Labeling of GPCRs with Fluorescent Dyes

Fluorescent tagging of proteins has become a broad applicable technique in GPCR research with respect to dynamic protein studies. However, the use of

GFP and its color variants is just one of the many ways in which to achieve this goal. Depending on the questions to be answered, a variety of different labeling approaches can be taken. In this section, we will briefly introduce several of the different approaches.

One of the earliest examples was chemical labeling of GPCRs using cyste-ine-reactive fluorescent probes on purified p2-adrenoceptors. The system employed purified and reconstituted ^-adrenoceptors that were modified on all but the essential cysteines [16]. The modified receptors can then be reacted with small cysteine-reactive fluorescent probes, which can be monitored for several different properties, like side-chain mobility, fluorescence intensity, or fluorescence lifetime [17, 18]. This system has been employed with great success to investigate conformational changes induced by ligands with varying degrees of efficacy and has helped to resolve some major questions of GPCR activation [18] .

The use of cysteine-reactive fluorescent probes has not only been limited to the p2- adrenoceptor but has also been used to study GPCRs that bind peptide ligands [19-21]. The studies used fluorescently modified ligands combined with receptors that were selectively modified on the extracellular domains to study the ligand binding mode by FRET. This technique was used successfully for the cholecystokinin receptor [19, 20] and the secretin receptor [21] . The approach uses a special methanethiosulfonate reagent that permits labeling of receptors in whole living cells, rather than purified receptors. A combination of several fluorophore attachment points on the ligand and receptor allowed measuring a number of distance constraints which were then used to generate a model of the ligand-receptor complex [21]. However, since steady - state fluorescence was used as the readout, no dynamic information of ligand binding could be obtained. Dynamic binding information was reported studying an N-terminally GFP-labeled parathyroid hormone (PTH) receptor construct and fluorescently modified PTH or PTH derivatives [22] in living cells. The studies revealed a two-step binding mode of the ligand to the receptor. The initial contact was made between the ligand and the receptor N-terminus, and a second binding phase involved contacts within the transmembrane domains of the receptor.

Successful chemical labeling of peptide ligands with fluorescent probes is more readily achieved compared to small biogenic amine ligands, as with biogenic amines the functional groups that could be used for chemical labeling are often also involved in receptor binding [23] . and chemical modifications of these groups can result in reduced binding affinities. These problems have been widely recognized and currently, more suitable fluorescent ligands are being developed [24, 25] .

An alternative labeling strategy for proteins utilizing small fluorescent dyes is the tetracystein labeling technology which employs a modified fluorescein derivative called FlAsH. FlAsH stands for fluorescein arsenical hairpin binder [26] and was originally introduced in 1998 [27]. This labeling strategy uses a genetically encoded sequence of minimally six amino acids with the sequence

Figure 10.1 Depicted are structures of moieties that are suitable to label proteins with fluorescent tags. The relative size of the depicted structures of an antibody (IgG, pdb 1IGT), green fluorescent protein (GFP, pdb 1emb), fluorescein arsenical hairpin binder (FlAsH), and O6 -alkylguanine-DNA alkyltransferease (AGT, pdb 1EHG) are shown for comparison. The color code depicts alpha-helical structures in red and beta strands in silver blue.

Figure 10.1 Depicted are structures of moieties that are suitable to label proteins with fluorescent tags. The relative size of the depicted structures of an antibody (IgG, pdb 1IGT), green fluorescent protein (GFP, pdb 1emb), fluorescein arsenical hairpin binder (FlAsH), and O6 -alkylguanine-DNA alkyltransferease (AGT, pdb 1EHG) are shown for comparison. The color code depicts alpha-helical structures in red and beta strands in silver blue.

CCPGCC [26, 28] . The advantages of this technique are the small size of the tag (see Fig. 10.1 for comparison of the different sizes of labeling tags) and that the sequence can be fused or inserted into the protein of interest [29] . The corresponding protein construct can be expressed and selectively labeled with the membrane permeable FlAsH in living cells [74, 75] . Thus, this technique can be viewed as a bridge, in between genetically encoded fluorescent proteins and fluorescent labeling of cysteines. This approach allowed the replacement of yellow fluorescent protein (YFP) as a FRET partner for cyan fluorescent protein (CFP) [29] and has been applied to several GPCRs to investigate receptor activation and signaling properties in living cells and in real time [30-34] . Currently, the fluorescein-based FlAsH is the only variant that has been applied to label GPCRs; however, a red resuferin-based color variant has been developed [26] and is available as resorufin arsenical hairpin binder (ReAsH). Additionally, more photo stable variants of FlAsH have been reported but are not yet commercially available for general use [35].

If one needs to achieve greater color variability and does not want to use genetically encoded proteins based on GFP [8, 10], an alternative approach is the use of an enzyme-based system like O6-alkylguanine-DNA alkyltransfer-ase (AGT) [4] or the acyl carrier protein (ACP) [5]. These enzymes are genetically encoded and can be fused to the protein of interest. The expressed protein is then labeled by activated small organic dyes, which get transferred to the fused protein by enzymatic transfer. However, the size of the AGT.

derived SNAP-tag is about 20kDa, and is thus closer to the size of GFP and significantly larger than the above-mentioned tetracystein technology (see Fig. 10.1). The SNAP-tag can be used with greater color variability since a selection of small dyes is available [36] . Furthermore, unlike for fluorescent proteins, the same construct can be used for labeling with different colors and thus, no additional cloning is needed to achieve different colors. SNAP-tag labeling has been applied in living cells to track the neuropeptide Y receptor [37] and gave similar results to those obtained with a GFP variant. When the ACP tag was employed to label the neurokinin- 1 (NK1) receptor [38, 39] . it provided a means to label surface-expressed receptors with very little background. Using two different colors and an elaborate labeling protocol, it was possible to investigate the NK1 receptor in single cells by FRET microscopy and to demonstrate that the receptors are monomeric and reside in microdomains in intact cells [39]. Receptor dimerization was further investigated using a time-resolved FRET approach involving the SNAP-tag technology [40] . and evidence was found that family A and family C GPCRs could not only dimerize, but could also occur in even higher oligomeric states. Recently, a further engineered variant of the SNAP-tag, named CLIP-tag, was reported. This CLIP-tag can be labeled with chemically different compounds compared to the SNAP-tag, and Gautier et al. reported four color labeling of two different proteins with two different colors at different time points [41]. Although this labeling was not done with GPCRs, it highlights the potential of this technology.

Any fluorescent label can potentially interfere with the function and fate of the labeled receptor. It is actually never possible to prove that a labeled receptor behaves in all aspect exactly as the nonlabeled receptors. Therefore, it is very important to carefully control for the function of the labeled receptor in respect to the specific aspect that is under investigation. In many cases, the trafficking and signaling of a given receptor is the focus of the research, and labeled receptor should be analyzed in detail regarding its signaling properties. There are many examples where C-terminal tags on receptors have had no detectable influence on the G protein-activating properties of the receptors; however, the C-terminal tags may influence desensitization mechanisms and arrestin-mediated signaling properties, and these properties have not always been investigated. In addition, for many receptors such as the PTH receptor [42] . the distal C-. erminus serves either as an anchoring point for adaptors such as NHERF1, or to exert other important functions such as targeting, which in turn may influence signaling [42, 43]. Careful consideration of appropriate functional controls is critical when fluorescent dyes have been used to label GPCRs.

0 0

Post a comment