The DEP domain determines subcellular targeting of the GTPase activating protein RGS9 in vivo

Abstract

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.

Figure 1.

Characterization of the retinas from DEP-less mice. A, Domain composition of the RGS9-Gβ5 complex. GGL, G-protein γ-subunit-like domain; RGS, RGS homology domain. The arrow marks the point of the DEP domain deletion in RGS9ΔDEP. B. Cross-sections (1 μm) of the retinas from 6-month-old wild-type and DEP-less mice stained by toluidine blue. OS, Outer segment layer; IS, inner segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cell layer. C, Western blot analysis of RGS9, Gβ5, and R9AP in the retinas of wild-type, DEP-less, and RGS9 knock-out (KO) mice. Each lane contained 15 pmol of rhodopsin. The lower band of the RGS9 staining of wild-type animals' retinas represents a C-terminal proteolytic fragment of RGS9 present in most RGS9 preparations (He et al., 2000; Lishko et al., 2002).

Figure 2.

Immunohistochemical analysis of RGS9 (top panel) and R9AP (bottom panel) distribution in rods of wild-type, DEP-less, and RGS9 knock-out (KO) mice. For details, see Materials and Methods. For abbreviations of the retina layers, see Figure 1 legend.

Figure 3.

Quantitative analysis of RGS9 and R9AP distribution in rods of wild-type and DEP-less mice by cryosectioning with Western blotting. A, Western blots of RGS9, R9AP, Gβ5, and two marker proteins, rhodopsin (Rho) and CoxIV. B, Densitometric profiles of the Western blots from A in which the densities of individual bands for RGS9, R9AP, rhodopsin, and CoxIV are expressed as a percentage of the total density of all bands representing each individual protein on the blot. C, A drawing illustrating the distribution of RGS9 and R9AP at their respective locations in rods of wild-type and DEP-less mice. The data are taken from one of three similar experiments.

Figure 4.

Flash responses of DEP-less rods resemble those of RGS9 knock-out rods. A, Representative mean single photon responses from wild-type, RGS9 knock-out (KO), and DEP-less rods. Amplitudes have been normalized to the mean DEP-less single photon response amplitude (0.49 pA) to aid comparison of recovery kinetics. B, Families of responses to increasing flash strengths from RGS9 knock-out (red) and DEP-less (blue) rods. Each trace is the average of 2 (bright) to 30 (dim) flashes. Dark currents were 9.2 pA (RGS9 knock-out) and 13.9 pA (DEP-less). Flash strengths ranged from 5.3 to 718 photons/μm² by factors of 4 (DEP-less) and 9.6 and from 18.1 to 733 photons/μm² by factors of 4 (RGS9 knock-out).

Figure 5.

The stimulation of RGS9 ability to activate transducin GTPase is abolished by deletion of the DEP domain. Multiple turnover transducin GTPase assays were performed as described in Materials and Methods. The basal GTPase activity of transducin measured without RGS9 or RGS9ΔDEP was subtracted from the values measured with RGS9, and the resulting values were plotted on the graph. Triangles represent the measurements conducted with full-length RGS9; circles represent the measurements conducted with RGS9ΔDEP; filled symbols represent the measurements conducted in the absence of R9AP; and open symbols represent the measurements conducted in the presence of R9AP. The data for the three bottom plots were fitted by straight lines, whereas the data obtained with RGS9 in the presence of R9AP were fitted by two straight lines (for the values of the rates, see Results). Data were averaged from three independent experiments; error bars represent SEM.

Figure 6.

R9AP shares structural similarities with SNARE protein family members. A, Schematic representation of R9AP domain composition in comparison with three canonical SNARE proteins. Hashed boxes represent transmembrane regions; dark boxes represent conservative domains participating in SNARE complex formation; light boxes represent domains with predicted coiled-coil folds; and wavy lines represent palmitoyl groups responsible for membrane attachment of SNAP-25. B, Sequence alignment of the coiled-coil regions involved in the formation of the SNARE complexes with the corresponding region of mouse R9AP. The position of the conserved ionic layer is marked with an asterisk. Gray boxes mark the conserved positions of the heptad repeats. Sx1a, Rat syntaxin 1a (GenBank accession number P32851); SNAP-25B, rat SNAP-25B (GenBank accession number P13795); Sx3, rat syntaxin 3 (GenBank accession number Q08849); sb2, rat synaptobrevin 2 (GenBank accession number M24105); sb7, mouse synaptobrevin 7 (GenBank accession number X96737); Sec22b, mouse vesicle trafficking protein Sec22b (GenBank accession number U91538).