Exposure of muscarinic receptors (mAChR) ()to agonist for short periods of time (seconds to minutes) causes receptors to become unavailable to binding of lipophobic ligands (Galper et al., 1982). This ``sequestration'' of receptors is thought to occur by the internalization of receptors from the cell surface into an intracellular compartment (Harden et al., 1985). When agonist is removed, the sequestered receptors are rapidly (minutes) returned to the cell surface (Maloteaux et al., 1983; Feigenbaum and El-Fakahany, 1985). In contrast, long term agonist exposure (hours) leads to a decrease in total mAChR number, and recovery requires de novo protein synthesis (Klein et al., 1979; Taylor et al., 1979; Hunter and Nathanson, 1984).

The molecular mechanisms responsible for mAChR sequestration are poorly understood, although progress has been made in the identification of receptor regions involved in the sequestration process. Moro et al.(1993) reported that replacing a serine- and threonine-rich region located in the third cytoplasmic loop of the m1 receptor with alanines abolished agonist-induced sequestration. However, deletion of these residues as well as surrounding residues does not always attenuate sequestration (Shapiro and Nathanson 1989; Lameh et al., 1992; Lee and Fraser, 1993). The serine- and threonine-rich region of the third cytoplasmic loop may therefore play a permissive role for successful interactions of other receptor regions with the cellular sequestration machinery. More recently, it has been demonstrated that mutation of a highly conserved lipophilic amino acid in the second extracellular loop of the m1 receptor causes a strong decrease in receptor sequestration (Moro et al., 1994). In addition, mutations of amino acids in the carboxyl- and amino-terminal ends of the third intracellular loop of the m1 receptor attenuate agonist-induced sequestration, suggesting that sequestration of mAChR involves a multisite domain (Moro et al., 1994).

The five subtypes of cloned mAChR can be divided into two broad functional categories: the m1, m3, and m5 subtypes preferentially couple to the G family of G-proteins, while the m2 and m4 subtypes preferentially couple to the G family of G-proteins. In this report, we examine the agonist-induced sequestration of the m1 and m2 mAChR transiently expressed in JEG-3 cells. We exploit the observation that the m2 but not the m1 receptor undergoes sequestration in response to agonist when expressed in these cells to identify regions of the m2 receptor which are necessary for subtype-specific receptor sequestration.

EXPERIMENTAL PROCEDURES

Construction of Chimeric and Mutant mAChR

Figure 4: Schematic of m1/m2 chimeric receptors. The m1 (stippled) and m2 (black) as well as the m1/m2 chimeric receptors are shown.

All other chimeric m1 and m2 mAChR receptors were constructed using the polymerase chain reaction (PCR). Oligonucleotides were synthesized by the University of Washington Molecular Pharmacology Facility or Genset (La Jolla, CA). Two primary PCR reactions were performed which utilized a 5`-terminal oligonucleotide and a 3`-inner oligonucleotide to yield a product corresponding to 5` sequence of the m1 or m2 receptor (primary 5`-PCR product) and a 5`-inner oligonucleotide and a 3`-terminal oligonucleotide to yield a product corresponding to 3` sequence of the m1 or m2 receptor (primary 3`-PCR product). 5`- and 3`-inner oligonucleotides contained complementary sequence such that 3` sequence of the primary 5`-PCR product overlaps with the 5` sequence of the primary 3`-PCR product. In some cases, inner oligonucleotides contained base changes as detailed below. Terminal oligonucleotides contained EcoRI sites adjacent to coding sequence to facilitate subsequent subcloning. The primary PCR reactions were performed with Pfu DNA polymerase (Stratagene, La Jolla, CA) utilizing reaction conditions described by the manufacturer with denaturation at 94 °C for 5 min, annealing at 60 °C for 2 min, and elongation at 70 °C for 3.5 min for a total of 30 cycles. PCR products were isolated by Qiagen (Chatsworth, CA) Qiaex DNA gel extraction. The primary 5`- and 3`-PCR products were then combined in a second PCR reaction with 5`-and 3`-terminal oligonucleotides, producing a m1/m2 chimeric receptor. The secondary PCR reactions were performed as above except with annealing at 50 °C for 13 cycles and 60 °C for 19 cycles. The secondary PCR products were isolated by Qiagen Qiaex DNA gel extraction and subcloned into the EcoRI site of pCD-PS. The validity of the m1 and m2 chimeric receptor junctions of the desired base pairs were verified by DNA sequencing using either Applied Biosystems, Inc. (Foster City, CA) Taq DyeDeoxy Terminator Cycle and automatic sequencing or U. S. Biochemical Corp. Sequenase Version 2.0 dideoxy manual sequencing analysis. The construction of the PCR-generated chimeric and mutant receptors is described below (Fig. 4).

