RGS8 Protein Is Distributed in Dendrites and Cell Body of Cerebellar Purkinje Cell Masayuki Itoh,* Megumi Odagiri,* Hideki Abe,† and Osamu Saitoh* ,1 *Department of Molecular Cell Signaling, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, Japan; and †Department of Physiology, Tokyo Medical and Dental University, Graduate School and Faculty of Medicine, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan Received July 19, 2001 RGS8 was originally identified as an RGS protein specifically expressed in neuronally differentiated P19 cells. We generated a polyclonal antibody specific to rat RGS8 using a synthetic peptide. When nonneu- ral cells (DDT1MF2, CHO, and NIH3T3) transfected with rat RGS8 cDNA were immuno-stained with this antibody, the RGS8 protein was mainly detected in the nuclei. Since RGS8 mRNA was exclusively expressed in Purkinje cells of the cerebellum in the rat brain, we further examined the cellular distribution of the RGS8 protein in Purkinje cells using cultured cerebellar cells and tissue sections of the cerebellum. The RGS8 protein was excluded from the nuclei and distributed in the cell body and dendrites, but not in the axons of Purkinje cells. These results demonstrate the pres- ence of a mechanism controlling the distribution of RGS8 protein in cerebellar Purkinje cells. © 2001 Academic Press Key Words: G protein; RGS; desensitization; recep- tor; Purkinje cells; nuclear localization. RGS (regulators of G protein signaling) proteins comprise a large family of more than 20 members, which modulate heterotrimeric G protein signaling (1, 2). This protein family was originally identified as a pheromone desensitization factor in yeast (3). Subse- quent studies have identified many RGS proteins by virtue of a common stretch of 120 amino acids termed the RGS domain in organisms ranging from yeast to humans (1, 2, 4, 5). It has been reported that several RGS proteins (RGS1, RGS3, RGS4, GAIP) attenuate the G protein signaling in cultures (4, 6, 7). Biochem- ical studies have demonstrated that RGS members function as GTPase-activating proteins (GAP) for the –subunits of heterotrimeric G proteins (8 –10). Hence, RGS proteins are thought to down-regulate G protein signaling in vivo by enhancing the rate of G GTP hydrolysis. However, our group and another have dem- onstrated that the RGS proteins significantly acceler- ated the turning on and off of the G protein-coupled inwardly rectifying K + channels (11–13). Since G subunits are usually associated with sig- naling events at the plasma membrane, the study of the subcellular distribution and its regulation of the RGS proteins will help to understand the nature of the G-protein signaling pathways that they regulate. Re- cently, several observations concerning the cellular distribution of RGS proteins have been reported. Druey et al. have reported that the majority of RGS4 is found as a soluble protein in the cytoplasm of mamma- lian cultured cells, and that the expression of a GTPase-deficient Gi resulted in the translocation of RGS4 to the plasma membrane (14). In yeast, the short N-terminal domain conserved in RGS4 and RGS16 was reported to be required for membrane localization and the ability to inhibit pheromone response (15, 16). In contrast, RGS3 has a long unique N-terminus, and it was reported to be predominant in the cytoplasm and to be translocated to the plasma membrane upon ago- nist stimulation. The N-terminal domain of RGS3 was also reported to be important for this translocation (17). A truncated isoform of RGS3, termed RGS3T, lacking a large portion of the N-terminus of RGS3 but retaining a core RGS domain and a smaller N-terminal tail has been identified by PCR analysis (18). Recently, it was found that RGS3T is localized in the nucleus and induces apoptosis (19). Thus, multiple RGS proteins within a given cell might be differentially localized and their intracellular localization might be regulated to determine a physiological response to a G protein- linked stimulus. Using a culture system of P19 cells, we have previ- ously isolated the cDNA of RGS8 and identified it as an RGS protein induced in neuronally differentiated P19 Abbreviations used: G proteins, heterotrimeric guanine nucleotide- binding proteins; RGS, regulators of G protein signaling. 1 To whom correspondence should be addressed. Fax: (+81) 423- 21-8678. E-mail: osaito@tmin.ac.jp. Biochemical and Biophysical Research Communications 287, 223–228 (2001) doi:10.1006/bbrc.2001.5489, available online at http://www.idealibrary.com on 223 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.