No abstract

(β-)Arrestins are important regulators of G-protein-coupled receptors (GPCRs)1–3. They bind to active, phosphorylated GPCRs and thereby shut off ‘classical’ signalling to G proteins3,4, trigger internalization of GPCRs via interaction with the clathrin machinery5–7 and mediate signalling via ‘non-classical’ pathways1,2. In addition to two visual arrestins that bind to rod and cone photoreceptors (termed arrestin1 and arrestin4), there are only two (non-visual) β-arrestin proteins (β-arrestin1 and β-arrestin2, also termed arrestin2 and arrestin3), which regulate hundreds of different (non-visual) GPCRs. Binding of these proteins to GPCRs usually requires the active form of the receptors plus their phosphorylation by G-protein-coupled receptor kinases (GRKs)1,3,4. The binding of receptors or their carboxy terminus as well as certain truncations induce active conformations of (β-)arrestins that have recently been solved by X-ray crystallography8–10. Here we investigate both the interaction of β-arrestin with GPCRs, and the β-arrestin conformational changes in real time and in living human cells, using a series of fluorescence resonance energy transfer (FRET)-based β-arrestin2 biosensors. We observe receptor-specific patterns of conformational changes in β-arrestin2 that occur rapidly after the receptor–β-arrestin2 interaction. After agonist removal, these changes persist for longer than the direct receptor interaction. Our data indicate a rapid, receptor-type-specific, two-step binding and activation process between GPCRs and β-arrestins. They further indicate that β-arrestins remain active after dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. Thus, GPCRs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signalling.

Aiming to design partial agonists as well as allosteric modulators for the M muscarinic acetylcholine (MAChR) receptor, two different series of bipharmacophoric ligands and their structural analogues were designed and synthesized. The hybrids were composed of the benzyl quinolone carboxylic acid (BQCA)-derived subtype selective allosteric modulator 3 and the orthosteric building block 4-((4,5-dihydroisoxazol-3-yl)oxy)-N,N-dimethylbut-2-yn-1-amine (base of iperoxo) 1 or the endogenous ligand 2-(dimethylamino)ethyl acetate (base of acetylcholine) 2, respectively. The two pharmacophores were linked via alkylene chains of different lengths (C4, C6, C8, and C10). Furthermore, the corresponding structural analogues of 1 and 2 and of modified BQCA 3 with varying alkyl chain length between C2 and C10 were investigated. Fluorescence resonance energy transfer (FRET) measurements in a living single cell system were investigated in order to understand how these compounds interact with a G protein-coupled receptor (GPCR) on a molecular level and how the single moieties contribute to ligand receptor interaction. The characterization of the modified orthosteric ligands indicated that a linker attached to an orthoster rapidly attenuates the receptor response. Linker length elongation increases the receptor response of bitopic ligands, until reaching a maximum, followed by a gradual decrease. The optimal linker length was found to be six methylene groups at the MAChR. A new conformational change is described that is not of inverse agonistic origin for long linker bitopic ligands and was further investigated by exceptional fragment-based screening approaches.

Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G q -coupled M 3 acetylcholine receptor (M 3 -AChR) with that of a constitutively active mutant receptor (M 3 -AChR-N514Y) using M 3 -AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M 3 -AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M 3 -AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-Gprotein interaction was measured by FRET between M 3 -AChRyellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-G␥ 2 . Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M 3 -AChR-N514Y reached only 12% of that of M 3 -AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged G␣ q and CFP-tagged G␥ 2 . Activation of G q was significantly slower than receptor activation and indistinguishable for the two agonists. However, G q deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agoniststimulated coupling to G q , agonist-stimulated G q activation by M 3 -AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M 3 -AChR by decreasing the rate of receptor deactivation, while having minimal effect on receptor activation.