G protein-coupled receptors (GPCRs) are the largest family of membrane proteins, involved in almost all physiological processes. Studying them can be difficult due to a lack of specific, high-affinity antibody reagents and the challenges associated with isolating GPCRs from the insoluble membrane environment.
What are GPCRs?
GPCRs, also known as seven-transmembrane receptors, are found at the surface of all eukaryotic cells, where they bind a vast array of signaling molecules. Each GPCR comprises a single polypeptide chain with an extracellular N-terminus and an intracellular C-terminus and features seven transmembrane α-helices, connected by three intracellular and three extracellular loops. The latter act as specific binding sites for ligands including hormones, neurotransmitters, and photons. As the name suggests, GPCRs interact with G proteins - a diverse group of proteins that bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP) - to perform their signaling function. In humans, almost a thousand different GPCRs have been discovered to date, many of which are the targets for approved drugs.
How do GPCRs work?
G proteins are fundamental to GPCR signaling, where they act as on/off switches for signal transduction. While some G proteins are monomeric, those that associate with GPCRs consist of three subunits - alpha, beta, and gamma - of which the alpha subunit is involved in nucleotide binding. When the alpha subunit is bound to GDP, it allows the intact (inactive) G protein heterotrimer to attach to a neighboring GPCR. However, when the GPCR recognizes and binds its specific ligand, it undergoes a conformational change, triggering the alpha subunit to exchange GDP for GTP. This causes the G protein to dissociate into two separate parts, the GTP-bound alpha subunit and a beta-gamma dimer, which can each interact with other membrane proteins to effect signal transduction. Well-known targets of activated G proteins include adenylyl cyclase, which catalyzes the synthesis of cyclic AMP from ATP; phospholipase C, which converts the membrane lipid phosphatidyl inositol to the secondary messengers diacylglycerol and inositol triphosphate; and various voltage-gated Ca2+ and Na+ channels. When the GTP is hydrolyzed back to GDP, the G protein complex reforms and reassociates with the GPCR, thereby switching off signaling
Figure 2: Activation of the G alpha subunit of a G-protein-coupled receptor
In unstimulated cells, the state of G alpha (orange circles) is defined by its interaction with GDP, G beta-gamma (purple circles), and a G-protein-coupled receptor (GPCR; light green loops). Upon receptor stimulation by a ligand called an agonist, the state of the receptor changes. G alpha dissociates from the receptor and G beta-gamma, and GTP is exchanged for the bound GDP, which leads to G alpha activation. G alpha then goes on to activate other molecules in the cell.
Nature.com - GPCR
Why are GPCRs difficult to study?
The study of GPCRs presents several challenges. First, because so many different GPCRs are known to exist, antibodies specific to the target of interest may simply not have been developed. Also, since GPCRs must often undergo numerous post-translational modifications (PTMs) in order to function, an antibody that recognizes a GPCR in one model system may not recognize the same GPCR from another source. These issues are compounded by the fact that generating antibodies against GPCRs is problematic due to protein insolubility, which makes releasing GPCRs from the plasma membrane for use as native antigens technically difficult. GPCR insolubility also creates issues for co-immunoprecipitation/Western blotting experiments, where the methods used for protein isolation have a high associated risk of disrupting protein-protein interactions or causing unnatural complexes to form, as well as for structural studies, where getting GPCRs to crystallize requires a significant level of expertise. Additionally, the ligands that activate many GPCRs are still unknown and, while GPCRs were originally thought to function as monomers, they are now known to form both homodimers and heterodimers; both these factors add yet further complexity when it comes to establishing the role of GPCRs in conditions of health and disease.
What tools and resources are available for studying GPCRs?
Various tools and techniques have been developed to facilitate the study of GPCRs. These include a steadily growing number of antibodies, which are increasingly produced using overexpression cell lines rather than isolated GPCR proteins as immunogens for improved affinity, and a broad range of specialized detergents. For example, Anatrace provides pre-mixed formulations of detergents that are commonly used for structural studies (e.g., 10% DDM/1% CHS ), which are designed to improve experimental reproducibility in applications like Lipidic Cubic Phase (LCP) crystallization. Biophysical methods have also been adapted to support GPCR research, including Fluorescence Resonance Energy Transfer (FRET), which generates a measurable signal when two biomolecules (e.g., a GPCR and its ligand, or a pair of GPCRs) are in close proximity to one another. Some reagent manufacturers are even dedicated to producing research tools primarily for membrane proteins, categorizing their products according to receptor type to help researchers identify a product that best meets their needs. One such example is Alomone Labs, which offers an extensive portfolio of antibodies, agonists, and antagonists for almost a hundred different types of GPCRs, including glutamate, frizzled, and muscarinic receptors.
Anatrace
Anatrace develops and supplies the industry’s finest high-purity detergents and lipids. They are an internationally recognized leader and specialist in manufacturing reagents for membrane protein studies and custom chemical synthesis.
| Cat-No. | Item | Size | Price (CHF) |
|---|---|---|---|
| D310-CH210-1ML | D310 (100 MG/ML)/CH210 (10 MG/ML) 10:1 PRE-MADE SOLUTION | 1 ml | 49.00 |
| D310-CH210-10ML | D310 (100 MG/ML)/CH210 (10 MG/ML) 10:1 PRE-MADE SOLUTION | 10 ml | 296.00 |
| D310-CH210-50ML | D310 (100 MG/ML)/CH210 (10 MG/ML) 10:1 PRE-MADE SOLUTION | 50 ml | 582.00 |
GeneTex
GeneTex offers primary & secondary antibodies validated with multiple methods. GeneTex utilizes its recombinant monoclonal antibody platform to target G protein-coupled receptors (GPCRs).
| Cat-No. | Item | Size | Price (CHF) |
|---|---|---|---|
| GTX636952-25UL | Dopamine Receptor D2 antibody [HL1478] | 25 ul | 196.00 |
| GTX637589-25UL | RAI3 antibody [HL1864] | 25 ul | 196.00 |
| GTX12583-25UG | GPCR GPR58 antibody | 25 ug | 395.00 |
| GTX12610-25UG | GPCR HM74 antibody | 25 ug | 477.00 |
Proteintech
Proteintech - The benchmark in antibodies - Primary antibodies, nanobodies, cytokines & growth factors - all made in-house. Strict validation by western blot, ELISA and siRNA testing.
| Cat-No. | Item | Size | Price (CHF) |
|---|---|---|---|
| 21180-1-AP-150UL | S1PR2 Polyclonal antibody | 150 ul | 528.00 |
| 13398-1-AP-150UL | GPR161 Polyclonal antibody | 150 ul | 528.00 |
| 25343-1-AP-150UL | AGTR1 Polyclonal antibody | 150 ul | 528.00 |
LubioScience represents some of the most trusted brands in research, including several companies with a proven track record in supporting GPCR research. To discuss how we can help to advance your project, contact us today.