Immunolabeling and autoradiography have traditionally been applied as the methods-of-choice to visualize and collect molecular information about physiological and pathological processes. Here, we introduce PharmacoSTORM super-resolution imaging that combines the complementary advantages of these approaches and enables cell-type- and compartment-specific nanoscale molecular measurements. We exploited rational chemical design for fluorophore-tagged high-affinity receptor ligands and an enzyme inhibitor; and demonstrated broad PharmacoSTORM applicability for three protein classes and for cariprazine, a clinically approved antipsychotic and antidepressant drug. Because the neurobiological substrate of cariprazine has remained elusive, we took advantage of PharmacoSTORM to provide in vivo evidence that cariprazine predominantly binds to D₃ dopamine receptors on Islands of Calleja granule cell axons but avoids dopaminergic terminals. These findings show that PharmacoSTORM helps to quantify drug-target interaction sites at the nanoscale level in a cell-type- and subcellular context-dependent manner and within complex tissue preparations. Moreover, the results highlight the underappreciated neuropsychiatric significance of the Islands of Calleja in the ventral forebrain.

β-arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and β-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether β-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Since heterologous phosphorylation is a common phenomenon among GPCRs, this mode of β-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, G_q/11-coupled GPCR or epidermal growth factor receptor stimulation promotes β-arrestin2 recruitment to unliganded AT₁ angiotensin receptor (AT₁R). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and β-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C-terminus and two conserved phosphate-binding lysines in the β-arrestin2 N-domain. Using improved FlAsH-based β-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-β-arrestin interaction, but also governs the structural rearrangements within β-arrestins. Furthermore, we found that β-arrestin2 binds to PKC-phosphorylated AT₁R in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of β-arrestins and reveal their novel role in receptor cross-talk.

Biased agonism on the type I angiotensin receptor (AT₁-R) can achieve different outcomes via activation of G protein–dependent and –independent cellular responses. In this study, we investigated whether the biased activation of AT₁-R can lead to different regulation and intracellular processing of the receptor. We analyzed β-arrestin binding, endocytosis, and subsequent trafficking steps, such as early and late phases of recycling of AT₁-R in human embryonic kidney 293 cells expressing wild-type or biased mutant receptors in response to different ligands. We used Renilla luciferase–tagged receptors and yellow fluorescent protein–tagged β-arrestin2, Rab5, Rab7, and Rab11 proteins in bioluminescence resonance energy transfer measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. β-arrestin binding and the internalization kinetics of the angiotensin II–stimulated AT₁-R differed from those stimulated by the biased agonists. Similarly, angiotensin II–stimulated wild-type AT₁-R showed differences compared with a biased mutant AT₁-R (DRY/AAY AT₁-R) with regards to β-arrestin binding and endocytosis. We found that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane phosphatidylinositol 4,5-bisphosphate depletion. Moreover, the stability of the β-arrestin binding is a major determinant of the later fate of the internalized AT₁-R receptor.

The role of the highly conserved ‘DRY’ motif in the signaling of the CB₁cannabinoid receptor (CB₁R) was investigated by inducing single-, double-, and triple-alanine mutations into this site of the receptor. We found that the CB₁R-R3.50A mutant displays a partial decrease in its ability to activate heterotrimeric G_oproteins (∼80% of WT CB₁R (CB₁R-WT)). Moreover, this mutant showed an enhanced basal β-arrestin2 (β-arr2) recruitment. More strikingly, the double-mutant CB₁R-D3.49A/R3.50A was biased toward β-arrs, as it gained a robustly increased β-arr1 and β-arr2 recruitment ability compared with the WT receptor, while its G-protein activation was decreased. In contrast, the double-mutant CB₁R-R3.50A/Y3.51A proved to be G-protein-biased, as it was practically unable to recruit β-arrs in response to agonist stimulus, while still activating G-proteins, although at a reduced level (∼70% of CB₁R-WT). Agonist-induced ERK1/2 activation of the CB₁R mutants showed a good correlation with their β-arr recruitment ability but not with their G-protein activation or inhibition of cAMP accumulation. Our results suggest that G-protein activation and β-arr binding of the CB₁R are mediated by distinct receptor conformations, and the conserved ‘DRY’ motif plays different roles in the stabilization of these conformations, thus mediating both G-protein- and β-arr-mediated functions of CB₁R.