title: Radiolabeling of receptor ligands by chemical incorporation of phosphorylation sites.
abstract: A chemical reagent, N-acetyl-cys((succinimidyl-6-(thioacetyl)amino) hexanoate)-ser-arg-arg-ala-ser-val-tyr-amide ("phosite NHS ester"), allowing the introduction of phosphorylation sites into proteins, peptides, or small molecules, has been synthesized and characterized. The phosite reagent enables the enzymatic radiolabeling of any protein, peptide, or small molecule containing a reactive amine using [(32)P] or [(33)P]ATP and protein kinase A. The utility of the reagent has been demonstrated in cytokine and G protein-coupled radioligand receptor binding assays using whole cell and immobilized receptor formats. Use of the reagent does not require genetic manipulation of the target ligand.
authors.affiliation: Pharmacopeia Inc., CN5350, Princeton, New Jersey, 08543, USA.
authors.forename: J
authors.initials: J
authors.lastname: Inglese
authors.forename: J F
authors.initials: JF
authors.lastname: Glickman
mesh_terms: D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D016207:Cytokines / Q000138:chemical synthesis / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods; D020738:Leptin / Q000138:chemical synthesis / Q000378:metabolism; D015288:Neurokinin A / Q000138:chemical synthesis* / Q000378:metabolism; D009842:Oligopeptides; D010748:Phosphopeptides / Q000138:chemical synthesis* / Q000378:metabolism; D010750:Phosphoproteins / Q000138:chemical synthesis* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011869:Radioligand Assay / Q000379:methods*; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D018041:Receptors, Neurokinin-2 / Q000032:analysis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D016207:Cytokines; D007202:Indicators and Reagents; D020738:Leptin; C402067:N-acetyl-cys((succinimidyl-6-(thioacetyl)amino)hexanoate)-Ser-Arg-Arg-Ala-Ser-Val-Tyr-amide; D009842:Oligopeptides; D010748:Phosphopeptides; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D018041:Receptors, Neurokinin-2; D011994:Recombinant Proteins; D015288:Neurokinin A; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins