title: The interaction network of rhodopsin involving the heterotrimeric G-protein transducin and the monomeric GTPase Rac1 is determined by distinct binding processes.
abstract: The monomeric G-protein Rac1, a member of the family of Rho/Rac/Cdc42 GTPases, is involved in light-induced photoreceptor degeneration, but its specific role remains elusive. In particular, reports on Rac1 interacting with the visual pigment rhodopsin are puzzling and need a more quantitative examination. We probed the presence of Rac1 in rod outer segments by immunohistochemical staining of bovine retinae and western blot analysis of isolated rod outer segments. Rac1 was present throughout the whole retina except in the outer and inner nuclear layers, but was strongly expressed in photoreceptor cells. Rac1 was distributed in three different fractions of rod outer segments: one fraction was soluble in detergents, a second fraction cosegregated with lipid rafts, and a third fraction was associated with lipid bilayer free axonemal/cytoskeletal structures. We also investigated the interaction between rhodopsin and Rac1 by using surface plasmon resonance spectroscopy under dark and light conditions. Biophysical interaction studies revealed that Rac1 could interact with rhodopsin, but in a light-independent manner, and kinetic analysis indicated that binding of Rac1 occurred with lower affinity and speed than the association of transducin and rhodopsin. Thus, in dark-adapted rod cells, Rac1 cannot compete with transducin for binding to rhodopsin, and signalling can proceed normally. Instead, the concentration of transducin has to drop significantly so that Rac1 can bind to rhodopsin; in the outer segment, this occurs only under intense illumination, when transducin is translocated to the inner segment.
authors.affiliation: Department of Neurosciences, Biochemistry Group, University of Oldenburg, Germany.
authors.forename: Maike
authors.initials: M
authors.lastname: Köster
authors.forename: Daniele
authors.initials: D
authors.lastname: Dell'Orco
authors.forename: Karl-Wilhelm
authors.initials: KW
authors.lastname: Koch
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D007150:Immunohistochemistry; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D012995:Solubility; D015293:Transducin / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012243:Rhodopsin; D015293:Transducin; D020830:rac1 GTP-Binding Protein
keywords: GTPases; Rac1; photoreceptor; retina; rhodopsin