Mass spectrometric identification of phosphorylation sites in bleached bovine rhodopsin

Abstract

Deactivation of the visual cascade is initiated by the phosphorylation of rhodopsin. We report here identification of the two major sites of phosphorylation in bleached bovine rhodopsin using tandem mass spectrometry in conjunction with synthetic phosphopeptide standards. Both bleached and unbleached rod outer segments were cleaved with endoproteinase Asp-N to release the C-terminal fragment, residues 330-348, containing seven potential sites of phosphorylation. High-performance liquid chromatographic separation of soluble cleavage products from both unbleached and bleached rod outer segments gave a peak which was identified by tandem mass spectrometry and comparison to synthetic standards as monophosphorylated (serine 338) DDEASTTVSKTETSQVAPA. Present only in the chromatogram of bleached ROS were two peaks identified as monophosphorylated (serine 343) and diphosphorylated (serines 338 and 343) derivatives of DDEASTTVSKTETSQVAPA. These results identify serines 338 and 343 as the major sites of phosphorylation within the C-terminal region of bleached bovine rhodopsin and constitute the first example of mass spectrometric characterization of phosphorylation sites in a G-protein coupled receptor.

Advances in Determination of a High-Resolution Three-Dimensional Structure of Rhodopsin, a Model of G-Protein-Coupled Receptors (GPCRs),

“…The disulfide bridge conserved among GPCRs is shown as purple filled circles (60). Two carbohydrate moieties at Asn 2 and Asp 15 and two palmitoyl groups at Cys 322 and Cys 323 (15,(64)(65)(66)(67)(68)(69)(70)(71) are shown as light yellow filled circles (32,72,73). Light red filled circles represent acidic residues, and blue filled circles represent basic residues.…”

“…The resultant 19-amino acid C-terminal peptide contained all of the phosphorylation sites. Heterogeneity and multiple phosphorylation of rhodopsin in vitro has been well documented (87)(88)(89)91,95,96). In vivo there are three sites, which were found to be phosphorylated by direct and quantitative methods after 20-40% bleaching of the protein (97,98).…”

“…In addition to the chromophore, discussed below, rhodopsin is modified by two palmitoyl groups at Cys 322 and Cys 323 in the cytoplasmic region (Figure 2) (15,(64)(65)(66)(67)(68)(69)(70)(71). Improved electron density for three of the four palmitoyl groups covalently attached to the two protein molecules in the asymmetric unit has allowed their identification during refinement (Figure 1, in green).…”

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

“…The identity of rhodopsin phosphorylation sites has been determined by analysis of a Cterminal peptide derived from isolated light-activated rod outer segments [68][69][70]. The initial sites of phosphorylation were identified at Ser$$) or Ser$%$.…”

A Segment Corresponding to Amino Acids Val170‐Arg182 of Bovine Arrestin is Capable of Binding to Phosphorylated Rhodopsin

“…In Fig , 1980). Several serine and threonine residues were identified as phosphorylated sites (Thompson and Findlay, 1984;McDowell et al, 1993;Ohguro et al, 1993;Papac et al, 1993). O n the basis of these findings, we assumed that an interaction of the peptide Va1170-Arg182 with phosphorylated residues in the carboxy-terminal domain of rhodopsin leads to formation of the oligomeric forms shown in Figs 4A, 5A and B. Phosphorylated rhodopsin specifically truncated at its carboxy-terminus by limited proteolysis should no longer tend to oligomerize in the presence of the peptide Vall70-Argl82.…”