Multi-Color, Single-Molecule Fluorescence Imaging Of Gpcr Signalosomes

G protein-coupled receptors (GPCRs) are the largest superfamily of membrane receptors in the human genome and they are targets for a quarter of all prescription drugs. Activation of a GPCR by an agonist ligand results in G protein-mediated downstream signaling, followed by kinase action and arrestin-mediated desensitization, internalization/sequestration, and recycling. Selective manipulation of these individual steps of the GPCR activation cycle is often desired when creating drugs targeting a given receptor. We are interested in the C-C chemokine receptor CCR5 that is the major HIV coreceptor used in person-to-person transmission. Globally, the HIV/AIDS pandemic has caused nearly 30 million deaths and a similar number of people are currently infected. Certain analogues of the chemokine RANTES/CCL5 are highly potent entry inhibitors against R5-tropic HIV-1 strains, in vitro and in vivo. Three such analogues, 5P12-, 5P14-, and 6P4-RANTES, are particularly interesting because while they differ only slightly in structure they show strikingly different pharmacological profiles (G protein-linked signaling activity, stimulation of receptor internalization). We have recently developed a general, simple, and robust method for stoichiometric, site-specific fluorescence labeling of expressed GPCRs. The method is based on bioorthogonal conjugation of a fluorescent reporter group to a genetically encoded azido group introduced into expressed GPCRs using amber codon suppression.[1] We have adopted a similar strategy for fluorescent labeling of chemokines with azido groups introduced by chemical synthesis. Here we present our progress towards automated, multi-color, single-molecule fluorescence studies of the compositional and conformational dynamics of GPCR signaling complexes ("signalosomes") using fluorescently labeled chemokines and receptors in biochemically defined systems. [1] H Tian, TP Sakmar, & T Huber (2013) Site-specific labeling of genetically encoded azido groups for multi-color, single-molecule fluorescence imaging of GPCRs. Methods in Cell Biology, 117, in press.