Introduction

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Agonist binding to G protein-coupled receptors stimulates the exchange of GTP for GDP bound to the  subunit of coupled heterotrimeric GTP binding proteins (Kaziro et al., 1991). Binding of the stable GTP analog, [³⁵S]guanosine-5'-O-(3-thio)triphosphate (GTP³⁵S) to membranes using rapid filtration techniques has been used as a functional assay for muscarinic receptors in cells and tissues (Hilf et al., 1989; Lazareno and Birdsall, 1993; Burford et al., 1995; Olianas and Onali, 1996). GTP binding coupled with immunoprecipitation using specific antibodies to G protein  subunits has been used as a method to identify which GTP binding proteins are coupled to receptors of interest (Okamoto et al., 1992; Offermanns et al., 1994; Murthy and Makhlouf, 1996; Barr et al., 1997). By using immunoprecipitation with anti-Gq/11, Reever et al. (1995) demonstrated an enhanced agonist-stimulated/basal binding ratio for GTP³⁵S binding to membranes from Chines hamster ovary (CHO) cells transfected with M₁ receptors compared with the rapid filtration method. Thus the use of specific antibodies to G protein  subunits may be used to identify which GTP binding proteins couple to individual subtypes of muscarinic receptors in cells and tissues, and may also improve signal to noise for GTP³⁵S binding under certain circumstances. In the present study we developed a GTP³⁵S binding assay using anti-G protein antibodies coupled with antibody capture via anti-IgG-coated scintillation proximity assay (SPA) beads. This approach is much more convenient than conventional immunoprecipitation and allows for development of medium throughput automated assays. In the present study we demonstrate the use of antibody capture GTP³⁵S binding to explore muscarinic receptor-G protein interaction in transfected cells and native brain tissue.

	Experimental Procedures

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Cell Culture. CHO cells transfected with human M₁-M₅ receptors were grown either in suspension or in monolayer. For suspension cultures cells were grown in roller bottles with constant agitation at 37°C and 5% CO₂ using Dulbecco's modified Eagles medium/F-12 (3:1) culture medium supplemented with 5% fetal bovine serum, 50 µg/ml tobramycin, and 20 mM HEPES. Monolayer cultures were grown in T-225 flasks at 37°C and 5% CO₂ in Dulbecco's modified Eagles medium supplemented with 10% fetal bovine serum and 100,000 U/liter of penicillin/streptomycin. Cells were harvested using trypsin-free dissociation media at 95% confluence and were collected by centrifugation and stored at 80°C. Cells stably expressing human muscarinic receptors were obtained from the National Institutes of Health.

Membrane Preparation. Cell pellets were thawed and resuspended in 20 volumes of 20 mM sodium phosphate buffer, pH 7.4, and were homogenized twice for 30 s at high speed using a Tissuemizer. Homogenates were centrifuged at 200g for 15 min at 4°C. The supernatant was removed and reserved on ice. This procedure was repeated twice and the pooled supernatants were then centrifuged at 40,000g for 45 min at 4°C. Membranes were suspended at 5 mg protein/ml and were stored at 80°C. Unless indicated otherwise in the figure legends, membranes from M₁, M₂, and M₄ cells were prepared from cells grown in suspension, whereas those from M₃ and M₅ cells were from cells grown in monolayer. Receptor densities (pmol mg¹ membrane protein) were 9.3, 0.7, 0.6, 0.9, and 4.8 for M₁-M₅ receptors, respectively.

