Analysis of GTP-Binding Protein–Coupled Receptor Assemblies by Flow Cytometry

GTP-binding protein–coupled receptors (GPCRs) represent the largest family of integral membrane signal-transducing molecules in the human genome, with estimates of at least 600 members. As such, they represent the targets of approximately 30%–50% of the prescription drugs on the market. They are involved in virtually every physiological process in the human body, with ligands including light, odorants, amines, peptides, proteins, lipids, and nucleotides. Binding of these ligands on the extracellular surface of the receptor leads to conformational changes within the receptor, resulting in a multitude of cellular responses. GPCRs, as their name implies, function through the actions of heterotrimeric GTP-binding proteins (G proteins). These G proteins then couple to a diverse array of effector molecules at the cell surface and inside the cell. GPCRs contain a common structural motif, with seven transmembrane alpha helices. With the recent description of the three-dimensional crystal structure of rhodopsin in its inactive state, a greater, though still incomplete, understanding of the functions of this receptor family has been achieved. In addition to the activation of G proteins, GPCRs undergo extensive regulation mediated primarily by a variety of kinases, including second messenger kinases and the family of G protein–coupled receptor kinases (GRKs). Following receptor phosphorylation by GRKs, additional proteins named arrestins associate with GPCRs. The traditional role of these molecules has been to serve as desensitizing agents, preventing further association of the receptor with G proteins. However, recent studies have demonstrated that arrestins can serve as adapters in the process of receptor internalization as well as scaffolds in the activation of numerous kinase pathways. Interactions between GPCRs and cellular proteins such as adaptins, rab GTPases, phosphatases, and ion channels have also been described. Thus, it has become apparent that understanding the interactions between GPCRs and their associated proteins is critical for any detailed understanding of receptor function. An overview of the activation and regulation of GPCRs is shown in figure 17.1 to provide a context for the approaches to be described in the remainder of this chapter.