Annexin VI Stimulates Endocytosis and Is Involved in the Trafficking of Low Density Lipoprotein to the Prelysosomal Compartment

Abstract

Annexins are calcium-binding proteins with a wide distribution in most polarized and nonpolarized cells that participate in a variety of membrane-membrane interactions. At the cell surface, annexin VI is thought to remodel the spectrin cytoskeleton to facilitate budding of coated pits. However, annexin VI is also found in late endocytic compartments in a number of cell types, indicating an additional important role at later stages of the endocytic pathway. Therefore overexpression of annexin VI in Chinese hamster ovary cells was used to investigate its possible role in endocytosis and intracellular trafficking of low density lipoprotein (LDL) and transferrin. While overexpression of annexin VI alone did not alter endocytosis and degradation of LDL, coexpression of annexin VI and LDL receptor resulted in an increase in LDL uptake with a concomitant increase of its degradation. Whereas annexin VI showed a wide intracellular distribution in resting Chinese hamster ovary cells, it was mainly found in the endocytic compartment and remained associated with LDL-containing vesicles even at later stages of the endocytic pathway. Thus, data presented in this study suggest that after stimulating endocytosis at the cell surface, annexin VI remains bound to endocytic vesicles to regulate entry of ligands into the prelysosomal compartment.Annexins are a family of highly conserved proteins, which are characterized by their Ca 2ϩ -dependent binding to phospholipids (1). Each annexin consists of a conserved core domain with four or eight repeats (70 amino acids) and a nonconserved, short, NH 2 -terminal domain. More than 10 different family members, several of which exist as multiple isoforms, have been described in higher vertebrates (2). Since annexins are expressed in many tissues and are located in the same cellular compartments, the understanding of the distinct physiological role of each annexin still remains elusive (1, 3). In recent years, the involvement of annexins in membrane traffic has emerged as one of their predominant functions (1, 4). Several annexins including annexin I, II, IV, VI, VII, and XIIIb have been directly implicated in different steps of the intracellular trafficking pathways (5-14) and, despite some controversy, essentially due to the variety of cells and antibodies used, they are all associated with the endocytic compartment.The enrichment of annexin VI in rat liver endosomes (12, 13), its polarized localization in the apical endosomes in rat hepatocytes (14) and WIF-B cells (15), and the colocalization with lgp120, a prelysosomal marker in normal rat kidney cells (15), indicate a potential role for annexin VI in the endocytic pathways of polarized and nonpolarized cells.In support of this hypothesis, annexin VI has been found to bind ␤-spectrin at the cell surface, which in turn recruits and activates a calpain-like protease. This cascade of events seems to open the actin-cortical cytoskeleton to facilitate the initial steps of endocytosis (16,17). The complexity of these interactions has recently...

Cytosolic Dynamics of Annexin A6 Trigger Feedback Regulation of Hypertrophy via Atrial Natriuretic Peptide in Cardiomyocytes

“…Participation of Anxa6 in vesicular trafficking has been well characterized, and the protein is known to associate with endosomes and secretory vesicles (37,42,43). Here we show that the cytosolic 4dynamics of Anxa6 confer sustained protection against cardiomyocyte hypertrophy by associating with and regulating pro-ANP traffic, an endogenous counterhypertrophic factor (44).…”

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

“…Furthermore, we identified AnxA6 as a major component of rat liver endosomes (Jackle et al , 1994; Pol et al , 1997). These findings correlate with the involvement of AnxA6 in low-density lipoprotein (LDL) receptor–mediated endocytosis (Kamal et al , 1998; Grewal et al , 2000) and the delivery of LDL-containing late endosomes to lysosomes for degradation (Pons et al , 2000). LDL-induced translocation of AnxA6 to late endosomes (Grewal et al , 2000) correlates with the recruitment of AnxA6 to cholesterol-enriched late endosomes, which establishes a connection between AnxA6 and cholesterol (de Diego et al , 2002).…”

“…Therefore we initially compared the localization of SNAP23 and syntaxin-4 in CHO wild-type (CHOwt) and the well-characterized CHO-A6 cell line (Grewal et al , 2000, 2005; Pons et al , 2001; Rentero et al , 2006; Cubells et al , 2007, 2008). CHOwt cells express low amounts of AnxA6, whereas AnxA6 levels in CHO-A6 cells are similar to AnxA6 expression levels observed in other commonly used cell lines, such as HeLa and NRK (Cubells et al , 2007).…”

“…Paraformaldehyde was from Electron Microscopy Sciences (Hatfield, PA), and Mowiol from Calbiochem (San Diego, CA). Polyclonal antibodies anti-SNAP23, anti–syntaxin-3, anti-VAMP2, anti–syntaxin-4, and anti-AnxA6 were prepared in our laboratories and have been described elsewhere (Grewal et al , 2000; Martin-Martin et al , 2000; de Diego et al , 2002; Torrejon-Escribano et al , 2002; Raptis et al , 2005). Polyclonal and monoclonal anti–caveolin-1, anti-flotillin, anti-GM130, and anti-Rab4 were from BD Transduction Laboratories (BD Biosciences, Franklin Lakes, NJ).…”

“…The generation of the stable AnxA6-overexpressing CHO cell line (CHO-A6) and A431 cell line (A431-A6) has been described in detail (Grewal et al , 2000, 2005; Pons et al , 2001; Cubells et al , 2007; Vila de Muga et al , 2009). To generate AnxA6-overexpressing HuH7, we transfected HuH7 cells with pcDNAanx6 (Grewal et al , 2000) and selected in the presence of 1 mg/ml G418. After 2 wk, G418-resistant and AnxA6-overexpressing HuH7 cells were identified.…”