GTP³⁵S Binding. Assays were run in 20 mM HEPES, 100 mM NaCl, and 5 mM MgCl₂ at pH 7.4 in a final volume of 200 µl in 96-well Costar plates at 25°C. One hundred microliters of membrane preparation (25 µg protein per well for cell membranes and 9-15 µg per well for brain membranes) containing the appropriate concentration of GDP was added followed by addition of 50 µl of buffer ± agonists and antagonists being tested followed by 50 µl of GTP³⁵S to provide a final concentration in the assay of 200 pM for CHO membranes and 500 pM for brain membranes. For CHO membranes, 0.1 µM GDP was used for M₁, M₃, and M₅ receptor assays, whereas 1 µM GDP was used for M₂ and M₄ assays. For brain membranes 0.1 µM GDP was used in assays carried out with anti-Gq/11, whereas 50 µM GDP was used for assays usng anti-Gi(1-3) and anti-Go. CHO cell membranes were incubated for 30 min at 25°C with agonists and antagonists followed by addition of GTP³⁵S and incubation for an additional 30 min. Brain membranes were incubated for 20 min at 25°C with agonists and antagonists followed by addition of GTP³⁵S and incubation for an additional 60 min. Preincubation was employed to ensure that agonists and antagonists were at equilibrium during the labeling period.

Materials. ³⁵S-GTPS (1000-1200 Ci/mmol), anti-rabbit-IgG and anti-mouse-IgG-coated SPA beads, and WGA-coated SPA beads were obtained from Amersham (Arlington Heights, IL). Rabbit anti-Gq/11 and rabbit anti-Gi(1-3) were from Santa Cruz Biotechnologies (Santa Cruz, CA). Mouse monoclonal anti-Go was from Chemicon (Temecula, CA). Oxotremorine M and pirenzepine were from Research Biochemicals Inc. (Natick, MA). 11-{[2-((Diethylamino)methyl)-1-piperidinyl]acetyl}-5,11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepin-6-one (AFDX 116) was synthesized at Eli Lilly. Complete protease inhibitor cocktail and 10% Nonidet P-40 were from Boehringer Mannheim (Indianapolis, IN).

Data Analysis. Concentration-response curves were fitted using sigmoidal nonlinear regression with variable slope in Graphpad Prism. Equilibrium K_is for data obtained via antagonist dose responses in the presence of a fixed agonist concentration were calculated from the general Cheng Prusoff relationship (Leff and Dougall, 1993):
where IC₅₀ is the half-maximal point on the antagonist dose-response curve, A is the fixed concentration of agonist used, EC₅₀ is the half-maximal point on the agonist dose-response curve, and n is the slope of the agonist dose-response curve. Equilibrium K_is for single shifts in oxotremorine M dose-response curves in the presence of a fixed antagonist concentration were calculated from:
where EC₅₀a and EC₅₀b are half-maximal points on the agonist dose-response curves in the absence and presence, respectively of a fixed concentration of antagonist [I].

