Site-specific fluorescence labeling of the beta2 adrenergic receptor amino terminus.
A modified human beta2 receptor, designated 0K-beta2, was developed for site-specific labeling at the amino terminus with amine reactive fluorescent probes. 0K-beta2 has the following modifications: (1) all 16 lysines in the wild-type beta2 receptor were mutated to arginines, (2) a FLAG epitope preceded by a cleaved hemagglutinin signal sequence was fused to the amino terminus, and (3) a hexahistidine tail was added to the carboxyl terminus. The FLAG epitope and hexahistidine tail were added to facilitate purification while lysine to arginine mutations eliminate potential labeling sites for amine-reactive fluorescent probes. The remaining primary amines in the 0K-beta2 receptor, the amino terminal amine and the epsilon-amine of Lys3, both reside in the amino-terminal FLAG epitope. The 0K-beta2 receptor expressed in Sf9 insect cells exhibited ligand binding and G-protein coupling characteristics similar to the wild-type beta2 receptor. The modified receptor was labeled with fluorescamine, an amine-reactive fluorescent probe. Proteolysis with factor Xa showed that labeling was confined to the amino terminus of the 0K-beta2 receptor. Our results demonstrate site-specific fluorescamine labeling at the amino terminus of the 0K-beta2 receptor, a lysine-depleted beta2 receptor that retains functional characteristics of the wild-type receptor.
Parola
A L
AL
Division of Cardiovascular Medicine, Stanford University, Stanford, California 94305, USA.
Lin
S
S
Kobilka
B K
BK
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D004789:Enzyme Activation; D015951:Factor Xa / Q000378:metabolism; D005450:Fluorescamine / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006388:Hemagglutinins / Q000235:genetics; D006639:Histidine; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D018411:Spodoptera
D005456:Fluorescent Dyes; D006388:Hemagglutinins; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D021382:Protein Sorting Signals; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D005576:Colforsin; C033223:polyhistidine; D005450:Fluorescamine; D006639:Histidine; C085706:FLAG peptide; D015951:Factor Xa; D000262:Adenylyl Cyclases; D007545:Isoproterenol