Cross-talk between ml Muscarinic Acetylcholine and &Adrenergic … · Receptor cross-talk or heterologous regulation refers to that form of regulation in which activation one receptor

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 11, Issue of April 15, pp. 7949-7957,1993 Printed in U.S.A.

Cross-talk between m l Muscarinic Acetylcholine and &Adrenergic Receptors cAMP AND THE THIRD INTRACELLULAR LOOP OF m l MUSCARINIC RECEPTORS CONFER HETEROLOGOUS REGULATION*

(Received for publication, September 29, 1992)

Norman H. Lee$§ and Claire M. FraserS From the Section on Molecuhr Neurobiolom. Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, ”“ I

Rockville, Maryland 20852

Genes encoding the m l muscarinic (ml mAChR) and &adrenergic receptors (&AR) were stably co-expressed into Chinese hamster ovary (CHO) cells to study receptor regulation and cross-talk. Persistent activation of the &AR/adenylate cyclase pathway by isoproterenol leads to heterologous desensitization, internalization, and down-regulation of the m l mAChR which is comparable, but smaller in magnitude, with that seen with persistent activation of the m l mAChR by carbachol. This heterologous effect was mimicked by dibutyryl cAMP and forskolin and antagonized by the protein kinase A (PKA) inhibitor H-8. A potential consensus sequence for phosphorylation by PKA ( L y ~ ~ ~ ’ - A r g - L y s - T h r ~ ~ ~ ) exists on the third intracellular loop of the m l mAChR, suggesting that receptor phosphorylation by PKA may be involved in heterologous regulation. The loss of m l mAChRs induced by carbachol was not reversed by H-8, indicating that homologous regulation is not dependent on PKA.

Recent evidence suggests that muscarinic agonist- mediated internalization of the m l mAChR involves the third intracellular loop (i3) (Maeda, S . , Lameh, J., Mallet, W. G., Philip, M., Ramachandran, J., and Sa- dee, w. (1990) FEBS Lett. 269,386-388). Three deletion mutant receptors were constructed in which the majority, or small regions, of i3 were eliminated but the membrane proximal portions of the loop were left intact. Each of the mutants was co-expressed with the BaAR in CHO cells. A small region in i3 was identified which is crucial for carbachol- and isoproterenol-promoted internalization and down-regulation. This region contains a series of 6 serine residues within an 8- amino acid stretch. A similar domain has been identified in the carboxyl tail of the BzAR and has been proposed to participate in receptor internalization (Hausdorff, W. P., Campbell, P. T., Ostrowski, J., Yu, S . S., Caron, M. G., and Lefkowitz, R. J. (1991) Proc. Ncttl. A C U ~ Sei. U. S. A. 88,2979-2983).

Two major signal transduction pathways are those involved

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘*advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

for Genomic Research, Gaithersburg, MD 20878. $ Current address: Dept. of Molecular and Cellular Biology, Inst.

8 TO whom correspondence should be addressed (see current address given above).

in the hydrolysis of inositol lipids by phospholipase C (PLC)’ and the formation of CAMP by adenylate cyclase. The cellular effects mediated by cAMP are subserved by protein kinase A (PKA), whereas those of diacylglycerol (a product resulting from the hydrolysis of inositol phosphates) are directed by protein kinase C (PKC). Many reports have indicated that “cross-talk’’ exists between these two signaling pathways in both neural and non-neural tissues (1). Activation of one pathway has been shown to be stimulatory or inhibitory on the other pathway. For example, treatment of frog erythro- cytes with 12-0-tetradecanoyl phorbol-13-acetate, a phorbol ester that activates PKC (2), enhances adenylate cyclase activity (3). This presumably is due to phosphorylation of adenylate cyclase by PKC (3). Likewise, for murine L cells transfected with the m5 muscarinic receptor gene, stimulation of this receptor which is coupled to PLC leads to a sensitiza- tion of adenylate cyclase (4). In contrast, PKC activation in NCBBO and NIH 3T3 cells inhibits the CAMP-generating system (5). Stimulation of the adenylate cyclase pathway has also been reported to interact in a regulatory fashion with the PLC effector system. In rat skeletal myoblasts, exogenously applied forskolin or dibutyryl cAMP (Bt2cAMP) has been shown to impair PKC activity (6). Similarly, activation of PKA impairs the coupling between PLC and a guanine nucleotide-binding protein (G-protein) in NG108-15 cells ( 7 ) . Examples of opposite actions involving the adenylate cyclase system on PLC signaling pathway are those observed in hepatocytes (8).

Less well characterized is the fate of G-protein-coupled receptors during cross-talk of signal transduction pathways. Receptor cross-talk or heterologous regulation refers to that form of regulation in which activation of one receptor by its agonist will functionally modify other receptors (9). By comparison, homologous regulation is agonist-specific, in that only those receptors which interact with the agonist are regulated (9). In both cases, regulation of receptor function can be at the level of transcription or translation. Examples on the molecular mechanisms of receptor cross-talk are limited to adrenergic receptors for the most part. In DDT1 MF- 2 vas deferens cells, activation of the &adrenergic receptor (&AR)/AC pathway elevates a*-adrenergic receptor mRNA and protein levels (10). Similarly, persistent activation of the

‘The abbreviations used are: PLC, phospholipase C; mAChR, muscarinic acetylcholine receptor; CHO, Chinese hamster ovary; G- protein, guanine nucleotide-binding protein; i3, third intracellular loop; QNB, quinuclidinyl benzilate; ICYP, iodocyanopindolol; KD, equilibrium dissociation constant; PMA, phorbol 12-myristate 13- acetate; BtzcAMP, dibutyryl CAMP PKA, protein kinase A PKC, protein kinase C; PBS, phosphate-buffered saline; CBC, carbachol; PI, phosphoinositide; P,AR, &adrenergic receptors.

7949

7950 Heterologous Regulation of the ml mAChR

A1 adenosine receptor increases the expression of P2ARs in the same cell line (11).

