R7BP Augments the Function of RGS7·Gβ5 Complexes by a Plasma Membrane-targeting Mechanism

Abstract

The RGS7 (R7) family of G protein regulators, G␤5, and R7BP form heterotrimeric complexes that potently regulate the kinetics of G protein-coupled receptor signaling. Reversible palmitoylation of R7BP regulates plasma membrane/nuclear shuttling of R7⅐G␤5⅐R7BP heterotrimers. Here we have investigated mechanisms whereby R7BP controls the function of the R7 family. We show that unpalmitoylated R7BP undergoes nuclear/cytoplasmic shuttling and that a C-terminal polybasic motif proximal to the palmitoylation acceptor sites of R7BP mediates nuclear localization, palmitoylation, and plasma membrane targeting. These results suggest a novel mechanism whereby palmitoyltransferases and nuclear import receptors both utilize the C-terminal domain of R7BP to determine the trafficking fate of R7⅐G␤5⅐R7BP heterotrimers. Analogous mechanisms may regulate other signaling proteins whose distribution between the plasma membrane and nucleus is controlled by palmitoylation. Lastly, we show that cytoplasmic RGS7⅐G␤5⅐R7BP heterotrimers and RGS7⅐G␤5 heterodimers are equivalently inefficient regulators of G proteincoupled receptor signaling relative to plasma membrane-bound heterotrimers bearing palmitoylated R7BP. Therefore, R7BP augments the function of the complex by a palmitoylation-regulated plasma membrane-targeting mechanism. G protein-coupled receptor (GPCR)3 signal transduction regulates a wide variety of physiological processes. For example in the nervous system, dopamine, serotonin, acetylcholine, and norepinephrine activate presynaptic GPCRs that regulate glutamate or ␥-aminobutyric acid release. GPCRs transduce signals via G protein-dependent and -independent mechanisms (reviewed in Refs. 1-3). GPCR signaling via heterotrimeric G proteins regulates second messenger production and ion channel activity (1-3), whereas G protein-independent signaling can activate mitogen-activated protein kinase pathways (4), nonreceptor tyrosine kinases (5, 6), and phosphatidylinositol 3-phosphate kinase (7,8). GPCR signaling from the plasma membrane to the nucleus may also occur by nuclear shuttling of ␤-arrestin 1 (9 -11).GPCR signaling is regulated potently by the RGS (regulator of G protein signaling protein) protein family (reviewed in Ref. 12). RGS proteins regulate the kinetics and amplitude of GPCR signaling by acting as GTPase-activating proteins (GAPs) for G␣ subunits (13-15). Certain RGS proteins also function as G␣ effectors or antagonists (reviewed in Ref. 12).Members of the RGS7 (R7) subfamily of RGS proteins (RGS6, RGS7, RGS9-1, RGS9-2, and RGS11) are of particular interest because of their biologic importance and unusual subunit structures. R7 family members are highly expressed in the visual and nervous systems (16 -19). They selectively deactivate G␣ subunits of the G i/o family (20 -22) that mediate phototransduction and the effects of modulatory neurotransmitters (reviewed in Ref. 23). Best understood is RGS9, which regulates GPCR signaling in the basal ganglia (24 -27) and phototransduction in retina (28). In contrast to oth...

The evolution of regulators of G protein signalling proteins as drug targets – 20 years in the making: IUPHAR Review 21

“…R7BP (for R7 binding protein) and R9AP (RGS9 associated protein, specifically in photoreceptor cells) are membrane tethered proteins that mediate targeting of R7 family RGS proteins to the plasma membrane, thereby enhancing proximity to the G protein target (Drenan et al, 2005;Grabowska et al, 2008). The functionality of all R7 family members is enhanced in the presence of R7BP (Drenan et al, 2006;Jayaraman et al, 2009). Gβ 5 interaction with the GGL domain of R7 protein regulates protein stability and is described below.…”

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

“…CellsInitial evidence suggesting that R7BP functions as a palmitoylation-regulated protein that shuttles GAP complexes (R7-G␤5 heterodimers) between the plasma membrane and nucleus was based on overexpression studies and steady state measurements of palmitoylation and localization (23,36). Although subsequent studies of brain extracts or slices failed to detect a significant pool of nuclear R7BP at steady state (19,26,27), these investigations did not test the shuttling hypothesis because they did not examine whether R7BP palmitoylation or localization is static or dynamic.…”

Bioinformatic profiling of the transcriptional response of adult rat cardiomyocytes to distinct fatty acids*

“…Expression of this gene is 553-fold higher in stearate-challenged cells, and 372-fold higher in palmitate-challenged cells, versus oleate-challenged cells; this is due to induction by both palmitate and stearate, as well as a slight repression by oleate (relative to control cells; see supplementary Table II). RGS7BP has been shown to modulate G protein signaling in the brain, although this function has not been investigated previously in the heart (43). Interestingly, RGS7BP requires reversible palmitoylation to anchor the protein at the cell surface, where it modulates G protein signaling; removal of palmitate from culture media causes relocalization of RGS7BP to the nucleus (43).…”

“…RGS7BP has been shown to modulate G protein signaling in the brain, although this function has not been investigated previously in the heart (43). Interestingly, RGS7BP requires reversible palmitoylation to anchor the protein at the cell surface, where it modulates G protein signaling; removal of palmitate from culture media causes relocalization of RGS7BP to the nucleus (43). Thus, challenging cardiomyocytes with palmitate would cause not only a dramatic induction of RGS7BP, but also anchoring of the protein to the cell surface.…”