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CELLULAR AND MOLECULAR

Cysteine Pairs in the Third Intracellular Loop of the Muscarinic M₁ Acetylcholine Receptor Play a Role in Agonist-Induced Internalization

From the Department of Biochemistry and Microbiology (G.W.S., C.A.S.), Center for Health Sciences, Oklahoma State University, Tulsa, Oklahoma; and the Department of Pharmacology (F.J.E.), School of Medicine, University of California, Irvine, California

Received for publication March 29, 2007
Accepted May 30, 2007.

	Abstract

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We determined the functional role of a small domain in the third intracellular loop of the human muscarinic M₁ (hM₁) receptor. Using site-directed mutagenesis, several mutant hM₁ receptors were made possessing either a deletion or point mutations within the third intracellular loop domain ²⁵²PETPPGRCCRCC²⁶³. Wild-type and mutant hM₁ receptors were transiently expressed in Chinese hamster ovary cells, and the effects of each mutation on radioligand binding, agonist-mediated phosphoinositide hydrolysis, and agonist-induced internalization were determined. The mutant receptors exhibited a modest reduction in affinity for [³H]N-methylscopolamine (pK_D = 9.0) and a moderately increased binding capacity relative to the wild-type receptor. This moderate increase in binding capacity was associated with small increases in the maximal response and potency of carbachol for eliciting phosphoinositide hydrolysis through the mutant receptors (pEC₅₀ = 5.5) relative to wild-type (pEC₅₀ = 5.35 ± 0.05). In contrast, carbachol-induced internalization of mutant hM₁ receptors possessing either C259A/C260A or C262A/C263A or both double point mutations was significantly reduced compared to the wild-type hM₁ receptor. Of the hM₁ receptor mutants tested, those possessing a C262D/C263D double point mutation had the least carbachol-induced internalization. The desensitization and down-regulation of receptors possessing either Cys/Ala or Cys/Asp double point mutations were similar to those observed for the wild-type hM₁ receptor. Collectively, these observations suggest that Cys pairs Cys259/Cys260 and Cys262/Cys263 play an important role in the agonist-induced internalization of hM₁ receptors.

The S/T-rich domain present in the i3 loop of hM₁ receptors is probably phosphorylated by G protein-coupled receptor kinase 2 (GRK2) in an agonist- and Gβ-dependent manner (Haga et al., 1996; Willets et al., 2005). Based on previous observations, this activity of GRK2 may lead to desensitization and arrestin-dependent internalization of the hM₁ receptor (see review, Pierce et al., 2002). Consistent with this expectation, the suppression of GRK2 expression in rat hippocampal neurons using an antisense strategy significantly reduced M₁ receptor phosphorylation and desensitization (Willets et al., 2005). However, the effect of depleting GRK2 on agonist-induced internalization of the M₁ receptor was not investigated.

Considering the numbers of sequence and structural motifs identified in GPCRs thus far, it is expected that other yet to be described receptor domains contribute to the agonist-dependent regulation of M₁ receptor signaling and plasma membrane expression (for review, see Bockaert et al., 2004). For example, binding sites for Gβ and β-arrestin probably exist on the M₁ receptor, because the presence of Gβ increases GRK2-mediated phosphorylation of affinity-purified M₁ receptors and M₁ receptors colocalize with β-arrestin (Haga et al., 1996; Tolbert and Lameh, 1996; Santini et al., 2000). To date, the binding sites for Gβ and β-arrestin have not been identified for the M₁ receptor, unlike the binding sites for both Gβ and β-arrestin on the M₃ receptor (Wu et al., 1997, 2000). It should also be noted that the functional role of the S/T-rich domain present in the i3 loop of the M₁ receptor may be to mediate down-regulation instead of internalization. Shockley et al. (1999) demonstrated that mutations within the S/T-rich domain decreased agonist-induced hM₁ receptor down-regulation and did not affect internalization in CHO cells. Although this finding could be a consequence of the cell type used (i.e., CHO cells versus HEK293 cells), it underscores the fact that our knowledge of the receptor domains that contribute to the agonist-dependent regulation of the hM₁ receptor is incomplete.

In this investigation, we determined the functional role of a novel domain in the i3 loop (²⁵²PETPPGRCCRCCR²⁶⁴) of the hM₁ receptor. This receptor domain possesses a putative SH-3 binding motif, a putative proline-dependent Ser/Thr kinase site, and two pairs of Cys residues with basic Arg residues on either side (i.e., putative palmitoylation sites). A part of this domain (²⁵⁴TPPGRCCR²⁶¹) was determined to mediate an interaction between "bait" and "prey" constructs made with the i3 loop sequence of the hM₁ receptor in yeast two-hybrid assays (G. W. Sawyer, unpublished observations). Deletion of ²⁵⁴TPPGRCCR²⁶¹ from the i3 loop of hM₁ receptors caused a significant decrease in the carbachol-induced internalization of the receptor. This decrease in internalization could not be attributed to the loss of either the putative SH3 binding motif or the Thr phosphorylation site but was rather caused by the deletion of Cys pair Cys259/Cys260. We also found that mutating Cys pair Cys262/Cys263 to an Asp pair almost completely inhibited carbachol-induced hM₁ receptor internalization. Thus, Cys pairs Cys259/Cys260 and Cys262/Cys263 appear to play an important role(s) in the agonist-dependent regulation of hM₁ receptor activity.