Muscarinic acetylcholine receptors (mAChRs), like 0- and &-adrenergic and adenosine receptors, belong to a gene super- family that couples to G-proteins (for review see Refs. 12 and 13). At present, five distinct mAChR subtypes have been cloned and are designated ml-m5 (14-17). Agonist activation of ml , m3, and m5 receptor subtypes results in the hydrolysis of phosphoinositides, whereas activation of m2 and m4 subtypes is linked to an inhibition of adenylate cyclase activity. Since both mAChRs and PARS are found co-localized in neural and non-neural tissues, and mAChRs can be phosphorylated by PKA, there is the potential for cross-regulation

In the present study we have examined whether activation of the PARIAC pathway would heterologously regulate the m l mAChR and compared this with homologous regulation of t he ml mAChR. This was done by stably co-transfecting the genes for the ml mAChR and &AR into Chinese hamster ovary (CHO) cells. We report that both m l mAChR and P2AR activation leads to internalization and down-regulation of the m l mAChR. To assess the structural basis for this phenom- enon, we examined the internalization and down-regulation characteristics of mutant forms of the m l mAChR that have deletions in the third intracellular loop (i3). A preliminary report of these findings has appeared previously in abstract form (22).

(18-21).

EXPERIMENTAL PROCEDURES

Materials-CHO-K1 cells were obtained from the American Type Culture Collection (Rockville, MD). [3H]Quinuclidinyl benzilate ([3H] QNB) (44.9 Ci/mmol), [1261]iodocyanopindolol (['"I]ICYP) (2200 Ci/ mmol), and myo-[3H]inositol (15.6 Ci/mmol) were from Du Pont- New England Nuclear. Carbachol, atropine, isoproterenol, and 1- propranolol were purchased from Sigma. The mammalian expression vector pSVL was from Pharmacia LKB Biotechnology Inc; pMSVneo was constructed as described previously (23). Restriction enzymes were purchased from New England Biolabs. Dulbecco's modified Eagle's medium, fetal calf serum, and Geneticin were from GIBCO.

Construction of Deletion Mutant Receptors-Deletion mutagenesis was employed to identify the region(s) on the third intracellular loop (i3) of the ml mAChR that plays a role in receptor internalization during homologous and heterologous regulation. A 1.6-kilobase XhoI- BamHI fragment of the ml mAChR gene (24), containing the entire coding region (plus 8 base pairs 5' to the initiation codon and 260 base pairs of 3'-untranslated region), was subcloned into pSP73 to yieldpSP73-ml. To delete amino acids 227-269 from the ml mAChR, pSP73-ml was digested with PstI and religated. The resulting mutant receptor, del 227-269, was in-frame and therefore did not require the reintroduction of nucleotide sequence. Del 275-348 was constructed by digesting pSP73-ml with PuuII and religating with a KpnI linker (5'-CTGGTACCAG-3'). In this deletion mutant, amino acids 275- 348 were replaced by a single tyrosine residue. To construct deletion mutant 227-341 (del 227-341), pSP73-mi was digested with PstI and PuuII and religated with a PstI cohesive end-PuuII blunt end adaptor (5'-GAAACCCCGAGGGAAGGAACAG-3' and complimentary strand 5'-CTGTTCCTTCCCTCGGGGTTTCTGCA-3'). The resulting in-frame construct deletes amino acids 227-341 in i3. Authenticity of all mutations was confirmed by dideoxy sequencing before inserting the mutant receptor genes into the expression vector pSVL.

Expression of Receptors in CHO Cells-Genes for the m l mAChR and the PIAR (23) were inserted into the mammalian expression vector pSVL and co-transfected with pMSVneo (which contains the selectable marker aminoglycoside phosphotransferase) into CHO cells using the calcium phosphate precipitation method (25). DeIetion mutant constructs of the m l mAChR gene were likewise co-transfected with the &AR gene into CHO cells. Cells were grown in selective medium containing Geneticin (500 pg/ml) where only those cells stably integrating pMSVneo into their genome survive. Colonies derived from single cells were isolated and expanded. Radioligand binding assays with [3H]QNB or ['261]ICYP were employed to assay for the presence of the ml mAChR and P2AR, respectively (23, 26).

Receptor Binding Assays with Intact Cells-To measure the total

number of cellular ml mAChR or &ARs, cells expressing both of these receptors were grown in monolayer and incubated without or with different agents for various times. Cells were harvested in PBS minus Caz+, centrifuged at 250 X g for 1 min at 4 "C, and washed three times in PBS. Intact cells were resuspended in 1 ml of assay buffer (Dulbecco's modified Eagle's medium supplemented with 25 mM HEPES) containing [3H]QNB or ['251]ICYP. Cells were then incubated for 90 min at 15 "C, during which time equilibrium was attained. The binding reaction was terminated by rapid filtration.

ICYP. Specific binding averaged 60-70% for [3H]QNB and 90% of ["'I]

Receptor Binding Assays in Plasma Membrane Homogenates-Two different protocols have been used to measure cell surface receptors. The first employs radioligand binding assays with a plasma membrane preparation (27, 28). In this procedure, cells are lysed by homogenization in ice-cold H20 and centrifuged at low speed. The resulting pellet contains mostly plasma membranes (27, 29). To recover cytoplasmic vesicles containing internalized receptors, the supernatant is subjected to high speed centrifugation (27, 30). If, however, cells are lysed by hypotonic shock in the absence of homogenization, then internalized receptors can be found associated with the plasma membrane fraction (27, 30).

A second protocol commonly utilized to measure cell surface receptors involves the binding of a hydrophilic ligand, such as the mAChR antagonist [3H]N-methylscopolamine, to whole cell preparations (30). We have found both methods equivalent when measuring cell surface ml mAChRs in desensitized and nondesensitized CHO cells.' In the present study, we utilized the plasma membrane protocol (27, 28) because in order to prevent recycling of the receptor, [3H]N-methylscopolamine binding assays must be carried out at 4 "C, necessitating long incubation (>3 h) to achieve equilibrium binding (30).

