Fluorescent ligands: A New Method to Label your GPCRs

Antibodies and GPCRs. Are they a versatile tool for GPCR pharmacology studies?

To date, it has been reported that about 800 GPCRs are encoded by the human genome. Some of them have known ligands available which led to the corresponding radioligands. However, in most cases they are orphan receptors, which means that no ligands are known, or there are only primary antibodies available for their study. When directly labelled or using labelled secondary antibodies (indirect immunofluorescence), primary Abs can be used to detect the GPCRs expression through a wide range of techniques, including immunohistochemistry, proximity ligation assays and immunoblotting.

Unfortunately, the generation of antibodies against unmodified GPCRs (as endogenously expressed GPCRs) is not a simple task because there are several significant hurdles to overcome [1]:

  • Low cell surface expression.
  • Need for the GPCR to be expressed in a membrane with the correct post-translational modifications.
  • Intrinsic conformational heterogeneity of GPCRs.
  • Low immunogenicity, with only the N-terminal domain and extracellular loops accessible as potential epitopes for extracellularly targeted antibodies.

GPCRs are often expressed in cellular backgrounds which do not reflect their natural environment, and often at expression levels far exceeding those found physiologically. Receptor over expression results in a shift in the relative abundance of interacting partners, including G proteins, β-arrestins, and potential dimer partners which are endogenously expressed in the recombinant cell. In synthesis, GPCR overexpression, removes the subtlety inherent in a physiological system.

An alternative solution to this problem is to study specific GPCR subtypes by cell-transfection. In this case human GPCRs genes are introduced into recombinant cells where they will be overexpressed. Sad to say that also in this scenario, there are several disadvantages:

GPCR antibodies, high variability in the results, small reproducibility in the experiments.

Generating high specific antibodies can be a complex task and, especially for GPCRs, it can be more challenging. Although the extracellular domains (N terminus and three extracellular loops), are the most immunogenic part of a GPCR, often these domains show low homology between species [2]. Unfortunately, the difficulties do not end with antibodies production, but also continue with quality consistency. Many anti-GPCR antibodies demonstrate batch-to-batch variations and thus, poor experimental reproducibility. Thanks to the extensive work performed by Grimsey et al. [3], several commercially available antibodies against the N terminus of the cannabinoid CB1 receptor were compared and tested with a wide range of immunological techniques. The results were impressive. They found a consistent batch-to-batch variations in the degree of CB1receptor staining not only histochemically, as in brain sections, but also when expressed in whole cells.

Taking a deeper look, Grimsey et al. used hemagglutinin tagged human CB1 receptors (HA-CB1R) expressed in HEK cells to directly assess the specific detection of cell surface, total CB1 receptor expression, as well as non-specific binding. When they tested cells expressing HA-CB1 receptor by live-cell-labelling they found that HA-CB1R is abundantly detected with the anti-HA antibody while ABR, BioSource, and Cayman CB1R antibodies completely failed to detect surface CB1 receptors (Fig.n.1). The Sigma Aldrich CB1R antibody detected some cell surface receptors, however, the staining was punctate and weaker compared to that observed with the HA antibody (Fig.n.1) suggesting that not all surface CB1 receptors were detected by the Sigma Aldrich antibody.


Fig.n.1. Immunocytochemical comparison of CB1 antibody specificity on human HA-tagged CB1receptors expressed in HEK cells. abundant HA-CB1 is detected with the anti-HA antibody. Also shown is the lack of staining detected with the ABR, BioSource, Cayman, and Sigma N-terminal CB1antibodies (all at 1:500 dilution except BioSource, used at 1:100) (3).

How to overcome Ab weakness: move to fluorescent ligands 

It is important to remember that one of the inherent properties of a GPCR is to bind small molecules or peptides. Using these innate physiological properties, fluorescently labelled GPCR ligands represent an alternative to antibodies to detect endogenously expressed receptors. These fluorescent tools are composed by a pharmacophore which binds the receptor of interest, a linker and a fluorophore.

Fig.n.2. Simplified structure of a fluorescent ligand.

