Phosphorylation of ovine rhodopsin. Identification of the phosphorylated sites.
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ABSTRACT:
SUBMITTER: Thompson P
PROVIDER: S-EPMC1153695 | biostudies-other | 1984 Jun
REPOSITORIES: biostudies-other
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Project description:Studies on the light-stimulated phosphorylation of rod outer segments by [gamma-32P]ATP showed that although nearly 1 mol of [32P]phosphate was incorporated/mol of total opsin, only a small fraction of the molecules were phosphorylated, and these contained at least 2-3 mol of phosphate/mol. Rod outer segments containing the phosphorylated opsin were incubated with 11-cis-retinal to generate phosphorylated rhodopsin and then digested with papain to produce a cleaved complex comprising three fragments, heavy (H), medium (M) and light (L). It was shown that L-fragment of apparent mol.wt. 6000 contained all the phosphorylation sites. This suggests that one specific domain of rhodopsin is susceptible to multiple phosphorylation.
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Project description:Arrestins constitute a family of small cytoplasmic proteins that mediate deactivation of G-protein-coupled receptors (GPCRs) and are known to be essential for cascade inactivation and receptor desensitization. Alternative splicing produces an array of arrestin gene products that have widely different specificities for their cognate receptors in vitro, but the differential functions of these splice variants in vivo are essentially unknown. Bovine rod photoreceptors express two splice variants of visual arrestin (p44 and p48) that display different affinities for the GPCR rhodopsin. To determine the functions of these splice variants in intact cells, we expressed a transgene encoding either a truncated form of murine arrestin (mArr(1-369), or m44) or the long (p48) isoform in mouse rods lacking endogenous arrestin (Arr-/-). Morphological analysis showed that expression of either variant attenuated the light-induced degeneration that is thought to result from excessive cascade activity in Arr-/-rods. Suction electrode recordings from individual rods indicated that the expression of either m44 or p48 splice variants could restore normal kinetics to Arr-/- dim flash responses, indicating that both isoforms can bind to and quench phosphorylated rhodopsin rapidly. To our surprise, only the full-length variant was able to alter the kinetics of responses in rods lacking both arrestin and rhodopsin kinase, indicating that p48 can also quench the activity of nonphosphorylated rhodopsin.
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Project description:Phosphorylation is an important post-translational modification that rapidly mediates many cellular events. A key to understanding the dynamics of the phosphoproteome is localization of the modification site(s), primarily determined using LC-MS/MS. A major technical challenge to analysis is the formation of phosphopeptide-metal ion complexes during LC which hampers phosphopeptide detection. We have devised a strategy that enhances analysis of phosphopeptides, especially multiply phosphorylated peptides. It involves treatment of the LC system with EDTA and 2D-RP/RP-nanoUPLC-MS/MS (high pH/low pH) analysis. A standard triphosphorylated peptide that could not be detected with 1D-RP-nanoUPLC-MS/MS, even if the column was treated with EDTA-Na2 or if 25 mM EDTA-Na2 was added to the sample, was detectable at less than 100 fmol using EDTA-2D-RP/RP-nanoUPLC-MS/MS. Digests of ?-casein and ß-casein were analyzed by EDTA-1D-RP-nanoUPLC, 2D-RP/RP-nanoUPLC, and EDTA-2D-RP/RP-nanoUPLC to compare their performance in phosphopeptide analysis. With the first two approaches, no tri- and tetraphosphopeptides were identified in either ?- or ß-casein sample. With the EDTA-2D-RP/RP approach, 13 mono-, 6 di-, and 3 triphosphopeptides were identified in the ?-casein sample, while 19 mono-, 8 di-, 4 tri-, and 3 tetraphosphopeptides were identified in the ß-casein sample. Using EDTA-2D-RP/RP-nanoUPLC-MS/MS to examine 500 ?g of a human foreskin fibroblast cell lysate a total of 1,944 unique phosphopeptides from 1,087 unique phosphoproteins were identified, and 2,164 unique phosphorylation sites were confidently localized (Ascore ?20). Of these sites 79% were mono-, 20% di-, and ?1% were tri- and tetraphosphopeptides, and 78 novel phosphorylation sites in human proteins were identified.
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Project description:Phosphorylation of keratinocyte transglutaminase occurs in its N-terminal extension and is stimulated several-fold by the protein kinase C agonist phorbol myristate acetate. In the present work, this stimulation was prevented by simultaneous treatment of the cells with the protein kinase C-selective inhibitor GF109203X. In contrast, phosphorylation occurring in the absence of phorbol ester was essentially unaffected by GF109203X, although it was decreased dramatically by the non-selective kinase inhibitor staurosporine. Four serine residues that are subject to phosphorylation have been identified by sequencing of radioactive tryptic peptides. Serines at positions 24 and 92, each containing 2-8% of the total radioactivity with or without phorbol ester stimulation, were minor sites of phosphorylation. Serine-82 was by far the dominant site of phosphorylation in the absence of phorbol ester treatment, and was also the major site in its presence. Serine-85 was phosphorylated to a high degree in the presence but not in the absence of phorbol ester stimulation. Thus the data indicate the influence of at least two different kinase activities in transglutaminase phosphorylation.
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