Methods to Investigate the β-Arrestin-Mediated Control of ARF6 Activation to Regulate Trafficking and Actin Cytoskeleton Remodeling.

ADP-ribosylation factors (ARF) are GTPases that act to control the activation of numerous signaling events and cellular responses. The ARF6 isoform, present at the plasma membrane, can be activated by the angiotensin II type 1 receptor (ATR), a process dependent upon β-arrestin recruitment to the activated receptor. Here, we describe classical methods used to assess β-arrestin-dependent activation of ARF6 following agonist stimulation of cells.

ARF6 protects sister chromatid cohesion to ensure the formation of stable kinetochore-microtubule attachments.

Sister chromatid cohesion, facilitated by the cohesin protein complex, is crucial for the establishment of stable bipolar attachments of chromosomes to the spindle microtubules and their faithful segregation. Here, we demonstrate that the GTPase ARF6 prevents the premature loss of sister chromatid cohesion. During mitosis, ARF6-depleted cells normally completed chromosome congression.

ARF GTPases control phenotypic switching of vascular smooth muscle cells through the regulation of actin function and actin dependent gene expression.

Vascular smooth muscle cells (VSMC) can exhibit a contractile or a synthetic phenotype depending on the extracellular stimuli present and the composition of the extracellular matrix. Uncontrolled activation of the synthetic VSMC phenotype is however associated with the development of cardiovascular diseases. Here, we aimed to elucidate the role of the ARF GTPases in the regulation of VSMC dedifferentiation.

The GTPase ARF6 Controls ROS Production to Mediate Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation.

High reactive oxygen species (ROS) levels and enhanced vascular smooth muscle cells (VSMC) proliferation are observed in numerous cardiovascular diseases. The mechanisms by which hormones such as angiotensin II (Ang II) acts to promote these cellular responses remain poorly understood. We have previously shown that the ADP-ribosylation factor 6 (ARF6), a molecular switch that coordinates intracellular signaling events can be activated by the Ang II receptor (AT1R).

β-Arrestin-mediated Angiotensin II Signaling Controls the Activation of ARF6 Protein and Endocytosis in Migration of Vascular Smooth Muscle Cells.

Angiotensin II (Ang II) is a vasopressive hormone but is also a potent activator of cellular migration. We have previously shown that it can promote the activation of the GTPase ARF6 in a heterologous overexpressing system. The molecular mechanisms by which receptors control the activation of this small G protein remain, however, largely unknown.

Differential β-arrestin-dependent conformational signaling and cellular responses revealed by angiotensin analogs.

The angiotensin type 1 receptor (AT1R) and its octapeptide ligand, angiotensin II (AngII), engage multiple downstream signaling pathways, including those mediated by heterotrimeric guanosine triphosphate-binding proteins (G proteins) and those mediated by β-arrestin. Here, we examined AT1R-mediated Gα(q) and β-arrestin signaling with multiple AngII analogs bearing substitutions at position 8, which is critical for binding to the AT1R and its activation of G proteins. Using assays that discriminated between ligand-promoted recruitment of β-arrestin to the AT1R and its resulting conformational rearrangement, we extend the concept of biased signaling to include the analog's propensity to differentially promote conformational changes in β-arrestin, two responses that were differentially affected by distinct G protein-coupled receptor kinases.