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Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation Melinda D Willard1,2,3 , Francis S Willard1,2,3 , Xiaoyan Li2 , Steven D Cappell1 , William D Snider2 and David P Siderovski1,2,3, *

1 Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA,

2 UNC Neuroscience Center, University of North Carolina, Chapel Hill, NC, USA and

3 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by heterotrimeric G-protein a subunits and thus inhibit signaling by many G protein-coupled recep- tors.

Several RGS proteins have a multidomain architec- ture that adds further complexity to their roles in cell signaling in addition to their GTPase-accelerating activity. RGS12 contains a tandem repeat of Ras-binding domains but, to date, the role of this protein in Ras-mediated signal transduction has not been reported. Here, we show that RGS12 associates with the nerve growth factor (NGF) receptor tyrosine kinase TrkA, activated H-Ras, B-Raf, and MEK2 and facilitates their coordinated signaling to prolonged ERK activation. RGS12 is required for NGF- mediated neurite outgrowth of PC12 cells, but not out- growth stimulated by basic ?broblast growth factor. siRNA-mediated knockdown of RGS12 expression also inhibits NGF-induced axonal growth in dissociated cul- tures of primary dorsal root ganglia neurons. These data suggest that RGS12 may play a critical, and receptor- selective, role in coordinating Ras-dependent signals that are required for promoting and/or maintaining neuronal differentiation. The EMBO Journal (2007) 26, 2029C2040. doi:10.1038/ sj.emboj.7601659;

Published online

22 March

2007 Subject Categories: signal transduction;

differentiation &

death Keywords: MEK;

Raf;

Ras;

RGS12;

TrkA Introduction Extracellular stimuli such as neurotransmitters, hormones, odorants and light are recognized by G protein-coupled receptors (GPCRs), one of the most abundant and diverse protein families in the nervous system (Davies et al, 2002b). Intercellular communication via G-protein-mediated signal- ing pathways in the nervous system is crucial for normal brain development and regulation of adult neural processes (Davies et al, 2002b). Thus, de?ning the molecular determinants that control GPCR signaling is important to understanding normal development and physiology of the nervous system and the pathophysiology of nervous system- related disorders. One level of regulation of GPCR signaling is mediated by '

regulators of G-protein signaling'

(RGS) proteins that accelerate Ga GTP hydrolysis and inhibit signaling by many GPCRs (Ross and Wilkie, 2000). For example, mice lacking RGS9 show enhanced responses to photonic stimulation and morphine administration, suggesting that RGS9 is a potent negative regulator of both rhodopsin and opioid receptor signal transduction in vivo (Chen et al, 2000;

Zachariou et al, 2003). In general, however, contributions by RGS proteins to physiological control of speci?c receptor signaling cascades are only now being elucidated. Multidomain RGS proteins add further complexity to the potential signaling roles played by RGS proteins in addition to their GTPase- accelerating activity (reviewed in Ross and Wilkie, 2000;

Siderovski and Willard, 2005). RGS12 is an example of a multidomain RGS protein with numerous signaling regulatory elements. In addition to a central RGS domain, RGS12 contains a PDZ (PSD-95/Discs- large/ZO-1 homology) domain, a phosphotyrosine-binding (PTB) domain, tandem Ras-binding domains (RBDs), and a GoLoco (Gai/o-Loco) interaction motif (Ponting, 1999;