G-protein coupled receptors (GPCRs) are versatile cellular sensors for chemical stimuli, but also serve as mechanosensors involved in various (patho)physiological settings like vascular regulation, cardiac hypertrophy and preeclampsia. However, the molecular mechanisms underlying mechanically induced GPCR activation have remained elusive. Here we show that mechanosensitive histamine H₁ receptors (H₁Rs) are endothelial sensors of fluid shear stress and contribute to flow-induced vasodilation. At the molecular level, we observe that H₁Rs undergo stimulus-specific patterns of conformational changes suggesting that mechanical forces and agonists induce distinct active receptor conformations. GPCRs lacking C-terminal helix 8 (H8) are not mechanosensitive, and transfer of H8 to non-responsive GPCRs confers, while removal of H8 precludes, mechanosensitivity. Moreover, disrupting H8 structural integrity by amino acid exchanges impairs mechanosensitivity. Altogether, H8 is the essential structural motif endowing GPCRs with mechanosensitivity. These findings provide a mechanistic basis for a better understanding of the roles of mechanosensitive GPCRs in (patho)physiology.

G protein-coupled receptors (GPCRs) activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and the formation of receptor–arrestin complexes. However, the impact of individual GRKs on arrestin binding is not clear. We report the creation of eleven combinatorial HEK293 knockout cell clones lacking GRK2/3/5/6, including single, double, triple and the quadruple GRK knockout. Analysis of β-arrestin1/2 interactions for twelve GPCRs in our GRK knockout cells enables the differentiation of two main receptor subsets: GRK2/3-regulated and GRK2/3/5/6-regulated receptors. Furthermore, we identify GPCRs that interact with β-arrestins via the overexpression of specific GRKs even in the absence of agonists. Finally, using GRK knockout cells, PKC inhibitors and β-arrestin mutants, we present evidence for differential receptor–β-arrestin1/2 complex configurations mediated by selective engagement of kinases. We anticipate our GRK knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and β-arrestin complex formation.

β-arrestins mediate regulatory processes for over 800 different G protein-coupled receptors (GPCRs) by adopting specific conformations that result from the geometry of the GPCR–β-arrestin complex. However, whether β-arrestin1 and 2 respond differently for binding to the same GPCR is still unknown. Employing GRK knockout cells and β-arrestins lacking the finger-loop-region, we show that the two isoforms prefer to associate with the active parathyroid hormone 1 receptor (PTH1R) in different complex configurations (“hanging” and “core”). Furthermore, the utilisation of advanced NanoLuc/FlAsH-based biosensors reveals distinct conformational signatures of β-arrestin1 and 2 when bound to active PTH1R (P-R*). Moreover, we assess β-arrestin conformational changes that are induced specifically by proximal and distal C-terminal phosphorylation and in the absence of GPCR kinases (GRKs) (R*). Here, we show differences between conformational changes that are induced by P-R* or R* receptor states and further disclose the impact of site-specific GPCR phosphorylation on arrestin-coupling and function.

Significance: Class A G protein−coupled receptors (GPCRs) can form dimers and oligomers via poorly understood mechanisms. We show here that the chemokine receptor CXCR4, which is a major pharmacological target, has an oligomerization behavior modulated by its active conformation. Combining advanced, single-molecule, and single-cell optical tools with functional assays and computational approaches, we unveil three key features of CXCR4 quaternary organization: CXCR4 dimerization 1) is dynamic, 2) increases with receptor expression level, and 3) can be disrupted by stabilizing an inactive receptor conformation. Ligand binding motifs reveal a ligand binding subpocket essential to modulate both CXCR4 basal activity and dimerization. This is relevant to develop new strategies to design CXCR4-targeting drugs.

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and their signal transduction is tightly regulated by GPCR kinases (GRKs) and β-arrestins. In this review, we discuss novel aspects of the regulatory GRK/β-arrestin system. Therefore, we briefly revise the origin of the “barcode” hypothesis for GPCR/β-arrestin interactions, which states that β-arrestins recognize different receptor phosphorylation states to induce specific functions. We emphasize two important parameters which may influence resulting GPCR phosphorylation patterns: (A) direct GPCR–GRK interactions and (B) tissue-specific expression and availability of GRKs and β-arrestins. In most studies that focus on the molecular mechanisms of GPCR regulation, these expression profiles are underappreciated. Hence we analyzed expression data for GRKs and β-arrestins in 61 tissues annotated in the Human Protein Atlas. We present our analysis in the context of pathophysiological dysregulation of the GPCR/GRK/β-arrestin system. This tissue-specific point of view might be the key to unraveling the individual impact of different GRK isoforms on GPCR regulation.

Ligand binding induces conformational changes in FZD ₅ , activation of heterotrimeric G proteins, and G _q -dependent signaling.