Quantification of GPCR internalization by single-molecule microscopy in living cells

Abstract

Receptor internalization upon ligand stimulation is a key component of a cell's response and allows a cell to correctly sense its environment. Novel fluorescent methods have enabled the direct visualization of the agonist-stimulated G-protein-coupled receptors (GPCR) trafficking in living cells. However, it is difficult to observe internalization of GPCRs in vivo due to intrinsic autofluorescence and cytosolic signals of fluorescently labeled GPCRs. This study uses the superior positional accuracy of single-molecule fluorescence microscopy to visualize in real time the internalization of Dictyostelium discoideum cAMP receptors, cAR1, genetically encoded with eYFP. This technique made it possible to follow the number of receptors in time revealing that the fraction of cytosolic receptors increases after persistent agonist stimulation and that the majority of the receptors were degraded after internalization. The observed internalization process was phosphorylation dependent, as shown with the use of a phosphorylation deficient cAR1 mutant, cm1234-eYFP, or stimulation with an antagonist, Rp-cAMPS that does not induce receptor phosphorylation. Furthermore, experiments done in mound-stage cells suggest that intrinsic, phosphorylation-induced internalization of cAR1 is necessary for Dictyostelium wild type cells to progress properly through multicellular development. To our knowledge, this observation illustrates for the first time phosphorylation-dependent internalization of single cAR1 molecules in living cells and its involvement in multicellular development. This very sensitive imaging of receptor internalization can be a useful and universal approach for pharmacological characterization of GPCRs in other cell types.

Direct Biochemical Measurements of Signal Relay during Dictyostelium Development

“…Similarly, in Dictyostelium, it has been shown that the cAMP-induced desensitization of cAR1 proceeds in at least two steps. The first step is rapid (min), is reversible, occurs at low cAMP concentrations, and involves a decrease in receptor affinity without down-regulation (8,38,47). The second step is essentially irreversible, requires prolonged cAMP exposure (hours), and leads to the down-regulation of receptors, which become either sequestered within the plasma membrane or internalized (10 -12).…”

Toxicity of an α-Pore-forming Toxin Depends on the Assembly Mechanism on the Target Membrane as Revealed by Single Molecule Imaging

“…2A). As the oligomerization kinetic takes place in the time frame of cell death and we observed labeled proteins in the cells surface adhered to the glass, toxin molecules in free solution are able to access such regions of the plasma membrane from the water interface between the adherent cells and the glass, and as a consequence this population can be considered as representative for EqtII effect (28).…”

Phosphorylation of chemoattractant receptors regulates chemotaxis, actin re-organization, and signal-relay

“…Exposure to a saturating dose of cAMP will downregulate CAR1 from the cell surface (Van Haastert, 1987a) and recent evidence, using single-particle imaging techniques, suggests that a fraction of cell surface CAR1 is internalized by a process that requires receptor phosphorylation and occurs within minutes of a global stimulus (Sergé et al, 2011). Although a group of proteins with arrestin-like domains has been identified in Dictyostelium (Guetta et al, 2010), none have been studied for associative roles with CAR1 or analyzed in the context of chemotaxis.…”

“…CAR1 phosphorylation/dephosphorylation oscillates concomitantly with the periodic rise and fall of extracellular cAMP during aggregation (Klein et al, 1985), yet CAR1 phosphorylation is non-adaptive and persists if cAMP concentrations are constant (Vaughan and Devreotes, 1988). Receptor downregulation in Dictyostelium , as defined by the loss of cAMP binding sites (Van Haastert, 1987a) or receptor internalization (Sergé et al, 2011), is observed in cells exposed to saturating (µM) levels of ligand. At sub-saturating, non-varying cAMP concentrations, which promotes adaptation/desensitization, binding sites are maintained, but with modified affinity (Van Haastert, 1987a).…”