Transfected CHO cells expressing PzAR with either wild-type or mutant ml mAChR were incubated without or with receptor agonists, phorbol 12-myristate 13-acetate (PMA), Bt'cAMP, or forskolin for various times. Cells were then washed three times with PBS (pH 7.4) and scraped into ice-cold 50 mM Na+/K+ phosphate buffer (pH 7.4). Cell suspensions were homogenized at 4 "C by Polytron (Brinkmann Instruments, setting 7, 30 s) followed by centrifugation for 20 min at 20,000 x g at 4 "C. The low-speed pellet was resuspended in buffer and used immediately for binding assays. 13H]QNB and ['z61]ICYP binding assays were carried out as described previously (23, 26). Reactions, performed in triplicate, were carried out for 60 min at 37 "C and terminated by rapid filtration through Whatman GF/C glass filters using a Brandel cell harvester. Nonspecific binding for [3H]QNB or ['*'I]ICYP was defined in the presence of 10 pM atropine or 2 p~ propranolol, respectively. Ligand binding data were analyzed using the computer program INPLOT (GraphPAD, San Diego, CAI. The ratio of cell surface receptors to total receptors ranged from 0.5 to 0.75 for both ml mAChRs and &ARs in CHO-transfected cells.

Phosphoinositide Hydrolysis-Transfected CHO cells expressing the P2AR along with wild-type or mutant ml mAChR were assayed for carbachol (CBC)-stimulated phosphoinositide (PI) hydrolysis as described previously (26). For studies involving agonist pretreatment, cells seeded onto 12-well plates were prelabeled with 1 pCi/ml m.yo- [3H]inositoI at 37 "C for 24 h with or without CBC or isoproterenol. This paradigm was employed to prevent the depletion of radiolabeled polyinositol phosphates during agonist pretreatment. Afterwards, cells were washed three times with PBS (pH 7.4) and rechallenged with CBC. &ARs were assayed for CAMP formation as described (23). Dose-response curves were analyzed by computerized iterative nonlinear least squares regression (GraphPAD INPLOT).

RESULTS

Pharmacologic Properties of Clonal CHO Cell Lines CO- expressing the P A R with Either the Wild-type or Deletion Mutant ml mAChR-Fig. 1 is a schematic diagram of the deletion mutant receptors constructed by restriction enzyme digestion of the m l mAChR gene. AI1 deletion constructs retain 19 and 18 amino acids on the amino- and carboxyl- terminal portions of i3, respectively, which have been shown previously to be important for m l mAChR/G-protein coupling (31). Del 227-269, del 275-348, and del 227-341 are missing 43, 14, and 115 amino acids out of 156 in i3, respectively.

N. H. Lee and C. M. Fraser, unpublished observation.

Heterologous Regulation of the ml mAChR 795 1

C D del 275-340 del 227-341

FIG. 1. Deletion mutations introduced into the m l mAChR. The illustrations of wild-type and deletion mutant m l mAChRs are shown in relation to the plasma membrane bilayer (shown as parallel horizontal lines). The amino (NH2)- and carboxyl (CO0H)-terminal regions are located on the extracellular and intracellular sides of the membrane, respectively. The darkened area of the receptor indicates the portion of the putative third intracellular loop that was deleted. *, location of consensus amino acid sequence for phosphorylation by PKA or PKC. A, wild-type m l mAChR. B , del 227-269 is a deletion mutant ml mAChR missing amino acids 227-269. C, del 275-348 is missing amino acids 275-348. D, del 227-341 is missing amino acids 227-341 from the third intracellular loop.

TABLE I Pharmacologicparameters of wild-type or deletion mutant ml mAChRs co-expressed with the &,AI? in CHO cells

Data are mean +- S.E. values of two to five independent experiments performed in triplicate. Receptor binding assays were performed with plasma membrane homogenates as described under “Experimental Procedures.”

(3H]QNB binding Receptor

CBC binding“ PI hydrolysis

&ax KD KH K L ECm Stimulation pmollmg PM P M PM -fold

Wild-type 1.05 t 0.10 26 t 7 6.0 t 1.4 (29 _t 3%)* 222 _t 29 (71 it 3%) 17 t 6 20 t 3

Del 227-269 2.06 1- 0.21 22 & 6 1.0 t 0.2 (20 1- 5%) 68 * 7 (80 + 5%) 6 t 2 19 t 3

Del 275-348 0.86 f 0.08 22 t 5 5.5 f 1.9 (27 f 3%) 182 t 62 (73 I 3 % ) 8 + 2 23 t 1

Del 227-341 1.06 k 0.14 23 +- 4 4.2 t- 0.8 (30 & 4%) 125 t 35 (70 t 4%) 20 t 1 22 t- 2

(1.20 1- 0.06)‘ (0.77 2 0.03)d

(1.53 +- 0.21) (0.69 k 0.07)

(1.25 f 0.13) (0.62 & 0.03)

(0.80 t 0.13) (0.68 t 0.01) KH (high affinity) and KL (low affinity) values for CBC binding were obtained from competition binding studies as described by Savarese

et al. (26). ’ Values in parentheses represent the percentage of total binding sites present in each affinity state. Values represent &AR levels in CHO cells expressing wild-type m l , del 227-269, del 275-348, or del 227-341. Values represent the Hill coefficients for binding data.

CHO cells were stably co-transfected with the genes for the PzAR plus either wild-type or mutant m l mAChR. Four clonal cell lines were isolated with the following patterns of recom- binant receptor expression: &AR/wild-type ml (MB-CHO cells), &AR/del 227-269, B2AR/del 275-348, and PeARldel 227-341. Saturation binding experiments revealed that all three mutant ml mAChRs exhibited wild-type binding affinity for antagonists; the KO value derived from Scatchard analysis of [3H]QNB binding to wild-type m l mAChR was 26 f 7 p~ (mean f S.E., n = 3) (Table I). Wild-type and mutant m l mAChR densities ranged from 1.05 to 2.06 pmol/mg protein in the four clonal cell lines (Table I). In addition, B2AR densities in these cell lines were similar and ranged from 0.80 to 1.53 pmol/mg protein (Table I).

