title: Arrestin-rhodopsin binding stoichiometry in isolated rod outer segment membranes depends on the percentage of activated receptors.
abstract: In the rod cell of the retina, arrestin is responsible for blocking signaling of the G-protein-coupled receptor rhodopsin. The general visual signal transduction model implies that arrestin must be able to interact with a single light-activated, phosphorylated rhodopsin molecule (Rho*P), as would be generated at physiologically relevant low light levels. However, the elongated bi-lobed structure of arrestin suggests that it might be able to accommodate two rhodopsin molecules. In this study, we directly addressed the question of binding stoichiometry by quantifying arrestin binding to Rho*P in isolated rod outer segment membranes. We manipulated the "photoactivation density," i.e. the percentage of active receptors in the membrane, with the use of a light flash or by partially regenerating membranes containing phosphorylated opsin with 11-cis-retinal. Curiously, we found that the apparent arrestin-Rho*P binding stoichiometry was linearly dependent on the photoactivation density, with one-to-one binding at low photoactivation density and one-to-two binding at high photoactivation density. We also observed that, irrespective of the photoactivation density, a single arrestin molecule was able to stabilize the active metarhodopsin II conformation of only a single Rho*P. We hypothesize that, although arrestin requires at least a single Rho*P to bind the membrane, a single arrestin can actually interact with a pair of receptors. The ability of arrestin to interact with heterogeneous receptor pairs composed of two different photo-intermediate states would be well suited to the rod cell, which functions at low light intensity but is routinely exposed to several orders of magnitude more light.
authors.affiliation: Institut für Medizinische Physik und Biophysik (CC2), Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
authors.forename: Martha E
authors.initials: ME
authors.lastname: Sommer
authors.forename: Klaus Peter
authors.initials: KP
authors.lastname: Hofmann
authors.forename: Martin
authors.initials: M
authors.lastname: Heck
mesh_terms: D000818:Animals; D019393:Arrestin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D008956:Models, Chemical; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000502:physiology*; D012243:Rhodopsin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014785:Vision, Ocular / Q000502:physiology*
chemical_list: D019393:Arrestin; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D012243:Rhodopsin