Phosphorylation of G protein–coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with β-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through β-arrestin–specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of β-arrestin engagement may aid in the discovery of therapeutic agents targeting individual β-arrestin functions. The β2-adrenergic receptor (β2AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the β2AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein–mediated and β-arrestin–mediated pathways or with a biased ligand that promotes signaling only through β-arrestin–mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of β-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a “barcode” that imparts distinct conformations to the recruited β-arrestin, thus regulating its functional activities.

Ubiquitously expressed seven-transmembrane receptors (7TMRs) classically signal through heterotrimeric G proteins and are commonly referred to as G protein-coupled receptors. It is now recognized that 7TMRs also signal through β-arrestins, which act as versatile adapters controlling receptor signaling, desensitization, and trafficking. Most endogenous receptors appear to signal in a balanced fashion using both β-arrestin and G protein-mediated pathways. Some 7TMRs are thought to be nonsignaling “decoys” because of their inability to activate typical G protein signaling pathways; it has been proposed that these receptors act to scavenge ligands or function as coreceptors. Here we demonstrate that ligand binding to the decoy receptor CXCR7 does not result in activation of signaling pathways typical of G proteins but does activate MAP kinases through β-arrestins in transiently transfected cells. Furthermore, we observe that vascular smooth muscle cells that endogenously express CXCR7 migrate to its ligand interferon-inducible T-cell alpha chemoattractant (ITAC), an effect that is significantly attenuated by treatment with either a CXCR7 antagonist or β-arrestin depletion by siRNA. This example of an endogenous “β-arrestin-biased” 7TMR that signals through β-arrestin in the absence of G protein activation demonstrates that some 7TMRs encoded in the genome have evolved to signal through β-arrestin exclusively and suggests that other receptors that are currently thought to be orphans or decoys may also signal through such nonclassical pathways.

Seven transmembrane receptors (7TMRs), commonly referred to as G protein-coupled receptors, form a large part of the "druggable" genome. 7TMRs can signal through parallel pathways simultaneously, such as through heterotrimeric G proteins from different families, or, as more recently appreciated, through the multifunctional adapters, ␤-arrestins. Biased agonists, which signal with different efficacies to a receptor's multiple downstream pathways, are useful tools for deconvoluting this signaling complexity. These compounds may also be of therapeutic use because they have distinct functional and therapeutic profiles from "balanced agonists." Although some methods have been proposed to identify biased ligands, no comparison of these methods applied to the same set of data has been performed. Therefore, at this time, there are no generally accepted methods to quantify the relative bias of different ligands, making studies of biased signaling difficult. Here, we use complementary computational approaches for the quantification of ligand bias and demonstrate their application to two well known drug targets, the ␤2 adrenergic and angiotensin II type 1A receptors. The strategy outlined here allows a quantification of ligand bias and the identification of weakly biased compounds. This general method should aid in deciphering complex signaling pathways and may be useful for the development of novel biased therapeutic ligands as drugs.