Involvement of Ras and Raf in the Gi-coupled Acetylcholine Muscarinic m2 Receptor Activation of Mitogen-activated Protein (IMAP) Kinase Kinase and MAP Kinase*

Stimulation of the acetylcholine muscarinic m2 receptor (m2R) expressed in Rat l a fibroblasts results in the activation of the cytoplasmic mitogen-activated protein kinase (MAF’K). Concomitant with carbachol stimula- tion of the m2R was the activation of MEK (MAPK kinase) and Raf. MEK is the dual function kinase that phosphorylates and activates MAPK. Raf is a serine/ threonine kinase capable of phosphorylating and activating MEK. Carbachol stimulation of the m2R also activated Ras. Pertussis toxin treatment of Rat la cells inhibited the m2R-mediated activation of Ras, Raf, MEK and MAPK. In contrast, epidermal growth factor recep-tor-mediated activation of Ras, Raf, MEK and MAPK was pertussis toxin-insensitive. m2R activation of Ras, Raf, and MAPK was insensitive to inhibition by genistein, while the epidermal growth factor receptor-induced responses were inhibited by genistein. The findings demonstrate that both Ras and Raf can be regulated by seven-membrane-spanning receptors that selectively couple to

G proteins found in fibroblasts and many other cell types. The classically defined Gi-regulated response is inhibition of adenylylcyclase activity (4). It is clear, however, that Gi a subunits (ai) are capable of regulating specific potassium channels (51, may be involved in controlling intra-Golgi vesicular transport (61, and are involved in mitogenic responses to thrombin and lysophosphatidic acid (7,8). Gi proteins are expressed at sufficient levels in many cell types that, when activated, the P-y subunits dissociated from ai2.GTP can also contribute to the regulation of effectors including specific isoforms of adenylylcyclase and phospholipase C (9,10).
Expression of the GTPase-inhibited ai2 polypeptide in Rat l a fibroblasts alters their normal growth control and induces a transformed phenotype (11). The transformation of Rat la cells by GTPase-inhibited ai2 can be dissociated from the regulation of adenylylcyclase, phospholipase Cp, and specific ion channels (11,12). It was found with Rat la cells that GTPase-inhibited ai2 polypeptide expression constitutively activated mitogen-activated protein kinases ( M A P & ) . M A P & are serinelthreonine kinases that phosphorylate and regulate the activity of several proteins including Rsk9O (131,cPLA2 (14,151,and the EGF receptor (17). Both tyrosine kinase-encoded growth factor receptors (Le. EGF receptor) and G protein-coupled receptors (i.e. thrombin receptor) are capable of stimulating the rapid activation of M A P & (18).
In order to define Gi-regulated signal transduction networks leading to MAPK activation, we expressed the Gi-coupled m2R in Rat la cells. In this report, we demonstrate that acute stimulation of the m2R leads to the activation of Ras and Raf in a Gi-dependent manner.
MATERIALS AND METHODS Expression of Muscarinic m2 Receptor in Rat l a Cells-Rat l a cells were co-transfected with the expression plasmid pCD, containing the cDNA for the muscarinic m2R, and LNCX, which contained the neomycin resistance gene. G418-resistant clones were isolated and seeded into 12-well dishes for screening with the muscarinic radioligand L-[N-methyl-3H]scopolamine methylchloride (NMS). Two positive clones were further characterized by saturation binding analysis and shown to express approximately 5 x lo6 and 1.4 x lo5 m2 receptordcell. Carbachol stimulation of the m2R expressed in each clone resulted in inhibition of cholera toxin-stimulated adenylylcyclase activity (not shown).
MAPK and MEK Assays-MAF'K activity was assayed as described previously (19). Immunoblotting of the fractions with anti-MAPK antibody showed that the peaks of activity corresponded with immunoreactive p42 and p44 MAF'K. MEK activity was assayed from cell lysates fractionated on a Mono S FPLC column (20).
Ras Activation Assays-Activation of Ras was determined by analyzing the ratio of GTP to GDP bound to immunoprecipitated Ras from control and stimulated Rat l a cells (21). Quantitation of radiolabeled GDP and GTP was accomplished using a Molecular Dynamics Phos-phorImager.