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Characteristics of Antibody Capture GTP³⁵S Binding. Figure 1 illustrates the effect of antibody concentration on oxotremorine M-stimulated GTP³⁵S binding determined with anti-Gq/11 (CHO M₁ membranes), anti-Gi(1-3) (CHO M₂ membranes), and anti-Go (rat striatal membranes). No agonist-stimulated binding was observed in the absence of added antibody. The cpm measured in the absence of antibody result from the nonproximity effect of ³⁵S in solution plus a small amount of nonspecific binding. Basal and agonist-stimulated binding increased to a plateau level with increasing antibody concentration. The antibody dilution curves shown in Fig. 1 were obtained using the SPA bead dilution stated in Experimental Procedures, i.e., each bottle of reagent obtained from the manufacturer diluted with 20 ml of assay buffer. Using the anti-Gq/11 dilution that produced maximal agonist-stimulated binding to CHO M₁ membranes as shown in Fig. 1, it was observed that, over the range of 25 ml/bottle to 10 ml/bottle, basal and agonist-stimulated binding signals increased with increasing bead density without major effect on signal to noise. A bead density was chosen that provides a sufficiently large agonist-stimulated signal to obtain reproducible concentration- response curves without using more reagent than necessary. We have observed that different lots of antibody may show differences in optimal antibody concentration required for maximal agonist-stimulated binding signal.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effect of antibody concentration on GTP35S binding to membranes from CHO cells and rat striatum. A, binding determined with rabbit anti-Gq/11 and CHO M1 membranes. B, binding determined with rabbit anti-Gi(1-3) and CHO M2 membranes. C, binding determined with mouse monoclonal antiGo and rat striatal membranes. No significant agonist-stimulated binding was observed in the absence of added antibody. Basal binding seen in the absence of antibody is a combination of nonproximity effect of 35S in solution plus a small amount of nonspecific binding. For C, cpm in the absence of antibody were 1570 ± 17 of which 1247 ± 49 were due to nonproximity effects (determined in the absence of added membranes). Data are means ± S.E. from a single experiment with each antibody run with eight replicates per data point.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Concentration-response curves for oxotremorine M-stimulated GTP35S binding mediated by M1-M5 receptors in membranes from stably transfected CHO cells. Data were obtained using antibody capture and rabbit antiserum indicated on each graph. Each curve was obtained using 25 µg of membrane protein per well. EC50 values for oxotremorine M were: M1: 7.9 ± 1.5 µM; M2: 0.17 ± 0.014 µM; M3: 16 ± 4 µM; M4: 1.7 ± 1.1 µM; and M5: 3.6 ± 0.5 µM. Slopes for curves were: M1: 1.01; M2: 0.83; M3: 0.92; M4: 0.84; and M5: 0.94. Number of experiments represented by each curve is indicated on each graph. Data are means ± S.E. Each experiment was run with two to four replicates per data point.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Basal and agonist-stimulated GTP35S binding mediated by M1-M5 receptors measured by WGA versus antibody capture. A, antibody capture assay. B, WGA capture assay. All assays were carried out with 25 µg of membrane protein per well. Data are from 2 to 15 experiments for each receptor. Number above each bar showing stimulation by 100 µM oxotremorine M is agonist stimulated/basal ratio. In antibody capture assay anti-Gq/11 was used for M1, M3, and M5 receptors and anti-Gi(1-3) for M2 and M4 receptors. Basal binding includes nonproximity effects. Numbers are means ± S.E. In each experiment, assays were run with two to four replicates per data point. Agonist-stimulated binding was significantly greater than basal binding (p < .05) in each case except for the M5 data obtained using the WGA assay.

Coupling of the M₁ Receptor in CHO Cells to Inhibitory G proteins. As shown in Fig. 4A, treatment of CHO M₁ cells for 18 h with 100 ng ml¹ pertussis toxin (PTX) before membrane preparation resulted in a significant 70% reduction in agonist-stimulated GTP³⁵S binding to whole membranes, suggesting that the M₁ receptor couples to Gi as well as Gq family GTP binding proteins in these cells. The data in Fig. 4B were obtained by antibody capture and demonstrate that M₁-mediated binding to Gq/11 was not significantly affected by PTX pretreatment, whereas binding determined using anti-Gi(1-3) was reduced by 76%. In contrast to the promiscuous coupling of the M₁ receptor in CHO cells, M₂ receptor-mediated GTP³⁵S binding was largely restricted to inhibitory G proteins (binding determined with anti-Gq/11 was only 5% of that determined with anti-Gi(1-3), (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of PTX on GTP35S binding to membranes from CHO M1 cells grown in monolayer culture. Data are binding stimulated by 100 µM oxotremorine M with basal values subtracted. Data are averaged from six individual experiments with eight replicates per data point in each. Data are means ± S.E. A, effect of PTX on GTPS binding to whole membranes determined using the WGA assay. B, effect of PTX on GTPS binding measured with anti-Gq/11 and anti-Gi(1-3) antibodies. PTX treatment of cells resulted in a significant 70% reduction in agonist-stimulated GTPS binding to whole membranes and a significant 76% reduction in agonist-stimulated binding measured with anti-Gi(1-3). PTX had no significant effect on agonist-stimulated GTPS binding determined with anti-Gq/11. *Significantly different from control, P < .01.