	Materials and Methods

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Site-Directed Mutagenesis. The hM₁ receptor cDNA, cloned into a modified Okayma-Berg expression vector (pCD), was a generous gift from Dr. Tom I. Bonner at the National Institute of Mental Health (Bethesda, MD). Mutant hM₁ receptors were made by either deleting sequences or introducing point mutations into the hM₁ receptor cDNA of pCD-hM₁ using the QuikChange II Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and various mutagenesis primers. A T254A point mutation was made in the hM₁ receptor sequence (pCD-hM₁T/A) using primers 5'-GAGGGCTCACCAGAGGCTCCTCCAGGCCGCTGC-3' (forward) and 5'-GCAGCGGCCTGGAGGAGCCTCTGGTGAGCCCTC-3' (reverse). pCD-hM₁P/A, a hM₁ receptor sequence with a P252A point mutation, was made using 5'-GCTGAGGGCTCAGCAGAGACTCCTCCAG-3' (forward) and 5'-CTGGAGGAGTCTCTGCTGAGCCCTCAGC-3' (reverse). 5'-GACTCCTCCAGGCCGCGCTGCTCGCTGCTGCCGGGCC-3' (forward) and 5'-GGCCCGGCAGCAGCGAGCAGCGCGGCCTGGAGGAGTC-3' (reverse) mutagenesis primers were used to make the C259A/C260A double point mutant (pCD-hM₁CC₂₅₉). The C262A/C263A double point mutant (pCD-hM₁CC262) was made using 5'-GGCCGCTGCTGTCGCGCCGCCCGGGCCCCCAGGCTG-3' (forward) and 5'-CAGCCTGGGGGCCCGGGCGGCGCGACAGCAGCGGCC-3' (reverse). pCD-hM₁2CC, a mutant possessing both C259A/C260A and C262A/C263A point mutation, was made using 5'-CTCCTCCAGGCCGCGCCGCTCGCGCCGCTCGGGCCCCCAGGCTG-3' (forward) and 5'-CAGCCTGGGGGCCCGAGCGGCGCGAGCGGCGCGGCCTGGAGGAG-3' (reverse). The C262D/C263D double point mutant (pCD-hM₁DD₂₆₂) was made using 5'-GGCCGCTGCTGTCGCGACGATCGGGCCCCCAGGCTG-3' (forward) and 5'-CAGCCT GGGGGCCCGATCGTCGCGACAGCAGCGGC-3' (reverse). pCD-hM₁2DD, a hM₁ receptor sequence possessing both C259D/C260D and C262D/C263D point mutations, was made using 5'-ACTCCTCCAGGCCGCGACGACCGCGACGACCGGGCCCCCAGGCTG-3' (forward) and 5'-CAGCCTGGGGGCCCGGTCGTCGCGGTCGCGCGGCCTGGAGGAGT-3' (reverse). The C263A point mutant (pCD-hM₁C₂₆₃) was made using 5'-TGCTGTCGCTGCGCCCGGGCCCCCAGG-3' (forward) and 5'-CCTGGGGGCCCGGGCGCAGCGACAGCA-3' (reverse) primers. The sequence encoding amino acids ²⁵⁴TPPGRCCR²⁶¹ was deleted from the hM₁ receptor (pCD-hM₁del) using 5'-GCTCACCAGAGTGCTGCCGGGC-3' (forward) and 5'-GCCCGGCAGCACTCTGGTGAGC-3' (reverse). All mutant hM₁ receptors were sequenced at the Oklahoma State University core DNA sequencing facility to verify the presence of the planned mutation and to ensure that no other mutations were acquired during PCR.

Cell Culture and Transient Transfections. CHO cells were maintained in growth medium (F-12K supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin) in a humidified incubator set at 37°C in an atmosphere of 5% CO₂-95% air and were subcultured every 2 to 3 days. For transient transfections, cells were trypsinized and plated in a 24-well plate format at 1.65 x 10⁵ cells/well in 500 µl of transfection medium (F-12K supplemented with 10% fetal bovine serum). Cells were transfected the following day using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the product protocol. Briefly, 19.2 µg of plasmid DNA/plate was incubated in 1200 µl of Opti-MEM I (Invitrogen) for 5 min at room temperature. Likewise, Lipofectamine 2000 (48 µl/plate) was incubated in 1200 µl of Opti-MEM I for 5 min at room temperature in a separate tube. The DNA and lipid mixtures were combined into a single tube and mixed gently. Lipid complexes were allowed to form during a 20-min incubation at room temperature. Complexes (100 µl/well) were added to each well of the 24-well plate, and the plate was placed into a humidified incubator. After a 6-h incubation, medium was replaced in each well with fresh transfection medium (500 µl). Cells were then incubated for an additional 18 h (24 h total) in a humidified incubator before experiments were conducted.

Receptor Internalization Assay. CHO cells transiently expressing either wild-type or mutant hM₁ receptors were washed with F-12K (three 500-µl washes) to remove serum and then incubated with the muscarinic receptor agonist carbachol (1 mM) in F-12K (500 µl) for up to 4 h (six wells for each time point) in a humidified incubator set at 37°C in an atmosphere of 5% CO₂-95% air. We chose this time period because we found that the internalization process was nearly complete in cells expressing the wild-type receptor over this time interval. Cells were washed extensively on ice with ice-cold PBS (three 500-µl washes) to remove carbachol and to prevent further receptor trafficking. Intact cell binding assays were then performed using a single concentration of the membrane-impermeable radioligand [³H]N-methylscopolamine ([³H]NMS; PerkinElmer Life and Analytical Sciences, Boston, MA) as described below under Receptor Binding Assays. This assay was used to determine the amount of wild-type and mutant hM₁ receptor expressed on the plasma membrane of CHO cells before and during incubation with carbachol.

