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Arrestins regulate cell spreading and motility via focal adhesion dynamics.

Cleghorn WM, Branch KM, Kook S, Arnette C, Bulus N, Zent R, Kaverina I, Gurevich EV, Weaver AM, Gurevich VV - Mol. Biol. Cell (2014)

Bottom Line: Clathrin exhibited decreased dynamics near FA in arrestin-deficient cells.In contrast to wild-type arrestins, mutants deficient in clathrin binding did not rescue the phenotype.Collectively the data indicate that arrestins are key regulators of FA disassembly linking microtubules and clathrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232.

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Arrestin interaction with clathrin contributes to arrestin-dependent regulation of cell morphology. (A–E) Representative images of DKO cells expressing HA-tagged arrestin stained with anti-HA antibodies (green) and rhodamine–phalloidin (red). (A) Control DKO MEFs transfected with HA-Rluc. (B) DKO MEFs transfected with WT arrestin-2. (C) DKO MEFs expressing clathrin binding–deficient A2CBD mutant. (D) DKO MEFs expressing WT arrestin-3. (E) DKO MEFS expressing A3CBD mutant. (F) Scatter plot showing the results of cell size analysis. The horizontal bars represent the medians. The cell size data were analyzed by Kruskal–Wallis analysis of variance, followed by posthoc pairwise comparison by Mann–Whitney test with Bonferroni correction for multiple comparisons. Data are from ∼200 cells/condition from four independent experiments. *p < 0.001, **p < 0.01; *p < 0.05, as compared with cells expressing RLuc; ap < 0.05; cp < 0.001, as compared with cells expressing corresponding WT arrestins.
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Figure 6: Arrestin interaction with clathrin contributes to arrestin-dependent regulation of cell morphology. (A–E) Representative images of DKO cells expressing HA-tagged arrestin stained with anti-HA antibodies (green) and rhodamine–phalloidin (red). (A) Control DKO MEFs transfected with HA-Rluc. (B) DKO MEFs transfected with WT arrestin-2. (C) DKO MEFs expressing clathrin binding–deficient A2CBD mutant. (D) DKO MEFs expressing WT arrestin-3. (E) DKO MEFS expressing A3CBD mutant. (F) Scatter plot showing the results of cell size analysis. The horizontal bars represent the medians. The cell size data were analyzed by Kruskal–Wallis analysis of variance, followed by posthoc pairwise comparison by Mann–Whitney test with Bonferroni correction for multiple comparisons. Data are from ∼200 cells/condition from four independent experiments. *p < 0.001, **p < 0.01; *p < 0.05, as compared with cells expressing RLuc; ap < 0.05; cp < 0.001, as compared with cells expressing corresponding WT arrestins.

Mentions: Arrestins promote GPCR internalization by virtue of their interaction with clathrin (Goodman et al., 1996) and the clathrin adaptor AP2 (Laporte et al., 2000). Mutations eliminating clathrin- or AP2-binding sites in the arrestin C-tail significantly reduce receptor internalization (Kim and Benovic, 2002). Clathrin was implicated in microtubule-dependent FA disassembly (Ezratty et al., 2009), suggesting that a defect in clathrin recruitment to FA in arrestin-2/3 DKO cells might contribute to their peculiar morphology. To test this idea, we compared DKO cell rescue by WT arrestin-2 and -3 and mutants with disabled clathrin-binding sites, A2CBD and A3CBD, respectively. These four-residue mutations do not perturb other arrestin functions (Kim and Benovic, 2002). DKO cells transfected with HA-tagged arrestins or HA-RLuc and plated on FN were stained for HA tag and actin (Figure 6A). HA-RLuc was used as a control to ensure appropriate selection of transfected cells and rule out the effect of transfection on cell morphology. We confirmed that the expression of WT arrestin-2 or -3 significantly reduced DKO cell size (Figure 6, A, B, D, and F). Arr2CBD had no effect, and the similar arrestin-3 mutant A3CBD reduced cell size much less effectively than WT arrestin-3 (Figure 6, C, E, and F). Partial effectiveness of this mutant might be the result of indirect clathrin binding via AP2. The AP2-binding site was not disabled because it includes an arginine that is part of the main phosphate sensor, and therefore AP2 binding cannot be completely eliminated without changing other functional characteristics of arrestins. The data suggest that arrestins are involved in microtubule-dependent FA regulation, and their binding to clathrin is necessary for the ability of arrestins to rescue the DKO phenotype.