GTP³⁵S Binding to Rat Striatal Membranes Using Antibody Capture. Basal and oxotremorine M-stimulated GTP³⁵S binding to rat striatal membranes determined with anti-Gq/11, anti-Go, and anti-Gi(1-3) is shown in Fig. 5A. Agonist-stimulated binding averaged 92%, 62%, and 49% over basal binding with these three antibodies, respectively. Basal binding determined with anti-Gq/11 was significantly lower than that obtained with the two antibodies against inhibitory G proteins (p < .01). Figure 5B compares the effect of 0.1 µM versus 50 µM GDP on agonist-stimulated GTP³⁵S binding to striatal membranes determined with anti-Gq/11 and anti-Go. Using anti-Gq/11, agonist-stimulated binding at 50 µM GDP was only 6% of that observed at 0.1 µM GDP. In contrast, with the anti-Go antibody no detectable agonist stimulation was observed at 0.1 µM GDP (and basal binding was very high), whereas a robust signal was obtained at 50 µM GDP. Concentration-response curves for oxotremorine M and for pirenzepine reversal of oxotremorine M-stimulated GTP³⁵S binding to striatal membranes determined with anti-Gq/11, anti-Gi(1-3), and anti-Go are shown in Fig. 6. Figure 7 demonstrates the rightward shifts of oxotremorine M dose responses due to a single concentration of AFDX 116 determined using the three antibodies. Table 1 compares the equilibrium K_is for pirenzepine and AFDX 116 calculated from the data in Figs. 6 and 7 with the constants determined for these antagonists based on data obtained for M₁-M₅ receptors in CHO cells. The antagonist constants obtained with CHO cell membranes were determined from concentration responses for pirenzepine- and AFDX 116-mediated reversal of oxotremorine M-stimulated binding.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   GTP35S binding to rat striatal membranes. Assays were carried out as described in Experimental Procedures. Data are means ± S.E. *Significantly different from basal p < .05. A, basal and stimulated binding determined with anti-Gq/11, anti-Go, and anti-Gi(1-3) antibodies. Data are from five independent experiments run with four replicates per data point. Basal and stimulated cpm for each antibody were: anti-Gq/11: basal, 3284 ± 246, stimulated, 6292 ± 337; anti-Go: basal, 6727 ± 631, stimulated, 10920 ± 433; anti-Gi(1-3): basal, 9842 ± 1484, stimulated, 14620 ± 1913. B, Effect of [GDP] on binding determined with anti-Gq/11 and anti-Go. Data are from a single experiment with 16 replicates per data point. Basal values include nonproximity effects.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Concentration responses for GTP35S binding to rat striatal membranes stimulated by oxotremorine M and pirenzepine block of oxotremorine M using anti-Gq/11, anti-Gi(1-3), and anti-Go. EC50 and IC50 values are shown on each graph along with curve slopes. Oxotremorine M curves were pooled from four experiments for each antibody. Pirenzepine concentration responses were from three experiments for anti-Gq/11 and anti-Go and from four experiments for anti-Gi(1-3). In each experiment with pirenzepine a concentration response for oxotremorine M was also run and EC50 and slope value from each agonist curve was used to calculate Ki for each experiment using the general Cheng-Prusoff equation shown in Experimental Procedures. Concentration responses for pirenzepine were determined in the presence of 100 µM oxotremorine M. Data are means ± S.E. from experiments run with four replicates per data point.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effect of 10 µM AFDX 116 on oxotremorine M-stimulated GTP35S binding to rat striatal membranes determined with anti-Gq/11, anti-Gi(1-3), and anti-Go. Data were pooled from three independent experiments. EC50 values are shown on each graph. Ki value for AFDX 116 was calculated in each experiment from equation shown in Experimental Procedures. Data are means ± S.E. from experiments run with four replicates per data point.