Receptor Down-Regulation Assay. CHO cells transiently expressing either wild-type or mutant hM₁ receptors were washed (three 500-µl washes) with F-12K to remove serum and then incubated with carbachol (1 mM) in F-12K (500 µl) for 24 h in a humidified incubator at 37°C and in an atmosphere of 5% CO₂-95% air. We chose this time interval because Shockley et al. (1997) showed that down-regulation is a slow process, requiring approximately 24 h for a substantial loss of receptor. Cells were washed extensively on ice with ice-cold PBS (three 500-µl washes) to remove carbachol. Intact cell binding assays were then performed using either a single concentration of [³H]NMS or the membrane-permeable radioligand [³H]quinuclidinyl benzylate ([³H]QNB; PerkinElmer Life and Analytical Sciences) as described below under Receptor Binding Assays. This assay was used to determine the effect of long incubations with carbachol on the expression of wild-type and mutant hM₁ receptors.

Receptor Binding Assays. Intact cell binding assays were performed using a single concentration of [³H]NMS (1.6 nM) or [³H]QNB (1.2 nM) to determine the amount of plasma membrane-expressed receptor or total receptor expressed, respectively. We chose these concentrations of the two radioligands because these conditions should yield receptor occupancies at the wild-type receptor of 86 and 87%, respectively. Washed CHO cells were incubated with either [³H]NMS or [³H]QNB in the absence (three wells for each time point or condition; total binding) and presence (three wells for each time point or condition; nonspecific binding) of atropine (1 µM) in 500 µl of binding buffer (25 mM HEPES, 113 mM NaCl, 6 mM dextrose, 3 mM CaCl₂, 3 mM KCl, 2 mM MgSO₄, and 1 mM NaH₂PO₄, pH 7.4) for either 1 h ([³H]NMS) or 18 h ([³H]QNB) at 4°C. After incubation, cells were rapidly and gently washed (three 1-ml washes) with icecold PBS to remove unbound [³H]NMS or [³H]QNB. Bound [³H]NMS or [³H]QNB was recovered as described previously (Griffin et al., 2003), and radioactivity was counted using a Beckman LS 6500 scintillation counter. Saturation binding assays were also performed on washed, intact CHO cells transiently expressing either wild-type or mutant hM₁ receptors. Cells were incubated for 2 h at 4°C with geometrically spaced (0.33 log units) concentrations of [³H]NMS (six wells of a 24-well plate for each concentration) in the absence (three wells; total binding) and presence (three wells; nonspecific binding) of atropine (1 µM) in binding buffer. Cells were rapidly and gently washed (three 1-ml washes) with ice-cold PBS to remove unbound [³H]NMS, and bound [³H]NMS was recovered as described previously (Griffin et al., 2003).

The average amount of protein expressed in CHO cells was determined for each radioligand binding assay performed. In brief, three wells of a 24-well plate were plated and transfected for each receptor construct and treatment condition as described above under Cell Culture and Transfections at the same time as experimental plates. The cells were washed two times with 500 µl of mannitol wash buffer (0.29 M mannitol, 0.01 M Tris, and 0.5 mM Ca(NO₃)₂, pH 7.4) to remove serum. The protein concentration was determined for each well using the bicinchoninic acid protocol as described previously (Goldschmidt and Kimelberg, 1989).

Phosphoinositide Hydrolysis and Desensitization Assays. Phosphoinositide hydrolysis assays were conducted on CHO cells transiently expressing wild-type and mutant hM₁ receptors as described previously (Sawyer et al., 2006). In some experiments, the method was modified to determine the effect of acetylcholine pretreatment on the phosphoinositide response elicited to carbachol (used as a measure of receptor desensitization). In brief, CHO cells transiently expressing wild-type hM₁, hM₁CC₂₆₂, or hM₁DD₂₆₂ receptors were washed (three 500-µl washes) with F-12K to remove serum and then incubated with myo-[³H]inositol (PerkinElmer Life and Analytical Sciences) in F-12K (500 µl) for 18 h in a humidified incubator. The cells were washed the following day with F-12K (three 500-µl washes) to remove unincorporated inositol, with a 10-min incubation in a humidified incubator between each wash. Cells were then incubated for 10 min in the absence or presence of acetylcholine (1 mM) in F-12K. Acetylcholinesterase (2 U/well; Sigma-Aldrich, St. Louis, MO) was added to all wells (regardless of whether they had been incubated with acetylcholine or not), and the cells were incubated for 2 min in a humidified incubator. F-12K supplemented with LiCl (10 mM) was added to each well after washing (one 500-µl wash) with F-12K, and the cells were incubated for 10 min in a humidified incubator. Cells were then incubated with geometrically spaced concentrations of carbachol (0.5 log unit) for 30 min in a humidified incubator. Total [³H]inositol phosphates were extracted from cells using perchloric acid as described previously (Sawyer et al., 2006). After extraction of [³H]inositol phosphates, 1.11 ml of chloroform/methanol/HCl (100:200:1, v/v) was added to each well of the plates, and the plates were allowed to stand for 15 min at room temperature. The solution was pipetted from each well into individual 15 x 100-mm polypropylene tubes, and 0.37 ml of water and 0.37 ml of chloroform were added to separate aqueous and organic phases. The tubes were capped, shaken, and then centrifuged at 2000g for 2 min. An aliquot of the organic phase (lower phase; 200 µl) was placed into scintillation vials, and the chloroform was allowed to evaporate. Scintillation cocktail (Scintiverse, 5 ml) was added to each vial after the chloroform had evaporated, and the radioactivity was measured with a Beckman LS6500 scintillation counter to determine the amount of [³H]phosphoinositides. The [³H]inositol phosphates are expressed relative to the total amount of [³H]phosphoinositides plus [³H]inositol phosphates to correct for variation between acetylcholine-treated and untreated cells.