The m1/m2(tail) Chimeric Receptor

The m1/m2(VT-6,7) Chimeric Receptor

The m1/m2(6,7) Chimeric Receptor

The m1/m2(VT) Chimeric Receptor

The m2/m1(6,7) Chimeric Receptor

The m2/m1(6) Chimeric Receptor

Cell Culture

Transfection

Sequestration and Down-regulation Assays

Ligand Binding in Membrane Homogenates

Analysis of Changes in Intracellular cAMP Levels

RESULTS

Agonist-induced Sequestration of the m1 and m2 Receptors

Figure 1: Agonist-induced sequestration of wild-type m1 and m2 mAChR. JEG-3 cells transiently transfected with wild-type m1 () or m2 () receptors were pretreated with the indicated concentrations of carbachol for 15 min (A) or with 10M carbachol for the indicated times at 37 °C (B), and the loss of [H]NMS binding sites was measured as described under ``Experimental Procedures.'' Data represent the mean ± S.E. of three to four experiments and is presented as a percentage of [H]NMS binding sites in untreated cells.

Figure 2: Agonist-induced sequestration of wild-type m1 and m2 mAChR expressed in Y1 cells. Y1 adrenal cells transiently transfected with wild-type m1 (black) or m2 (white) receptors were pretreated with 10M carbachol for 1 h at 37 °C and the loss of [H]NMS binding sites was measured as described under ``Experimental Procedures.'' Data represent the mean ± S.D. of triplicate cultures and is presented as a percentage of sequestration in untreated cells.

The limited sequestration of the m1 receptor is not due to a generalized lack of functional activity when expressed in JEG-3 cells. We have previously reported that the m1 receptor mediates a large increase in cAMP-regulated gene transcription in these cells due to ectopic coupling of the receptor to the stimulatory G-protein G (Migeon and Nathanson, 1994). The m1 receptor also exhibits long term agonist-induced down-regulation of total receptor number to a similar level as the m2 receptor when these receptors are expressed in JEG-3 cells (Fig. 3). These results suggest that the m1 and m2 receptors utilize different mechanisms or cellular machinery to undergo agonist-induced sequestration and that the JEG-3 cells are lacking or limiting in the m1-specific pathway.

Figure 3: Agonist-induced down-regulation of wild-type m1 and m2 mAChR. JEG-3 cells transiently transfected with wild-type m1 () or m2 () receptors were pretreated with 10M carbachol for the indicated times at 37 °C and the loss of [H]QNB binding sites was measured as described under ``Experimental Procedures.'' Data represent the mean ± S.E. of three to ten experiments and are presented as a percentage of [H]QNB binding sites in untreated cells.

Construction and Expression of Mutant and Chimeric mAChR

[H]QNB saturation binding and carbachol/[H]QNB competition binding experiments were performed to ensure that exchanging the sixth and seventh transmembrane domains between the m1 and m2 receptors did not significantly affect antagonist or agonist binding. Swapping the sixth and seventh transmembrane domains between the m1 and m2 receptors resulted in only minor differences in binding to antagonist and agonist (Table 1). However, these difference are not surprising considering the importance of the sixth and seventh transmembrane domains in ligand binding (Wess et al., 1991).