The recent and fast spread of well-validated selective tools to study GPCRs as fluorescent ligands has brought with it new advantages as:

  • Combine a panel of different applications as Flow cytometry and FRET to detect the expression of endogenous GPCRs in complex samples of heterogeneous expression (e.g., tumours, immune cells).
  • Compare at the same time different cell surface proteins expression and potential interactions of GPCRs with other proteins (cell surface and intracellular).
  • Study GPCR pharmacology in real time, to potentially reveal molecular mechanisms underlying health and disease.

Table n.1. Differences between Antibodies and Fluorescent ligands to detect endogenous GPCRs 

The advantages to use fluorescent ligands in combination with different techniques

One of the biggest advantages of using fluorescent ligands is the possibility to combine different techniques in the same experiment. An example is the combination of Fluorescence resonance energy transfer (FRET) and flow cytometry. FRET/flow cytometry has the advantage of combining the relative spatial sensitivity of FRET to measure protein–protein interactions with the much greater throughput possible with flow cytometry. Moreover, the major advantage of using flow cytometry to quantify receptor/ligand engagement is that no washing steps need to be made to separate bound/unbound fluorescent ligand, like in other ligand binding assays. This is a consequence of the narrow sample volume used so that only a small volume of sample fluid that surrounds the cell is excited. This minimises excitation of unbound fluorescent molecules that are also in solution, diminishing the background fluorescence signal.

Taking advantage of this approach, Banning C. et al. combined these two techniques to investigate the entry mechanism of human immuno virus HIV-1. Thanks to their assay, it was possible to map interacting domains within proteins of the human and simian immunodeficiency virus (HIV and SIV) Vpu protein with the restriction factor CD137 (Bst-2 or tetherin). Using the VpuRD (URD), a mutant that is defective in the enhancement of HIV-1 particle release [4], they demonstrated how Vpu was unable to bind to CD317 but fully retained its ability to interact with CD4. Thanks to this research Banning C. et al. provided the first evidence that the Vpu/CD317 interaction is mediated by specific residues in the TM region, suggesting that interaction of Vpu with CD317 is functionally required to overcome its restricting activity on HIV-1 release [5].

New architecture designs for fluorescent ligands

Recent advancements in fluorescent ligands design, with improved subtype selectivity, affinities and physicochemical properties, has made them a valid alternative to antibodies to specifically detect endogenous GPCRs. In the study presented by Hatse S. et al, they developed a new valuable and more convenient alternative version, CXCL12AF647, to the traditional radiolabelled ligand. This new fluorescent ligand was specifically designed for ligand/receptor interaction studies aimed for the identification of new CXCR4-interacting molecules. Thanks to this innovative design, which is characterized by favourable spectral properties, such as superior fluorescence intensity, pH insensitivity, and minor change in absorbance or fluorescence spectra when conjugated, the new fluorescently labelled CXCL12 has been used to identify CXCR4 positive T lymphoid SupT1 cells [6].

Fig.n.2. A:Concentration-dependent bind-ing of CXCL12AF647in human T-lymphoidSupT1 cells. The blue curve (negative con-trol) shows the background fluorescence  SupT1 cells in channel FL4 on a logarithmic scale; the red, green, and orange curves represent SupT1 cells incubated with 4, 20, and 100 ng/ml CXCL12AF647, respectively, and were analyzed immediately after the negative control with identical instrument settings. B:CXCR4-specific interaction of CXCL12AF647. CXCR4-,CXCR3-, and CCR5-transfected human astro-glioma U87. CD4 cells were incubated in the presence (red curves) or absence (blue curves) of 20 ng/ml CXCL12AF647 (6).