Data Analysis. The Hill slope, the maximal response (E_max), and the concentration of carbachol eliciting half-maximal response (EC₅₀) were estimated from phosphoinositide hydrolysis data using nonlinear regression analysis according to a logistic equation as described by Bowen and Jerman (1995). The same equation was used to estimate the Hill slope, maximal binding capacity (B_max), and the concentration of [³H]NMS eliciting half-maximal receptor occupancy (K_D) from saturation binding data (Bowen and Jerman, 1995). Significance values (P values) were calculated using either one- or two-way ANOVAs (GraphPad Prism, version 4.03; GraphPad Software Inc., San Diego, CA). One-way ANOVAs were performed with a Dunnett's post-hoc test on the E_max, B_max, K_D, and EC₅₀ values estimated from the binding and phosphoinositide hydrolysis data obtained for mutant and wild-type hM₁ receptors. Two-way ANOVAs were performed with a Bonferroni post-hoc test on the data obtained for the carbachol-induced internalization of mutant and wild-type hM₁ receptors. Estimates of the rate constants for wild-type and mutant hM₁ receptor internalization were made by fitting data using a single-phase exponential decay equation (GraphPad Prism, version 4.03).

	Results

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Effects of Deleting ²⁵⁴TPPGRCCR²⁶¹ from the i3 Loop of hM₁ Receptors. We determined that a small domain in the i3 loop of the hM₁ receptor, ²⁵⁴TPPGRCCR²⁶¹, interacts with itself in yeast two-hybrid assays (G. W. Sawyer, unpublished observations). In an effort to determine the functional role of this domain, we deleted it from the i3 loop of the hM₁ receptor and characterized the resulting mutant receptor, hM₁del, in CHO cells.

To determine whether deletion of ²⁵⁴TPPGRCCR²⁶¹ affects the expression of the hM₁ receptor, we measured the binding of [³H]NMS to intact CHO cells transiently expressing the deletion mutant or wild-type receptor (Fig. 1A). [³H]NMS is a quaternary ammonium muscarinic ligand that does not cross the plasma membrane, and, consequently, it can be used in the intact cell to measure the expression of muscarinic receptors at the cell surface. Deletion of ²⁵⁴TPPGRCCR²⁶¹ from the hM₁ receptor caused a moderate 1.6-fold increase in both the dissociation constant (K_D) and binding capacity (B_max) of [³H]NMS relative to those measured for the wild-type receptor (Table 1). These data suggest a small enhancing effect of the deletion on the expression of the hM₁ receptor at the plasma membrane.

View larger version (15K): [in this window] [in a new window]   Fig. 1. The effect of deleting 254TPPGRCCR261 from the i3 loop of hM1 receptors on [3H]NMS binding (A) and carbachol-mediated phosphoinositide hydrolysis (B). A, intact cell [3H]NMS binding assays were performed on CHO cells transiently expressing either wild-type hM1 or hM1del receptors. Each data point represents the mean ± S.E.M. of three experiments conducted in triplicate. B, CHO cells were transiently transfected with wild-type hM1 or hM1del receptors 24 h before measuring carbachol-mediated phosphoinositide hydrolysis. Each data point represents the mean ± S.E.M. of four experiments conducted in triplicate.

To investigate the signaling properties of the deletion mutant, we measured agonist-mediated phosphoinositide hydrolysis in CHO cells transiently expressing the hM₁del or wild-type receptor (Fig. 1B). The potency of carbachol for eliciting phosphoinositide hydrolysis increased 1.4-fold in CHO cells expressing the deletion mutant compared with that measured for the wild-type receptor. There was little difference in the maximal effect of carbachol (E_max) in cells expressing either hM₁ or hM₁del (Table 2). The small increase in the potency of carbachol at hM₁del relative to wild-type is consistent with the greater expression of this deletion mutant at the plasma membrane (see Fig. 1A).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Effect of deleting 254TPPGRCCR261 from the i3 loop of hM1 receptors on carbachol-induced internalization. A, CHO cells transiently expressing either wild-type hM1 or hM1del receptors were incubated with the agonist carbachol (1 mM) for up to 4 h. The amount of plasma membrane expressed receptor was determined at various times during the incubation by conducting intact cell [3H]NMS binding assays. Each data point represents the mean ± S.E.M. of three experiments performed in triplicate. *, significantly different from the carbachol-induced internalization of wild-type hM1 receptors, P < 0.01. **, significantly different from the carbachol-induced internalization of wild-type hM1 receptors, P < 0.001. B, CHO cells transiently expressing either wild-type hM1 or hM1del receptors were incubated with increasing concentrations of carbachol for 2 h. Cells were then washed and used in intact cell [3H]NMS binding assays. Each data point represents the mean ± S.E.M. of four experiments conducted in triplicate.

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To determine whether the deletion of ²⁵⁴TPPGRCCR²⁶¹ affects the potency of carbachol-induced hM₁ receptor internalization, we incubated CHO cells transiently expressing either wild-type hM₁ or hM₁del receptors with various concentrations of carbachol for 2 h and subsequently measured specific [³H]NMS binding to these intact cells. Carbachol caused a concentration-dependent decrease in specific [³H]NMS binding to cells expressing the wild-type hM₁ receptor as well as those expressing the hM₁del receptor (see Fig. 2B). As described above, the maximal decrease was significantly greater for the wild-type hM₁ receptor (36.3 ± 3.6%) compared with that observed for the hM₁del receptor (14.9 ± 5.8%) (F_1,30 = 24.3; P < 0.01). The EC₅₀ value for carbachol-induced internalization of the hM₁del receptor was 1.5-fold greater than that estimated for the wild-type receptor (pEC₅₀ = 4.44 ± 0.02), although this estimate is not significantly different from wild type because of the difficulty in obtaining an accurate estimate of EC₅₀ for such a small response.

