Fig 2: Schematic illustration indicating GRK2 (G protein-coupled receptor kinase 2) is associated with ADRB1 (adrenergic receptor beta 1) internalization and cardiac hypertrophy. A, In hypertension, ADRB1 is activated via interacting with circulating catecholamines, eg, epinephrine. GRK2 participates in the regulation of ADRB1 by triggering ADRB1 desensitization from G proteins complex and internalization, thus contributing to signal propagation. B, Paroxetine suppresses ADRB1 internalization via functioning as the GRK2 inhibitor, thus enhances the stabilization and activity of ADRB1 on the surface of cardiomyocytes. Therefore, catecholamines in circulation decreases and reduces the stimulation of cardiomyocytes resulting in alleviation of cardiac hypertrophy. ADRB1 indicates adrenergic receptors beta 1; and GRK2, G-protein-coupled receptor kinase 2.

Fig 3: GRK2 (G protein-coupled receptor kinase 2) inhibitor promotes beta-1 blockers (metoprolol) therapeutic effect in spontaneously hypertensive rat (SHR). A, The dynamic change in systolic blood pressure (upper panel) and heart rate (lower panel) of SHR and SHR treated with paroxetine, metoprolol or both (n=6 rats per group) at the age from 10 to 22 weeks. B, Up, overall comparison of representative whole-heart tissue (first panel; scale bar, 1 cm), hematoxylin and eosin staining of whole-heart left ventricular mid–papillary muscle plane sections (second panel; scale bar, 5 mm) and picrosirius red staining of the perivascular area (third panel; scale bar, 50 µm) and interstitial area (fourth panel; scale bar, 25 µm) in the indicated groups (n=6 rats per group). Bottom, quantitative results pertaining to LV collagen volumes in the indicated groups (n=20 fields per group). C, Comparison of the heart weight, heart weight to body weight ratio and heart weight to tibia length ratio in the indicated groups (n=6 per group) at the age of 22 weeks (n=6 rats per group). D, Comparison of the echocardiographic parameters interventricular septal thickness at diastole, interventricular septal thickness in systole, ejection fraction in the indicated groups at the age from 10 to 22 weeks (n=6 rats per group). Data shown are mean±SD (A through D). Data were first analyzed and passed normality test (Shapiro-Wilk test in A through D). P values were shown and assessed by unpaired 2-tailed t-test (A and D) and by 1-way ANOVA with Tukey test (B and C). In (A and D), *P<0.05 (compared with the SHR group), # P<0.05 (compared with the SHR+metoprolol group). BW indicates body weight; EF, ejection fraction; CTGF, connective tissue growth factor; H&E, hematoxylin and eosin; HR, heart rate; HW, heart weight; IVSd, interventricular septal thickness in diastole; IVSs, interventricular septal thickness in systole; PRS, picrosirius red stain; SHR, spontaneously hypertensive rats; and TL, tibia length.

Fig 4: 19F-NMR spectra revealed the different structural states of ß-arrestin-1 in response to different phospho-binding patterns.(a) Common structural changes that occur after ß-arrestin-1 activation induced by the binding of functional phosphopeptides. These results suggest the existence of common conformational states representing ß-arrestin-1 activation after functional phosphopeptide binding. (b) Specific structural rearrangements after GRK2pp binding. (c) GRK6-encoded specific structural rearrangements at F277 position. (d) V2Rpp specifically stimulated conformational change at Y209 position. (e) Proposed models illustrating the receptor phospho-barcode-encoded distinct arrestin conformations that dictate specific downstream signalling. The phosphorylated C-tail of the receptor (in orange) extends into the cytoplasm. Interaction with the receptor phospho-C-tail induces a structural rearrangement of ß-arrestin-1 that includes residues in the finger loop (such as F75) and the middle loop (such as T136); these residues (in purple) might participate in the receptor transmemberane core interaction. Different GRKs encode different conformations in the C-lobe of ß-arrestin-1. GRK2 encodes specific conformations of Y249 and L338 (blue triangles), which are recognized by clathrin (in blue). GRK6 encodes distinct ß-arrestin-1 conformations, such as the conformation of F277 (red triangle), which might be recognized by signalling proteins such as SRC (in red). Specific V2Rpp-induced conformational change is localized at Y209 (in orange).

Fig 5: Different phospho-barcodes encode different arrestin functions and conformations.(a) Activation of ß2-AR-induced conformational changes in ß-arrestin-1 as measured using intramolecular BRET. Plasmids encoding a ß-arrestin-1 BRET biosensor (Luc-ß-arrestin-1-YFP) and Flag-ß2-AR were transiently co-transfected in HEK293 cells. After starvation, the cells were stimulated with ISO (10 µM) for 10 min. The effects of GRK phosphorylation of ß2-AR on ISO-induced ß-arrestin-1 conformational change were probed using siRNA-mediated GRK knockdown. (b) A representative western blotting is shown for the effects of knockdown of specific GRKs in ß2-AR-mediated proto-oncogene tyrosine-protein kinase (SRC) activation. Left panel: HEK293 cells that were transiently transfected with Flag-ß2-AR were stimulated with ISO for 1, 5 and 15 min. To examine the roles of GRK2 and GRK6 in ß2-AR-mediated SRC activation, siRNAs that were selective for specific GRKs were transfected into the cells. A western blot representative of at least three independent experiments is shown on the left. Right panel: western blot signals of phospho-SRC 416 (a) were quantified using densitometry and are expressed as fold change versus basal level. Whereas GRK6 is required for SRC activation downstream of ß2-AR activation, GRK2 is a negative regulator of ß2-AR-induced SRC activation. (a,b) At least three independent experiments were performed. #P<0.05, ISO-stimulated cells are compared with unstimulated cells. *P<0.05; **P<0.01; ***P<0.005; GRK siRNA-treated cells are compared with control siRNA-treated cells. (c,d) Effects of phospho-peptide binding on ß-arrestin-1/clathrin complex formation (c) or ß-arrestin-1/SRC-SH2-SH3-kinase-three domain (SRC3D) complex formation (d). ß-arrestin-1 (300 nM) was incubated with an equal concentration of various phosphopeptides and either GST-clathrin 1–494 (c) or GST-SRC3D (d). The complexes were pulled down using GST beads and the amount of ß-arrestin-1 bound to clathrin or SRC3D was monitored using a specific ß-arrestin-1 antibody (left panel in each figure). At least three independent experiments for each assay were performed. Western blot signals of ß-arrestin-1 bound to clathrin (c) or SRC3D (d) from the left panels were quantified using densitometry and are shown as columns (right panel). **P<0.01; ***P<0.005; phosphopeptide stimulated ß-arrestin-1/clathrin complex formation (c) or ß-arrestin-1/SRC complex formation (d) are compared with the basal state.