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

Abstract

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 ϳ50% 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 g/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. Seven-transmembrane helical G protein-coupled receptors (GPCRs)7 comprise a superfamily of cell surface proteins that participate in the most important and diverse signaling pathways in nature (1). The human genome encodes ϳ725 such receptors, and mutations in them cause a variety of diseases. GPCRs are, therefore, the target of a large fraction of drugs on the market (2, 3). Activation of GPCRs requires the binding of a ligand. Understanding the molecular mechanism of ligand recognition, GPCR activation, and subsequent receptor interaction with G proteins is an important goal in studies of GPCRs. The most informative model system to date has been the visual pigment, rhodopsin (activated by light), which is especially well suited for various spectroscopic studies that can be interpreted in the context of high resolution crystal structures (4 -6).Current dynamic studies of other GPCRs rely on fluorescence techniques. However, methods currently available for dynamic studies of fluorescently labeled GPCRs in reconstituted model systems have inherent limitations. The two most common methods to introduce site-specific fluorescent labels, maleimide chemistry targeting cysteine residues and the use of green fluorescent protein fusions, are cases in point. The former method requires either detailed exploration of labeling conditions to control the precise labeling site with wild-type receptors in the native membrane environment (7), or replacement of naturally occurring cysteines with unreactive amino acids by site-directed mutagenesis (6,8). One obvious technical problem with the mutagenesis approach is that the su...

Probing the role of the cation–π interaction in the binding sites of GPCRs using unnatural amino acids

“…An early study incorporated the fluorescent unnatural amino acid NBD-Dap into the NK2 receptor and showed that exposure to tachykinin did produce measurable electrophysiological currents in Xenopus oocytes (due to opening of Ca 2ϩ -activated Cl Ϫ channels that are endogenous to the oocyte) (29). A very recent study used an orthogonal tRNAsynthetase pair to incorporate a benzophenone-containing unnatural amino acid into the Ste2p GPCR (30) and the CCR5 receptor (31). Certainly, extensive conventional mutagenesis studies on GPCRs have provided a wealth of valuable information about which residues are important to receptor function (32).…”

Selective chemical protein modification

“…For example, hydroxylamines have been used to generate glycoprotein mimics 155 , to label G-protein-coupled receptors 156 , antibodies 157 and therapeutic proteins for increased pharmacokinetics 6 , and for dual protein tagging and formation of bifunctional antibodies 158,159 . However, despite its widespread use, the reactions of aldehydes and ketones suffer from a number of key drawbacks.…”

Mapping Substance P Binding Sites on the Neurokinin-1 Receptor Using Genetic Incorporation of a Photoreactive Amino Acid

“…Mutations were constructed by the use of the QuikChange lightning site-directed mutagenesis kit according to the manufacturer's protocol (Agilent Technologies). Amber suppressor tRNA Tyr (Bst-Yam) and tRNA synthetase from Escherichia coli were engineered to solely recognize BzF as previously described (23).…”

“…The site-specific incorporation of BzF relies on the expression of an orthogonal pair of a suppressor tRNA and an engineered aminoacyl-tRNA synthetase (23). We characterized the capacity for 34 BzF-NK1 mutants to interact with and cross-link to a fluorescent SP analog in a cell-based assay.…”

“…We used amber codon suppression to introduce the photoreactive unnatural amino acid p-benzoyl-L-phenylalanine (BzF) at 11 selected individual positions in the Nt tail (residues 11-21) and 23 The neurokinin (NK) 2 receptors belong to the rhodopsinlike class A 7-transmembrane receptors and are divided in three subtypes; NK1, NK2, and NK3. NK1 is widely expressed in both the central and the peripheral nervous systems, whereas NK2 is preferentially expressed in the peripheral system (1).…”