The effect of deleting ²⁵⁴TPPGRCCR²⁶¹ on carbachol-induced down-regulation of the hM₁ receptor was also investigated. CHO cells transiently expressing either wild-type hM₁ or hM₁del receptors were incubated with carbachol (1 mM) for 24 h before intact cell binding assays were conducted with a single concentration of [³H]NMS (1.6 nM) or [³H]QNB (1.2 nM). [³H]QNB crosses the plasma membrane, and, consequently, it binds to both intra- and extracellular receptors. A reduction in [³H]QNB binding is indicative of a reduction in the total number of receptors, consistent with receptor down-regulation. In CHO cells transiently expressing the wild-type hM₁ receptor, specific [³H]QNB binding decreased by 58.6 ± 5.0% after a 24-h incubation with carbachol (Fig. 3). A similar decrease was also observed for the hM₁del receptor after 24-h carbachol treatment (65.6 ± 4.6%) (Fig. 3). Under the same conditions, specific [³H]NMS binding decreased by 80.3 ± 2.1% in CHO cells expressing the wild-type hM₁ receptor and by 71.4 ± 3.9% in cells expressing the hM₁del receptor (Fig. 3). The intracellular pool of receptor (determined by subtracting specific [³H]NMS binding from specific [³H]QNB binding) in CHO cells expressing wild-type hM₁ receptors decreased by 27% from a control value of 0.22 pmol/mg protein after carbachol treatment. In contrast, the intracellular pool of hM₁del receptor decreased by 52% from a control value of 0.48 pmol/mg protein after carbachol treatment. These data suggest that the i3 loop domain ²⁵⁴TPPGRCCR²⁶¹ of the hM₁ receptor does not play a role in agonist-induced down-regulation.

View larger version (14K): [in this window] [in a new window]   Fig. 3. The effect of either deleting or mutating amino acid residues in the i3 loop of hM1 receptors on carbachol-induced down-regulation. CHO cells were transiently transfected with the indicated receptor constructs 24 h before conducting experiments. Cells expressing wild-type hM1, hM1del, hM1CC262, or hM1DD262 receptors were incubated in the absence (control) and presence of carbachol (1 mM) for 24 h and then washed and used in either intact cell [3H]NMS or [3H]QNB binding assays. Each bar represents the mean ± S.E.M. of four experiments conducted in triplicate.

View larger version (18K): [in this window] [in a new window]   Fig. 4. The effect of mutating a putative-SH3 motif and proline-dependent Thr kinase site on carbachol-induced hM1 receptor internalization. A, point mutations were introduced into the i3 loop domain 252PETPPGRCCRCC263 of hM1 receptors to disrupt the putative SH3 motif and Pro-dependent Thr kinase site. The domain deleted from hM1del receptors is in boldface in the wild-type hM1 receptor sequence. Also indicated in the WT-hM1 receptor sequence is the putative SH3 binding motif (italics) and the putative kinase site (underlined). B, CHO cells transiently expressing wild-type hM1, hM1P/A, or hM1T/A receptors were incubated with carbachol (1 mM) for up to 4 h. The cells were then washed and used in intact cell [3H]NMS binding assays. Each data point represents the mean ± S.E.M. of three experiments performed in triplicate. The data for wild-type hM1 receptors are from Fig. 2.

Effect of Cys to Ala Point Mutations. We investigated Cys pairs within and adjacent to the i3 loop domain ²⁵⁴TPPGRCCR²⁶¹ to determine their potential role in receptor internalization. We made three hM₁ receptor mutants, the first containing a C259A/C260A double point mutation (hM₁CC₂₅₉), the second containing a C262A/C263A double point mutation (hM₁CC₂₆₂), and the third having both of these double point mutations (hM₁2CC) (Fig. 5A). The results of [³H]NMS binding experiments on intact CHO cells transiently expressing these mutants showed that the mutations caused a modest reduction in the affinity (1.7–3.5-fold increase in K_D) of [³H]NMS and a substantial 2.2 to 2.4-fold increase in the binding capacity of [³H]NMS when expressed relative to the binding parameters of the wild-type receptor (Table 1). The largest effects were observed in cells expressing the hM₁CC₂₆₂ receptor mutant. These increases in binding capacity were associated with corresponding 1.8 to 2.2-fold increases in potency and 1.2 to 1.3-fold increases in the E_max of carbachol-stimulated phosphoinositide hydrolysis relative to that for the wild type (Table 2).

View larger version (21K): [in this window] [in a new window]   Fig. 5. The effects of various Cys to Ala or Cys to Asp point mutations on carbachol-induced hM1 receptor internalization. A, the position of the deletion in hM1del receptors and the positions of various Cys to Ala or Cys to Asp point mutations. B, CHO cells transiently expressing wild-type hM1, hM1del, hM1CC259, hM1CC262, hM12CC, or hM1C263 receptors were incubated with carbachol for up to 4 h. Cells were then washed and used in intact cell [3H]NMS binding assays. Each data point represents the mean ± S.E.M. of three to five experiments performed in triplicate. C, CHO cells transiently expressing wild-type hM1, hM1DD262, or hM12DD receptors were incubated with carbachol for up to 4 h, washed extensively, and used in intact cell [3H]NMS binding assays. Each data point represents the mean ± S.E.M. of three to four experiments performed in triplicate. The data for shown for wild-type hM1 receptors in B and C are from Fig. 2.

