Antibodies for G Protein-Coupled Receptors (GPCRs)

Bio-Techne Antibodies for G protein-coupled receptors (GPCRs).

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G protein-coupled receptors (GPCRs) are the foundation of cell signaling. They play a role in a many diseases, causing them to be important targets for drug development. As such, there is considerable demand for anti-GPCR antibodies. Antibodies are invaluable tools in the drug discovery process as they are used for target identification and validation, assay development, evaluation of experimental models, and patient selection.

Bio-Techne’s brand R&D Systems has been a pioneer in the development and manufacturing of antibodies that target GPCRs. We offer the largest selection (Figure 1) of highly validated (Figure 2) GPCR antibodies in the industry. Visit our G Protein-Coupled Receptors (GPCRs) page, or a specific G Protein-Coupled Receptors by Research Area page (listed below) to learn which GPCRs are involved in different research areas and find antibodies and other tools for GPCR research and drug development.

R&D Systems offers antibodies for over 70% of the GPCR receptor families.

Figure 1. Mapping of R&D Systems GPCR Antibody Offerings to the GPCR Classification Tree. R&D Systems offers over 2000 antibodies for GPCRs, covering over 70% of GPCR receptor families. View a larger version of this image to see our GPCR antibody offerings across receptor families.

R&D Systems offers antibodies for over 70% of the GPCR receptor families.

Figure 2. R&D Systems GPCR Antibodies are Validated for Multiple Applications. About 95% of the R&D Systems GPCR antibodies are validated for flow cytometry. Additional applications for our antibodies include mass cytometry (CyTOF-ready), immunocytochemistry/immunohistochemistry (ICC/IHC), Western blot, blocking/neutralization, Simple Western, ELISA, immunoprecipitation, and agonist activity (the latter four applications are included in Other Applications portion of chart).


What are GPCRs?

GPCRs are the largest, and most diverse, family of cell-surface receptors in the human genome. They are integral membrane proteins that possess seven membrane-spanning domains, which are arranged as a barrel-like structure within the membrane to form a cavity that serves as a ligand-binding domain. GPCRs transduce extracellular stimuli to intracellular signals through activation of heterotrimeric G proteins. GPCRs are activated by a range of diverse ligands including hormones, neurotransmitters, ions, photons, and odorants, which activate several different signaling cascades, allowing for functional diversities. Thus, they are crucial for numerous physiology functions, and play a role in many diseases, such as cancer, metabolic diseases, and cardiac and inflammatory disorders.


GPCR Signaling

GPCRs transduce extracellular stimuli to intracellular signals through heterotrimeric G proteins. Heterotrimeric G proteins are composed of three different subunits: a α subunit (Gα), β subunit, and γ subunit. The Gα subunit binds either GTP or GDP depending on whether the G protein is active (GTP) or inactive (GDP). In the absence of a receptor agonist, GDP is bound to Gα, and the entire G protein is joined to a GPCR. When an agonist binds to a GPCR, the receptor undergoes a conformational change, causing the exchange of GDP on the Gα subunit for a GTP molecule. The heterotrimeric G protein then dissociates from the GPCR and separates into 2 parts: the Gα-GTP complex and the Gβγ dimer. Each part independently activates downstream signaling pathways. GPCR signaling will continue until the Gα subunit, which is a GTPase, hydrolyzes GTP. Gα-GDP then binds to the Gβγ dimer and the inactive heterotrimer reassociates with a GPCR.


GPCRs as Drug Targets

GPCRs are crucial for numerous physiological functions and play a role in many diseases, such as cancer, metabolic diseases, and cardiac and inflammatory disorders. As such, these receptors serve as an important target for drug discovery. In fact, approximately 35% of all FDA-approved drugs are against GPCRs.1 However, only a small proportion of these 800 receptors are targeted by drugs because many GPCRs are still incompletely characterized.2 Characterization of GPCRs require determining their cell surface expression as the surface density of GPCRs fluctuates during various physiological and pathophysiological conditions.3,4 It is best to monitor GPCR protein levels as GPCR mRNA and cell-surface protein expression do not always correlate.3 While there are several methods for detecting GPCR protein levels, only anti-GPCR antibodies can selectively bind and modulate native receptors, allowing for the specific quantification of endogenous receptor expression.


