Abstract:Phosphorylation of proteins at multiple sites creates different phosphorylation patterns that are essential for their biological activity. For example, such patterns contribute to the redirection of signalling to alternative pathways. Multi-phosphorylated peptides are excellent tools to systematically study the impact of unique phosphorylation patterns on signalling, but their synthesis is extremely difficult. Here we present an efficient and general method for the synthesis of multi-phosphorylated peptides, u… Show more
“…Based on these findings, we designed a series of multiply phosphorylated peptides aimed at delineating the role of each site and the importance of the phosphorylation pattern in controlling arrestin affinity and activation (Table 1). The phosphopeptides were produced by a synthesis strategy that allowed us to synthesize peptides with several phosphorylation sites in very close proximity 32 .Fig. 1High-throughput screening of potential phosphorylation sites.…”
Section: Resultsmentioning
confidence: 99%
“…Peptides were synthesized using combinatorial Fmoc Solid Phase Synthesis strategy. The coupling methods applied were specific for each amino acid and were determined based on the phosphorylation pattern as described by Samarasimhareddy et al 32 .…”
Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: ‘key sites’ required for arrestin binding and activation, an ‘inhibitory site’ that abrogates arrestin binding, and ‘modulator sites’ that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.
“…Based on these findings, we designed a series of multiply phosphorylated peptides aimed at delineating the role of each site and the importance of the phosphorylation pattern in controlling arrestin affinity and activation (Table 1). The phosphopeptides were produced by a synthesis strategy that allowed us to synthesize peptides with several phosphorylation sites in very close proximity 32 .Fig. 1High-throughput screening of potential phosphorylation sites.…”
Section: Resultsmentioning
confidence: 99%
“…Peptides were synthesized using combinatorial Fmoc Solid Phase Synthesis strategy. The coupling methods applied were specific for each amino acid and were determined based on the phosphorylation pattern as described by Samarasimhareddy et al 32 .…”
Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: ‘key sites’ required for arrestin binding and activation, an ‘inhibitory site’ that abrogates arrestin binding, and ‘modulator sites’ that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.
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