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 ß(2)-adrenergic receptor (ß(2)AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged ß(2)AR FRET sensor. In this ß(2)AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard "full" agonist isoproterenol. Furthermore, norepinephrine-induced changes in the ß(2)AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial ß(2)AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of G(s) and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering ß-arrestin2 recruitment to the cell surface and its interaction with ß(2)AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the ß(2)AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to G(s) and less efficient in signaling to ß-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the ß(2)AR and link it to distinct conformational changes in the receptor.

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. Using the cell surface receptor for parathyroid hormone and its cytosolic binding protein, beta-arrestin2, we show their selective visualization in intact cells and analyze their interaction by colocalization and fluorescence resonance energy transfer (FRET). We propose that this method may be widely applied to label intracellular proteins and to study their interactions in intact cells with minimal disturbance of their function.

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. The corresponding receptor constructs were expressed in human embryonic kidney (HEK)-293 cells and stimulated with 100 muM ADP. Rapid receptor internalization was observed for the wild-type receptor and from those constructs mutated in the third intracellular loop and the proximal C terminus. However, the construct lacking phosphorylation sites at the distal C terminus did not show receptor internalization upon stimulation. The microscopic data were validated by HA-tagged receptor constructs using a cell surface enzyme-linked immunosorbent assay. P2Y(1)-receptor stimulated beta-arrestin-2-yellow fluorescent protein (YFP) translocation followed the same pattern as receptor internalization. Hence, no beta-arrestin-2-YFP translocation was observed when the distal C-terminal phosphorylation sites were mutated. Individual mutations indicate that residues Ser352 and Thr358 are essential for receptor internalization and beta-arrestin-2-YFP translocation. In contrast, protein kinase C (PKC)-mediated receptor desensitization was not affected by mutation of potential phosphorylation sites in the distal C terminus but was prevented by mutation of potential phosphorylation sites in the proximal C terminus. P2Y(1)-receptor internalization in HEK-293 cells was not blocked by inhibitors of PKC and calmodulin-dependent protein kinase. Thus, we conclude that P2Y(1)-receptor desensitization and internalization are mediated by different phosphorylation sites and kinases.

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. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor-beta-arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of beta-arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and beta-arrestin2 translocation. A beta2-adrenergic receptor truncated distal to residue 381 interacted normally with beta-arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and beta-arrestin, but its last eight amino acids facilitate receptor internalization in concert with beta-arrestin2.

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. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2-YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally co-transfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin-2-YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.