Activity-Dependent Internalization of Smoothened Mediated by ß-Arrestin 2 and GRK2

Abstract

Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.

Identification of select glucocorticoids as Smoothened agonists: Potential utility for regenerative medicine

“…1 G-J). Hit compound agonist activities in U2OS cells were also confirmed using a βarr2-GFP assay with WT Smo (16) (Fig. S1).…”

“…1B). The overexpression of Smo (16) or Smo-633 caused a redistribution of βarr2-GFP to intracellular vesicles/ aggregates (Fig. 1C).…”

“…Activated Smo shares important behaviors with canonical G proteincoupled receptors (GPCRs), including an ability to undergo GPCR kinase phosphorylation and to recruit β-arrestin2 (βarr2) proteins for endocytosis, as shown in our previous study (16). Cyclopamine, a naturally occurring steroid alkaloid, inhibits the constitutive activity of Smo via direct antagonism, preventing its phosphorylation and interaction with βarr2.…”

“…It contains 68 glucocorticoids or structurally related steroid compounds, including cortisone and dexamethasone. The primary screening assay employed U2OS cells, chosen for adherence, flatness, and stable expression of βarr2-GFP, and a tail substitution mutant of Smo, Smo-633, which provided better sensitivity than WT Smo (16,17). Images of Smo-633/βarr2-GFP complexes were obtained at the rate of 5,000 per day using an automated confocal-based plate reader (ImageXpress Ultra; Molecular Devices).…”

Expression of β-arrestin 1 in Gastric Cardiac Adenocarcinoma and its Relation with Progression

“…β-arrestins translocate from the cytoplasm to the nucleus and associate with transcription cofactors such as p300 and cAMP-response element-binding protein (CREB) at the promoters of target genes to promote transcription (Kang et al, 2005). β-arrestins are also involved in the development (Chen et al, 2004;Wilbanks et al, 2004) and cellular movement (Ge et al, 2004;Hunton et al, 2005; This study aims to analyze the expression of β-arrestin 1 in gastric cardiac adenocarcinoma and its relationships with clinicopathologic characteristics and survival status, which might help to investigate the functions of β-arrestin 1 in the differentiation and metastasis progression of gastric cardiac adenocarcinoma.…”