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To investigate the possibility that Cys residues in Cys pair Cys262/Cys263 function in an all-or-none manner, we made an hM₁ mutant (hM₁C₂₆₃) possessing a single Ala point mutation in this pair (C623A) and characterized its behavior (Fig. 5A). When transiently expressed in CHO cells, the hM₁C₂₆₃ mutant bound [³H]NMS in the intact cell with moderately reduced affinity (2.1-fold increase in K_D) and a slightly increased binding capacity (1.3-fold increase in B_max) (Table 1). This modest increase in expression relative to wild type was associated with increases in the potency (1.2-fold increase in pEC₅₀) and maximal response (1.3-fold increase in E_max) of carbachol-stimulated phosphoinositide hydrolysis (Table 2).

Carbachol-mediated internalization of hM₁C₂₆₃ was slightly attenuated compared with that for the wild-type receptor, although this difference was not significant (Fig. 5B). The data were consistent with a first-order decay model, having a 1.36-fold greater rate constant (k₁ = 0.015 min^–1) but a higher plateau value (51.6%) relative to that for the wild-type receptor. Overall, these data suggest that the presence of one Cys residue in Cys pair Cys262/Cys263 at least partially, or perhaps fully, rescues carbachol-induced internalization of the hM₁ receptor.

Effect of Cys to Asp Point Mutations. We also investigated the effect of mutating Cys residues to Asp on carbachol-induced internalization of the hM₁ receptor. We made two hM₁ receptor mutants, one possessing a C262D/C263D double point mutation (hM₁DD₂₆₂) and one possessing a C259D/C260D and C262D/C263D quadruple point mutation (hM₁2DD) (Fig. 5A). In [³H]NMS binding assays performed on intact CHO cells transiently expressing either receptor mutant, a 4.2-fold reduction in affinity and a substantial increase in binding capacity (2.5–3.0-fold) was observed (Table 1). These increases in binding capacity were associated with corresponding increases in potency (1.8–2.1-fold) and E_max (1.6–1.7-fold) of carbachol-stimulated phosphoinositide hydrolysis relative to those for the wild type (Table 2).

Carbachol-induced internalization of hM₁DD₂₆₂ and hM₁2DD receptors showed the greatest impairment of all of the mutants tested. [³H]NMS binding decreased only 18 ± 0.8 and 16 ± 3.5% for hM₁DD₂₆₂ and hM₁2DD, respectively, after 4 h of carbachol treatment (Fig. 5C). These decreases in binding exhibited half-times (t_-estimate, 588 and 590 min, respectively) and rate constants (k_1-estimate, 0.0012 and 0.002 min^–1, respectively) for hM₁DD₂₆₂ and hM₁2DD receptors that were 9-fold greater than those relative to wild-type, assuming the same plateau value (i.e., 34.2% of control).

Effect of the C262A/C263A and C262D/C263D Double Point Mutations on hM₁ Receptor Desensitization and Down-Regulation. Desensitization of GPCRs is thought to precede internalization and is probably initiated by the agonist-dependent phosphorylation of the receptor. To investigate whether internalization of the hM₁ receptor is necessary for desensitization to occur, we compared the desensitization of hM₁CC₂₆₂ and hM₁DD₂₆₂ receptors with that of the wild-type hM₁ receptor. These receptor mutants were chosen for this experiment because they had the largest reduction in carbachol-induced internalization (see Fig. 5, B and C), with the fewest amino acid residues mutated (see Fig. 5A).

CHO cells transiently expressing wild-type hM₁, hM₁CC₂₆₂, or hM₁DD₂₆₂ receptors were incubated in the absence or presence of acetylcholine (1 mM) for 10 min before measuring carbachol-stimulated phosphoinositide hydrolysis. Acetylcholine was used to induce receptor desensitization because its action can be rapidly terminated by the addition of purified acetylcholinesterase (see Materials and Methods). In cells expressing the wild-type hM₁ receptor, the EC₅₀ value of carbachol for stimulating phosphoinositide hydrolysis was 1.6-fold greater in cells pretreated with acetylcholine than that measured in untreated cells (pEC₅₀ = 5.44 ± 0.05; Fig. 6A). Acetylcholine treatment also caused an 18.4% reduction in the E_max of the response mediated by the wild-type hM₁ receptor (Fig. 6A). Similar results were obtained for the mutant receptors. In cells expressing the hM₁CC₂₆₂ receptor, acetylcholine treatment caused a 1.9-fold increase in the EC₅₀ value of carbachol (control pEC₅₀ = 5.59 ± 0.09) and a 15.1% reduction in E_max relative to untreated cells (Fig. 6B). In cells expressing the hM₁DD₂₆₂ receptor, acetylcholine treatment caused a 2.0-fold increase in EC₅₀ (control pEC₅₀ = 5.41 ± 0.13) and a 37.6% reduction in E_max relative to untreated cells. These data indicate that hM₁CC₂₆₂ and hM₁DD₂₆₂ receptors desensitize to an extent similar to that observed for the wild-type hM₁ receptor.

View larger version (14K): [in this window] [in a new window]   Fig. 6. The effect of mutagenizing Cys pair Cys262/Cys263 on the desensitization of the phosphoinositide response elicited by hM1 receptors. CHO cells were transiently transfected with either wild-type hM1 (A), hM1CC262 (B), or hM1DD262 (C) receptor constructs 24 h before an 18-h incubation with myo-[3H]inositol (0.2 µM). The cells were washed extensively to remove unincorporated myo-[3H]inositol and then incubated either in the absence or presence of acetylcholine (1 mM) for 10 min in a humidified incubator set at 37°C in an atmosphere of 5% CO2-95% air. Acetylcholinesterase (2 U/well) was added to both control and treated cells, and the cells were incubated for 2 min in a humidified incubator. Phosphoinositide hydrolysis assays were then performed. Each data point represents the mean ± S.E.M. of three experiments performed in triplicate.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The i3 loop of muscarinic receptors is quite large relative to other GPCRs, and it is 158 amino acid residues long in the hM₁ sequence (Tyr208/Thr366). Deletion of most of the i3 loop (Gly232/Tyr358) was shown to inhibit carbachol-induced internalization of the hM₁ receptor in U293 cells without inhibiting phosphoinositide hydrolysis (Maeda et al., 1990). Lameh et al. (1992) found that agonist-mediated internalization of the hM₁ receptor is complex and probably involves the contribution of multiple i3 loop sequences.