Although fluorescent ligands are gaining popularity as tools to aid GPCR research, the in vivo application of such tools is hampered due to their short excitation wavelengths in the visible range and lack of fluorogenic switch. Recent interesting advancements in fluorescent ligand design and synthesis, more precisely innovative approaches used for tuning the physicochemical properties of the linker region, allowed to improve ligand affinity and solubility making them suitable for GPCRs imaging in vivo. One example of this new approach was performed by Maet al. [101], designing and using an infrared-emitting α1-adrenoceptor antagonist to label endogenous α1-adrenoceptors in ex vivo slices of murine prostate tissue and in vivo, administered intravenously in mice, to image the distribution of α1-adrenoceptors in several tissues. This innovative approach represents a simple and non-invasive way to detect receptor localisation without the need for lengthy and costly RNA-sequencing or northern blotting.

The explosion of new fluorescent ligand designs not only allowed to expand the limits of in vitro and in vivo studies, but also opened new frontiers in GPCR heterogeneity definition in Single-cell profiling. Very recent studies conducted by Astet al. [7] presented the implementation of a panel of new fluorescently labelled GLP-1 agonists (LUXendins) to localise endogenous GLP-1 receptors in mice using two-photon microscopy. These fluorescent ligands demonstrated high affinities for the GLP-1 receptor with exceptional signal-to-noise ratios. Thanks to their action, it was possible to identify a distinctive pattern in murine-cells of GLP-1 receptor expression in pancreatic islets. Additionally, the good imaging characteristics of these ligands, best in class for tissue penetration and signal-to-noise ratio, made them flexible to be implemented in super-resolution approaches to study subcellular receptor localisations and understand how, in pancreatic b-cells, GLP-1 receptors tend to cluster in nanodomains.


GPCR receptors can couple to more than one signalling pathway and some ligands, named biased agonists, can stimulate selectively only one of them. The emergence of this new concept is crucial in medicinal pharmacology since side-effects of some drugs have been linked to the activation of secondary signalling pathways. New strategies based on fluorescence measurement constitute excellent alternatives to the traditional radioactive assays. Less hazardous, faster and cheaper, these methods also exhibit particularly good sensitivity and can be used on various biological models such as heterologous expression systems or native tissues. Although antibodies represented historically the common solution for protein structure and function studies, their application for GCPR characterization is limited by the fact that this group is characterized by low immunogenicity and high heterogeneity. Thanks to new emerging technologies and tuneable architectures, fluorescent ligands represent a new alternative for GPCR characterisation studies. At Celtarys we count on more than 20 years of experience in GPCRs, and we have generated antagonist and agonist fluorescent probes for GPCR families validated in a variety of assays such as High Content Screening, Fluorescence Polarization, Flow Cytometry and Fluorescence microscopy. Thanks to our expertise, we can also support in Custom Development Services when existing off-the-shelf solutions are not enough.


  1. Hutchings CJ, Koglin M & Marshall FH (2010) Therapeutic antibodies directed at G protein-coupled receptors.mAbs2, 594–606.
  2. Strasser A, Wittmann HJ, Buschauer A, Schneider EH& Seifert R (2013) Species-dependent activities of G-protein-coupled receptor ligands: lessons from histamine receptor orthologs. Trends Pharmacol Sci34,13–32.
  3. Grimsey NL, Goodfellow CE, Scotter EL, Dowie MJ,Glass M & Graham ES (2008) Specific detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal! J Neurosci Methods171,78–86.
  4. Schubert U, Bour S, Ferrer-Montiel AV, Montal M, Maldarell F, et al. (1996) The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains. J Virol 70: 809–819.
  5. Banning C, Votteler J, Hoffmann D, Koppensteiner H,Warmer M, Reimer R, Kirchhoff F, Schubert U,Hauber J & Schindler M (2010) A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells. PLoS One5, e9344.
  6. Hatse S, Princen K, Liekens S, Vermeire K, De Clercq E & Schol D (2004) Fluorescent CXCL12AF647 as a novel probe for non radioactive CXCL12/CXCR4 cellular interaction studies. Cytometry A61, 178–188.
  7. Ast J, Arvaniti A, Fine NHF, Nasteska D, Ashford FB, Stamataki Z, Koszegi Z, Bacon A, Jones BJ, Lucey MAet al. (2020) Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics. Nat Commun11, 467.   

New at rhenium - analytical Chemistry