Muscarinic acetylcholine receptor subtypes as agonist-dependent oncogenes.

Abstract

We have evaluated the muscarinic acetylcholine family of G protein-coupled receptors (mAChRs) for their oncogenic potential. These receptors are preferentially expressed in postmitotic cells, transducing signals specified by their endogenous agonist, the neurotransmitter acetylcholine. Cells transfected with individual human mAChR genes were morphologically indistinguishable from parental NIH 3T3 cells in the absence of agonist. In contrast, when cultures were supplemented with carbachol, a stable analog of acetylcholine, foci of transformation readily appeared in ml, m3, or m5 but not in m2 or m4 mAChRs transfectants. Receptor expression was verified by ligand binding and was similar for each transfected culture. Transformation was dose-dependent and required only low levels of receptor expression. In transformation-competent cells, agonist induced phosphatidylinositol hydrolysis, whereas in m2 or m4 transfectants, receptors were coupled to the inhibition of adenylyl cyclase. These findings demonstrate that mAChRs linked to phosphatidylinositol hydrolysis can act as conditional oncogenes when expressed in cells capable of proliferation.Receptors for polypeptides, such as epidermal growth factor and platelet-derived growth factor, can induce cellular transformation when constitutively activated (1-4). Structural mutations or unregulated availability of ligand are mechanisms known to account for their transforming activity. These receptors are prototypes of a class that mediate signal transduction by virtue of an intrinsic protein-tyrosine kinase activity (2,5,6). When the mas oncogene was discovered, a class of cell-surface receptors lacking protein-tyrosine kinase domains was also implicated in cellular transformation. The mas oncogene product has a structural motif characteristic of receptors that mediate signal transduction by coupling to GTP-binding proteins (G proteins) (7). Although mas has a weak focus-inducing activity in vitro, cells transfected with this gene are highly tumorigenic in nude mice (7). More recently, G protein-coupled serotonin receptors have been shown to convert fibroblasts to a tumorigenic state (8). Because in these latter cases exogenous ligand is not required for transformation, either these genes encode aberrant receptors or endogenous ligands are responsible for their activation. Thus, ligand independence has limited the study of the mechanism by which these receptors mediate transformation.In the present study, we have directly tested the hypothesis that normal G-protein-coupled receptors can induce agonistdependent neoplastic transformation. We chose a family of cell-surface neurotransmitter receptors, human muscarinic acetylcholine receptors (mAChRs), which possess sequence homology with both mas and the serotonin receptor (9-12). Muscarinic receptors are preferentially expressed in neurons and other postmitotic cells, and they transduce signals specified by their endogenous agonist, the neurotransmitter acetylcholine. The mAChR family consists of five distinct but high...

Regulation of cell proliferation by G proteins

“…It has been observed that the microinjection of PLC triggers transformation in NIH3T3 cells (Smith et al, 1989). In addition, it has been noticed that the expression of m1, m2, m5 muscarinic- (Gutkind et al, 1991), a1b adrenergic-or 5HT 1c -receptors (Allen et al, 1991) that are known to be coupled to PLC pathways leads to NIH3T3 cell transformation. These observations suggest that the activation of PLC is involved in the mitogenic pathway regulated by Ga q .…”

“…All of the signaling pathways regulated by the a-subunits can be equally or more potently activated by the bgsubunits (Clapham and Neer, 1993;Gutkind, 1998). In spite of its ability to potently activate diverse signaling pathways ± the pathways used by the Ga-subunits to activate cell proliferation ± the expression of Gbg fails to activate cell proliferation.…”

“…The mechanism by which bg-subunit activate the Ras-dependent ERK pathway has been the focus of many laboratories. The ability of bg-subunits to activate PLC and hence PKC or Ca 2+ -dependent pathways suggest that bg can activate the ERK pathway through more than one mechanism Dhanasekaran and Vara Prasad, 1998;Gutkind, 1998).…”

The ras-related GTPase rac1 regulates a proliferative pathway selectively utilized by G-protein coupled receptors

“…The m1 muscarinic receptor, which transforms NIH3T3 cells in response to agonist stimulation (Gutkind et al, 1991), has been shown to activate both the MAP kinase and Jun kinase cascades (Coso et al, 1995b), which are each preferentially regulated by ras and rac, respectively (Denhardt, 1996;Coso et al, 1995;Minden et al, 1995). The closely related m5 muscarinic receptor also transforms via a ras-dependent pathway (Mattingly et al, 1994).…”

“…The Receptor Selection and Ampli®cation Technology (R-SAT TM ) assay is based on protocols previously used to evaluate induction of foci by ligand-occupied muscarinic receptors (Gutkind et al, 1991). In this assay technology, ligands select and amplify cells that express functional receptors.…”