Agonist displacement binding studies revealed that each mutant receptor displayed both high and low affinity sites for CBC; with the KH and KL values and the proportion of binding sites present in each affinity state being similar to that seen for the wild-type receptor (Table I) . Furthermore, the potency and efficacy of CBC to stimulate PI turnover in clonal cell lines containing the deletion mutants were comparable with those of the wild-type ml mAChR (ECbo value and -fold stimulation for CBC at the wild-type receptor were 17 WM and

20, respectively, n = 3) (Table I). All responses could be blocked by the muscarinic receptor antagonist, atropine, at a concentration of 10 pM. Hence, the deletion mutant constructs represent functional receptors that retained their ability to couple to PLC.

Agonist-specific Regulation of ml mAChR Number in MB- CHO Cells-CBC-mediated loss of cell surface and total m l mAChRs in MB-CHO cells was monitored by [3H]QNB binding to plasma membrane homogenates and whole cells, respectively. Cell surface m l mAChR number steadily declined to 43% of control values during the first 6 h of 1 mM CBC incubation, after which time receptor levels slowly declined to approximately 35% o f control levels (Fig. 2 A ) . By comparison, total cellular m l mAChR content decreased only mar- ginally, by <5%, during 1-6 of h exposure to CBC (Fig. 2 A ) . However, agonist incubations between 12 and 24 h resulted in a 65-75% down-regulation of total ml mAChRs (Fig, 2 A ) . The receptors remaining after CBC treatment did not exhibit a change in affinity for [3H]QNB. Scatchard analysis revealed that plasma membrane homogenates from CBC-pretreated cells exhibited a single class of binding sites with a KO of 19 f 3 PM and a B,,, (maximum binding capacity) of 0.44 f 0.03 pmol/mg protein ( n = 2); by comparison, control cells showed


I AI CBC-pretreated -

20 ' 0 4 8 12 16 20 24

Time hours)

FIG. 2. Time course of homologous and heterologous regulation in MB-CHO cells. A, homologous regulation of m l mAChR function. Intact cells were pretreated with 1 mM carbachol for 24 h at 37 "C. Cells were then washed three times with PBS and assayed for PI hydrolysis (O), cell surface m l mAChR number (W), and total ml mAChR content (0) as detailed under "Experimental Proce- dures." Values shown are the average f S.E. of two to ten independent experiments performed in triplicate and are expressed as a percent of control. B, heterologous regulation of m l mAChR function. Intact cells were pretreated with 2 PM isoproterenol for 24 h at 37 "C. Cells were assayed for PI hydrolysis (0), cell surface ml mAChR number (A), and total ml mAChR content (A). Values shown are the average 2 S.E. of three to ten independent experiments performed in triplicate and are expressed as a percent of control.

a single class of sites with a KD of 18 k 3 p~ and a BmSx of 1.22 k 0.05 pmol/mg protein (n = 3).

The reduction of m l mAChRs by muscarinic agonists could be mimicked with the phorbol ester PMA, an activator of PKC (2). Hence, pretreatment of MB-CHO cells with 1 pM PMA for 5 h resulted in a 35% decrease in cell surface m l mAChR number compared with a 50% loss with 1 mM CBC for 5 h (n = 5 for each treatment group). When mAChR number was assessed in intact cells with [3H]QNB after pretreatment with 1 mM CBC or 1 p~ PMA for 5 h, total receptor number down-regulated 30 and 35%, respectively (n = 3 for each treatment group).

Actiuation of the PAR Regulates m l mAChR Number in MB-CHO Cells-The effects of prolonged PzAR activation on m l mAChR number and affinity was monitored in MB-CHO cells. Heterologous regulation of m l mAChR number was time-dependent. Isoproterenol, a t 2 p ~ , steadily decreased the levels of cell surface m l mAChRs from 3 to 12 h with maximum decreases (45-48%) occurring after 12 h (Fig. 2B). In contrast, total m l mAChR content did not appreciably decline for the first 6 h (Fig. 2B). This strongly suggests that the initial response to PzAR activation was internalization of m l mAChRs. Longer incubations with isoproterenol, from 12 to 24 h, led to a 45-50% reduction (down-regulation) in total m l mAChRs (Fig. 2B). The loss of m l mAChRs induced by isoproterenol was not accompanied by a change in the KD of [3H]QNB for the remaining receptors in plasma membrane homogenates. Scatchard analysis showed that isoproterenol- pretreated cells (24 h) exhibited a single class of binding sites with a calculated KO of 17 f 1 PM and a Bmax of 0.74 f 0.10

pmol/mg protein ( n = 3). The loss of [3H]QNB sites induced by isoproterenol was independent of PzAR density over a tested range of 0.4-2.0 pmol/mg protein (data not shown). Decreases in m l mAChR number were dependent on isoproterenol concentrations. For example, m l mAChR number declined by approximately 20-50% when pretreated with isoproterenol a t concentrations ranging from 0.2 nM up to 2 pM (Fig. 3). m l mAChR loss induced by low isoproterenol concentrations was completely blocked by 2 JLM propranolol, indicating that the P2AR activation was responsible for the deficits (Fig. 3).

To determine whether membrane-permeable cAMP ana- logues as well as other agents which elevate intracellular levels of cAMP could mimic the effects of isoproterenol on m l mAChR number, MB-CHO cells were incubated with 1 mM BtzcAMP or 100 p~ forskolin for 24 h, and 13H]QNB binding assays were performed. In either case, BtzcAMP (n = 3) or forskolin (n = 7) decreased cell surface m l mAChR number by 38 and 49%, respectively. A comparable decrease in total cellular m l mAChR number induced by these agents also could be seen in radioligand binding experiments with intact MB-CHO cells (data not shown). These observations suggest a potential role for PKA in heterologous control of m l mAChR levels. Thus, the effects of inhibiting PKA were investigated. As shown in Fig. 4, pretreatment of MB-CHO cells with the PKA inhibitor H-8 (39) almost completely abolished the observed losses of m l mAChRs following addition of 0.2 and 2 pM isoproterenol. Unlike isoproterenol treatment, CBC-induced decreases in m l mAChR number could not be inhibited by H-8 (Fig. 4). Likewise, decreases in PzAR number mediated by 24-h isoproterenol pretreatment, at concentrations of 0.2 and 2 p ~ , could not be reversed by H-8 (Fig. 4).

