Ancient Ubiquitous Protein 1 Binds to the Conserved Membrane-proximal Sequence of the Cytoplasmic Tail of the Integrin α Subunits That Plays a Crucial Role in the Inside-out Signaling of αIIbβ3

Abstract

Modification of the cytoplasmic tails of the integrinIntegrin ␣ IIb ␤ 3 (GPIIb-IIIa) is one of the receptors on the cellular surface of platelets and megakaryocytes. It binds to various adhesive proteins including fibrinogen, von Willebrand factor, vitronectin, and fibronectin that contain a core amino acid sequence of arginine-glycine-aspartic acids (RGD). Binding of fibrinogen to ␣ IIb ␤ 3 leads to platelet aggregation and finally to thrombus formation at the injured vascular sites. A pivotal role of ␣ IIb ␤ 3 in hemostasis is supported by the clinical observation that the congenital deficiency of ␣ IIb ␤ 3 , Glanzmann's thrombasthenia, results in lifelong bleeding tendency (1). Whereas ␣ IIb ␤ 3 on resting platelets does not bind soluble fibrinogen, once platelets are activated, conformation of the extracellular domains of the ␣ IIb ␤ 3 is altered and its ligandbinding affinity is increased (affinity modulation) (2). This process of the inside-out signaling is considered to be mediated by modification of the short cytoplasmic tails of ␣ IIb and ␤ 3 subunits; however, the mechanism remains to be elucidated.The nuclear magnetic resonance structural analysis of the ␣ IIb cytoplasmic tail revealed a closed conformation where the highly conserved N-terminal membrane-proximal region forms an ␣-helix followed by a turn, and the acidic C-terminal loop interacts with the N-terminal helix (3). Deletion of almost the entire ␣ IIb -cytoplasmic tail and mutations in its N-terminal sequence (GFFKR) conserved among the integrin ␣ subunits enhance the affinity of ␣ IIb ␤ 3 for ligands (4 -6). The cytoplasmic tail of the ␤ 3 subunit also has an amino acid sequence that is conserved among integrin ␤ subunits: a stretch of 8 amino acids (KLLITIHD) adjacent to the transmembrane domain. In a similar fashion to the ␣ IIb subunit, deletion or mutation in this conserved region induces activation of ␣ IIb ␤ 3 (6, 7). These observations suggest that membrane-proximal regions of the cytoplasmic domains of both subunits exert a negative regulatory function and lock ␣ IIb ␤ 3 in a low affinity state. Negative regulation may be mediated by the interaction between ␣ IIb and ␤ 3 cytoplasmic tails, possibly through a salt bridge between Arg-995 in ␣ IIb and Asp-723 in ␤ 3 (6), or binding of intracellular proteins to ␣ IIb and/or ␤ 3 subunits. Two candidates for the modulator proteins have been reported: calcium-and integrinbinding protein (CIB) 1 (8) and ␤ 3 -endonexin (9, 10), which bind to ␣ IIb and ␤ 3 cytoplasmic tails, respectively. Although CIB is unlikely to have a regulatory effect on ␣ IIb ␤ 3 ligand binding function (11), ␤ 3 -endonexin fused to GST protein induces the conformational change of ␣ IIb ␤ 3 and activates it when co-transfected with ␣ IIb and ␤ 3 subunits in Chinese hamster ovary cells. Another mechanism of modification has been recently suggested: an interaction between cytoplasmic tails of ␣ IIb ␤ 3 and the actin cytoskeleton. ␣ IIb ␤ 3 and the actin cytoskeleton are physically linked by binding of talin to the ␤ 3 cy...

Ancient Ubiquitous Protein 1 (AUP1) Localizes to Lipid Droplets and Binds the E2 Ubiquitin Conjugase G2 (Ube2g2) via Its G2 Binding Region

“…to detect AUP1 localization to LDs in former studies (30,32,48) is likely due to the cell lines used in those studies and their respective metabolic state. Although some cell lines, e.g.…”

“…Independently, AUP1 was found as a binding partner of adenoviral proteins (28,29). AUP1 was also reported as a cytosolic protein that binds to integrin ␣ subunits and supports inside-out signaling in platelets (30,31). Very recently, AUP1 was identified as a component of the Sel1l complex at the ER (32,33), indicating an involvement of AUP1 in protein degradation processes.…”

SEL1L nucleates a protein complex required for dislocation of misfolded glycoproteins

“…AUP1 has a CUE domain, involved in ubiquitin binding or in recruitment of ubiquitin-conjugating enzymes to the site of dislocation (24). AUP1 has an N-terminal membrane anchor, with the remainder predicted to be in the cytosol (23). AUP1 has not been implicated in any aspect of (glyco) protein quality control and lacks an obvious homologue in yeast.…”

“…Ancient ubiquitous protein 1 is proposed to interact with integrins (23), but its function is obscure. AUP1 has a CUE domain, involved in ubiquitin binding or in recruitment of ubiquitin-conjugating enzymes to the site of dislocation (24).…”

ICln, a Novel Integrin αIIbβ3-Associated Protein, Functionally Regulates Platelet Activation

“…Many integrin-binding cytosolic proteins have been identified and can be broadly divided into structural, cytoskeletal proteins such as talin (10) or ␣-actinin (11), cytosolic proteins such as CIB (12), or ␤ 3 -endonexin (13) and membrane proteins such as CD9 (14) and CD153 (15). New integrin-binding proteins are continuously being identified (16), but the precise molecular mechanisms by which they regulate integrin activation remain unknown.…”