Arrestins regulate cell spreading and motility via focal adhesion dynamics.

Cleghorn WM, Branch KM, Kook S, Arnette C, Bulus N, Zent R, Kaverina I, Gurevich EV, Weaver AM, Gurevich VV - Mol. Biol. Cell (2014)

Arrestin interaction with clathrin contributes to arrestin-dependent regulation of cell morphology. (A–E) Representative images of DKO cells expressing HA-tagged arrestin stained with anti-HA antibodies (green) and rhodamine–phalloidin (red). (A) Control DKO MEFs transfected with HA-Rluc. (B) DKO MEFs transfected with WT arrestin-2. (C) DKO MEFs expressing clathrin binding–deficient A2CBD mutant. (D) DKO MEFs expressing WT arrestin-3. (E) DKO MEFS expressing A3CBD mutant. (F) Scatter plot showing the results of cell size analysis. The horizontal bars represent the medians. The cell size data were analyzed by Kruskal–Wallis analysis of variance, followed by posthoc pairwise comparison by Mann–Whitney test with Bonferroni correction for multiple comparisons. Data are from ∼200 cells/condition from four independent experiments. *p < 0.001, **p < 0.01; *p < 0.05, as compared with cells expressing RLuc; ap < 0.05; cp < 0.001, as compared with cells expressing corresponding WT arrestins.
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Figure 6: Arrestin interaction with clathrin contributes to arrestin-dependent regulation of cell morphology. (A–E) Representative images of DKO cells expressing HA-tagged arrestin stained with anti-HA antibodies (green) and rhodamine–phalloidin (red). (A) Control DKO MEFs transfected with HA-Rluc. (B) DKO MEFs transfected with WT arrestin-2. (C) DKO MEFs expressing clathrin binding–deficient A2CBD mutant. (D) DKO MEFs expressing WT arrestin-3. (E) DKO MEFS expressing A3CBD mutant. (F) Scatter plot showing the results of cell size analysis. The horizontal bars represent the medians. The cell size data were analyzed by Kruskal–Wallis analysis of variance, followed by posthoc pairwise comparison by Mann–Whitney test with Bonferroni correction for multiple comparisons. Data are from ∼200 cells/condition from four independent experiments. *p < 0.001, **p < 0.01; *p < 0.05, as compared with cells expressing RLuc; ap < 0.05; cp < 0.001, as compared with cells expressing corresponding WT arrestins.
Mentions: Arrestins promote GPCR internalization by virtue of their interaction with clathrin (Goodman et al., 1996) and the clathrin adaptor AP2 (Laporte et al., 2000). Mutations eliminating clathrin- or AP2-binding sites in the arrestin C-tail significantly reduce receptor internalization (Kim and Benovic, 2002). Clathrin was implicated in microtubule-dependent FA disassembly (Ezratty et al., 2009), suggesting that a defect in clathrin recruitment to FA in arrestin-2/3 DKO cells might contribute to their peculiar morphology. To test this idea, we compared DKO cell rescue by WT arrestin-2 and -3 and mutants with disabled clathrin-binding sites, A2CBD and A3CBD, respectively. These four-residue mutations do not perturb other arrestin functions (Kim and Benovic, 2002). DKO cells transfected with HA-tagged arrestins or HA-RLuc and plated on FN were stained for HA tag and actin (Figure 6A). HA-RLuc was used as a control to ensure appropriate selection of transfected cells and rule out the effect of transfection on cell morphology. We confirmed that the expression of WT arrestin-2 or -3 significantly reduced DKO cell size (Figure 6, A, B, D, and F). Arr2CBD had no effect, and the similar arrestin-3 mutant A3CBD reduced cell size much less effectively than WT arrestin-3 (Figure 6, C, E, and F). Partial effectiveness of this mutant might be the result of indirect clathrin binding via AP2. The AP2-binding site was not disabled because it includes an arginine that is part of the main phosphate sensor, and therefore AP2 binding cannot be completely eliminated without changing other functional characteristics of arrestins. The data suggest that arrestins are involved in microtubule-dependent FA regulation, and their binding to clathrin is necessary for the ability of arrestins to rescue the DKO phenotype.

Bottom Line: Clathrin exhibited decreased dynamics near FA in arrestin-deficient cells.In contrast to wild-type arrestins, mutants deficient in clathrin binding did not rescue the phenotype.Collectively the data indicate that arrestins are key regulators of FA disassembly linking microtubules and clathrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232.

Show MeSH
Related in: MedlinePlus