Differential signaling of the endogenous agonists at the beta2-adrenergic receptor.

The concept of "functional selectivity" or "biased signaling" suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the β(2)-adrenergic receptor (β(2)AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged β(2)AR FRET sensor.

Site-specific, orthogonal labeling of proteins in intact cells with two small biarsenical fluorophores.

The fusion of fluorescent proteins to proteins of interest has greatly advanced fluorescence microscopy, but is often limited by their large size. Here, we report site-specific, orthogonal labeling of two cellular proteins in intact cells with two small fluorescent dyes: fluorescein arsenical hairpin binder, FlAsH, and its red analogue, ReAsH, which bind to tetracysteine motifs. Development of a sequential labeling method to two different motifs, CCPGCC and FLNCCPGCCMEP, allowed site-specific labeling with FlAsH and ReAsH, respectively.

beta-Arrestin-2 interaction and internalization of the human P2Y1 receptor are dependent on C-terminal phosphorylation sites.

The nucleotide receptor P2Y(1) regulates a variety of physiological processes and is involved in platelet aggregation. Using human P2Y(1)-receptors C-terminally fused with a fluorescent protein, we studied the role of potential receptor phosphorylation sites in receptor internalization and beta-arrestin-2 translocation by means of confocal microscopy. Three receptor constructs were generated that lacked potential phosphorylation sites in the third intracellular loop, the proximal C terminus, or the distal C terminus.

Dual role of the beta2-adrenergic receptor C terminus for the binding of beta-arrestin and receptor internalization.

Homologous desensitization of beta2-adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind beta-arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by beta-arrestins, we have investigated beta-arrestin interaction and internalization of a set of mutants of the human beta2-adrenergic receptor.

Agonist-selective, receptor-specific interaction of human P2Y receptors with beta-arrestin-1 and -2.

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern.