TY - JOUR
T1 - Specific binding of RGS9-Gβ5L to protein anchor in photoreceptor membranes greatly enhances its catalytic activity
AU - Lishko, Polina V.
AU - Martemyanov, Kirill A.
AU - Hopp, Johnathan A.
AU - Arshavsky, Vadim Y.
PY - 2002/7/5
Y1 - 2002/7/5
N2 - 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 β subunit (RGS9-Gβ5L) 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-Gβ5L 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-Gβ5L 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-Gβ5L, which prevents the binding of exogenous recombinant RGS9-Gβ5L to these sites. However, treating membranes with urea or high pH buffers causes either removal or denaturation of the endogenous RGS9-Gβ5L, allowing for high affinity binding of recombinant RGS9-Gβ5L to these sites. This binding results in a striking -70-fold increase in the RGS9-Gβ5L ability to activate transducin GTPase. The DEP (disheveled/EGL-10/pleckstrin) domain of RGS9 plays a crucial role in the RGS9-Gβ5L 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-Gβ5L 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.
AB - 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 β subunit (RGS9-Gβ5L) 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-Gβ5L 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-Gβ5L 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-Gβ5L, which prevents the binding of exogenous recombinant RGS9-Gβ5L to these sites. However, treating membranes with urea or high pH buffers causes either removal or denaturation of the endogenous RGS9-Gβ5L, allowing for high affinity binding of recombinant RGS9-Gβ5L to these sites. This binding results in a striking -70-fold increase in the RGS9-Gβ5L ability to activate transducin GTPase. The DEP (disheveled/EGL-10/pleckstrin) domain of RGS9 plays a crucial role in the RGS9-Gβ5L 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-Gβ5L 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.
UR - http://www.scopus.com/inward/record.url?scp=0037025306&partnerID=8YFLogxK
U2 - 10.1074/jbc.M203237200
DO - 10.1074/jbc.M203237200
M3 - Article
C2 - 12006596
AN - SCOPUS:0037025306
SN - 0021-9258
VL - 277
SP - 24376
EP - 24381
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 27
ER -