View this table: [in this window] [in a new window]   TABLE 1 Comparison of equilibrium Kis for pirenzepine and AFDX 116 for block of GTP35S binding mediated by muscarinic receptors in rat striatal and CHO cell membranes

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The present study describes for the first time the use of SPA technology in conjunction with anti-G protein antibodies to measure GTP³⁵S binding mediated through activation of G protein- coupled receptors. The method provides a convenient alternative to labor-intensive conventional immunoprecipitation for investigating receptor coupling to specific classes of GTP binding proteins. We have shown, as previously described by Reever et al. (1995) for the muscarinic M₁ receptor, that the signal to noise for GTP³⁵S binding mediated by muscarinic receptors primarily coupled to phosphatidylinositol hydrolysis (M₁, M₃, and M₅) is markedly increased by isolation of labeled Gq/11 from the total pool of G proteins present in whole membranes. The specificity of the antibodies used in the present investigation is illustrated by the improved signal to noise obtained with anti-Gq/11, by the PTX block of M₁-mediated binding determined with anti-Gi(1-3) without effect on M₁-mediated binding determined with anti-Gq/11, and by the much greater magnitude of agonist-stimulated binding mediated by M₂ receptors (which are coupled to inhibition of adenylate cyclase in CHO cells) when measured with anti-Gi(1-3) than with anti-Gq/11. The rank order of potencies determined by antibody capture for oxotremorine M-stimulated GTP³⁵S binding mediated by M₁-M₄ receptors shown in Fig. 2 (M₂ > M₄ > M₁ > M₃) was the same as reported by Lazareno and Birdsall (1993) for acetylcholine-stimulated GTP³⁵S binding to CHO cell membranes measured by rapid filtration. It is noteworthy that the slopes of agonist dose-response curves shown in Fig. 2 for M₁-M₅ receptor-mediated GTP³⁵S binding were universally steeper than those obtained in the study by Lazareno and Birdsall (1993) using binding to whole membranes, probably reflecting a more restricted variety of G proteins involved in binding due to the use of antibody capture. The direct demonstration of M₁ receptor coupling to inhibitory G proteins in CHO cells shown with anti-Gi(1-3) in this study confirms and extends the observations of Burford et al. (1995), which were based only on PTX effects in CHO cells. The difference in M₁ versus M₂ promiscuity observed in these studies may well be due to the fact that M₁ receptor expression was 10-fold greater than M₂ in our cell lines because we have shown that removal of 80% of [³H]N-methylscopolamine binding to CHO M₁ membranes by partial alkylation had no significant effect on Gq/11 binding while completely eliminating Gi binding (results not shown). It should be pointed out that not all commercially available antibodies will work in the GTP³⁵S binding assay because we have tried a number of anti-Gq/11 and anti-Gi/o antibodies that did not allow demonstration of appreciable agonist-stimulated signals.

