ABSTRACT
G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the ß2 adrenoceptor. These receptors co-localize with a variety of G proteins even before receptor activation, and activated receptors are largely sequestered from G proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as a potential regulator of ß2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.
Subject(s)
Ascorbate Peroxidases/chemistry , Receptor, Angiotensin, Type 1/analysis , Receptor, Angiotensin, Type 1/metabolism , Signal Transduction , Staining and Labeling/methods , Ascorbate Peroxidases/metabolism , Biotin/chemistry , GTP-Binding Proteins/analysis , HEK293 Cells , Humans , Oligopeptides/pharmacology , Protein Engineering , Receptor, Angiotensin, Type 1/agonists , beta-Arrestins/chemistryABSTRACT
For reasons that remain insufficiently understood, the brain requires among the highest levels of metals in the body for normal function. The traditional paradigm for this organ and others is that fluxes of alkali and alkaline earth metals are required for signaling, but transition metals are maintained in static, tightly bound reservoirs for metabolism and protection against oxidative stress. Here we show that copper is an endogenous modulator of spontaneous activity, a property of functional neural circuitry. Using Copper Fluor-3 (CF3), a new fluorescent Cu(+) sensor for one- and two-photon imaging, we show that neurons and neural tissue maintain basal stores of loosely bound copper that can be attenuated by chelation, which define a labile copper pool. Targeted disruption of these labile copper stores by acute chelation or genetic knockdown of the CTR1 (copper transporter 1) copper channel alters the spatiotemporal properties of spontaneous activity in developing hippocampal and retinal circuits. The data identify an essential role for copper neuronal function and suggest broader contributions of this transition metal to cell signaling.