Inhibition of CBC-stimulated PI Turnover by Prolonged ml mAChR and PAR Activation-In MB-CHO cells exposed to 1 mM CBC for 24 h, the subsequent ability of the agonist to maximally stimulate PI hydrolysis was reduced by approximately 65% ( n = 3). However, there was no apparent change in the ECSo of CBC to stimulate PI turnover in CBC-pretreated cells (25 k 7 p ~ ) as compared with control cells (19 k 3 p ~ ) ( n = 3 for each treatment group). In addition, basal

"p E

0 . 2 - t

2.0 - t

20 - t

200 2000 - t - 1 -

FIG. 3. Concentration-dependent effect of &AR activation on m l mAChR number and reversal by propranolol. Intact cells were pretreated with the indicated concentrations of isoproter-

propranolol (Prop) for 24 h at 37 "C. Cells were then washed three enol (Zso) at the indicated times in the absence or presence of 2 p M

times with PBS, and the ml mAChR number was determined by ['HI QNB binding to plasma membranes using a saturating concentration of 400 p ~ . Data shown are the average f S.E. of three to five separate experiments performed in triplicate and are expressed as a percent of receptor number in untreated control cells studied in parallel.

Heterologous Regulation of the ml mAChR 7953

iOOrml mAChR

CBC (PM) 10 100

ISO - -

i

- -

0.2 2.0

B 2AR

0.2 2 . 0

FIG. 4. H-8 impairs isoproterenol-induced, but not carbachol-induced, effects on m l mAChR number. Intact cells were pretreated with the indicated concentrations of carbachol (CBC) or isoproterenol (Iso) in the absence (0) and presence (a) of 20 p M H- 8 for 24 h at 37 "C. Cells were then washed three times with PBS. m l mAChR and &AR numbers were determined by [3H]QNB and ['"I] ICYP binding to plasma membranes, respectively, using a saturating concentration of 400 PM for each radioligand. Data shown are the average f S.E. of three to eight separate experiments performed in triplicate and are expressed as a percent of receptor number in untreated control cells studied in parallel. *, significantly different from cells not treated with H-8.

lipid labeling of MB-CHO cells was not affected by the preincubation with muscarinic agonist for up to 24 h (data not shown). The time course for desensitization of CBC- stimulated release of inositol phosphates after prolorged activation of t he ml mAChR is shown in Fig. 2A. CBC-simulated P I turnover was reduced by 26 and 60% in cells pretreated with the agonist for 3 and 24 h, respectively.

Preincubation of MB-CHO cells with 2 p~ isoproterenol for 24 h resulted in a subsequent reduction in the ability of CBC to stimulate P I turnover when compared with untreated MB-CHO cells. The loss of responsiveness was such that the efficacy of CBC was reduced by 40% ( n = 3). As mentioned above CBC pretreatment led to a 65% decrement in mAChR- stimulated PI hydrolysis. Hence, heterologous desensitization of m l mAChR function was smaller in magnitude as compared with homologous desensitization. As was the case for CBC pretreatment, no change was observed in the ECS0 value for CBC-mediated PI hydrolysis in cells pretreated with isoproterenol (33 k 9 p ~ ) as compared with control cell value ( n = 3 for each treatment group). Furthermore, no effect of isoproterenol on basal lipid labeling was observed (data not shown). The time course for desensitization of CBC-stimulated release of inositol phosphates after prolonged activation of the pzAR is shown in Fig. 2B. CBC-simulated P I turnover was reduced by 20 and 40% in cells pretreated with isoproterenol for 3 and 24 h, respectively.

Mutational Analysis Identifies a Small Region on i3-con- trolling ml rnAChR Internalization following Homologous and Heterologous Receptor Actiuation-The third intracellular loop of the m l mAChR has been shown previously to be important for muscarinic agonist-mediated internalization of the receptor (32). In order to determine whether this region of t he ml mAChR might also be important in heterologous internalization induced by P2AR activation, a detailed time course depicting agonist-mediated loss of [3H]QNB binding to plasma membranes was performed.

Deletion of amino acids 227-341 produced a mutant m l mAChR (del 227-341) that did not internalize significantly following CBC pretreatment for up to 6 h, as compared with wild-type receptor which exhibited a 50 and 62% loss after 3-

and 6-h CBC exposure, respectively (Fig. 5, A and D ) . Longer CBC incubations of 24 h resulted in a decline of cell surface del 227-341 which mimicked wild-type losses (Fig. 5, A and D ) . By comparison, the same i3 mutation essentially abolished the ability of P2AR activation to trigger m l mAChR internalization for up to 24 h (Fig. 6, A and D ) . These findings suggest that a region within amino acids 227-341 may control CBC- and isoproterenol-mediated m l mAChR internalization.

Smaller deletions were constructed to better define the domain(s) on i3 which mediates m l mAChR internalization. Del 227-269, like the large deletion mutant del 227-341, was refractory to CBC-induced receptor internalization (Fig. 5, A and B ) . This was especially true for CBC incubations of 3 and 6 h where del 227-269 was essentially unresponsive. After 12-h CBC pretreatment, deficits in cell surface del 227-269 levels approached wild-type losses; however, internalization was still significantly ( p < 0.05) impaired as compared with wild-type receptor (Fig. 5, A and B ) . Also of note was the finding that the same deletion completely impaired m l mAChR internalization triggered by heterologous receptor activation for up to 24 h (Fig. 6, A and B ) . In contrast to the results obtained with del 227-269, deletion of amino acids 275-348 from the m l sequence had no effect on homologous- or heterologous-mediated internalization (Figs. 5, A and C, and 6, A and C). These results suggest the motif(s) responsible for m l mAChR internalization mediated by homologous and heterologous receptor activation is the same or at the very least localized within amino acids 227-269.

