Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis.
G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.
Ye
Shixin
S
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10065.
Köhrer
Caroline
C
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Huber
Thomas
T
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10065.
Kazmi
Manija
M
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10065.
Sachdev
Pallavi
P
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10065.
Yan
Elsa C Y
ECY
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10065.
Bhagat
Aditi
A
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10065.
RajBhandary
Uttam L
UL
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. Electronic address: bhandary@mit.edu.
Sakmar
Thomas P
TP
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10065. Electronic address: sakmar@rockefeller.edu.
D000596:Amino Acids / Q000378:metabolism*; D049148:Aminoacylation; D001577:Benzophenones / Q000378:metabolism; D002460:Cell Line; D004926:Escherichia coli / Q000201:enzymology; D001411:Geobacillus stearothermophilus / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000378:metabolism; D019713:Receptors, CCR5 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism
C476995:3-(4-acetylphenyl)-2-aminopropanoic acid; C488060:4-benzoylphenylalanine; D000596:Amino Acids; D001577:Benzophenones; D050505:Mutant Proteins; D012365:RNA, Transfer, Tyr; D019713:Receptors, CCR5; D043562:Receptors, G-Protein-Coupled; D010649:Phenylalanine; D012243:Rhodopsin; D008156:Luciferases; D014448:Tyrosine-tRNA Ligase