Challenges with Generating High-Quality Anti-GPCR Antibodies

Limited Immunogenic Epitopes

For antibodies to bind to their native protein, they must be generated from an accessible region of the protein. The most accessible areas of molecules are the parts that are exposed or on the outside of the structure. GPCRs are multispan transmembrane proteins. They exist either embedded within the lipid membrane or intracellularly. Thus, the only accessible regions that are exposed and can be used as immunogenic epitopes are the extracellular loop regions and the N-terminal domain.5

Difficult to Isolate Full GPCRs

Due to limited immunogenic epitopes, it may be beneficial to use the full receptor. However, GPCRs are difficult to isolate. Their expression is tightly regulated due to their role in many physiological processes. As a result, they are typically expressed at low levels, making it difficult to isolate the protein from whole cell extracts. It is also difficult to solubilize recombinantly expressed receptors as a native folded antigen. The detergent that is needed to extract GPCRs from their lipophilic environment needs to be present in all subsequent studies to prevent aggregation of the solubilized receptor. However, the detergent can cause problems with the stability of the receptor. This can make functionally relevant epitopes be disordered, and antibodies raised against such proteins are usually non-functional.

GPCR Specificity

GPCRs are dynamic structures. Ligand binding to GPCRs causes the receptors to undergo a conformational change, which exposes intracellular sites that effectively engage and activate a range of signaling complexes. Further research has suggested that GPCR activation isn’t a simple "on-off" state, but in fact is a highly dynamic situation where the receptors exist in many functionally distinct conformations.6 GPCRs can also bind to multiple ligands, undergo multiple post-translational modifications, and form oligomers with other proteins. This heterogeneity in structure and ligand-binding complicates the development of specific anti-GPCR antibodies. GPCRs also exhibit a high degree of sequence homology between closely related receptors and receptor subtypes, adding to the complexity of generating specific anti-GPCR antibodies.7


Our Approach to Manufacturing Anti-GPCR Antibodies

We Select the Best Immunogen for the Desired Antibody

R&D Systems has long recognized the need for reliable anti-GPCR antibodies and the difficulties in developing them. We believe that generating anti-GPCR antibodies that display the maximum immunogenicity with minimal antibody cross-reactivity begins with using the right antigenic epitope.

When generating our antibodies for GPCRs, we utilize a combination of immunization techniques to get the best selection of antibodies. We use short and long synthetic peptides to generate antibodies against specific domains and extracellular loops, respectively. Using immunogenic peptides, though, is not ideal for generating antibodies for applications that require the GPCR’s native structure, such as flow cytometry, as the peptides often lack the conformation and relevant post-translational modifications. Additionally, these antibodies tend to not be suitable for receptor mapping due to their lack of specificity and functional activity. To produce antibodies against the full, native GPCR, we pioneered the use of whole cell transfectants as immunogens (Figure 3).

Many commercial GPCR antibodies are made only using peptides; however, we have been using whole cell transfectants to generate anti-GPCR antibodies for decades. This commitment is illustrated by a 1999 paper published in The Journal of Biological Chemistry and through ongoing efforts in a 2017 Nature paper. Authors of the papers partnered with R&D Systems to develop antibodies to intricately map the epitopes of the CCR5 chemokine receptor and PAR2, respectively, to study receptor structure and function.8,9

R&D Systems Mouse Anti-Human CCR7 Monoclonal Antibody was generated using whole cell transfectants as an immunogen.

Figure 3. R&D Systems CCR7 Antibody Detects the GPCR on Human PBMCs by Flow Cytometry. R&D Systems Mouse Anti-Human CCR7 Monoclonal Antibody (Catalog # MAB197) was generated by immunizing mice with whole cell transfectants expressing the GPCR. Human peripheral blood mononuclear cells (PBMCs), stained with a PE-Conjugated Mouse Anti-Human CD4 Monoclonal Antibody (Catalog # FAB3791P), were stained with either (A) this CCR7 antibody or a (B) Mouse IgG2A Isotype Control (Catalog # MAB003), followed by an APC-Conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0101B). All products listed are from R&D Systems.


We Screen Using Native GPCR-Expressing Cells

Antibody screening is performed to identify which animals or hybridoma clones produce high levels of antigen-specific antibodies. To screen for anti-GPCR antibodies, we use samples that naturally express GPCRs to ensure that our antibodies recognize the native protein (Figure 4). Antibody specificity is then verified using transfected cells and irrelevant transfectants. (Figure 5).

Human blood lymphocytes, which express endogenous CCR7, was used to confirm binding of  R&D Systems Mouse Anti-Human CCR7 Monoclonal Antibody to the native GPCR.

