes contained a range of concentrations of isoproterenol (2 × 10⁹-2 × 10⁴ M), 4 mM ATP, and permeabilized cells (25 µg protein) in a final volume of 100 µl. Reactions were initiated by the addition of cells and the reactions allowed to proceed for 10 min at 37°C. Stimulations were terminated and samples neutralized as described above for Ins(1,4,5)P₃ determination and cAMP content determined as previously described (Brown et al., 1971).

	Results

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Immunodetection of -Adrenergic Receptor in CHO-₂/m3 Cells. Western blots using an anti-₂-adrenergic receptor antibody revealed an immunoreactive band of ~64 kDa in membranes prepared from CHO cells expressing the ₂-adrenergic receptor (CHO-₂/m3 cells) but not in CHO cells expressing either the m1- or m3-muscarinic receptors (Fig. 1A). The apparent molecular mass of the ₂-adrenergic receptor corresponded to the glycosylated receptor, which has been reported previously (Benovic et al., 1984). In phosphorylation studies, the anti-₂-adrenergic receptor antiserum was used to immunoprecipitate the ₂-adrenergic receptor from CHO-₂/m3 cells labeled with [³²P]orthophosphate. In these studies the receptor ran as an ~64-kDa phosphoprotein that demonstrated a rapid (measured in seconds) increase in its phosphorylation state after agonist stimulation (1 µM isoproterenol; Fig. 1B). In control experiments, using immunoprecipitates from cells expressing only the m3-muscarinic receptor, the ~64-kDa band was not present (Fig. 1C). Note that in addition to the ₂-adrenergic receptor, the antiserum was able to immunoprecipitate a high molecular mass band (>200 kDa) and a band running at ~42 kDa. Both of these phosphoproteins were present in the control immunoprecipitation (Fig. 1C) and are therefore distinct from the ₂-adrenergic receptor.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Immunodetection of the 2-adrenergic receptor expressed in CHO-2/m3 cells. A, Western blot of membranes prepared from CHO cells expressing the m1- or m3-muscarinic receptors or coexpressing the m3-muscarinic and 2-adrenergic receptors (2m3). Fifty micrograms of membrane protein was loaded per well and the blot probed with anti-2-adrenergic receptor antiserum at a 1:500 dilution. B, time course for phosphorylation of the 2-adrenergic receptor immunoprecipitated from CHO-2/m3 cells labeled with [32P]-orthophosphate. The cells were stimulated with 1 µM isoproterenol for the times indicated. The receptors were then solubilized and immunoprecipiated using the anti-2-adrenergic receptor antiserum. C, control immunoprecipation using the 2-adrenergic receptor antiserum and CHO cells expressing only the m3-muscarinic receptor. The positions of molecular mass markers are shown in kilodaltons.

Identification of PKA- and Non-PKA-Mediated Components of ₂-Adrenergic Receptor Phosphorylation in CHO-₂/m3 Cells. The established mechanism of -adrenergic receptor phosphorylation is that at low agonist concentrations the -adrenergic receptor is phosphorylated solely by PKA and at high concentrations by both PKA and GRKs (Clark et al., 1988; Johnson et al., 1990; Pippig et al., 1993). It was important to confirm that there were two components in the phosphorylation of the ₂-adrenergic receptor in the CHO-₂/m3 cells.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Agonist-mediated phosphorylation of the 2-adrenergic receptor. CHO-2/m3 cells were labeled with [32P]orthophosphate and stimulated with vehicle or isoproterenol (ISO; 1 µM or 100 µM) for 10 min. In the appropriate experiments the PKA amide inhibitor (20 µM) was added 10 min before agonist. A, a representive autoradiograph. The position of molecular mass markers are shown in kilodaltons. B, cumulative data form three experiments in which the data have been normalized to the basal phosphorylation of the receptor. C, displacement of [3H]CGP 12177 (~0.5 nM) binding by isoproterenol from intact CHO-2/m3 cells was conducted in the presence of vehicle (), PKA amide inhibitor (20 µM; ), or the PKA inhibitor H-89 (25 µM; ). Data are means (±S.E.M.) of three experiments carried out in duplicate.