The ability of the wild-type m1 and m2 receptors and the m1/m2(6,7) and m2/m1(6,7) to activate cAMP accumulation was measured by assay of a luciferase receptor gene under the transcriptional control of a CRE as described previously (Migeon and Nathanson, 1994). We have previously reported that both G-coupled and G-coupled mAChR can stimulate the expression of the luciferase gene, presumably through coupling to G (Migeon and Nathanson, 1994; Migeon et al., 1995), thus providing a convenient assay for determining if chimeric receptors are functional. The mAChR were cotransfected with the CRE-luciferase reporter gene and a constitutive expression plasmid for -galactosidase (to normalize for slight differences in transfection efficiency). Fig. 5shows that exchanging the sixth and seventh transmembrane domains between the m1 and m2 receptors does not affect the ability of the receptors to stimulate luciferase expression. Additional experiments demonstrated that the m2/m1(6) and m1/m2(VT-6,7) receptors also exhibit a similar ability to stimulate CRE-luciferase expression. ()

Figure 5: Luciferase activity of wild-type m1 and m2 and chimeric mAChR. JEG-3 cells transiently transfected with m1 (black), m1/m2(6,7) (striped), m2 (white), or m2/m1(6,7) (stippled) were incubated with 10M carbachol for 4 h at 37 °C. Luciferase activity was assayed as described under ``Experimental Procedures'' and is presented as a -fold increase in the luciferase activity of control cells receiving no carbachol. All data were normalized by assay of -galactosidase activity and represent the mean ± S.D. of a representative experiment.

Agonist-induced Sequestration of Chimeric Receptors

Figure 6: Agonist-induced sequestration of wild-type m1 and m2 mAChR, m1/m2, and m1/m2(tail). JEG-3 cells transiently transfected with wild-type m1 (), m2 (), m1/m2 (), or m1/m2(tail) () were pretreated with the indicated concentrations of carbachol for 15 min (A) or with 10M carbachol for the indicated times at 37 °C (B), and the loss of [H]NMS binding sites was measured as described under ``Experimental Procedures.'' Data represent the mean ± S.E. of three to six experiments and are presented as a percentage of [H]NMS binding sites in untreated cells.

When we replaced the intracellular carboxyl-terminal tail of the m1 receptor with the carboxyl-terminal tail of the m2 receptor, the resulting chimera (m1/m2(tail)) did not undergo sequestration (Fig. 6), indicating that the m2 carboxyl-terminal tail is not sufficient to allow sequestration of the m1 receptor. In addition, the m1/m2(VT-6,7) chimeric receptor exhibits sequestration in response to agonist (Fig. 7), indicating that the carboxyl-terminal tail of the m2 receptor is not required for m2 receptor sequestration.

Figure 7: Agonist-induced sequestration of wild-type m1 and m2 mAChR, m1/m2(VT-6,7), and m1/m2(6,7). JEG-3 cells transiently transfected with wild-type m1 (), m2 (), m1/m2(VT-6,7) (), or m1/m2(6,7) () were pretreated with the indicated concentrations of carbachol for 15 min (A) or with 10M carbachol for the indicated times at 37 °C (B) and the loss of [H]NMS binding sites was measured as described under ``Experimental Procedures.'' Data represent the mean ± S.E. of three to seven experiments and are presented as a percentage of [H]NMS binding sites in untreated cells.

We next examined the role of the seven proximal amino acids in the carboxyl-terminal third of the m2 receptor third cytoplasmic loop in receptor sequestration. As shown in Fig. 8, there are only two amino acid differences between the m2 and the m1 receptors in this region: the m2 receptor contains a valine (Val-385) and a threonine (Thr-386) in the corresponding position of Ala-363 and Ala-364 of the m1 receptor. Val-385 and Thr-386 of the m1/m2(VT-6,7) chimeric receptor were replaced with alanines, producing a m1 chimeric receptor containing only the sixth and seventh transmembrane domains of the m2 receptor. This chimeric receptor, m1/m2(6, 7) , does not undergo agonist-induced sequestration (Fig. 7), indicating that the m2 Val-385 and Thr-386 residues are necessary for conferring sequestration upon the m1 receptor. However, the m2 Val-385 and Thr-386 residues are not sufficient in causing receptor sequestration since replacing Ala-363 and Ala-364 in the m1 receptor with a valine and threonine, respectively, does not cause the m1 receptor to sequester (Fig. 9).