In this investigation, we characterized the functional role of a novel domain (i.e., ²⁵²PETPPGRCCRCC²⁶³) in the i3 loop of the hM₁ receptor. Deletion of most of this domain (²⁵⁴TPPGRCCR²⁶¹) decreased carbachol-induced internalization to a rate only one-fourth of that of the wild-type receptor (see Fig. 2A). This effect could not be attributed to disruption of a putative SH3 binding motif or a putative Pro-dependent Thr kinase site within this domain (see Fig. 4). In contrast, alanine mutagenesis of Cys pair Cys259/Cys260 could account for the defect in internalization of the hM₁del mutant.

It seems that attenuated receptor internalization should cause an increase in receptor expression and function. This reasoning can explain why the receptor mutants with attenuated internalization exhibited a moderate increase in [³H]NMS binding and agonist-mediated phosphoinositide hydrolysis relative to wild type when CHO cells were transiently transfected. It has been shown previously that the potency and E_max for carbachol-stimulated phosphoinositide hydrolysis are proportional to hM₁ receptor density in murine fibroblasts (Mei et al., 1989), illustrating that there is a receptor reserve for the phosphoinositide response. In contrast, we expect that internalization would be proportional to receptor occupancy. Accordingly, we found that the pEC₅₀ value of carbachol (4.5) for eliciting internalization is approximately equal to its binding affinity (pK_D) for M₁ receptors expressed in CHO K1 cells (Savarese et al., 1992) and murine fibroblasts (Mei et al., 1989).

It seems unlikely that receptor internalization is rate-limiting and that the increased expression of hM₁del and hM₁CC₂₅₉ receptors, per se, causes a decrease in receptor internalization. Three lines of evidence argue against this hypothesis. First, the increases in the plasma membrane expression of hM₁del and hM₁P/A receptors were similar (i.e., 1.6- and 1.4-fold, respectively), yet the former showed a marked decrease in internalization, whereas the latter behaved like the wild-type receptor (see Table 1; Fig. 6). Second, Lameh et al. (1992) demonstrated that carbachol-induced internalization of the hM₁ receptor was independent of receptor density over at least a 10-fold range in U293 cells; the maximal plasma membrane expression of the mutant receptors tested in this study fell well within this range (see Table 1). Third, Shockely et al. (1999) measured a rate of internalization of the hM₁ receptor in stably transfected CHO cells similar to that reported here, yet the expression of the hM₁ receptor in their study was approximately 3-fold greater than that used here. Therefore, we believe it is unlikely that the increased expression of the mutants investigated in this study (i.e., hM₁del, hM₁CC₂₅₉, hM₁CC₂₆₂, hM₁2CC, hM₁DD₂₆₂, and hM₁2DD) is the cause of their decreased agonist-induced internalization.

Mutagenesis of Cys pair Cys262/Cys263 (hM₁CC₂₆₂) yielded a receptor with an internalization rate only half of that of the hM₁del, hM₁CC₂₅₉, and hM₁2CC receptors (see Fig. 5B). This result suggests that the loss of internalization function cannot be attributed to the loss of a disulfide bond between Cys pairs Cys259/Cys260 and Cys262/Cys263 because one would expect that mutagenesis of either pair would result in equivalent defects in carbachol-induced internalization. It was anticipated that mutation of both Cys pairs Cys259/Cys260 and Cys262/Cys263 (i.e., hM₁2CC) would create a mutant that had internalization kinetics similar to or slower than hM₁CC₂₆₂ receptors. Instead, hM₁2CC receptors had internalization kinetics that were more consistent with hM₁del and hM₁CC₂₅₉ receptors. Changing Cys pair Cys262/Cys263 or both Cys pairs Cys259/Cys260 and Cys262/Cys263 to Asp pairs (i.e., hM₁DD₂₆₂ and hM₁2DD, respectively) further inhibited the carbachol-induced internalization of hM₁ receptors (9-fold slower than wild type). Overall, our data indicate that both Cys pairs Cys259/Cys260 and Cys262/Cys263 play a role in hM₁ receptor internalization, although the roles may not be equivalent.

It is interesting to note that both Cys pairs in the i3 loop of hM₁ receptors are surrounded by basic Arg residues (i.e., ²⁵⁸RCCRCCR²⁶⁴) and, thus, may be palmitoylated to anchor this region of the receptor to the membrane (for review, see el-Husseini and Bredt, 2002). To date, several GPCRs (including the M₂ receptor) are known to be palmitoylated on Cys residues in the C-terminal tail, and palmitoylation has been shown to affect GCPR activity, phosphorylation, and/or trafficking (for reviews, see el-Husseini and Bredt, 2002; Torrecilla and Tobin, 2006). However, we found that 2-bromopalmitate (50 µM; 18-h treatment) did not inhibit carbachol-mediated internalization of wild-type, hM₁CC₂₅₉, and hM₁CC₂₆₂ receptors. 2-Bromopalmitate is known to inhibit palmitoylation of substrates in intact cells. Instead, we found that 2-bromopalmitate caused a 50% inhibition of the expression of all of the receptors tested (G. W. Sawyer, unpublished observations). Thus, palmitoylation does not appear to play a role in the agonist-dependent internalization of hM₁ receptors but instead plays a role in the plasma membrane delivery and expression of the receptor. This observation is consistent with that of Sadeghi et al. (1997), who found that mutagenesis of C-terminal Cys residues Cys341/Cys342 of the vasopressin V2 receptor to serines caused a decrease in the plasma membrane expression of the receptor. The latter Cys residues are known to be palmitoylated.