Can Changes in Intracellular Free Calcium and PIP₂ Levels Influence Homologous ₂-Adrenergic Receptor Phosphorylation? Previous work from our laboratory and others (Tobin et al., 1992; Fisher et al., 1994; Willars et al., 1996) have demonstrated that stimulation of the PLC-coupled m3-muscarinic receptor results in a biphasic increase in Ins(1,4,5)P₃ and a corresponding fall in PIP₂. The biphasic rise in Ins(1,4,5)P₃ also correlates with a biphasic increase in intracellular free calcium (Tobin et al., 1992). The experimental protocol employed in this study was to stimulate the CHO-₂/m3 cells for 30 s with a maximally effective concentration of the muscarinic agonist carbachol (1 mM) followed by stimulation with 100 µM isoproterenol to induce GRK-mediated ₂-adrenergic receptor phosphorylation.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Signaling through the m3-muscarinic receptor. Determination of Ins(1,4,5)P3 (A), PIP2 (B), and intracellular free calcium (C) in CHO-2/m3 cells stimulated with 1 mM carbachol () or 1 mM carbachol and 100 µM isoproterenol (). The isoproterenol was added 30 s after carbachol addition. The Ins(1,4,5)P3 and PIP2 data are the means (±S.E.M) of four experiments carried out in duplicate. The PIP2 data is expressed as disintegrations per minute associated with the 3H-deacylated product of PIP2 per well. The calcium data are the means of 17 cells. CCH, carbachol; ISO, isoproterenol.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effects of muscarinic receptor stimulation on GRK-mediated 2-adrenergic receptor phosphorylation. CHO-2/m3 cells labeled with [32P]orthophosphate were stimulated with carbachol (1 mM) or isoproterenol (100 µM) only, or with carbachol (1 mM) for 30 s then isoproterenol (100 µM). The reactions were allowed to continue for 10 mins after which receptors were solubilized and immunoprecipitated with the anti-2-adrenergic receptor antiserum. A, the experiment was conducted in the absence of the PKC inhibitor Ro 31-8220. B, in the experiments indicated, Ro 31-8220 (RO; 10 µM) was added 10 min before agonist stimulation. C, cells were treated with Ro 31-8220 (10 µM) and the PKA amide inhibitor (20 µM) 10 min before agonist stimulation. The gels shown are representive of at least three experiments. Next to each autoradiograph is the cummulative data (±S.E.M) as determined by densitometric anaylsis. The positions of molecular mass markers are shown in kilodaltons.

Characterization of m3-Muscarinic Receptor-Mediated Phosphorylation of ₂-Adrenergic Receptor. The second possible mechanism of receptor cross talk under investigation in this study was the possibility of direct ₂-adrenergic receptor phosphorylation after m3-muscarinic receptor activation. As demonstrated above, stimulation of m3-muscarinic receptors mediated heterologous phosphorylation of the ₂-adrenergic receptor via PKC (Fig. 4).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Characterization of the m3-muscarinic receptor-mediated phosphorylation of the 2-adrenergic receptor. A, effects of the muscarinic receptor antagonist atropine (10 µM), the -adrenergic receptor antagonist timolol (5 µM), and the PKC inhibitor Ro 31-8220 (10 µM) on carbachol (1 mM) -mediated phosphorylation of the 2-adrenergic receptor. The effect of phorbol 12,13,-dibutyrate (1 µM) on 2-adrenergic receptor phosphorylation is also shown. B, time course for carbachol (1 mM)-stimulated phosphorylated of the 2-adrenergic receptor. The gels are representive of at least two experiments. Stimulation with agonists and phorbol esters were for 10 min. Antagonists and Ro 31-8220 were added 10 min before agonist stimulation. The positions of molecular mass markers are shown in kilodaltons. ATR, atropine; TIM, timolol.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Concentration dependence of muscarinic receptor-mediated phosphorylation of the 2-adrenergic receptor. A, representive gel of carbachol-mediated phosphorylation of the 2-adrenergic receptor. B, cumulative results from three experiments. The data shown represent the means ± S.E.M. The positions of molecular mass markers are shown in kDa. CCH=carbachol.