Figure 8: Amino acid sequence of the carboxyl-terminal fifth of the m1 and m2 mAChR. Amino acid residues are identified by single-letter codes. The amino acid differences (Ala-363 and Ala-364 of m1 versus Val-385 and Thr-386 of m2) in the carboxyl-terminal end of the third cytoplasmic loop and the sixth transmembrane and seven transmembrane domains are highlighted.

Figure 9: Agonist-induced sequestration of wild-type m1 and m2 mAChR, m1/m2(VT), m2/m1(6,7), and m2/m1(6). JEG-3 cells transiently transfected with m1 (), m2 (), m1/m2(VT) (), m2/m1(6,7) (), or m2/m1(6) () were pretreated with the indicated concentrations of carbachol for 15 min (A) or with 10M carbachol for the indicated times at 37 °C (B), and the loss of [H]NMS binding sites was measured as described under ``Experimental Procedures.'' Data represent the mean ± S.E. of three to six experiments and are presented as a percentage of [H]NMS binding sites in untreated cells.

Replacing the sixth and seventh transmembrane domains of the m2 receptor with those of the m1 receptor abolishes agonist-induced sequestration of the m2 receptor as shown in Fig. 9. Likewise, replacing only the sixth transmembrane domain of the m2 receptor with that of the m1 receptor eliminated the sequestration of the m2 receptor in response to agonist (Fig. 9).

Specificity of Functional Coupling of Chimeric Receptors

Figure 10: Regulation of forskolin-stimulated CRE-luciferase expression of wild-type m1 and m2 and chimeric mAChR in presence of G. JEG-3 cells transiently cotransfected with G and either m1 (), m2 (), m1/m2(VT-6,7) (), m2/m1(6) () or m2/m1(6,7) () were incubated with increasing concentrations of carbachol, and luciferase activity was assayed as described under ``Experimental Procedures.'' Data are presented as the percent of luciferase activity in cells not treated with carbachol. All data were normalized by assay of -galactosidase activity and represent the mean ± S.D. of two experiments, each performed in triplicate.

DISCUSSION

Utilizing a JEG-3 cell transient expression system, we show that distinct mechanisms are involved in the agonist-induced sequestration of the m2 and m1 mAChR receptor subtypes. When transiently expressed in JEG-3 human choriocarcinoma cells (Fig. 1) or COS-7 monkey kidney cells (Goldman and Nathanson, 1992), the m1 receptor does not undergo agonist-induced sequestration. Agonist treatment results in the sequestration of the m1 receptor when stably expressed in Y1 adrenal cells (Shapiro and Nathanson, 1989; Scherer and Nathanson, 1990) and U293 human kidney cells (Moro et al., 1993), or when transiently expressed in Y1 cells (Fig. 2), suggesting that a component necessary for the agonist-induced sequestration of the m1 receptor is absent in JEG-3 and COS-7 cells. The ability of the m1 receptor to down-regulate after prolonged exposure to agonist (Fig. 3) indicates that the long term down-regulation and the rapid sequestration of the m1 receptor are independent processes involving distinct molecular mechanisms, an observation we have also made with the m2 receptor (Goldman and Nathanson, 1994).