A

loo r

& n J

s c 2 100 r LT

:!li z w 20

0 3 6 12 24

"i M

0

D

1 3 6

1 0 0 r L

12 24

3 6 12 24

Time (hours) FIG. 5. Homologous-induced losses of wild-type and dele-

tion mutant m l mAChRs. Intact cells containing the &AR and either the wild-type or deletion mutant m l mAChR were incubated with 1 mM carbachol at the indicated times at 37 "C. Cells were washed three times with PBS, plasma membranes prepared, and receptor binding assays performed with a saturating concentration of 400 p~ [3H]QNB. Data shown are the average 5 S.E. of three to ten independent experiments performed in triplicate and are expressed as a percentage of receptor number in untreated cells studied in parallel. *, significantly different when compared with the pretreated wild-type m l mAChR. A, wild-type m l mAChR. B, mutant m l mAChR missing amino acids 227-269 (del 227-269). C, mutant m l mAChR missing amino acids 275-348 (del 275-348). D , mutant m l mAChR missing amino acids 227-341 (del 227-341). Insets, at the top are schematic representations of the fifth and sixth transmembrane domains and the third intracellular loop of various m l mAChR constructs. The darkened area indicates the portion of the third intracellular loop that was deleted.


t 3 6 12 24

T

3 6 12 24 il 6

12 24

Time (hours) FIG. 6. Heterologous-induced losses of wild-type and dele-

tion mutant m l mAChRs. Intact cells containing the ,&AR and either the wild-type or deletion mutant m l mAChR were incubated with 2 FM isoproterenol at the indicated times at 37 "C. Cells were washed three times with PBS, plasma membranes prepared, and receptor binding assays performed with a saturating concentration of 400 p~ r3H1QNB. Data shown are the average S.E. of three to ten independent experiments performed in triplicate and are expressed as a percentage of receptor number in untreated cells studied in parallel. *, significantly different when compared with the pretreated wild-type m l mAChR. A , wild-type m l mAChR. B, mutant ml mAChR missing amino acids 227-269 (del 227-269). C , mutant m l mAChR missing amino acids 275-348 (del 275-348). D, mutant m l mAChR missing amino acids 227-341 (del 227-341). Insets, at the top of each panel are schematic representations of the fifth and sixth transmembrane domains and the third intracellular loop of various ml mAChR constructs. The darkened area indicates the portion of the third intracellular loop that was deleted.

Homologous- and Heterologous-induced Uncoupling of the ml mAChR-Persistent activation of m l mAChRs or &ARs in MB-CHO cells desensitizes CBC-stimulated P I turnover which closely parallels m l mAChR internalization (Fig. 2). The molecular basis for G-protein-coupled receptor desensitization (uncoupling) has been shown to involve receptor phosphorylation and/or internalization away from the effector enzyme (9). However, it is unlikely that desensitization of the PI response is due to the loss of cell surface m l mAChRs in CHO cells, since varying m l mAChR density from 0.4 to 1 pmol/mg protein has little to no effect on the maximal level of PI turnover elicited by CBC (44). Inasmuch as del 227-269 (and del 227-341) did not markedly internalize (Figs. 5 and 6) or down-regulate (data not, shown) following early agonist treatment, we were interested in determining whether the i3 mutant receptors would stili undergo agonist-induced uncoupling. In order to examine this, clonal cell lines co-expressing the &AR and either wild-type or i3 mutant ml mAChRs were preincubated with 1 mM CBC or 2 p M isoproterenol from 3 to 24 h. Afterwards, intact cells were washed to remove agonist and rechallenged with 3 mM CBC, which maximally activates PI turnover in wild-type and i3 mutant m l mAChRs. Chronic exposure of MB-CHO cells expressing wild-type m l mAChRs to CBC or isoproterenol leads to a time-dependent reduction

turnover (Fig. 7, A and B ) . For cells expressing del 227-269, the deletion of amino acids 227-269 had no effect on CBC- or isoproterenol-induced desensitization (Fig. 7, A and B) . Likewise, deletion of amino acids 227-341 did not prevent agonist-induced desensitization (Fig. 7, A and B) . The reduced magnitude of CBC rechallenge to maximally stimulate PI turnover does not appear to be due to a defect in the coupling of G-protein and PLC, since the efficacy and potency of NaF to stimulate PI turnover was not modified by CBC or isoproterenol pretreatment (Fig. 8). Hence, amino acids 227-269 of the m l mAChR sequence appear to be involved in receptor internalization but not uncoupling. Therefore, these results suggest that a separate motif outside of residues 227-341 may be responsible for agonist-induced uncoupling (see "Discus- sion").

DISCUSSION

Cross-talk Involving the ml mAChR-Although numerous reports have appeared on the interactions of multiple signaling pathways at the post-receptor level, information on cross- talk at the level of the receptor is limited. This is especially true for the mAChR gene family. To date, mAChRs in cardiac tissue (m2 subtype), intestinal smooth muscle (m2 and m3 subtypes), and SK-N-SH neuroblastoma cells (m3 subtype) have been shown to desensitize upon activation of the PAR/ adenylate cyclase system (33-35). However, the exact loci and molecular mechanisms of cross-talk between the mAChR and PAR are not. yet resolved.

It has been demonstrated that short term activation of mAChRs with muscarinic agonists ( 4 h) leads to their rapid internalization/sequestration away from the cell surface and into an intracellular compartment (36). This agonist-specific process (homologous regulation) is not accompanied by a

I A) CEIC-pretreated

eo A d e l 227-269

P) m U m c 40

: : ? a' 5 20-

-

dol 20' 0 4 8 12 16 20 24

Time (hours] FIG. 7. Time course of wild-type and deletion mutant m l

mAChR uncoupling induced by homologous and heterologous receptor activation. Cells, seeded in 12-well plates, were prelabeled with rny~-[~H]inositol for 24 h in the absence and presence of 1 mM carbachol ( A ) or 2 p~ isoproterenol ( B ) , for the indicated times at 37 "C. Cells were washed three times with PBS and rechallenged with 3 mM carbachol for 30 min in the presence of 10 mM Licl for PI hydrolysis assays. Values shown are the average t- S.E. of three independent experiments performed in triplicate and expressed as a

in the ability of CBC rechallenge to maximally stimulate PI percentage of control.