Functional Significance of Heterologous Phosphorylation of ₂-Adrenergic Receptor. To test whether PKC-mediated phosphorylation of the ₂-adrenergic receptor driven by the m3-muscarinic receptor resulted in receptor desensitization, we analyzed agonist binding curves generated from membranes derived from CHO-₂/m3 cells. In control membranes isoproterenol displaced [³H]CGP-12177 binding in a biphasic fashion with computer-assisted curve fitting revealing two sites with a high affinity (K_H ~8.4 nM) and a low affinity (K_L ~1.34 µM). In the presence of the nonhydrolyzable GTP analog (GppNHp, 100 µM), the agonist binding curves shifted exclusively to the low-affinity binding state (K_L ~1.2 µM; Fig. 7A). Membranes prepared from CHO-₂/m3 cells pre-exposed to carbachol (1 mM, 10 min) exhibited ligand-binding curves that only had a low-affinity component (K_L ~1.6 µM), and no significant shift in the binding curve was observed after addition of GppNHp (Fig. 7B). These results demonstrate the inability of the receptor to form a high-affinity ternary complex and is indicative of a reduced coupling efficiency (or desensitization; e.g., Strasser and Lefkowitz, 1985; Samama et al., 1993). The ability of carbachol to desensitize the ₂-adrenergic receptor could be prevented by inhibition of PKC with Ro 31-8220 in intact cells during agonist treatment. The ligand binding curve of membranes prepared from cells exposed to carbachol and Ro-318220 showed both high- and low-affinity components together with a guanine nucleotide induced rightward shift (Fig. 7C).

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Effects of muscarinic receptor stimulation on agonist binding curves for isoproterenol at the 2-adrenergic receptor. Intact CHO-2/m3 cells were incubated with vehicle (A), 1 mM carbachol (B), or 1 mM carbachol + 10 µM Ro 31-8220 (C). Reactions were stopped by washing cells in ice-cold PBS/EDTA. Cells were harvested and membranes prepared. Displacement of [3H]CGP 12177 (~0.5 nM) binding by isoproterenol was conducted in the presence () or absence () of GppNHp (100 µM). The data shown are the means (±S.E.M) of four experiments carried out in duplicate.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Concentration-response curves for cAMP production in permeabilized CHO-2/m3 cells prestimulated with carbachol () or vehicle (). Intact CHO-2/m3 cells were stimulated with vehicle or carbachol (1 mM) for 10 mins before permeabilization. After -escin permeabilization, cells were stimulated with isoproterenol for 10 min and cAMP levels determined as described in the text. The data shown are the means ±S.E.M of three experiments carried out in duplicate.

	Discussion

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The present study investigated two possible mechanisms of receptor cross talk between m3-muscarinic and ₂-adrenergic receptors coexpressed in CHO cells. The first mechanism investigated addresses the possibility that homologous phosphorylation of ₂-adrenergic receptors may be regulated by changes in PIP₂ and intracellular free calcium (Chuang et al., 1996; DebBurman et al., 1996; Pitcher et al., 1996; Pronin et al., 1997). The second mechanism studied centers on the possibility that m3-muscarinic receptor stimulation activates heterologous phosphorylation of the ₂-adrenergic receptor in a GRK-independent manner. We clearly demonstrate that elevation of intracellular free calcium and decrease in PIP₂ pools mediated by m3-muscinic receptor activation has no effect on the ability of endogenous receptor kinases, possibly GRKs, to mediate ₂-adrenergic receptor phosphorylation. In contrast, m3-muscarinic receptor stimulation does regulate ₂-adrenergic receptor function via heterologous phosphorylation of the receptor by PKC.

