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J. Biol. Chem., Vol. 283, Issue 3, 1525-1533, January 18, 2008

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Site-specific Incorporation of Keto Amino Acids into Functional G Protein-coupled Receptors Using Unnatural Amino Acid Mutagenesis^*

Shixin Ye¹², Caroline Köhrer¹, Thomas Huber, Manija Kazmi, Pallavi Sachdev³, Elsa C.Y. Yan⁴, Aditi Bhagat⁴, Uttam L. RajBhandary⁵, and Thomas P. Sakmar⁶

From the Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York 10065, and the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

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 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 µg/10⁷ 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.

Received for publication, August 31, 2007 , and in revised form, November 7, 2007.

^* This work was supported in part by the Ellison Medical Foundation and the Allene Reuss Memorial Trust (to T. P. S.) and by National Institutes of Health Grant GM67741 and U.S. Army Research Office Grant W911NF-04-1-0353 (to U. L. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and Tables S1 and S2.

¹ Both authors contributed equally to this work.

² Supported by a C.H. Li Fellowship of The Rockefeller University.

³ A Murray Foundation fellow.

⁴ Current address: Dept. of Chemistry, Yale University, New Haven, CT 06520.

⁵ To whom correspondence may be addressed: Dept. of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: 617-253-4702; Fax: 617-252-1556; E-mail: bhandary{at}mit.edu. ⁶ To whom correspondence may be addressed: Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, 1230 York Ave., New York, NY 10065. Tel.: 212-327-8288; Fax: 212-327-7904; E-mail: sakmar{at}rockefeller.edu.

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