Our observations in yeast two-hybrid assays suggest that Cys pair Cys259/Cys260 may promote hM₁ receptor dimerization/oligomerization. In these assays, bait and prey fusion proteins possessing the full-length i3 loop sequence of hM₁ receptors interacted with one another, and this interaction was lost when ²⁵⁴TPPGRCCR²⁶¹ was deleted from the prey i3 loop fusion construct (G. W. Sawyer, unpublished observations). Dimerization/oligomerization of muscarinic M₂ and M₃ receptors has been described previously, although the functional consequence of such interactions is unclear but may affect receptor signaling and/or ligand affinity (Maggio et al., 1996; Zeng et al., 1999; Zeng and Wess, 2000; Park et al., 2001). In a more recent investigation using bioluminescence resonance energy transfer, M₁, M₂, and M₃ receptors were observed to form homodimers/oligomers in HEK 293 cells (Goin and Nathanson, 2006). The functional consequence of M₁ receptor dimerization/oligomerization was not determined; however, the formation of M₂/M₃ heterodimers enhanced carbachol-induced M₃ receptor down-regulation (Goin and Nathanson, 2006). Dimerization/oligomerization of hM₁ receptors may result in the efficient delivery of receptor to clathrin-coated pits; a mechanism involved in hM₁ receptor internalization (Tolbert and Lameh, 1996).

Alternatively, one or both Cys pairs may act as a conformational switch, controlling the accessibility of other critical i3 loop domains to the internalization mechanism. Disulfide bonds are known to form between the side chains of adjacent Cys residues, forming an eight-membered cysteinyl-cysteine ring (Creighton et al., 2001; Carugo et al., 2003). The peptide bond between these adjacent residues can assume either a cis-ora trans-conformation depending upon whether the Cys residues are oxidized or reduced, respectively (Carugo et al., 2003). This change in the conformation of the peptide bond is known to alter the conformation of peptides containing them, and, thus, the conformation of the i3 loop of hM₁ receptors may change in a redox-dependent manner (Creighton et al., 2001; Carugo et al., 2003). Given the close proximity of Cys pairs Cys259/Cys260 and Cys262/Cys263 to the S/T rich domain (i.e., 20 amino acid residues away) described by Lameh et al. (1992), either one or both of the Cys pairs may fold the i3 loop of hM₁ receptors to hide this critical internalization domain in an agonist-reversible manner. It is interesting to note that the presence of one Cys residue in Cys pair Cys262/Cys263 (i.e., hM₁C₂₆₃; see Fig. 5A) almost completely rescued carbachol-induced internalization of hM₁ receptors (see Fig. 5B). Perhaps this Cys residue is forming a disulfide linkage with either Cys residue in Cys pair Cys259/Cys260, forming the conformational switch necessary to control the exposure of other critical i3 loops domains.

A role for Cys residues in GPCR internalization has been demonstrated previously, although the residues were in the C-terminal tail of the receptor. Preisser et al. (1999) showed that mutating Cys pair Cys371/Cys372 in the C terminus of the vasopressin V1a receptor to an Ala pair significantly inhibited agonist-induced internalization. Moreover, the mutagenesis of the C-terminal sequence ³³⁵CNC³³⁷ of thyrotropin-releasing hormone receptor to either ³³⁵SNS³³⁷ or ³³⁵GNG³³⁷ also inhibited agonist-induced internalization (Nussenzveig et al., 1993). In the case of the V1a receptor, the Cys pair was eight amino acids downstream of a dileucine motif; which when mutated also significantly inhibited the agonist-induced internalization (Preisser et al., 1999). A dileucine motif is also implicated to mediate the internalization of β₂-adrenergic receptors, and it is adjacent to a Cys residue that is palmitoylated (Gabilondo et al., 1997). Interestingly, there is a dileucine motif four amino acids downstream of Cys pair Cys262/Cys263 in the i3 loop of the hM₁ receptor. It is possible that either or both Cys pairs are controlling the accessibility of this motif.

After 24 h of carbachol treatment, the hM₁ receptor mutants hM₁del, hM₁CC₂₆₂, and hM₁DD₂₆₂ were observed to down-regulate in a manner consistent with wild-type hM₁ receptors, even though both of these mutants have greatly reduced internalization (see Fig. 3). This is consistent with the observations of Shockley et al. (1999), who showed that the domain(s) involved in the agonist-induced internalization of hM₁ receptors appear to be separate from the domain(s) necessary for hM₁ receptor down-regulation.

	Footnotes

 
This work was supported by an Oklahoma Health Research award for project number HR03-107S from the Oklahoma Center for the Advancement of Science and Technology

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.123695.

ABBREVIATIONS: GPCR, G protein-coupled receptor; S/T, Seradn Thr-rich; CHO, Chinese hamster ovary; GRK, G protein-coupled receptor kinase; hM₁, human muscarinic M₁; i3, third intracellular; [H]NMS, [³H]N-methylscopolamine; [³H]QNB, [³H]quinuclidinyl benzylate; ANOVA, analysis of variance; PBS, phosphate-buffered saline; SH3, Src homology 3.

Address correspondence to: Dr. Gregory W. Sawyer, Department of Biochemistry and Microbiology, Center for Health Sciences, Oklahoma State University, 1111 W. 17th Street, Tulsa, OK 74107-1898. E-mail: gwsawyer{at}chs.okstate.edu

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