The DEP domain determines subcellular targeting of the GTPase activating protein RGS9 in vivo.
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.
Martemyanov
Kirill A
KA
Howe Laboratory of Ophthalmology, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, USA.
Lishko
Polina V
PV
Calero
Nidia
N
Keresztes
Gabor
G
Sokolov
Maxim
M
Strissel
Katherine J
KJ
Leskov
Ilya B
IB
Hopp
Johnathan A
JA
Kolesnikov
Alexander V
AV
Chen
Ching-Kang
CK
Lem
Janis
J
Heller
Stefan
S
Burns
Marie E
ME
Arshavsky
Vadim Y
VY
D000818:Animals; D004594:Electrophysiology; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D051379:Mice; D008822:Mice, Transgenic; D010775:Photic Stimulation; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D020710:RGS Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D012160:Retina / Q000378:metabolism* / Q000528:radiation effects; D013347:Subcellular Fractions / Q000737:chemistry
D044387:GTP-Binding Protein beta Subunits; C094668:Gnb5 protein, mouse; D046911:Macromolecular Substances; D020710:RGS Proteins; C116925:regulator of g-protein signaling 9; D020558:GTP Phosphohydrolases