G-Protein Linked Cell Surface Receptors (GPCRs)

GPCRs are the membrane proteins that constitute the largest superfamily of the protein. In the human genome, there are more than 800 G-protein linked cell surface receptors that are categorized into four superfamilies; Rhodopsin-like GPCRs, secretin-like/adhesion-like GPCRs, glutamate, and frizzled/Tas2 GPCRs superfamilies.

All G-protein linked cell surface receptors share a conserved serpentine transmembrane domain (seven membrane-spanning transmembrane domains) and play varieties of physiological and pathological roles. About 50% of the drugs that are currently used in the treatment of neurodegenerative diseases, cancer, pain, viral infection, allergy, etc. are designed to target the G-protein linked cell surface receptors.

Elucidation of bovine rhodopsin G-protein cell surface receptor for the first time in 2000 has led to the determination of several GPCRs including a β2-adrenergic receptor, chemokine receptor, and A2A adenosine receptor, etc.

Using the protein engineering strategies such as cleavage of unstructured domains, systematic or point stabilizing mutations and structural data of the majority of the GPCRs available from published articles, it was concluded that ligand-dependent stabilization is one of the fundamental properties of the receptor that is a structural and functional characteristics of the GPCR.

Signal transduction through G-protein linked cell surface receptor

G-protein linked cell surface receptor signaling cycle. Image: Repapetilto via Common Wikimedia

It has been found that all GPCR structures have a bound ligand either in the orthosteric or in the allosteric binding sites that confer the presence of bound ligand stabilizes the structure of the GPCR. Ligand recognition and binding to the GPCR binding site promote the correct conformation locking the receptor into a stable state.

However, researchers have also found that ligand not only helps to attain the receptor stable conformation but also promotes the expression of some of the G-protein linked cell surface receptors to the membrane surface. During which ligand helps to reduce the internalization of the receptors and enhances the membrane trafficking and receptor folding.

For this purpose, researchers gathered published data for about 48 GPCR ligands and crystal structures of their receptors bound to the ligands. They analyzed the properties including molecular weight, binding affinity, the thermal stability of all the GPCRs bound to the ligand. The result revealed that ligands potentially influenced the GPCR crystallization process.

The ligand properties related to the GPCR crystallization process are due to the thermal stability of the receptor achieved by the ligand binding. However, there are some more approaches to measure the properties of the ligands by both cell-based approaches as well as protein-based approaches.

Researchers used the fluorescent thermal stability assay to assess and rank the ligands in the context of improving thermal stability. They found that the melting temperature of the purified receptors without ligand-bound in the detergent solution ranging from 40-45°C. However, Tm values and crystal diffraction resolution data of the ligand-bound GPCRs showed Tm values higher than 55°C in the micro-scale fluorescent thermal stability assay.

Factors that stabilizes the ligand-receptor interaction

From a structural perspective, receptors have ligand-binding sites for non-peptide ligands in the transmembrane domain and, therefore the larger ligand may not access the binding site.

The affinity of the ligand to the receptor is another factor that plays an important role in the stability of the ligand-bound receptors. The receptor binding affinity of the ligand is based on the association and dissociation rates of the ligand and receptor. Good ligand-receptor stabilization is achieved only when ligands have high affinity and a slow rate of dissociation of the receptor.

Based on this principle of receptor binding ligand, a ligand with the ability to form a covalent bond with the receptor may further improve the receptor stability slowing the dissociation rate and fastening the rate of association.

The solubility of the ligand in the aqueous environment is another property that can affect the stability of the ligand-bound receptors. The aqueous solubility of the ligand helps to determine whether it can easily be manipulated in the receptor-bound ligand purification. Therefore, a ligand must be soluble sufficient to remain the soluble state necessary to provide the complete occupancy of the ligand-binding site of the receptor.

H-bonding between the ligand and receptor is another factor that influences the formation of a stable molecular interaction of ligand and receptor in the ligand-binding pocket.

While reviewing the ligand properties of ligand-bound GPCR co-crystal structures, researchers had tried to understand the relationship between the receptor and ligand and they found some other principles that are involved in stabilizing the receptor bound to the ligand such as the influence of receptor variation

It was found that altering the sequence of GPCR improves receptor stability. To prove that researchers designed the GPCR constructs with removing the entire or partial fragment from the N- or C- terminal and considering some features like the presence of cysteine residues, Glycosylation/phosphorylation sites, and the presence of conserved structural motifs and ligand binding sites on the receptor.

They found improved folding kinetics and stable conformation of the GPCR constructs with removed N- or C-terminal sequence while GPCR constructs with soluble fusion partner apocytochrome b562RIL (BRIL) and T4 lysozyme (T4L) more thermo-stabilized than the natural counterpart.

Researchers also employed a systematic alanine/leucine scanning approach to construct a library with individual residues mutated to either alanine or leucine and then they selected the construct by thermal stability assay.

Conclusion

All these approaches were intended to improve the expression level of the GPCR and to increase receptor stability. Receptor stability is therefore related to the ligand-induced conformational changes in the receptor and requires a mutual fit between the receptor and the ligand.

Therefore, the relation between receptor and ligand is an important area of drug discovery where screening of the compounds against a target G-protein linked cell surface receptor is a good way to find a compound that recognizes the receptor.