32] Identification of phosphorylation sites within vertebrate and invertebrate rhodopsin

Abstract

Publisher Summary Studies of vertebrate and invertebrate rhodopsin have been applicable to our understanding of amplification and desensitization processes of Gprotein-coupled receptors in general. In vertebrate rod outer segments, photoisomerization of rhodopsin (Rho*) initiates activation of hundreds of G-protein (G t ) molecules, which in turn stimulate cGMP phosphodiesterase, resulting in closure of cGMP gated channels in response to the decrease in cytosolic cGMP concentrations. To study the enzymatic and functional roles of Rho phosphorylation, it was necessary to develop protocols for the identification of phosphorylation sites within vertebrate and invertebrate Rho. This chapter describes a method that employs mass spectrometry in conjunction with specific proteolysis, and should be applicable to the study of phosphorylation in other systems. Rho phosphorylation is carried out on rod outer segment (ROS) membranes isolated from fresh bovine retinas by discontinuous sucrose density gradient centrifugation in 10 mM Bis-Tris propane (BTP) buffer, pH 7.5, containing 5 mM MgCl2 and 1 mM ATP at 30° for 0–45 minutes under illumination from a 150-W lamp at a distance of 10 cm. Phosphorylated forms of the peptide could, in principle, be analyzed by tandem mass spectrometry (MS/MS) to identify the specific residues phosphorylated.