Green fluorescent proteins in receptor research: An emerging tool for drug discovery

In the last five years, green fluorescent protein (GFP) has emerged from being a mere curiosity to become a reliable tool for molecular pharmacological research. GFP produces an intense and stable green fluorescence noncatalytically by absorbing blue light maximally at 395 nm and emitting green light with a peak at 509 nm. It consists of 238 amino acids and its molecular mass is 27-30 kDa. GFP fluorescence occurs without cofactors and this property allows GFP fluorescence to be utilised in nonnative organisms, wherein it can be used as a reporter. This use of GFP permits real-time analysis of receptor dynamics. The emitted fluorescence can be used as a nontoxic marker and detected using fluorescence-activated cell sorting (FACS), thus avoiding any staining procedure, expensive mRNA analysis or hazardous radiolabeled binding assays. The potential value of GFP has also been recognized in orphan receptor research, where various GFP-tagged therapeutic proteins have been constructed in an attempt to identify the endogenous ligand(s). These chimeric proteins have been used to determine the site and time course of receptor expression and to relate receptor dynamics with therapeutic outcome. The preparation of new GFP constructs for identifying germ layer cells (endodermal, ectodermal, and mesodermal), as well as neuronal, haematopoietic, endothelial, and cartilage cells, has provided a useful battery of tissue/receptor-specific screening assays for new chemical entities. Genetically engineered cells with GFP expression have provided a valuable tool for automated analysis, and can be adapted for high-throughput systems. GFP is being increasingly utilised for the study of receptor dynamics, where, having already proved beneficial, it will likely continue to contribute towards the search for new classes of drugs, as well as to “de-orphaning” orphan receptors.