It is proven that fluorescence techniques are powerful tools for investigation of the very dynamic family of GPCRs to understand their subcellular localisation and to further elucidate key elements in GPCR trafficking and interaction with other signal pathways.
However, to obtain physiologically relevant results, some considerations have to be made. First, it is of utmost importance that the investigated GPCR, ligand or interacting protein is not influenced in its functionality by the fluorescent modification. Therefore careful characterizations are needed and to exclude interferences it might be helpful to apply different labels at different sites of the protein for data evaluation [212
]. Additionally, the label should be as small as possible, the recent and ongoing development and optimization of self-labeling tags will be advantageous in this field. Using unnatural amino acid mutagenesis the site-specific incorporation of reactive keto groups, such as p
-benzoyl-L-phenylalanine (Bzp) or p
-acetyl-L-phenylalanine (Acp), into functional GPCRs and their ability to react with a variety of spectroscopic and other probes was previously described [213
]. Because of their excellent fluorescent properties quantum dots are very attractive for labeling, however the full potential of QDs for cellular imaging has not yet been realized because of problems with large QD size, QD multivalency and the difficulty of delivering QDs into the cytosol. Recently, monovalent and reduced-size quantum dots were generated and successfully applied for receptor imaging in living cells [214
Fluorescent antibodies provide a powerful tool for examining the cellular distribution of GPCRs. However, quantification is highly depended on the accessibility of – in most cases – the small epitope by the large antibody. The challenge is to develop even more high-affinity fluorophore- or enzyme-conjugated primary antibodies for one-step labeling assays on living cells. The generation of bright and stable dyes as well as pH sensitive ones, such as CypHer 5 [34
], will lead to further insights into the life of GPCRs and will enable high-throughput screening applications. A new group of molecules, called affibody molecules, is especially interesting for imaging applications because of their small size (7–15 kDa) compared to antibodies. These proteins are composed of a three-helix bundle of 58 amino acids and are derived from the scaffold of one of the IgG-binding domains of the staphylococcal protein A [215
]. The binding site is equivalent to an antibody with respect to the surface area. The size, the simple structure, the specific target recognition, the ease of production and the high stability give affibody molecules significant advantages over antibodies. These molecules can be labeled with fluorophores but also with radionuclides which make them promising candidates for GPCRs associated tumor diagnosis and therapy [216
Recombinant DNA technologies have highly advanced fluorescence labeling as well as transfection and transgenic techniques that enable simple DNA delivery to cells that results in covalent labeling by using the protein expression system of the cell. However, expression levels in cell cultures may significantly differ from those in natural systems. Concerning the signaling and trafficking behavior of GPCRs the relationship between the occupation of the receptor by physiological levels of agonists and the initiation of translocation is an important issue. The general use of very high concentrations of agonist leaves open the possibility that the investigated processes are more pharmacological than physiological.
A major criticism of FRET/BRET studies used to investigate protein-protein-interactions, is that the required protein overexpression can result in RET attributed to a high incidence of random collisions, rather than direct protein-protein-interactions. If low expression levels can not be obtained by varying DNA amounts within transient cell transfections, stable cell transfections will provide an alternative, since there is a homogenous population of cells expressing the protein of interest at the same level. Another possibility is the baculovirus expression system which enables protein expression levels to be controlled more closely than with transient transfection, because protein expression can be titrated by adjusting the multiplicity of viral infection [217
Since protein co-localization is the first prerequisite for interactions, this should be proven by fluorescence microscopy, and by using parallel labeling strategies to locate subcellularly the interaction of interest. For correct evaluation of FRET and BRET data appropriate controls have to be used to demonstrate the specificity of the interactions and to establish levels of RET considered to be background in any given experiment. The additional application of a biochemical approach might support the results. To validate the physiological role of the detected interaction studies in other, more natural cell systems, e.g. cell lines endogenously expressing one protein of interest, as well as investigations on tissues and animals will be indispensable in proving the relevance of the interactions in the future. For example, the in vivo
co-expression of GPCRs has to be demonstrated in the same tissue, and ideally in the same cell for establishing the physiological relevance of receptor oligomerization. Functional cross-talk between the receptor signaling pathways as well as novel pharmacological and/or functional properties will provide evidence for the mechanism by which receptor-receptor-interactions modulate cellular activity [218
An exciting application of GPCR-GFP chimeras involves their use in genetic screens in genetically tractable organisms such as yeast, e.g. to identify mutant yeast strains in which the receptor is mis-localized. Such strategies contribute greatly to the identification of new components involved in GPCR targeting and trafficking in additional model organisms [219
]. New approaches using whole organisms, in which the GFP-chimera can be expressed under the control of the endogenous promoter, e.g. invertebrates as C. elegans
or mouse models, allow cell biological, molecular and biochemical results to be interpreted in a physiologically relevant context and to be compared to those observed in cultured cells [220
]. GFP and its variants as reporters represent the next step in mouse genome engineering technology by opening up the possibility of combinatorial non-invasive reporter usage within a single animal, e.g. for gene-expression, as well as for co-visualization and FRET assays [222
In summary, many issues concerning the life of GPCRs can be addressed by fluorescence techniques, however many remain challenging. Further rapid advances in labeling and imaging technology can be expected and their parallel as well as their combined application will provide novel insights that will also broaden the range of new therapeutic interventions.