Using the anti-Gq/11, anti-Gi(1-3), and anti-Go antibodies employed in this study, we were able to measure significant oxotremorine M-stimulated GTP³⁵S binding to membranes prepared from rat striatum. The large difference in GDP requirement for agonist-stimulated binding determined with anti-Gq/11 compared with anti-Gi(1-3) and anti-Go strongly indicates that these two classes of antibodies are able to separate signaling mediated by receptors in striatal membranes linked to stimulation of phosphatidylinositol hydrolysis from those coupled to the inhibition of adenylate cyclase. With regard to muscarinic receptors, rat striatum is known to be dominated by M₁ and M₄ receptors (Purkerson and Potter, 1998), but also has significant numbers of M₂ receptors, which are found on large cholinergic interneurons in this tissue (Levey et al., 1991). Muscarinic M₄ receptors have been shown to be responsible for mediating muscarinic agonist-induced inhibition of adenylate cyclase in membranes from rat striatum (Olianas et al., 1996). The calculated equilibrium K_is for pirenzepine and AFDX 116 shown in Table 1 for inhibiting oxotremorine M-stimulated GTP³⁵S binding to striatal membranes determined with anti-Gq/11 are quite similar to those obtained for binding mediated by M₁ receptors in CHO membranes, strongly suggesting that this binding response is primarily due to the muscarinic M₁ receptor. It is unlikely that M₂ or M₄ receptors contributed to the response measured with anti-Gq/11 based on the equilibrium K_i obtained for AFDX 116 and the fact that these receptors do not normally couple to Gq/11. Any significant contribution of M₃ and M₅ receptors to the response measured with anti-Gq/11 would have increased the observed K_i for pirenzepine (Table 1). Although we and others have shown that M₁ receptors in transfected cells can couple to inhibitory G proteins, such coupling is inhibited by high concentrations of GDP as for coupling to Gq/11 (data not shown). For this reason it is unlikely that M₁ receptors made any significant contribution to the binding responses in striatum measured with anti-Gi(1-3) or anti-Go, which were determined at 50 µM GDP. The potency for pirenzepine block of GTP³⁵S binding mediated by M₄ receptors in CHO cells shown in Table 1 is very similar to the value reported by Lazareno and Birdsall (1993) for GTP³⁵S binding in CHO M₄ cell membranes and also to the value obtained in these cells for pirenzepine block of M₄-mediated displacement of N-methylscopolamine binding (Dorje et al., 1990). These values for pirenzepine block of M₄-mediated responses in CHO cells are, however, much lower than the numbers reported in the literature (180-313 nM) for pirenzepine blockade of muscarinic M₄ receptor-mediated inhibition of adenylate cyclase in striatal membranes (DeLapp et al., 1996; Olianas and Onali, 1991; Ehlert et al., 1989; McKinney et al., 1989). This discrepancy may reflect the differences in buffer ionic strengths used for radioligand and GTPS binding compared with conditions used to measure adenylate cyclase activity because M₄-mediated inhibition of adenylate cyclase in N1E-115 neuroblastoma cells (McKinney et al., 1991) was blocked by pirenzepine with an affinity (214 nM) in the same range as reported for the values obtained in rat striatum. The K_i for pirenzepine block of oxotremorine M-stimulated GTP³⁵S binding to rat striatal membranes determined with anti-Go in this study (81 nM) lies between the numbers reported in the literature for M₄ receptor-mediated responses in cells versus rat striatum, and is significantly lower than the pirenzepine value for the cloned M₂ receptor (Table 1). Because pirenzepine blocked the response determined with anti-Go with a potency three times greater than observed for AFDX 116, whereas the reverse order of potency was found for these antagonists at the cloned M₂ receptor (Table 1), we concluded that this response was mediated primarily by M₄ receptors. Because the values for pirenzepine and AFDX 116 determined with anti-Gi(1-3) did not differ significantly, it is reasonable to suggest that this response can be attributed to both M₂ and M₄ receptors. This interpretation of the data suggests that M₂ and M₄ receptors in striatum may couple differentially to members of the inhibitory G protein family as has been shown for these receptors transfected into JEG-3 cells (Migeon et al., 1995).

Reports of receptor-mediated GTP³⁵S binding to whole brain membranes measured by rapid filtration have employed high micromolar concentrations of GDP in the assay to obtain significant agonist stimulation (Olianas and Onali, 1996; Alper and Nelson, 1998). Figure 5B shows that it was not possible in striatal membranes to demonstrate agonist stimulation with the anti-Go antibody at 0.1 µM GDP because of the high basal binding. The same observation has been made with striatal preparations for whole membrane binding (determined with WGA beads) and for binding determined with anti-Gi(1-3) (data not shown). The data in Fig. 5B obtained with anti-Gq/11 also demonstrate that, at least for muscarinic agonist-stimulated GTP³⁵S binding, high micromolar concentrations of GDP inhibit coupling to Gq family proteins. For this reason one cannot reliably measure agonist-stimulated GTP³⁵S binding mediated by receptors coupled to stimulation of phospholipase C in striatal membranes using conventional rapid filtration techniques. This is one clear advantage of the antibody capture method which, as we have shown, allowed us to determine oxotremorine M-stimulated binding in striatal membranes using anti-Gq/11 at 0.1 µM GDP, which was likely mediated primarily through activation of the phospholipase C-coupled M₁ receptor. As indicated by the present study, the antibody capture method employing specific antisera to individual G proteins also has the potential for developing receptor subtype-specific binding assays in complex native tissues in situations where differential coupling of receptor subtypes to individual G protein members of the same family may occur.