Subcellular Targeting of RGS9-2 Is Controlled by Multiple Molecular Determinants on Its Membrane Anchor, R7BP

Abstract

RGS9-2, a member of the R7 regulators of G protein signaling (RGS) protein family of neuronal RGS, is a critical regulator of G protein signaling. In striatal neurons, RGS9-2 is tightly associated with a novel palmitoylated protein, R7BP (R7 family binding protein). Here we report that R7BP acts to target the localization of RGS9-2 to the plasma membrane. Examination of the subcellular distribution in native striatal neurons revealed that both R7BP and RGS9-2 are almost entirely associated with the neuronal membranes. In addition to the plasma membrane, a large portion of RGS9-2 was found in the neuronal specializations, the postsynaptic densities, where it forms complexes with R7BP and its constitutive partner G␤5. Using site-directed mutagenesis we found that the molecular determinants that specify the subcellular targeting of RGS9-2⅐G␤5⅐R7BP complex are contained within the 21 C-terminal amino acids of R7BP. This function of the C terminus was found to require the synergistic contributions of its two distinct elements, a polybasic motif and palmitoylated cysteines, which when combined are sufficient for directing the intracellular localization of the constituent protein. In differentiated neurons, the C-terminal targeting motif of R7BP was found to be essential for mediating its postsynaptic localization. In addition to the plasma membrane targeting elements, we identified two functional nuclear localization sequences that can mediate the import of R7BP into the nucleus upon depalmitoylation. These findings provide a mechanism for the subcellular targeting of RGS9-2 in neurons.G protein signaling pathways mediate a wide range of critical neuronal processes (1). The normal functioning of these pathways is dependent on the tight control of signal duration mediated by regulators of G protein signaling (RGS) 2 proteins (2, 3). RGS proteins act to control the inactivation of heterotrimeric G proteins by dramatically accelerating the rate of their GTP hydrolysis (4, 5). Much recent attention has been focused on the R7 subfamily of RGS proteins due to their emerging roles in the regulation of a variety of critical neuronal functions (6, 7). The R7 subfamily contains four highly homologous proteins, RGS6, RGS7, RGS9, and RGS11, which are expressed exclusively in the nervous system (8) as constitutive complexes with a type 5 G protein ␤ subunit (G␤5) (9 -11).RGS9, with its two splice isoforms, is one of the best-studied members of the R7 subfamily. The short isoform RGS9-1 is expressed in the photoreceptor neurons where it regulates the visual signal transduction cascade (12, 13). The long splice isoform RGS9-2 is predominantly expressed in the striatum and the central nervous system structures, mediating nociceptive responses where it was shown to regulate dopamine and -opioid pathways (14 -17). Disruption of RGS9-2 in mice resulted in the stimulation of locomotor and reward behavior, increased physical dependence and analgesic effects of morphine, and movement disorders (15)(16)(17). In photoreceptors, the function ...

GIRK channel modulation by assembly with allosterically regulated RGS proteins

“…We focus on elucidating the function of R7 RGS binding protein (R7BP), a palmitoylated SNARE-like protein that has been suggested to function as a membrane anchor, scaffold, or allosteric regulator for R7-RGS/Gβ5 complexes (24)(25)(26)(27)(28). Our studies support a model in which GIRK modulation occurs by channel assembly with R7-RGS/Gβ5 complexes that are allosterically regulated by R7BP.…”

Gi/o Signaling and the Palmitoyltransferase DHHC2 Regulate Palmitate Cycling and Shuttling of RGS7 Family-binding Protein

“…Thus, R9AP is essential for normal photoresponse kinetics in both cell types (10,20,21). In contrast, R7BP is expressed widely in the nervous system, binds each R7 isoform, lacks a transmembrane domain, and associates with membranes by covalent attachment of the fatty acid palmitate to two C-terminal cysteine residues (22)(23)(24). In striatum, R7BP stabilizes RGS9 expression and function as a regulator of motor coordination and locomotor response to morphine (25).…”

“…On the one hand, R7BP is palmitoylated efficiently because at steady state it associates nearly exclusively with membrane fractions of brain extracts (24,26). On the other, steady state nuclear accumulation of RGS7 and G␤5 in neurons appears to depend partially on R7BP (27).…”

“…In striatum, R7BP stabilizes RGS9 expression and function as a regulator of motor coordination and locomotor response to morphine (25). Intriguingly, blockade of R7BP palmitoylation in cultured cells enables the protein to use a conserved C-terminal polybasic sequence resembling a classical nuclear localization sequence to import R7-G␤5 complexes into the nucleus (23,24). Because palmitate attachment to proteins occurs via reversible thioester bonds, R7BP potentially functions as a palmitoylation-regulated protein that shuttles R7-G␤5 heterodimers between the plasma membrane and nucleus.…”

β-Arrestin2 Plays Permissive Roles in the Inhibitory Activities of RGS9-2 on G Protein-Coupled Receptors by Maintaining RGS9-2 in the Open Conformation

“…R7BP is a neuronal protein that anchors RGS proteins of the R7 family as a complex with G␤5 (12). Previously it was reported that R7BP interacts with RGS9-2 in the DEP domain and renders its localization on the plasma membrane (42). As shown in Fig.…”