DEP (for Disheveled, EGL-10, Pleckstrin) homology domains are present in numerous signaling proteins, including many in the nervous system, but their function remains mostly elusive. We report that the DEP domain of a photoreceptor-specific signaling protein, RGS9 (for regulator of G-protein signaling 9), plays an essential role in RGS9 delivery to the intracellular compartment of its functioning, the rod outer segment. We generated a transgenic mouse in which RGS9 was replaced by its mutant lacking the DEP domain. We then used a combination of the quantitative technique of serial tangential sectioning-Western blotting with electrophysiological recordings to demonstrate that mutant RGS9 is expressed in rods in the normal amount but is completely excluded from the outer segments. The delivery of RGS9 to rod outer segments is likely to be mediated by the DEP domain interaction with a transmembrane protein, R9AP (for RGS9 anchoring protein), known to anchor RGS9 on the surface of photoreceptor membranes and to potentiate RGS9 catalytic activity. We show that both of these functions are also abolished as the result of the DEP domain deletion. These findings indicate that a novel function of the DEP domain is to target a signaling protein to a specific compartment of a highly polarized neuron. Interestingly, sequence analysis of R9AP reveals the presence of a conserved R-SNARE (for soluble N-ethylmaleimide-sensitive factor attachment protein receptor) motif and a predicted overall structural homology with SNARE proteins involved in vesicular trafficking and fusion. This presents the possibility that DEP domains might serve to target various DEP-containing proteins to the sites of their intracellular action via interactions with the members of extended SNARE protein family.

The complex between the short splice variant of the ninth member of the RGS protein family and the long splice variant of type 5 G protein ␤ subunit (RGS9-G␤5L) plays a critical role in regulating the duration of the light response in vertebrate photoreceptors by activating the GTPase activity of the photoreceptor-specific G protein, transducin. RGS9-G␤5L is tightly associated with the membranes of photoreceptor outer segments; however, the nature of this association remains unknown. Here we demonstrate that rod outer segment membranes contain a limited number of sites for high affinity RGS9-G␤5L binding, which are highly sensitive to proteolysis. In membranes isolated from bovine rod outer segments, all of these sites are occupied by the endogenous RGS9-G␤5L, which prevents the binding of exogenous recombinant RGS9-G␤5L to these sites. However, treating membranes with urea or high pH buffers causes either removal or denaturation of the endogenous RGS9-G␤5L, allowing for high affinity binding of recombinant RGS9-G␤5L to these sites. This binding results in a striking ϳ70-fold increase in the RGS9-G␤5L ability to activate transducin GTPase. The DEP (disheveled/EGL-10/pleckstrin) domain of RGS9 plays a crucial role in the RGS9-G␤5L membrane attachment, as evident from the analysis of membrane-binding properties of deletion mutants lacking either N-or C-terminal parts of the RGS9 molecule. Our data indicate that specific association of RGS9-G␤5L with photoreceptor disc membranes serves not only as a means of targeting it to an appropriate subcellular compartment but also serves as an important determinant of its catalytic activity.Vertebrate photoreceptors produce rapid electrical responses to light and rapidly recover from excitation upon extinction of illumination (see Refs. 1-4 for recent reviews). Essential for the high speed of the photoresponse are the rates at which the G protein, transducin, is first activated by photoexcited rhodopsin and at which it is then inactivated by RGS9-G␤5L. 1 The rates of activation and inactivation were both documented to be at the high end of the range observed in the characterized G protein-based signaling pathways (Refs. 5 and 18; reviewed in Ref. 4). The rapid rates of both processes may be explained, at least in part, by the membrane association of most of the signaling proteins participating in phototransduction (reviewed in Ref. 1). Membrane association may increase the probability that these proteins encounter each other in orientations optimized for their productive interactions. The membrane association of most phototransduction proteins results from either their transmembrane position (e.g. rhodopsin) or their posttranslational modifications by fatty acids of isoprenoids (e.g. the subunits of transducin) (reviewed in Ref. 1). However, the nature of RGS9-G␤5L membrane attachment remains unknown. Neither RGS9 nor G␤5L has been shown to contain transmembrane domains or lipophilic posttranslational modifications. However, RGS9-G␤5L is bound to photoreceptor membranes so t...