title: Specific binding of RGS9-Gbeta 5L to protein anchor in photoreceptor membranes greatly enhances its catalytic activity.
abstract: 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 beta subunit (RGS9-Gbeta5L) 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-Gbeta5L 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-Gbeta5L 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-Gbeta5L, which prevents the binding of exogenous recombinant RGS9-Gbeta5L to these sites. However, treating membranes with urea or high pH buffers causes either removal or denaturation of the endogenous RGS9-Gbeta5L, allowing for high affinity binding of recombinant RGS9-Gbeta5L to these sites. This binding results in a striking approximately 70-fold increase in the RGS9-Gbeta5L ability to activate transducin GTPase. The DEP (disheveled/EGL-10/pleckstrin) domain of RGS9 plays a crucial role in the RGS9-Gbeta5L 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-Gbeta5L 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.
authors: Department of Ophthalmology, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, USA.
authors: Polina V
authors: PV
authors: Lishko
authors: Kirill A
authors: KA
authors: Martemyanov
authors: Johnathan A
authors: JA
authors: Hopp
authors: Vadim Y
authors: VY
authors: Arshavsky
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D020710:RGS Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*
chemical_list: D020710:RGS Proteins; D011994:Recombinant Proteins; C116925:regulator of g-protein signaling 9