Blog Understanding why GPCRs are difficult to study Published on July 11th, 2019 In this blog post we delve into the 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 ~3-5% of gene encoding proteins. 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/tast2 and Secretin. Approximately 30-40% of drugs prescribed for heart failure, hypertension, diabetes, prostate cancer and bronchial asthma (to name a few) are targeting GPCRs. 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. 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 downstream signaling molecules such as the trimeric GTP-binding protein, or G protein and other proteins called β-arrestins. 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/or ability for the GCPR to bind other molecules, alters as a result. It is for this reason, that in order to understand receptor signaling and regulation, and to design drugs that target GPCR, it is necessary to characterize the conformational and structural receptor dynamics. 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 challenges have risen that include: 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. 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. However, one disadvantage in the study of GPCR dimerization/oligomerization in native systems is the lack 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. This carries a risk which could lead to artificial protein–protein associations being formed or possibly destroying existing associations. Blue Native PAGE (BN-PAGE) is another method that exhibits if GPCRs exist as monomers, dimers or higher order oligomers. This method works the same as SDS PAGE, except here SDS is substituted by Coomassie Blue. This is because SDS is a highly denaturing detergent and Coomassie Blue gives proteins an overall negative charge resulting in rapid electrophoretic mobility. However, this method also requires cell disruption and membrane solubilization. It is worth noting that under the right conditions it is a technique that can be used to preserve oligomeric structure. 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, biophysical techniques such as FRET, BRET and PCA as well as Microfluidic Diffusional Sizing (MDS).