Heterologous Regulation of the ml mAChR 7955

15 r

0 Untreated 0 Iso-pretreated A CBC-pretreated 10 -

5 -

0-8- 6

n I " -4 -3 -2 -1

Log [NaFl , (MI FIG. 8. Carbachol and isoproterenol treatment does not

modify NaF stimulation of phosphoinositide hydrolysis. Intact cells, containing the P2AR and wild-type m l mAChR, were prelabeled with rny~-[~H]inositol for 24 h in the absence and presence of 1 mM carbachol (CBC) or 2 PM isoproterenol at 37 "c. Cells were washed three times with PBS and rechallenged with the indicated concentrations of NaF plus 10 mM AlCl3 in the presence of 10 m M Licl. Values shown are the average k S.E. of three independent experiments performed in triplicate and expressed as a -fold basal stimulation of PI hydrolysis.

reduction in total cellular mAChR sites. Upon removal of agonist, internalized receptors return rapidly to the cell surface. However, continued muscarinic agonist exposure leads to down-regulation (a decrease in total cellular mAChR levels), presumably due to an increased rate of receptor degra- dation. For mAChRs coupled to PLC, the generation of phosphoinositide-derived second messengers appears not be required for either receptor internalization or down-regulation (37, 38).

In this report, we describe a similar series of events that occur during heterologous regulation, where m l mAChRs initially internalize and then down-regulate as a consequence of P2AR activation. For both homologous and heterologous receptor activation, down-regulation of m l mAChRs did not occur for at least 6 h, after which time losses of total m l mAChRs proceeded in a time-dependent manner. A recent study has indicated that agonist-mediated internalization of mAChRs is a prerequisite for down-regulation in SK-N-SH neuroblastoma cells (38) and requires G-protein involvement (38). We do not know at this time whether a similar chain of events is obligatory for heterologous down-regulation of mAChRs. Experiments are currently underway to address this issue.

Heterologous Control of mAChR Number by CAMP May Involve PKA-PAR-mediated Regulation of ml mAChR Num- ber Appears to Involve PKA-This premise is supported by the following findings. First, forskolin, a diterpene that directly stimulates adenylate cyclase, and Bt,cAMP, a membrane-permeable cAMP analogue, mimic the effects of isoproterenol pretreatment. Second, prior exposure of MB-CHO cells to H-8, a PKA inhibitor (39), reversed isoproterenol- induced, but not CBC-induced, losses in m l mAChR number (Fig. 4). Consistent with this notion is the finding that mAChRs purified from brain tissue, which is abundant in the m l subtype (40), can be phosphorylated directly by the cata- lytic subunit of PKA i n vitro (19). The stoichiometry of 32P incorporation was -1 mol of phosphate/mol of receptor in these studies, which is in agreement with the number of PKA phosphorylation sites on the m l mAChR based on primary structure analysis (see below). It is noteworthy that PKA also has been demonstrated to covalently modify other muscarinic

RECEPTOR LOCATION SEQUENCE SUBTYPE MOTIF

B B X=

P2 AR i3 G L

P 2 m COOH c L

ml mAChR i3 L A

m2 mAChR i2 P V

m3 mAChR i3 K R

m4 mAChR i2 P A

m5 mAChR i3 s s

FIG. 9. Consensus sequence motifs for PKA phosphorylation on the mAChR subtypes and the D2AR. PKA sequence motif is boxed and generalized as follows: B-B-X-S/T, where B represents the basic amino acids arginine or lysine, X represents any amino acid, and S/T represents the phosphoacceptor site of either serine or threonine. These sites are located on the second intracellular loop (iZ), third intracellular loop (D), or the carboxyl terminus (COOH) of the receptor. The sequence motif for the ml mAChR represents amino acids 351-354 of the rat sequence (24).

receptor subtypes such as the m2 mAChR (21). Primary structure analysis of the ml mAChR reveals a potential PKA phosphorylation sequence motif L y ~ ~ ~ l - A r g - L y s - T h r ~ ~ ~ on i3 (Fig. 9) (41), in an area proposed to be important for receptor- G-protein coupling (31). Sites for PKA are also present in the second intracellular loop of the m2 and m4 subtypes and i3 of the m3 and m5 subtypes (Fig. 9).

The role of PKA-mediated phosphorylation has been exten- sively examined in the hamster P2AR (42, 43). Two PKA phosphorylation motifs are found in the receptor, one of these sites being located on i3 and the other on the COOH-terminal domain (Fig. 9). Removal of these motifs by deletion or site- directed mutagenesis produces mutant receptors that are no longer phosphorylated by PKA and exhibit an impaired ability to undergo rapid heterologous desensitization and down-regulation (42, 43). Taken together, these results suggest that PKA may serve a similar role in m l mAChR function. Iden- tification of these motifs in the m2 and m3 mAChRs, previously shown to undergo modulation upon PAR activation (33- 35), underscores PKA's potential involvement in cross-regu- lating these subtypes; an unappreciated mechanism at the time these studies were performed. However, direct confir- mation of PKA phosphorylation of these consensus sites during heterologous regulation of mAChRs awaits future mutagenesis experiments.

Agonist-specific Regulation of mAChR Number Does Not Involve PKA-mAChRs coupled to PLC are known to weakly stimulate cAMP formation in CHO cells (44), presumably via a calmodulin-dependent process (45), although this may not be the sole mechan i~m.~ Inasmuch as CBC-mediated down- regulation was not blocked by H-8, PKA appears not to be required for homologous regulation of the ml mAChR in MB- CHO cells. Likewise, PKA activity was not required for isoproterenol-induced losses of PzARs in the present study, not an unexpected finding (46).

