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. 2023 Jan 26;34(4):590–606. doi: 10.1681/ASN.0000000000000082

Figure 4.

Figure 4

Grk4 does not require kinase activity during cilium formation. (A) Verification that Grk4 K216,217M resembles a kinase-dead version of Grk4 and that Grk4 c.580-585del is unable to modify GPCRs. Graph shows concentration-dependent recruitment of βArrestin 2 (βArr2) to the PTH1R on stimulation with PTH peptide. Recruitment was assessed using BRET. GRK5 served as positive control. n=4 (EV, Grk4, GRK5) and n=3 (Grk4 K216,217M, Grk4 c.580-585del). (B) Immunofluorescence of fibroblasts after nucleofection with siRNAs and plasmids. Reconstitution of siGRK4 cells successfully rescues cilium length. Red: cilia (acTub.), green: centrioles (gTub.), DAPI: nucleus. Scale bar: 10 µm. (C) Kinase-dead Grk4 rescues cilium length in human fibroblasts. n=98 (siCTRL), 95 (siGRK4), and 104 (siGRK4+grk4 K216,217M) cilia. Kruskal-Wallis test with Dunn multiple comparison test, ****P< 0.0001, **P=0.0026 (siCTRL versus siGRK4+grk4 K216,217M). Red line: median. (D) Cyst formation can be partially rescued using kinase-dead GRK4 (GRK4 K216,217M). Mean±SEM. Circles show individual experiments. n=8 experiments with 197–253 embryos in total. RM one-way ANOVA with Holm-Šídák multiple comparison test. ****P< 0.0001. (E) Kinase-dead GRK4 ameliorates edema formation upon Grk4 knockdown. Mean±SEM. Circles show individual experiments. n=4 experiments with 110–121 embryos in total. Brown-Forsythe and Welch ANOVA test. ***P=0.0004 (CTRL MO versus Grk4 MO), **P=0.0061 (Grk4 MO versus Grk4 MO+GRK4 K216,217M). (F) Cilia projecting into the lumen of mouse IMCD3 spheroids are longer after Grk4 depletion and can be rescued by wt zebrafish Grk4 and a kinase-dead variant. Representative z-stacks of spheroids, apical cell borders stained for ZO1 (green), and cilia in magenta. n=266 (siCTRL), 360 (siGRK4), 284 (siGrk4+grk4), and 292 (siGRK4+grk4 K216,217M) cilia. Kruskal-Wallis test with Dunn multiple comparison test, ****P< 0.0001. Red line: median. EV, empty vector; gTub., γTubulin.