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 (G5).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...
Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein  subunit (G5). RGS9⅐G5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or G5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9⅐G5 complex, and therefore all three proteins, RGS9, G5, and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP.Timely termination of the light response in retinal photoreceptors is essential for normal vision (reviewed in Refs. 1 and 2). On the molecular level, the normal time course of the light response requires rapid deactivation of the G protein transducin, which relays the visual signal to the effector, cyclic GMP phosphodiesterase. Deactivation of transducin occurs when the transducin ␣ subunit hydrolyzes its bound GTP. In normal rods, GTP hydrolysis is catalyzed by the complex of the regulator of G protein signaling protein (RGS9) 1 with type 5 G protein  subunit (G5) (reviewed in Refs. 2 and 3). Recent studies have demonstrated that photoreceptors lacking RGS9 or G5 produce light responses that recover at an abnormally slow rate (4, 5).In photoreceptors, the RGS9⅐G5 complex is tightly associated with the transmembrane protein R9AP (RGS9 anchor protein), which anchors RGS9⅐G5 on the surface of the disc membranes of the outer segment, which is the subcellular compartment where visual transduction occurs (6 -8). R9AP is a 25-kDa protein structurally related to members of the SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) protein family, which are involved in vesicular trafficking and exocytosis (8 -10). In mammals, R9AP is expressed predominantly in the retina (6, 9), whereas in chicken it is also present in cochlear hair cells and dorsal root ganglion neurons (9). R9AP dramatically enhances the ability of RGS9⅐G5 to stimulate transducin GTPase (7,8,10) and participates in the delivery of RGS9⅐G5 to photoreceptor outer segment (10).In this study, we analyzed visual signaling in rods of R9AP knockout mice. The knockout did not affect the overall retinal morphology or photoreceptor development. However, light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or G5 knockout rods. The effect of the R9AP knockout on the photoresponse recovery was explained by a...
Photoreceptors are compartmentalized neurons in which all proteins responsible for evoking visual signals are confined to the outer segment. Yet, the mechanisms responsible for establishing and maintaining photoreceptor compartmentalization are poorly understood. Here we investigated the targeting of two related membrane proteins, R9AP and syntaxin 3, one residing within and the other excluded from the outer segment. Surprisingly, we have found that only syntaxin 3 has targeting information encoded in its sequence and its removal redirects this protein to the outer segment. Furthermore, proteins residing in the endoplasmic reticulum and mitochondria were similarly redirected to the outer segment after removing their targeting signals. This reveals a pattern where membrane proteins lacking specific targeting information are delivered to the outer segment, which is likely to reflect the enormous appetite of this organelle for new material necessitated by its constant renewal. This also implies that every protein residing outside the outer segment must have a means to avoid this “default” trafficking flow.
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