Activation of the MAP kinase cascade by exogenous calcium-sensing receptor Susan A. Hobson, Jay Wright, Fred Lee, Scott E. McNeil 1 , Timothy Bilderback, Karin D. Rodland * Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, OR 97201-3098, USA Received 17 September 2001; accepted 26 November 2001 Abstract In Rat-1 fibroblasts and ovarian surface epithelial cells, extracellular calcium induces a proliferative response which appears to be mediated by the G-protein coupled calcium-sensing receptor (CaR), as expression of the nonfunctional CaR-R795W mutant inhibits both thymidine incorporation and activation of the extracellular-regulated kinase (ERK) in response to calcium. In this report we utilized CaR-transfected HEK293 cells to demonstrate that functional CaR is necessary and sufficient for calcium-induced ERK activation. CaR-dependent ERK activation was blocked by co-expression of the Ras dominant-negative mutant, Ras N17, and by exposure to the phosphatidyl inositol 3? kinase inhibitors wortmannin and LY294002. In contrast to Rat-1 fibroblasts, CaR- mediated in vitro kinase activity of ERK2 was unaffected by tyrosine kinase inhibitor herbimycin in CaR-transfected HEK293 cells. These results suggest that usage of distinct pathways downstream of the CaR varies in a cell-type specific manner, suggesting a potential mechanism by which activation of the CaR could couple to distinct calcium-dependent responses. # 2002 Published by Elsevier Science Ireland Ltd. Keywords: Calcium-sensing receptor; MAP Kinase; PI3? Kinase; Proliferation; HEK293 cells 1. Introduction It has been recognized for some time that extracellular calcium can modulate the proliferative state of various cell types. In calcium-sensitive epithelial cells, extracel- lular calcium can act as a differentiation factor, as exemplified in breast epithelial cells, intestinal epithelial cells, and keratinocytes (Hennings et al., 1980; Black and Smith, 1989; Yuspa et al., 1989; Soule et al., 1990). In contrast, several mesenchymal cell types, including Rat-1 fibroblasts and ovarian surface epithelial cells, show a proliferative response to increased extracellular calcium (Huang et al., 1995; McNeil et al., 1998a,b). Expression of the G protein-coupled calcium-sensing receptor (CaR) has been demonstrated in these calcium- responsive epithelial and mesenchymal cell types, sug- gesting a possible mechanism by which extracellular calcium could induce changes in proliferation. In sup- port of this model, we have previously demonstrated that expression of the dominant interfering mutant CaR-R795W in Rat-1 fibroblasts results in the inhibi- tion of calcium-sensitive proliferation (McNeil et al., 1998a,b). Similar results were obtained in rat ovarian surface epithelial cells, which also proliferate in response to extracellular calcium (Hobson et al., 2000). However, interpretation of these results is complicated by the presence of endogenous wild-type CaR in these cells. In this report, we express the wild-type CaR in HEK293 cells, which are characterized as null for the CaR (Bai et al., 1996; Ruat et al., 1996). This model system has been used for structure-function studies of the CaR (Bai et al., 1996), and facilitates rigorous testing of the specificity of extracellularsignal-regulated-kinase (ERK) activation by extracellular calcium while simpli- fying investigation of downstream signaling events. We demonstrate here that calcium-induced ERK activation requires expression of functional CaR in HEK293 cells, as calcium could not induce ERK activation in HEK * Corresponding author. Tel.:  /1-503-494-7812; fax:  /1-503-494- 4253. E-mail address: karin.rodland@pnl.gov (K.D. Rodland). 1 Present address: Science Applications International Corporation, Frederick, MD 21702, USA. Molecular and Cellular Endocrinology 200 (2003) 189  /198 www.elsevier.com/locate/mce 0303-7207/02/$ - see front matter # 2002 Published by Elsevier Science Ireland Ltd. PII:S0303-7207(01)00749-3