Why G protein-coupled receptors (GPCRs) are difficult to study

In this blog post we delve into the definition of GPCRs, reasons behind why elucidating GPCR structures are so important as well as why they are hard to study.

Why are GPCRs important?

G protein-coupled receptors (GPCRs) make up the largest family of membrane proteins, compromising ~1% of human gene encoding proteins [1]. There are more than 800 GPCRs in humans and around 1000 just involved with the sense of smell in mice. In humans these GPCRs have been categorized into 5 groups based on their ligand-binding characteristics: glutamate, rhodopsin, adhesion, frizzled/taste2 and secretin [2]. Approximately 35% of drugs approved by the FDA target GPCRs, impacting treatment across a wide range of indications including cancer, heart failure, hypertension, diabetes, and bronchial asthma (to name a few) [3].

One key characteristic of GPCRs is their ability to interact with a plethora of chemically diverse ligands. Subsequently, GPCRs can mediate a wide range of physiological processes including vision, olfaction and signaling in organs, the endocrine system and the central nervous system.

What are GPCRs and what are their basic structure?

Figure 1: The 7 transmembrane helices of bovine rhodopsin. Source: Wikimedia Commons

Despite the chemical and functional diversity of the signal molecules that activate them, all GPCRs have a similar structure. They are made up of a single polypeptide chain that crosses back and forth through the lipid bilayer seven times [4]. These 7 membranes spanning segments are connected by intra and extracellular loops. However, it should be noted that the amino terminus is always extracellular facing and the carboxyl terminus is always intracellular facing.

When an extracellular molecule binds to a GPCR, the receptor undergoes a conformational change. This change induces interactions between the intracellular domains of the GPCR and the downstream signaling transducers G-protein, a trimeric GTP-binding protein, or β-arrestin.

The physiological outcome of GPCR-mediated signaling depends on the molecules with which the receptors interact. GPCR posttranslational modifications including phosphorylation, acetylation, glycosylation and many others can all induce tertiary/quaternary structural changes. These changes regulate function and receptor association and so alter the ability of the GCPR to bind other signals and transmit signals. It is thus necessary to characterize the conformational and structural dynamics of GPCRs to understand their signaling and regulation, and hence to discover and develop drugs that target any particular GPCR target.

So why is it hard to study GPCRs using biological methods?

X-ray crystallography has been described as the gold standard for investigating the structures of proteins and protein complexes. However, when this technique is employed to study GPCRs, there are several challenges:

• Difficulties in protein crystallization due to the proteins being insoluble: This is a major bottleneck in X-ray crystallography for drug discovery since so many drug targets are anchored on the cell membrane.
• Unresolved protein dynamics and conformational diversity
• Limited detection of post-translational modifications, which as explained above, are a crucial aspect to GPCR signaling [5].

Co-immunoprecipitation (Co-IP) along with Western blotting were one of the first techniques that were used to prove that the β2 adrenergic receptor (β2-AR) could form homodimers that could be stabilized by agonists [6]. However, one challenge in the study of GPCR dimerization/oligomerization in native systems is the paucity of specific and high-affinity antibodies to GPCRs themselves. Another disadvantage of this method is that the sample lysis/solubilization required for releasing the protein of interest from its insoluble membrane environment risks generation of artefactual protein–protein interactions or possible disruption of authentic associations.

Blue Native PAGE (BN-PAGE) is another method that can be used to determine whether GPCRs exist as monomers, dimers or higher order oligomers [7]. This method works the same way as SDS-PAGE, except substituting SDS with Coomassie Blue. This works as negatively charged Coomassie Blue binds efficiently to proteins and so imparts bound proteins with an overall negative charge and hence electrophoretic mobility. Unlike SDS Coomassie Blue does not denature proteins and so BN-PAGE can be used to detect oligomeric state of a protein. This method does, however, require cell disruption and membrane solubilization and so like Co-IP risks formation of artefactual associations or disruption of natural interactions.

Conclusion

Despite all the progress that has been made, studies into GPCR structures remain challenging. This is largely because it is very difficult to express and purify a sufficient quantity of a GPCR in an intact and functionally active form. While biological methods have proven effective as a starting point, other methodologies have now been pioneered to shed new light on GPCR structure. These include mass spectrometry, and biophysical techniques such as Förster Resonance Energy Transfer (FRET), Bioluminescence Resonance Energy Transfer (BRET) and Protein fragment Complementation Assays (PCAs).

Microfluidic Diffusional Sizing (MDS) as a tool to study GPCRs

Microfluidic Diffusional Sizing (MDS) is a new protein interaction technology that is ideally suited to work with GPCRs. Measurement of protein size (hydrodynamic radius) takes place in solution and can be used to robustly and directly detect protein interactions. There is no need to immobilize proteins and so the associated challenges and artefacts are eliminated. Furthermore, MDS is able to work with complex mixtures and can even detect binding in crude membrane protein libraries.

Follow the link below to learn more about MDS or contact us to discuss how MDS can assist your GPCR study.

References 

1. Dijkman, P.M.; Muñoz-García, J.C.; Lavington, S.R.; Kumagai, P.S.; dos Reis, R.I.; Yin, D.; Stansfeld, P.J.; Costa-Filho, A.J.; Watts, A. Conformational Dynamics of a G Protein–Coupled Receptor Helix 8 in Lipid Membranes. Sci. Adv. 2020, 6, eaav8207. https://doi.org/10.1126/sciadv.aav8207
2. Fredriksson, R.; Lagerström, M.C.; Lundin, L.-G.; Schiöth, H.B. The G-Protein-Coupled Receptors in the Human Genome Form Five Main Families. Phylogenetic Analysis, Paralogon Groups, and Fingerprints. Mol. Pharmacol. 2003, 63, 1256–1272. https://doi.org/10.1124/mol.63.6.1256
3. Sriram, K.; Insel, P.A. G Protein-Coupled Receptors as Targets for Approved Drugs: How Many Targets and How Many Drugs? Mol. Pharmacol. 2018, 93, 251–258. https://doi.org/10.1124/mol.117.111062
4. Kroeze, W.K.; Sheffler, D.J.; Roth, B.L. G-Protein-Coupled Receptors at a Glance. J. Cell Sci. 2003, 116, 4867–4869. https://doi.org/10.1242/jcs.00902
5. Chun, E.; Thompson, A.A.; Liu, W.; Roth, C.B.; Griffith, M.T.; Katritch, V.; Kunken, J.; Xu, F.; Cherezov, V.; Hanson, M.A.; et al. Fusion Partner Toolchest for the Stabilization and Crystallization of G Protein-Coupled Receptors. Struct. Lond. Engl. 1993 2012, 20, 967–976. https://doi.org/10.1016/j.str.2012.04.010
6. Hebert, T.E.; Moffett, S.; Morello, J.P.; Loisel, T.P.; Bichet, D.G.; Barret, C.; Bouvier, M. A Peptide Derived from a Beta2-Adrenergic Receptor Transmembrane Domain Inhibits Both Receptor Dimerization and Activation. J. Biol. Chem. 1996, 271, 16384–16392. https://doi.org/10.1074/jbc.271.27.16384
7. Suda, K.; Filipek, S.; Palczewski, K.; Engel, A.; Fotiadis, D. The Supramolecular Structure of the GPCR Rhodopsin in Solution and Native Disc Membranes. Mol. Membr. Biol. 2004, 21, 435–446. https://doi.org/10.1080/09687860400020291