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Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR–G protein–β-arr ‘megaplex’. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine β-arr to the core and phosphorylated tail, respectively, of a single active human chimeric β 2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (β 2V 2R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and β-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or “megaplexes” more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

G-protein-coupled receptors (GPCRs) are conventionally considered to function at the plasma membrane, where they detect extracellular ligands and activate heterotrimeric G proteins that transmit intracellular signals. Consequently, drug discovery efforts have focused on identification of agonists and antagonists of cell surface GPCRs. However, β-arrestin (ARR)-dependent desensitization and endocytosis rapidly terminate G protein signaling at the plasma membrane. Emerging evidence indicates that GPCRs can continue to signal from endosomes by G-protein- and βARR-dependent processes. By regulating the duration and location of intracellular signaling events, GPCRs in endosomes control critically important processes, including gene transcription and ion channel activity. Thus, GPCRs in endosomes, in addition to at the cell surface, have emerged as important therapeutic targets.

Luminal fluid reabsorption plays a fundamental role in male fertility. We demonstrated that the ubiquitous GPCR signaling proteins Gq and β-arrestin-1 are essential for fluid reabsorption because they mediate coupling between an orphan receptor ADGRG2 (GPR64) and the ion channel CFTR. A reduction in protein level or deficiency of ADGRG2, Gq or β-arrestin-1 in a mouse model led to an imbalance in pH homeostasis in the efferent ductules due to decreased constitutive CFTR currents. Efferent ductule dysfunction was rescued by the specific activation of another GPCR, AGTR2. Further mechanistic analysis revealed that β-arrestin-1 acts as a scaffold for ADGRG2/CFTR complex formation in apical membranes, whereas specific residues of ADGRG2 confer coupling specificity for different G protein subtypes, this specificity is critical for male fertility. Therefore, manipulation of the signaling components of the ADGRG2-Gq/β-arrestin-1/CFTR complex by small molecules may be an effective therapeutic strategy for male infertility.

β-Arrestins (βarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR-βarr complexes: the "tail" conformation, with βarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the "core" conformation, where, in addition to the phosphorylated C-terminal tail, βarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of βarrs is unknown. Here, we created a mutant form of βarr lacking the "finger-loop" region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and βarr signaling but not desensitization of G protein signaling. Thus, the two GPCR-βarr conformations can carry out distinct functions.

Acute hormone secretion triggered by G protein-coupled receptor (GPCR) activation underlies many fundamental physiological processes. GPCR signalling is negatively regulated by β-arrestins, adaptor molecules that also activate different intracellular signalling pathways. Here we reveal that TRV120027, a β-arrestin-1-biased agonist of the angiotensin II receptor type 1 (AT1R), stimulates acute catecholamine secretion through coupling with the transient receptor potential cation channel subfamily C 3 (TRPC3). We show that TRV120027 promotes the recruitment of TRPC3 or phosphoinositide-specific phospholipase C (PLCγ) to the AT1R-β-arrestin-1 signalling complex. Replacing the C-terminal region of β-arrestin-1 with its counterpart on β-arrestin-2 or using a specific TAT-P1 peptide to block the interaction between β-arrestin-1 and PLCγ abolishes TRV120027-induced TRPC3 activation. Taken together, our results show that the GPCR-arrestin complex initiates non-desensitized signalling at the plasma membrane by coupling with ion channels. This fast communication pathway might be a common mechanism of several cellular processes., Angiotensin II type 1 receptor (AT1R)-mediated acute catecholamine release is modulated by β-arrestin. Here the authors show that β-arrestin-1 recruits the Ca2+ channel TRPC3 and the PLCγ to the AT1R-β-arrestin complex, triggering G protein-independent calcium influx and catecholamine secretion.

G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.