Figure 4. Confirmation of Binding of R&D Systems CCR7 Antibody to Endogenously Expressed CCR7. Human peripheral blood lymphocytes were stained with an APC-Conjugated Mouse Anti-Human CD4 Monoclonal Antibody (Catalog # FAB3791A) and either a (A) PE-Conjugated Mouse Anti-Human CCR7 Monoclonal Antibody (Catalog # FAB197P) or a (B) PE-Conjugated Mouse IgG2A Isotype Control (Catalog # IC003P). All products listed are from R&D Systems.

True and irrelevant transfectants were used to verify specificity of the R&D Systems antibody to the GPCR CCR7.

Figure 5. Specificity of R&D Systems CCR7 Antibody Verified Using True and Irrelevant Transfectants. The specificity of R&D Systems PE-Conjugated Mouse Anti-Human CCR7 Antibody (Clone 150503; Catalog # FAB197P) was demonstrated by its ability to react with (A) HEK293 human embryonic kidney cell line transfected with human CCR7 and not react with (B) irrelevant HEK293 transfectants. Antibody binding was monitored using a PE-Conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0102B). All products listed are from R&D Systems.


Our Validation Protocol is Unparalleled

R&D Systems has always taken rigorous steps to test our antibodies. By developing and manufacturing over 90% of our antibodies, we can not only ensure we use optimized products during product development, but we also can also execute quality tests to ensure lot-to-lot consistency and outstanding performance, which most antibody suppliers are unable to do since they don't manufacture their own product. Additionally, we continuously confirm the specificity of our antibodies by testing using a variety of applications such as true and irrelevant transfectants (Figure 5) and knockout cell lines (Figure 6). We also perform additional tests for select GPCR antibodies, such as live cell staining, which confirms detection of low expressing GPCRs (Figure 7). These tests ensure that we provide the highest quality and most consistent anti-GPCR antibodies.

A knockout cell line was used to verify specificity of the R&D Systems antibody to the GPCR Calcitonin Receptor.

Figure 6. Calcitonin Receptor Specificity is Shown by Flow Cytometry in a Knockout Cell Line. The Calcitonin Receptor knockout MCF-7 human breast cancer cell line was stained with a Mouse Anti-Human Calcitonin Receptor Monoclonal Antibody (Catalog # MAB4614; filled histogram) or a Mouse IgG2A Isotype Control (Catalog # MAB003; open histogram), followed by a PE-Conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0102B). No staining in the Calcitonin Receptor knockout MCF-7 cell line was observed. All products listed are from R&D Systems.


R&D Systems GPCR antibodies are tested at physiological temperatures to ensure optimal flow staining.

Figure 7. R&D Systems CCR7 Antibody is Tested at Different Temperatures to Ensure Optimal Staining at Physiological Conditions. CCR7, like all GPCRs, are fluid structures. These receptors move within the lipid membrane as well as undergo internalization and endocytic recycling. These factors can mask the antibody binding site, resulting in no staining by flow cytometry. We examined the effect of temperature on CCR7 staining to determine if our antibody exhibits optimal staining at physiological temperatures (37 °C). Human whole blood was stained with a Mouse anti-Human CCR7 APC-conjugated Monoclonal Antibody (Catalog #AB197A) and a Mouse anti-Human CD4 PE-conjugated Monoclonal Antibody (Catalog #FAB3791P) at 4 °C, room temperature (RT), or 37 °C. Cells were washed and red blood cells were lysed with Flow Cytometry Human Lyse Buffer (10X) (Catalog #FC002) prior to the final wash and flow analysis. All products listed are from R&D Systems.


1. Insel, P.A. et al. (2019) Trends Pharm. Sci. 40:378.
2. Ayoub, M.A. (2017) MAbs 9:735.
3. Michel, M.C. et al. (2009) Naunyn Schmiedebergs Arch. Pharmacol. 379:385.
4. Insel, P.A. et al. (2007) Biochim. Biophys. Acta. 1768:994.
5. Jo, M. and S.T. Jung (2016) Experimental & Molecular Medicine 48:e207.
6. Hilger, D. et al. (2018) Nat. Struct. Mol. Biol. 25:4.
7. Grupta, A. et al. (2008) Comb. Chem. High Throughput Screen 11:463.
8. Lee, B. et al. (1999) J. Biol. Chem. 274:9617.
9. Cheng, R.K.Y. et al. (2017) Nature 545:112.