Abstract
G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays.
Highlights
G protein-coupled receptors (GPCRs) contain the characteristic seven-transmembrane domain and represent one of the largest families of signal-transducing receptors [1]
The interaction of phosphorylated GPCRs with ß-arrestins followed by receptor accumulation in clathrin-coated pits (CCPs) is the critical event that initiates agonist-induced GPCR internalization. ß-arrestins are scaffolding molecules recruited from the cytosol to the activated receptors at the cell surface [4]. ß-arrestins inhibit the interaction of GPCRs with G proteins for signal desensitization, and promote GPCR internalization by binding to the activated receptors and to structural components of the CCPs
We developed a novel GPCR internalization assay utilizing HaloTag technology
Summary
G protein-coupled receptors (GPCRs) contain the characteristic seven-transmembrane domain and represent one of the largest families of signal-transducing receptors [1]. SS-arrestins inhibit the interaction of GPCRs with G proteins for signal desensitization, and promote GPCR internalization by binding to the activated receptors and to structural components of the CCPs. The endocytic trafficking pathway of individual GPCRs is remarkably varied. Some GPCRs have been shown to be stable after prolonged and/or repeated ligand stimulation, suggesting that they immediately recycle back to the plasma membrane after endocytosis. This second type of GPCR has the ability to recover quickly from a period of desensitization and thereby maintains cellular responsiveness to the cognate ligands. Distinct endocytic trafficking routes of otherwise similar GPCRs lead to different functional effects on cellular responsiveness to GPCR ligands
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