Regulation of Endocytosis of Activin Type II Receptors by a Novel PDZ Protein through Ral/Ral-binding Protein 1-dependent Pathway

Abstract

Using yeast two-hybrid screening, we have identified a mouse Postsynaptic density 95/Discs large/Zona occludens-1 (PDZ) protein that interacts with activin type II receptors (ActRIIs). We named the protein activin receptor-interacting protein 2 (ARIP2). ARIP2 was found to have one PDZ domain in the NH 2 -terminal region and interact specifically with ActRIIs among the receptors for the transforming growth factor ␤ family by the PDZ domain. Interestingly, overexpression of ARIP2 enhances endocytosis of ActRIIs and reduces activin-induced transcription in Chinese hamster ovary K1 cells. In addition, immunofluorescence co-localization studies indicated the direct involvement of ARIP2 in the intracellular translocation of ActRIIs by PDZ domain-mediated interaction. Moreover, we have identified that the COOH-terminal region of ARIP2 interacts with Ralbinding protein 1 (RalBP1). RalBP1 is a potential effector protein of small GTP-binding protein Ral and regulates endocytosis of epidermal growth factor and insulin receptors. The studies using deletion mutants of RalBP1 and constitutively GTP and GDP binding forms of Ral indicate that ARIP2 regulates endocytosis of ActRIIs through the Ral/RalBP1-dependent pathway, and the GDP-GTP exchange of Ral is critical for this regulation.Activin, a member of the TGF-␤ 1 superfamily, has a broad range of physiological activities including hematopoiesis, bone morphogenesis, neurogenesis, and hormone action (1-5). These various actions on cell proliferation, differentiation, and apoptosis are dependent upon target cells. Activin transduces its signal via heteromeric complexes composed of two different serine/threonine kinase receptors, termed type I and type II (6 -8). Upon ligand binding, the type II receptor transphosphorylates and activates the type I receptor kinase at the membrane region. Then, the type I receptor cytoplasmic domain interacts with intracellular signaling molecules, Smads, which regulate transcription of selected genes in a cell-specific manner (9, 10).In the current model, the functions of the ActRIIs are limited to ligand binding, type I receptor recruitment, and transphosphorylation. However, it is speculated that there are specific roles of the ActRIIs in activin signaling. For example, the serine/threonine kinase domains of ActRIIs are constitutively activated, and ActRIIs exist as homooligomers (probably homodimers) even in the absence of ligands (11,12). These data suggest the existence of a strict monitoring mechanism for type II receptor kinase activity. Furthermore, multiple forms of ActRIIs exist; two subtypes of activin type II receptors, ActRIIA and ActRIIB, each of which is encoded by individual genes, are known (13,14). Several alternative splicing variants have also been found in each subtype (14 -16). For example, activin type IIA-N receptor, a splicing product of ActRIIA, is specifically expressed in neural cells and is thought to mediate neuronalspecific activin action. In a previous report, we noted the identification of a PDZ protein called a...

Endocytic regulation of TGF-β signaling

“…Internalization of the type II activin receptor (ActRII) was shown to be promoted by activin receptor-interacting protein 2 (ARIP2) [113]. Overexpression of ARIP2 attenuated the activin-induced reporter expression, indicating that internalization of ActRII negatively regulates activin signaling.…”

Regulation of activin receptor-interacting protein 2 expression in mouse hepatoma Hepa1-6 cells and its relationship with collagen type IV

“…However, ARIP1 mainly distributed in nerve tissues, ARIP2 widely existed in tissues. The high expression of ARIP2 mRNA could be detected in liver tissue by Northern blot [7] . In order to investigate the kinetic changes of ARIP2 expression, we used activin A and endotoxin LPS to stimulate Hepal-6 cells, and then examined the expression of ARIP2 mRNA.…”

“…ARIPs have obvious expression and distribution diversity, which are key factors to the histological specificity of activin action [6][7][8] . It has been demonstrated that both ARIP1 and ARIP2 are inhibitors of activin signal transduction.…”

“…ARIP2 can enhance ActRII endocytosis and reduce ActRIIA receptor expression on cell membranes via Ral/RalBP1-depending pathway, and has a capability of suppressing activin-induced signal transduction. There was high expression of ARIP2 mRNA in liver tissues tested by Northern blot [7] . Therefore, we reason out that ARIP2 may participate in the functional regulation of hepatocytes treated by activin.…”

“…It has been found that the tissue-specificity might depend on a new group of intracellular signal proteins, activin receptor-interacting proteins (ARIPs) [6][7][8] . ARIPs have four forms at least, all of which can specifically interact with activin type Ⅱ receptor (ActRII) and regulate intracellular signal transduction induced by activin [6][7][8][9][10] . It has been demonstrated that not only the expression and distribution but also the biological activities of ARIPs were obviously different in various tissues.…”