Calcium- and pH-dependent localization of annexin A6 isoforms in Balb/3T3 fibroblasts reflecting their potential participation in vesicular transport.
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.
Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Agnieszka
A
Strzelecka-Kiliszek
Malgorzata E
ME
Buszewska
Paulina
P
Podszywalow-Bartnicka
Slawomir
S
Pikula
Katarzyna
K
Otulak
Rene
R
Buchet
Joanna
J
Bandorowicz-Pikula
D016475:3T3 Cells; D000818:Animals; D017317:Annexin A6 / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002118:Calcium; D002462:Cell Membrane / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D020033:Protein Isoforms; D017384:Sequence Deletion
D017317:Annexin A6; D020033:Protein Isoforms; D002118:Calcium