Phosphorylation Analysis of G Protein-Coupled Receptor by Mass Spectrometry: Identification of a Phosphorylation Site in V2 Vasopressin Receptor

Wu, Shilan; Birnbaumer, Mariel; Guan, Ziqiang

doi:10.1021/ac8008548

Cited by 22 publications

(12 citation statements)

References 24 publications

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“…Both parent proteins are G protein-coupled receptors (GPCR) in which the phosphorylation patterns in the selected regions play a key role in activity and binding. [20,34] To achieve optimal temperature control during the synthesis of the model HPPs, the synthesis was performed on the Figure 1. Methods for the synthesis of multi phosphorylated peptide (MPPs).…”

Section: Resultsmentioning

confidence: 99%

Automated Synthesis of Heavily Phosphorylated Peptides

2021

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Multi phosphorylated peptides are key tools in understanding the biological roles of protein phosphorylation patterns. In this work, we focused on multi phosphorylated peptides with over four, clustered, phosphorylation sites that are termed herein heavily phosphorylated peptides (HPPs). The synthesis of heavily phosphorylated peptides is extremely difficult and requires the use of a wide temperature range. Standard peptide synthesizers are incapable of both cooling and heating, which impedes the automated synthesis of those peptides. Herein, we used the oligosaccharide synthesizer Glyconeer 2.1 to develop a protocol for the automated synthesis of heavily phosphorylated peptides. The Glyconeer 2.1 is able to both cool and heat, which enabled the development of highly controlled coupling and deprotection conditions that were used for the automated synthesis of four different heavily phosphorylated peptides with five or more, clustered, phosphorylation sites. Our approach paves the way for an easy automated synthesis of a variety of heavily phosphorylated peptides.

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Section: Resultsmentioning

confidence: 99%

Automated Synthesis of Heavily Phosphorylated Peptides

2021

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“…Sixth , phosphorylation plays a pivotal role in the regulation of GPCR function. Previous studies identified putative sites which are clustered in the C terminus and in ICL3 and are phosphorylated by specific GPCR kinases (GRK) and by protein kinase A (PKA), respectively [41] – [43] . Whereas abundant putative Ser/Thr phosphorylation sites are found in the C terminus of all V2R orthologs the putative PKA phosphorylation site within ICL3 at Ser 255 [41] is substituted by Asn, Gly and Asp in many V2R orthologs implicating that this position is, if at all, a substrate for PKA in only a few species.…”

Section: Resultsmentioning

confidence: 99%

Involvement of the V2 Vasopressin Receptor in Adaptation to Limited Water Supply

et al. 2009

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Mammals adapted to a great variety of habitats with different accessibility to water. In addition to changes in kidney morphology, e.g. the length of the loops of Henle, several hormone systems are involved in adaptation to limited water supply, among them the renal-neurohypophysial vasopressin/vasopressin receptor system. Comparison of over 80 mammalian V2 vasopressin receptor (V2R) orthologs revealed high structural and functional conservation of this key component involved in renal water reabsorption. Although many mammalian species have unlimited access to water there is no evidence for complete loss of V2R function indicating an essential role of V2R activity for survival even of those species. In contrast, several marsupial V2R orthologs show a significant increase in basal receptor activity. An increased vasopressin-independent V2R activity can be interpreted as a shift in the set point of the renal-neurohypophysial hormone circuit to realize sufficient water reabsorption already at low hormone levels. As found in other desert mammals arid-adapted marsupials show high urine osmolalities. The gain of basal V2R function in several marsupials may contribute to the increased urine concentration abilities and, therefore, provide an advantage to maintain water and electrolyte homeostasis under limited water supply conditions.

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“…Whereas site-directed mutagenesis was considered the primary method for identifying the potential phosphorylation residues in early studies, multiple novel techniques have been ND (Wu et al, 2008) CHO, Chinese hamster ovary cells; CK2, casein kinase II; COS7, derived from CV-1 in origin with SV40 genes monkey kidney cells; CPM, chymotryptic phospho-peptide mapping; HEK293, human embryonic kidney 293 cells;…”

Section: Phosphorylation Of the Gpcr At Multiple Sitesmentioning

confidence: 99%

Phosphorylation of G Protein-Coupled Receptors: From the Barcode Hypothesis to the Flute Model

Yang

Zhang

et al. 2017

Molecular Pharmacology

125

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Seven transmembrane G protein-coupled receptors (GPCRs) are often phosphorylated at the C terminus and on intracellular loops in response to various extracellular stimuli. Phosphorylation of GPCRs by GPCR kinases and certain other kinases can promote the recruitment of arrestin molecules. The arrestins critically regulate GPCR functions not only by mediating receptor desensitization and internalization, but also by redirecting signaling to G protein-independent pathways via interactions with numerous downstream effector molecules. Accumulating evidence over the past decade has given rise to the phospho-barcode hypothesis, which states that ligand-specific phosphorylation patterns of a receptor direct its distinct functional outcomes. Our recent work using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (F-NMR) spectroscopy led to the flute model, which provides preliminary insight into the receptor phospho-coding mechanism, by which receptor phosphorylation patterns are recognized by an array of phosphate-binding pockets on arrestin and are translated into distinct conformations. These selective conformations are recognized by various effector molecules downstream of arrestin. The phospho-barcoding mechanism enables arrestin to recognize a wide range of phosphorylation patterns of GPCRs, contributing to their diverse functions.

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Phosphorylation Analysis of G Protein-Coupled Receptor by Mass Spectrometry: Identification of a Phosphorylation Site in V2 Vasopressin Receptor

Cited by 22 publications

References 24 publications

Automated Synthesis of Heavily Phosphorylated Peptides

Automated Synthesis of Heavily Phosphorylated Peptides

Involvement of the V2 Vasopressin Receptor in Adaptation to Limited Water Supply

Phosphorylation of G Protein-Coupled Receptors: From the Barcode Hypothesis to the Flute Model

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