By utilizing chimeric m1 and m2 receptors, we have taken advantage of the fact that the m2 but not the m1 receptor sequesters in response to agonist when expressed in JEG-3 cells to identify regions of the m2 receptor that are involved in the m2 receptor specific sequestration pathway. This approach has the advantage that, instead of attempting to create mutations which simply eliminate sequestration, one can look for regions of the m2 receptor that are sufficient to confer sequestration on a chimeric receptor. While initial observations indicated that the last 86 amino acids of the m2 receptor (consisting of the carboxyl-terminal end of the third cytoplasmic loop, the sixth and seventh transmembrane domains, and the carboxyl-terminal tail) were sufficient to confer sequestration upon the m1 receptor (Fig. 6), additional chimeric receptors showed that the intracellular carboxyl-terminal tail of the m2 receptor is not required for the m2 sequestration pathway ( Fig. 6and Fig. 7). However, the amino acids Val-385 and Thr-386 at the carboxyl-terminal end of the third cytoplasmic loop of the m2 receptor are necessary (Fig. 7), but not sufficient (Fig. 9) in conferring sequestration upon the m1 receptor. Thus, in addition to m2 Val-385 and Thr-386, the sixth and seventh transmembrane domains or regions therein are involved in subtype-specific m2 receptor sequestration. Indeed, the sixth and seventh transmembrane domains of the m2 receptor are necessary for agonist-induced sequestration of the m2 receptor, since replacing this region with that of the m1 receptor eliminates the ability of the m2 receptor to sequester (Fig. 9). Exchanging the sixth and seventh transmembrane domains between the m1 and m2 receptors does not profoundly affect either antagonist or agonist binding (Table 1). In addition, chimeric receptors which have impaired abilities to sequester in response to agonist are able to couple to G to stimulate CRE-luciferase expression as effectively as wild-type receptors, making it unlikely that the observed effects on sequestration are due to gross conformational changes created by the construction of the chimeric receptors.

Tsuga et al.(1994) reported that coexpression of the m2 receptor with a G-protein receptor kinase in COS-7 or BHK-21 cells increased the ability of the receptor to undergo agonist-induced sequestration, while coexpression of a catalytically inactive G-protein receptor kinase reduced agonist-induced sequestration. The authors concluded that the agonist-induced sequestration of the m2 receptor requires G-protein receptor kinase-mediated phosphorylation. However, because the G-protein receptor kinase phosphorylation sites of the m2 receptor (Nakata et al., 1994) are in regions of the third cytoplasmic loop which are not present in the m1/m2 and related chimeras which do undergo sequestration, the differences in sequestration between the m1 and m2 receptors observed here are unlikely to be related to phosphorylation of m2 receptor-specific sequences by G-protein receptor kinase.

Both the amino (Lechleiter et al., 1990)- and the carboxyl (Shapiro et al., 1993)-terminal ends of the third cytoplasmic loop have been demonstrated to be involved in G-protein coupling. Based on secondary structure prediction algorithms, the membrane-proximal regions of the third cytoplasmic loop are thought to form -helical extensions from the fifth and sixth transmembrane domains (Strader et al., 1989). In support of this theory, interruption of the predicted -helical structure of the amino-terminal end of the third cytoplasmic loop via mutagenesis disrupts G-protein coupling (Duerson et al., 1993; Blüml et al., 1994). Blin et al.(1995) have shown that sequences in the carboxyl-terminal end of the third cytoplasmic loop and the adjacent region of the sixth transmembrane domain are involved in the G-protein coupling specificity of the mAChR. Moro et al.(1994) have shown that mutations in the proximal regions of the third cytoplasmic loop of the m1 receptor affect G-protein coupling and that the same mutations attenuate agonist-induced receptor sequestration. Since some domains involved in sequestration overlap with domains involved in G-protein coupling, it has been hypothesized that the agonist-induced conformation of the mAChR, which allows coupling to G-proteins, might also be the conformation which allows sequestration of receptor protein (Moro et al., 1994). Heterotrimeric G-proteins (Traub and Sagi-Eisenberg, 1991), as well as small GTP-binding proteins (Donaldson et al., 1991; Shpetner and Vallee, 1992; Pimplikar and Simons, 1993), have been implicated in cellular trafficking. As shown in Fig. 10, however, the ability of a chimeric receptor to couple to G and mediate inhibition of adenylyl cyclase is unrelated to its ability to be competent to undergo m2-specific sequestration in JEG-3 cells.