Recent in vitro studies have demonstrated that GRK activity has an absolute requirement for phospholipids (Onorato et al., 1995; DebBurman et al., 1996; Pitcher et al., 1996) and that PIP₂ levels can modulate purified GRK activity in a concentration-dependent manner (DebBurman et al., 1996). In addition, studies on GRK-2, GRK-3, and GRK-5 have demonstrated that Ca²⁺/calmodulin is a potent inhibitor of GRK activity in vitro (Chuang et al., 1996; Pronin, et al., 1997). These studies suggest that cellular changes in free calcium and membrane PIP₂ levels may be physiological regulators of GRK activity, however, this has never been tested in intact cells. Using a CHO cell line coexpressing the ₂-adrenergic and the m3-muscarinic receptors we were able to induce rapid changes in intracellular free calcium and PIP₂ by stimulation of the m3-muscarinic receptor. Despite elevating intracellular free calcium by ~10-fold and decreasing membrane PIP₂ levels by >70% there was no change in the ability of endogenous kinases to phosphorylate the ₂-adrenergic receptor in CHO-₂/m3 cells. Our data, therefore, demonstrates that the non-PKA component of ₂-adrenergic receptor phosphorylation, which is possibly mediated by the GRKs, is not regulated by changes in intracellular calcium or PIP₂.

Furthermore, our data dispel the notion that PLC-coupled receptors, by virtue of the ability to mobilize intracellular calcium stores and decrease PIP₂, may regulate ₂-adrenergic receptor phosphorylation. This is particularly important because PLC-coupled receptors are coexpressed with ₂-adrenergic receptors in many cell types, for example, m3-muscarinic and ₂-adrenergic receptors in smooth muscle (Eglen et al., 1994).

The large reserves of the lipid precursors phosphatidylinositol 4-phosphate and phosphatidylinositol ensure that on PLC-coupled receptor stimulation PIP₂ is never completely depleted (Willars et al., 1998). It is therefore possible that there is always sufficient PIP₂ to enable proteins that have an absolute requirement for this low abundance phospholipid to operate. In addition to the GRKs there is now a growing number of signaling proteins such as protein kinases. (e.g., Bruton's tyrosine kinase), exchange factors, and GTPase-activating proteins (e.g., Ras GTPase-activating protein) that have been shown to interact with PIP₂, either through pleckstrin homology domains or other protein motifs (e.g., the phosphotyrosine binding-domain on Shc; Harlan et al., 1994; Rameh et al., 1997). The notion that all of these proteins may be sensitive to receptor-mediated changes in membrane PIP₂ levels seems unlikely. The data we present here demonstrate that despite there being a potential for regulating these proteins by receptor-mediated changes in PIP₂, certainly for the GRKs this appears not to be the case. Further studies to establish the concentration of cellular PIP₂ necessary to sustain GRK activity and the size of the "PLC-insensitive" PIP₂ pool will be needed to further address this question. An additional explanation for our results is that GRK activity in intact cells is maintained by phospholipids other than PIP₂ for which the concentrations do not change after PLC-coupled receptor stimulation. For example, in vitro studies have demonstrated that phosphatidylserine and phosphatidylinositol can support GRK activity in vitro (Onorato et al., 1995; DebBurman et al., 1996). This explanation does not, however, detract from the fact that changes in the cellular levels of PIP₂ appear not to regulate the phosphorylation of ₂-adrenergic receptors.

The possibility that changes in intracellular free calcium might regulate GRK activity has a precedent in the interaction of GRK-1 with recoverin. GRK-1 is localized exclusively to the retinal rod outer segments and phosphorylates and desensitizes rhodopsin in response to light stimulation (Lorenz et al., 1991). Recoverin, a calcium binding protein almost exclusively found in photoreceptors, has been shown to bind to and inhibit the activity of GRK-1 in a calcium-sensitive manner, and that this binding and inhibition has a physiological role in photoreceptor light adaptation (Chen et al., 1995). Recent studies have demonstrated that Ca²⁺/calmodulin can inhibit GRK-2, GRK-3, and GRK-5 activity in vitro (Chuang et al., 1996; Pronin et al., 1997), raising the possibility that, like GRK-1, the other GRKs may be physiologically regulated by changes in free intracellular calcium. This, however, has never been tested in intact cells. We report here that m3-muscarinic receptor-mediated increases in intracellular free calcium concentrations have no effect on ₂-adrenergic receptor phosphorylation mediated by endogenous receptor kinase(s), possibly the GRKs. Our results indicate, therefore, that despite in vitro experiments demonstrating the ability of Ca²⁺/calmodulin to regulate GRK activity, changes in intracellular calcium concentrations in the CHO-₂/m3 cell line have no regulatory effect on homologous ₂-adrenergic receptor phosphorylation.

