Calcium- and pH-dependent localization of annexin A6 isoforms in Balb/3T3 fibroblasts reflecting their potential participation in vesicular transport
✍ Scribed by Agnieszka Strzelecka-Kiliszek; Malgorzata E. Buszewska; Paulina Podszywalow-Bartnicka; Slawomir Pikula; Katarzyna Otulak; Rene Buchet; Joanna Bandorowicz-Pikula
- Publisher
- John Wiley and Sons
- Year
- 2008
- Tongue
- English
- Weight
- 350 KB
- Volume
- 104
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
Abstract
Annexin A6 (AnxA6), calcium‐ and membrane‐binding protein, is involved in membrane dynamics. It exists in the cell in two isoforms, AnxA6‐1 and AnxA6‐2, varying only by the VAAEIL sequence. In most cells, AnxA6‐1 predominates. A limited number of observations suggests that both isoforms differ from each other functionally. The EGF‐dependent Ca^2+^ influx in A431 cells is inhibited only by AnxA6‐1. Moreover, AnxA6‐2 was found to exhibit higher affinity for Ca^2+^. In this report we addressed the potential significance of the VAAEIL deletion in AnxA6‐2. For this purpose, we expressed AnxA6 isoform cDNAs in bacteria or mouse Balb/3T3 fibroblasts. The recombinant AnxA6‐2 was characterized by a less extended molecular shape than that of AnxA6‐1 and required a narrower [Ca^2+^] range to bind liposomes. Upon lowering pH in the presence of EGTA recombinant AnxA6‐2 became less hydrophobic than AnxA6‐1 as revealed by the Triton X‐114 partition. Furthermore, AnxA6‐2 revealed stronger F‐actin binding than that of AnxA6‐1. Immunofluorescence microscopy showed that the EGFP‐tagged AnxA6 isoforms expressed in Balb/3T3 fibroblasts relocate in a Ca^2+^‐ and H^+^‐sensitive manner to the vesicular structures in a perinuclear region or in cytosol. Cell fractionation showed that in resting conditions AnxA6‐1 is associated with early endosomes and AnxA6‐2 with late endosomes, and an increase in [Ca^2+^] and/or [H^+^] induced their opposite distribution. These findings suggest a potentially independent regulation, localization, and function of AnxA6 isoforms in Balb/3T3 fibroblasts. More generally, our findings suggest distinct functions of AnxA6 isoforms in membrane dynamics. J. Cell. Biochem. 104: 418–434, 2008. © 2007 Wiley‐Liss, Inc.