Linked Life Data

17993461

Source:http://linkedlifedata.com/resource/pubmed/id/17993461

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2008-1-14
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/3-(4-acetylphenyl)-2-aminopropanoic..., http://linkedlifedata.com/resource/pubmed/chemical/4-benzoylphenylalanine, http://linkedlifedata.com/resource/pubmed/chemical/Amino Acids, http://linkedlifedata.com/resource/pubmed/chemical/Benzophenones, http://linkedlifedata.com/resource/pubmed/chemical/Luciferases, http://linkedlifedata.com/resource/pubmed/chemical/Mutant Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Phenylalanine, http://linkedlifedata.com/resource/pubmed/chemical/RNA, Transfer, Tyr, http://linkedlifedata.com/resource/pubmed/chemical/Receptors, CCR5, http://linkedlifedata.com/resource/pubmed/chemical/Receptors, G-Protein-Coupled, http://linkedlifedata.com/resource/pubmed/chemical/Rhodopsin, http://linkedlifedata.com/resource/pubmed/chemical/Tyrosine-tRNA Ligase
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
18
pubmed:volume
283
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1525-33
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
2008
pubmed:articleTitle
Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis.
pubmed:affiliation
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, NY 10065, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural