Trudy A. Kohout

β-Arrestin1 Mediates Insulin-like Growth Factor 1 (IGF-1) Activation of Phosphatidylinositol 3-Kinase (PI3K) and Anti-apoptosis

β-arrestins (1 and 2) are widely expressed cytosolic proteins that play central roles in G protein-coupled receptor signaling. β-arrestin1 is also recruited to the insulin-like growth factor 1 (IGF-1) receptor, a receptor tyrosine kinase, upon agonist binding. Here we report that, in response to IGF-1 stimulation, β-arrestin1 mediates activation of phosphatidylinositol 3-kinase in a pathway that leads to the subsequent activation of Akt and anti-apoptosis. This process is independent of both Gi and ERK activity. The pathway fails in mouse embryo fibroblasts lacking both β-arrestins and is restored by stable transfection of β-arrestin1. Remarkably, this pathway is insensitive to chemical inhibition of IGF-1 receptor tyrosine kinase activity. These results suggest that, in addition to their roles in G protein-coupled receptor signaling, β-arrestins couple the IGF-1 receptor tyrosine kinase to the phosphatidylinositol 3-kinase system and suggest that this mechanism is operative independently of the tyrosine kinase activity of the receptor.

Endocytosis of the Viral Chemokine Receptor US28 Does Not Require Beta‐Arrestins But Is Dependent on the Clathrin‐Mediated Pathway

Arrestins bind phosphorylated G‐protein coupled‐receptors (GPCR) and inhibit agonist‐induced signal transduction by uncoupling the receptors from their cognate G‐proteins. β‐arrestins also act as adaptors that target GPCR to endocytic clathrin‐coated vesicles. Unlike cellular GPCRs, the human cytomegalovirus GPCRs and chemokine receptor, US28, shows constitutive signal transduction activity and undergoes constitutive endocytosis. To determine the role of β‐arrestins in US28 trafficking, we used embryonic fibroblasts derived from β‐arrestin knockout mice. In these cells, the internalization of transfected β2‐adrenergic receptor and of the cellular chemokine receptor CCR5 was impaired. By contrast, US28 distribution was unaffected, and US28‐mediated RANTES internalization was similar in normal and knockout cell lines. To investigate whether a clathrin‐mediated pathway is involved in US28 endocytosis, we developed small interfering RNA against the μ2‐adaptin subunit of the AP‐2 adaptor complex. In cells transfected with μ2 small interfering RNA transferrin endocytosis was severely inhibited. Antibody‐feeding experiments and biochemical analysis showed that US28 internalization was also inhibited. Together, these data indicate that US28 endocytosis occurs via a clathrin‐mediated mechanism but is independent of β‐arrestins.

The Adaptor Protein β-Arrestin2 Enhances Endocytosis of the Low Density Lipoprotein Receptor

Endocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor β-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of β-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.05), but had no effect on high density lipoprotein cholesterol levels. We therefore tested whether β-arrestins could affect the constitutive endocytosis of the LDLR. When overexpressed in cells, β-arrestin1 and β-arrestin2 each associated with the LDLR, as judged by co-immunoprecipitation, and augmented LDLR endocytosis by ∼70%, as judged by uptake of fluorescent LDL. However, physiologic expression levels of only β-arrestin2, and not β-arrestin1, enhanced endogenous LDLR endocytosis (by 65%) in stably transfected β-arrestin1/β-arrestin2 double-knockout mouse embryonic fibroblasts (MEFs). Concordantly, when RNA interference was used to suppress expression of β-arrestin2, but not β-arrestin1, LDLR endocytosis was reduced. Moreover, β-arrestin2–/– MEFs demonstrated LDLR endocytosis that was 50% less than cognate wild type MEFs. In fusion protein pull-down assays, β-arrestin2 bound to the LDLR cytoplasmic tail stoichiometrically, and binding was abolished by mutation of LDLR Tyr807 to Ala. Mutation of LDLR cytoplasmic tail Ser833 to Asp enhanced both the affinity of LDLR fusion protein binding to β-arrestin2, and the efficiency of LDLR endocytosis in cells expressing β-arrestin2 physiologically. We conclude that β-arrestin2 can bind to and enhance endocytosis of the LDLR, both in vitro and in vivo, and may thereby influence lipoprotein metabolism.