Role of i3 i n Receptor Cross-talk-The third intracellular loop (i3) of t he ml mAChR contains 156 amino acids, of which only 11 and 21 residues at the NH2- and COOH-

D. Gunvitz, C. M. Fraser, A. Fisher, unpublished observations.


terminal ends, respectively, are necessary for conferring receptor-G-protein coupling (31). The central portion of i3 appears to play a role in muscarinic agonist-mediated internalization, as deletion of up to 130 amino acids impaired this process (32). The results presented here, where we have deleted amino acids 227-341 but left intact the membrane proximal portions of i3, are in agreement with the above studies. Moreover, our results reveal that the middle portion of i3 is also required for m l mAChR internalization during heterologous regulation. By deleting smaller regions within amino acids 227-341, we have identified a 43-residue domain (amino acids 227-269) that is crucial for both homologous- and heterologous-promoted internalization. Of interest was the finding that del 227-341 exhibited impaired ability to internalize during the first 12 h of CBC incubation but not at 24 h. This was not the case during PzAR activation, where del 227-341 exhibited impaired ability to internalize for up to 24 h. Recent findings in our laboratory indicate that long term exposure to CBC (>6 h) destabilizes m l mAChR mRNA.4 Such a regulatory event explains the loss of m l mAChRs at later CBC incubations.

In terms of homologous internalization, del 275-348 was indistinguishable from wild-type m l mAChR. This result was somewhat surprising in light of a recent report implicating amino acids 284-292 in CBC-mediated internalization of the human m l mAChR (47). It should be noted that the sequence identity of i3 in the rat m l mAChR (present study) and its human homolog (47) is 100%. We do not have an explanation at this time for the discrepancies between our results and those of Lameh et al. (47). It is possible that the different cell lines utilized in the transfection experiments in the two studies are a contributing factor. Another possibility is that an additional domain located inside residues 275-348 partic- ipates in receptor recycling (47). Regardless, these two studies when taken together implicate multiple i3 domains in m l mAChR internalization.

Residues 227-269 and 284-269, as postulated by Lameh et al. (47), contain a series of polar residues that may contribute to ml mAChR internalization. Within amino acids 227-269 are a string of 5 adjacent Ser residues and a total of 6 within an 8-amino acid stretch (residues 237-244). A similar Ser/ Thr-rich domain, found on the cytoplasmic tail of P2ARs, is known to participate in receptor internalization (48). Replace- ment of these residues with Ala/Gly impairs pzAR internalization apparently by altering the conformation of the cytoplasmic tail (48). We propose that the secondary structure of amino acids 237-244 in i3 may likewise be pivotal for m l mAChR internalization. Whether this region is necessary for the recognition of a cytosolic factor that promotes receptor internalization, analogous to the role of p-arrestin in &AR/ G. uncoupling (49), remains to be seen.

Agonist-induced Receptor Uncoupling-Chronic treatment of MB-CHO cells with CBC or isoproterenol leads to a reduction in the efficacy, but not EC50, of CBC to stimulate PLC. The molecular basis for this defect appears to be at the level of the receptor itself for several reasons. First, NaF was capable of maximally stimulating PI hydrolysis in CBC- and isoproterenol-treated cells, indicating that post-receptor mechanisms were uneffected by agonists. Second, the efficacy of CBC to stimulate PI turnover in CHO cells is independent of m l mAChR densities ranging from 0.4 to 1 pmol/mg protein (44). Finally, recent findings suggest that mAChR phosphorylation modulates receptor-G-protein coupling (50).

Since the cytoplasmic loops of t he ml mAChR contain consensus sites for PKC and PKA, we were interested in

' N. H. Lee and C. M. Fraser, submitted for publication.

testing the ability of our i3 mutants to undergo agonist- induced uncoupling. Not surprisingly, removal of amino acid residues 227-269 or 227-341, but leaving intact the consensus PKA site at position 351-354, did not reverse m l mAChR uncoupling promoted by heterologous receptor activation. It is apparent from our data that the single PKA site on the m l mAChR functions in a manner analogous to the two PKA sites on the PZAR (42, 43), where phosphorylation of these sites leads to receptor uncoupling and internalization/down- regulation. Uncoupling of m l mAChRs induced by CBC, like isoproterenol, was not dependent on residues 227-269 and 227-341. The lack of effect when deleting residues 227-341, containing two consensus PKC sites at positions 325-328 and 329-332, suggests that other sites on the receptor facilitate muscarinic agonist-induced uncoupling. Indeed, a third PKC site (residue 136-141) is situated on i2, a region of the ml mAChR shown to be important for receptor-G-protein interactions (51).

In conclusion, we have investigated the mechanism of heterologous regulation of the m l mAChR. Activation of the PARladenylate cyclase pathway results in internalization and down-regulation of m l mAChRs via a CAMP- and PKA- dependent mechanism. These findings, the identification of a consensus PKA site on the m l mAChR, and previous studies showing that this subtype can be phosphorylated in vitro by PKA (19), suggest that phosphorylation may trigger internalization and down-regulation. We also provide evidence for the role of multiple domains in i3 participating in m l mAChR internalization. Specifically, amino acids 227-269 (present study) and 284-292 (47) are involved in homologous internalization, whereas amino acids 227-269 and 351-354 (PKA site) are required for heterologous internalization. These domains, with the exception of amino acids 351-354, do not appear to participate in agonist-mediated m l mAChR uncoupling. Hence, it seems likely that additional domains (e.g. PKC sites) are responsible for receptor uncoupling. Clearly, the role, if any, of in situ phosphorylation of m l mAChRs in receptor uncoupling remains to be clarified and awaits future experiments.

Acknowledgments-We thank T. Miller and E. Cheng for expert technical assistance.

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Documents

Cross-talk between ml Muscarinic Acetylcholine and &Adrenergic … · Receptor cross-talk or heterologous regulation refers to that form of regulation in which activation one receptor