Site-specific in vitro and in vivo incorporation of molecular probes to study G-protein-coupled receptors

Chemical modification of proteins has a rich history in biochemistry and chemical biology. However, studies of membrane protein function, especially in cases where functional expression is low and purification and reconstitution are not feasible, present unique challenges. Heptahelical G-protein-coupled receptors (GPCRs) are a particularly important class of cell-surface receptors that represent targets of more than a quarter of all therapeutic drugs. Understanding with chemical precision how GPCRs function in biological membranes remains a central problem in biology. Recently a number of creative strategies have been developed that allow site-specific attachment of chemical probes or tags directly on expressed receptors or on biologically active peptide ligands or substrates. One particularly important advance is the genetic encoding of unnatural amino acids (UAAs) with unique small bioorthogonal tags using amber codon suppression in mammalian cells. This method should allow site-specific labeling of GPCRs with various molecular probes to facilitate cell-based studies of protein–protein or protein–ligand interactions and the visualization of conformational changes using fluorescence spectroscopy or single-molecule imaging.

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► Several creative strategies are now available to label expressed GPCRs.
► Genetically encoded unnatural amino acids can be introduced into expressed GPCRs.
► Site-specific bioorthogonal labeling reactions are possible.
► Selective photoactivatable crosslinkers can be introduced in cell-based systems.
► Labeling strategies can be combined to prepare fluorescently-labeled GPCRs.