In addition to the membrane-proximal carboxyl-terminal end of the third cytoplasmic loop, our studies also implicate the involvement of the sixth and seventh transmembrane domains of the m2 in receptor sequestration. The transmembrane domains of a variety of membrane proteins have been demonstrated to be important in cellular trafficking. For example, transmembrane domains are necessary determinants for the retention of membrane proteins in the Golgi (Nilsson et al., 1991; Burke et al., 1992; Machamer et al., 1993). In addition, an aspartic acid residue in the seventh transmembrane domain of the KDEL receptor is involved in the retrograde transport of this receptor from the Golgi to the endoplasmic reticulum (Townsley et al., 1993). Recently, Keefer et al.(1994) have demonstrated that targeting of the 2-adrenergic receptor to the basolateral membrane in Madin-Darby canine kidney cells also involves the transmembrane domain regions.

The transmembrane domains of the mAChR subtypes have a high degree of amino acid identity (Fig. 8). Conserved residues located in the sixth and seventh transmembrane domains of the mAChR have been demonstrated to be important in ligand binding and functional coupling (Wess et al., 1991, 1992; Wess, 1993). Our results are the first to suggest that regions in the sixth and seventh transmembrane domains of the mAChR may be involved in the sequestration process and that these regions are subtype-specific. Preliminary attempts to identify a more specific region involved in agonist-induced sequestration of the mAChR has shown that the sixth transmembrane domain of the m2 receptor can not be replaced with that of the m1 receptor and still maintain the ability to sequester (Fig. 9). While there are six amino acid residues in the sixth transmembrane domain which differ between the m1 and m2 receptors, four of these are conservative changes (Fig. 8). One of the nonconservative differences (Ser-368 of the m1 receptor and Leu-390 of the m2 receptor) is near the carboxyl-terminal end of the third cytoplasmic loop and might be part of a recognition sequence which includes Ala-363 and Ala-364 of the m1 receptor and Val-385 and Thr-386 of the m2 receptor. The other nonconservative difference in the sixth transmembrane domain residue is a threonine (Thr-379) in the m1 receptor and an alanine (Ala-401) in the corresponding position of the m2 receptor (Fig. 8). Interestingly, the threonine residue is present in the m1, m3, m4, and m5 subtypes with only the m2 receptor possessing an alanine residue. The only nonconservative amino acid difference between the m1 and m2 receptors in the seventh transmembrane domain is a methionine (Met-416) in the m1 receptor compared with an alanine (Ala-438) in the m2 receptor (Fig. 8). Additional studies will be required to ascertain the importance of the amino acid differences in the sixth and seventh transmembrane domains of the m1 and m2 receptor in the sequestration process.

In conclusion, we have shown that there are cell-type specific differences in the agonist induced sequestration of the m1 and m2 receptors, suggesting that these receptors utilize different cellular mechanisms or pathways for this process. The m2 receptor requires sequences both in the end of the third cytoplasmic loop and the adjacent sixth transmembrane domain in order to undergo this subtype-specific sequestration pathway. These contiguous regions may thus form a single domain which interacts with a cell-specific component in an agonist-dependent manner that targets the receptor for sequestration.

FOOTNOTES

*

This work was supported by National Institutes of Health Grant HL44948 and Training Grants GM07750 and GM07270. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR 97201-3098.

¶

Present address: Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Ave., Seattle, WA 98121.

**

To whom correspondence should be addressed: Dept. of Pharmacology, K536A HSB, University of Washington, Box 357750, Seattle, WA 98195-7750; Tel.: 206-543-9457; Fax: 206-616-4230.

()

The abbreviations used are: mAChR, muscarinic acetylcholine receptor; PCR, polymerase chain reaction; QNB, quinuclidinyl benzilate; NMS, N-methylscopolamine; CRE, cAMP response element.

()

P. S. Goldman and N. M. Nathanson, unpublished observations.

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