RESULTS
Rat l a cells express the pertussis toxin-sensitive G protein a subunits ai2 and ai3 but not a, (not shown). For this reason the m2R was expressed in Rat l a cells to promote selective coupling and activation of Gi-regulated response pathways including the MAPK regulatory network (1,2,12). Fig. 1 shows that carbachol treatment of Rat l a cells, expressing approximately m2 Receptor Activation of Ras and Raf 19197 1.4 x lo6 m2Wcel1, activated MAPK to levels similar to those observed with stimulation of the EGF receptor. m2R stimulation of MAPK activity was completely inhibited by pretreatment of the cells with pertussis toxin, consistent with the selective coupling of the m2R with Gi proteins, which are pertussis toxin substrates (Fig. 1). Carbachol stimulation of the m2R did not activate phospholipase C activity in Rat la cells (not shown). Similar to previous reports (241, the ability of the EGF receptor to activate MAPK was completely pertussis toxin-insensitive and does not require a functional Gi protein. MEK, the dual function tyrosine and threonine kinase that phosphorylates MAPK (221, was similarly activated by the stimulation of the m2R or the EGF receptor (Fig. 2). MEK activation by the m2R was inhibited by treatment of Rat la cells with pertussis toxin, whereas the EGF receptor regulation of MEK was again pertussis toxin-insensitive (not shown). + EGF +PTa 10  (clone 61) was serum-starved overnight in medium containing 0.1% bovine serum albumin in the presence or absence of 100 ng/ml pertussis toxin (PTx). Cells were challenged with 100 PM carbachol or 30 ng/ml EGF for 3 min. Carbachol had no effect on MAF' K activity in wild-type Rat l a cells, and atropine (10 PM) completely blocked the m2R response but had no effect on EGF (not shown). The results are representative of three independent experiments and are similar to that of two independent m2R-expressing Rat l a cell clones. Cell extracts were prepared from carbachol and EGFstimulated, m2R-expressing Rat l a cells as described for Fig. 1. A, MEK resolved by Mono S chromatography. The results are representative of three independent experiments. E , immunoblotting with a rabbit antiserum recognizing the C terminus of MEK-1 specifically recognized a 45-kDa protein that co-eluted with MEK enzymatic activity.
The regulation of MEK and MAPK activity in response to tyrosine kinases has been shown to involve the activation of Raf (23). It was unclear, however, if selective Gi-coupled receptors such as the m2R could also regulate Raf activity. Using purified recombinant MEK as a phosphorylation substrate for Raf. it was found that carbachol activated Raf in a manner similar to that observed with EGF (Fig. 3). The m2R-mediated activation of Raf was completely inhibited by pertussis toxin treatment of the cells, but the EGF receptor response was refractory to pertussis toxin. This is the first demonstration that a Gi-coupled seven-membrane-spanning receptor such as the m2R is capable of activating Raf.
An interaction between Raf and Ras has been defined (25), strongly implicating a linkage between Ras activation and Raf activation (26). Fig. 4 demonstrates that carbachol stimulation of the m2R in Rat la cells caused a time-dependent activation of Ras. Ras activation measured by the formation of Ras-GTP A. rum-starved wild-type ( W T ) Rat la cells and m2R-expressing Rat la cells were stimulated with 100 p~ carbachol or 30 ndml EGF for 3 min as described in Fig. 1. Cell lysates were prepared and Raf-1 was immunoprecipitated. Control refers to immunoprecipitation in the absence of Raf antibody. Purified recombinant kinase inactive MEK wan used an a (10 pCi). After incubation for 10 min. the reaction was stopped with Raf substrate in an in vitro kinase assay in the presence of ly"'P1ATP

