G-protein-coupled receptors (GPCRs), the largest family of integral membrane proteins, participate in the regulation of many physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases (approximately 30% of currently marketed drugs). However, despite their pharmacologic potential, clinically useful ligands do not exist for the majority of GPCRs. GPCRs are extremely hydrophobic, characterized by seven transmembrane-spanning Î±-helices. These receptors require a lipid environment to maintain the native structure, conformation and activity. Typically, GPCRs are expressed with low levels in native tissues.
The first challenge in the study of the binding activity of GPCRs is the development of a suitable expression system to correctly produce these proteins. A cell-free expression system has been developed and optimized by Synthelis for producing folded membrane proteins into liposomes to form proteoliposomes.
The second challenge is the development of biochemical or biophysical protocols to measure GPCR-ligand interactions. Surface Plasmon Resonance imaging (SPRi) represents an ideal method to directly study GPCR-ligand interactions. Also, this technology offers the opportunity for high-throughput label-free screening. The bottleneck is the immobilization of proteoliposomes containing membrane proteins to the sensor surface, conditions that retain the active conformation of the protein. Ligand binding is a good indication of correct conformation of a membrane protein.
We have developed a GPCR biosensor assay protocol using as model a Chemokine Receptor type 4 (CxCR4) which has great potential in drug therapy development. CxCR4 is involved in many diseases including cancer and immunodeficiency disorders. The functional proteoliposomes CxCR4 was tested by the binding of native chemokine ligand stromal cell-derived factor 1α (SDF1-1α) and two others specific ligands (injected molecules).