R7BP, a Novel Neuronal Protein Interacting with RGS Proteins of the R7 Family

Abstract

The R7 subfamily of the regulators of G protein signaling (RGS) proteins is represented by four members broadly expressed in the mammalian nervous system. Here we report that in the brain all four R7 proteins form tight complexes with a previously unidentified protein, which we call the R7-binding protein or R7BP. We initially identified R7BP as a protein co-precipitating with the R7 protein, RGS9, from extracts obtained from the striatal region of the brain. We further showed that R7BP forms a tight complex with RGS9 in vitro and that this binding occurs via the N-terminal DEP domain of RGS9. R7BP is expressed throughout the entire central nervous system but not in any of the tested nonneuronal tissues. All four R7 RGS proteins co-precipitate with R7BP from brain extracts and recombinant R7 proteins bind recombinant R7BP with high efficiency. The closest homolog of R7BP is R9AP which was previously found to interact with RGS9 in photoreceptors. Both R7BP and R9AP are related to the syntaxin subfamily of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins involved in vesicular trafficking and exocytosis. In photoreceptors R9AP regulates several critical properties of RGS9 including its intracellular targeting, stability and catalytic activity. This suggests that R7BP interactions with R7 proteins in the brain may also bear major functional significance. RGS1 proteins regulate the duration of G protein signaling by stimulating the rate of the GTP hydrolysis on G protein ␣ subunits (reviewed in Refs. 1 and 2). RGS proteins are classified in six to nine subfamilies based on the homology within the RGS catalytic domain (1-3). Most RGS proteins contain additional noncatalytic domains that modulate the properties of their catalytic domains and allow them to interact with their partners in a broad range of signaling pathways. The R7 (also called "C") subfamily of RGS proteins contains four members expressed in the mammalian nervous system, RGS6, RGS7, RGS9, and RGS11 (reviewed in Ref. 4). R7 proteins share common domain composition and exist as constitutive complexes with the type 5 G protein ␤ subunit (G␤5).Marked progress in the understanding of the function of the R7 protein came from the studies of the short splice variant of RGS9, RGS9-1, which regulates signal duration in the phototransduction pathway in vertebrate rod and cone photoreceptors. Several functional properties of RGS9-1 in photoreceptors are regulated by its interacting partner, a SNARE-like protein R9AP. R9AP is responsible for targeting RGS9-1 to the photoreceptor outer segment, an intracellular compartment where phototransduction takes place (5). It also determines the RGS9-1 expression level by protecting RGS9-1 from proteolytic degradation in the cell (6). Finally, R9AP enhances the ability of RGS9-1 to stimulate G protein GTPase activity thus allowing the visual signal to be terminated on the physiologically rapid time scale (5,7,8). RGS9-1 interaction with R9AP is mediated by the N-terminal domain of RGS9-1 c...

R7BP Modulates Opiate Analgesia and Tolerance but not Withdrawal

“…Interestingly, the R7BP phenotype is not identical to the one observed in mice lacking RGS9, as blockade of RGS9-2 actions enhances morphine analgesia but also exacerbates morphine withdrawal, whereas genetic deletion of R7BP affects analgesia but not withdrawal. As R7BP interacts with RGS6/7 and RGS9-2 in the brain (Martemyanov et al, 2005), and it is present in the LC, we speculate that actions of R7 complexes in the LC oppose the actions of R7 complexes in the striatum and for this reason, the overall withdrawal expression does not change in R7BP mutants. Our study provides solid evidence on a role of R7BP protein in morphine tolerance, using several paradigms of morphine administration in pain-free as well as in Figure 6 R7BP does not affect morphine withdrawal.…”

“…For western blot analysis studies, Gb5 (a protein shown not to be regulated by opiate administration) was used as a loading control. The following antibodies were used for IP and western blot assays: rabbit protein A-purified anti-RGS9-antibody (1 : 10 000; Psifogeorgou et al, 2007) and a rabbit anti-Gb5 (C-terminus) antibody (1 : 20 000; W Simonds, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD), a rabbit anti-RGS6/7 antibody (Wyeth-Ayerst Pharmaceuticals, Madison, NJ, USA), and a rabbit anti-R7BP antibody (Martemyanov et al, 2005). For synaptosomal preparations, striatum was dissected and homogenized in 1 ml 0.32 M sucrose 0.1 mM CaCl 2 (containing phosphatase-NaF, Na 3 VO 4 -, and protease inhibitors) using a glass homogenizing tube and a Teflon pestle.…”

“…The R7-Gb5 association is essential for the protein's stability and function (He et al, 2000;Chen et al, 2003). R7 proteins also share a DEP domain (Martemyanov et al, 2003;Ballon et al, 2006), which is required for their interaction with the adaptor molecules R9AP in the retina (Hu and Wensel, 2002) and R7 family binding protein (R7BP) in the brain (Martemyanov et al, 2005). The R7-R7BP interaction is essential for the cellular localization and function of R7 proteins, as it determines the localization of the protein at the cell membrane (Drenan et al, 2005;Anderson et al, 2009;Jayaraman et al, 2009).…”

Functional comparison of RGS9 splice isoforms in a living cell

“…The same rule should apply to both RGS9 isoforms because they share an identical R9AP-binding domain and both bind to R9AP with very high affinity (8,27). Therefore, in the rest of this study we concentrated on comparing the wild-type-like group of transgenic rods with wild-type rods.…”

“…Little is known about the functional roles of these non-catalytic domains, although they are thought to contribute to the specificity of RGS interactions (reviewed in 1, 2). RGS9 is one of the better-studied multidomain RGS proteins and exists in two splice isoforms (3)(4)(5), both of which form constitutive complexes with the type 5 G protein ␤ subunit, G␤5 (6)(7)(8). The difference between these isoforms resides in the structure of their C-termini: a short 18-aa sequence in RGS9-1 is replaced with 209 residues in RGS9-2 (Fig.…”