8.
Laemmli SDS sample buffer and the proteins resolved by SDS-polyacrylamide gel electrophoresis (10% acrylamide). Purified recombinant wild-type ( WT) MEK was autophosphorylated and used as a standard. complexes was rapid, with maximal activation occurring within 1-3 min and returning to basal levels by 30 min. m2R activation of Ras was pertussis toxin-sensitive. whereas the EGF receptor activation of Rae was insensitive to pertussis toxin treatment (Fig. 4).
Genistein. a tyrosine kinase inhibitor (271, inhibited the EGF receptor activation of Ras, Raf, and MAPK in Rat la cells (Fig.  6). However, even 300 PM genistein, a high concentration that completely inhibited the EGF receptor regulation of this pathway, had no inhibitory effect on the m2R activation of Rae, Raf, and MAPK in Rat la cells. If anything, genistein pretreatment enhanced carbachol stimulation of Raf activity. This result indicates that the Gi-dependent regulation of Ras, Raf, and MAPK involves a genistein-insensitive pathway in Rat la cells. If a tyrosine kinase is involved in the Gidependent. m2R activation of Ras, Raf, and MAPK, it must be very insensitive to genistein relative to the EGF receptor or EGF receptor-regulated tyrosine kinases involved in regulating this pathway.

DISCUSSION
It has been generally assumed that heterotrimeric G p r e teins coupled to seven-membrane-spanning receptors utilized signal transduction pathways that were largely independent of Ras. This assumption was based primarily on the known Rasindependent effectors regulated by heterotrimeric G proteins such as adenylylcyclase. phospholipase Cp, and K' channels (4,6,9,10). There is a growing awareness, however, that G proteincoupled signal transduction pathways can exert a strong growth regulatory control in selected cell types (12). This is particularly true for the G protein-coupled thrombin and LPA receptor control of mitogenesis in fibroblasts (7,8). Both the thrombin-and LPA receptor-mediated responses seem to involve more than one G protein. For example, thrombin receptor responses have been elegantly shown to involve both Gi and G,(28,29). The LPA receptor appears to be similarly coupled to both Gi and G, (8). The acetylcholine muscarinic m2 receptor (m2R) is significantly more selective than either the thrombin or LPA receptors and couples primarily to Gi (1). The m2R did not activate phospholipase C activity in the Rat la cells used in this study. For this reason we used the m2R to selectively study the ability of Gi-regulated signal transduction pathways to regulate the MAPK regulatory network. Our results unequivocally demonstrate that the m2R receptor, in a Gi-dependent manner, is capable of mediating Rae and Raf activation in Rat la cells. Interestingly, expression of the acetylcholine muscarinic m l receptor, which selectively couples to G, and activates phospholipase Cp (301, in Rat la cells did not activate Ras or Raf (not shown).
The linkage between Gi, Ras, and Raf is presently unclear. The most obvious candidate would be a tyrosine kinase activated by Gi. In fact, this has been proposed recently for the LPA and thrombin receptor activation of Ras in Rat 1 and CCL39 cells (31). This response was inhibited 50-75% by 50 p~ genistein pretreatment. In a second study with Rat 1 cells, treatment with 10 p~ herbimycin, a different tyrosine kinase inhibitor, inhibited LPA-stimulated DNA synthesis and only partially inhibited MAPK activation (32). Our studies in Rat la cells indicate that the m2R activation of Rae, Raf, and MAPK are genistein-insensitive. The basis for the difference between receptors and cell types in sensitivity to tyrosine kinase inhibitor influences on Gi-coupled activation of Ras is not apparent.
The ability of Gi-coupled seven-membrane-spanning recep Based on the multiple proteins required for tyrosine able that this response will not be observed in all cell types. This would predict a unique effector for Gi expressed in some, but not all, cell types capable of integrating Gi with the Ras activation pathway. The contribution of Ras and Raf regulation by Gi will have to be considered when defining the physiological responses of Gi-coupled seven-membrane-spanning receptors.