In drawing conclusions from the data present here, we have made the assumption that the PKA-independent phosphorylation of the ₂-adrenergic receptor is mediated via the GRKs. This is based on a very extensive literature including a number of studies that have focused on endogenous GRK activity in intact CHO cells (e.g., Bouvier et al., 1988; Moffett et al., 1993; Pippig et al., 1993). Due to the lack of specific inhibitors to the GRKs, it is not, however, possible to categorically assign the PKA-independent phosphorylation identified in this study to the GRKs. As such we cannot discount the possibility that in our cells the ₂-adrenergic receptor may be phosphorylated by a kinase distinct from the GRKs. We have, for example, recently reported that rhodopsin and the m3-muscarinic receptor is phosphorylated in an agonist-sensitive manner by casein kinase 1 (Tobin et al., 1996, 1997). It is possible that the ₂-adrenergic receptor is also phosphorylated by casein kinase 1, or another kinase that is distinct from the GRKs and is not regulated by calcium nor PIP₂. At present, however, the overwhelming evidence from the literature (Pitcher et al., 1998) is that this is not the case and that GRKs represent the receptor-specific kinases responsible for ₂-adrenergic receptor phosphorylation in cell lines and intact tissues.

Although activation of the PLC pathway appears to have no effect on homologous ₂-adrenergic receptor phosphorylation, we show in this study that stimulation of the m3-muscarinic receptor can influence -adrenergic receptor function through heterologous receptor phosphorylation via PKC. It has been shown previously that the -adrenergic receptor can be desensitized by phorbol ester treatment (Johnson et al., 1990; Yuan et al., 1994), however the present study is the first to demonstrate that ₂-adrenergic receptors are phosphorylated in a heterologous fashion after PLC-coupled receptor activation through a mechanism that involves PKC. Because receptor-mediated PKC activation represents the physiologically relevant signaling pathway for this kinase, our data indicate that PKC may play a physiological role in ₂-adrenergic receptor regulation. Furthermore, the rapid time course of m3-muscarinic receptor-evoked phosphorylation of the ₂-adrenergic receptor suggests a rapid functional role for phosphorylation.

Previous studies have demonstrated that PKC can phosphorylate and, by promoting membrane translocation, activate GRK-2 (Winstel et al., 1996). This may, therefore, provide for an indirect mechanism of stimulating ₂-adrenergic receptor phosphorylation. We, however, do not favor this possibility in the context of m3-muscarinic receptor stimulation of ₂-adrenergic receptor phosphorylation because the GRKs are known to phosphorylate only the agonist-occupied ₂-adrenergic receptor (Pitcher et al., 1998) and m3-muscarinic receptors are able to induce phosphorylation of the agonist unoccupied ₂-adrenergic receptor. It appears more likely that PKC is able to directly phosphorylate the ₂-adrenergic receptor.

The functional consequence of m3-muscarinic receptor-mediated phosphorylation of the ₂-adrenergic receptor was to reduce the coupling efficiency of the ₂-adrenergic receptor, as determined by a loss of high-affinity agonist binding and guanine nucleotide-induced shift. This desensitization response could be prevented by pharmacological inhibition of PKC activity, indicating that the loss of coupling and heterologous receptor phosphorylation are linked. Furthermore, the maximal adenylyl cyclase response was reduced after m3-muscarinic receptor pretreatment, indicative of a partial desensitization of the receptor. This may be of profound physiological and pathophysiological importance because m3-muscarinic and ₂-adrenergic receptors are coexpressed in many smooth muscle types, for example airway smooth muscle (Eglen et al., 1994), where they regulate smooth muscle tone. Furthermore, the ₂-adrenergic receptor in airway smooth muscle is the therapeutic site for -adrenergic receptor agonists in obstructive airway diseases such as asthma. The ability of the m3-muscarinic receptor to phosphorylate and desensitize the ₂-adrenergic receptor via PKC may, therefore, have a significant role in the control of smooth muscle tone under normal and pathological conditions.