Intact Ca(2+)-binding sites are required for targeting of annexin 1 to endosomal membranes in living HeLa cells.
Annexin 1 is a Ca(2+)-regulated membrane binding protein and a major substrate of the epidermal growth factor receptor kinase. Because of its properties and intracellular distribution, the protein has been implicated in endocytic trafficking of the receptor, in particular in receptor sorting occurring in multivesicular endosomes. Up to now, however, the localization of annexin 1 to cellular membranes has been limited to subcellular fractionation and immunocytochemical analyses of fixed cells. To establish its localization in live cells, we followed the intracellular fate of annexin 1 molecules fused to the Green Fluorescent Protein (GFP). We show that annexin 1-GFP associates with distinct, transferrin receptor-positive membrane structures in living HeLa cells. A GFP chimera containing the Ca(2+)/phospholipid-binding protein core of annexin 1 also shows a punctate intracellular distribution, although the structures labeled here do not resemble early but, at least in part, late endosomes. In contrast, the cores of annexins 2 and 4 fused to GFP exhibit a cytoplasmic or a different punctate distribution, respectively, indicating that the highly homologous annexin core domains carry distinct membrane specificities within live cells. By inactivating the three high-affinity Ca(2+) binding sites in annexin 1 we also show that endosomal membrane binding of the protein in live HeLa cells depends on the integrity of these Ca(2+) binding sites. More detailed analysis identifies a single Ca(2+) site in the second annexin repeat that is crucially involved in establishing the membrane association. These results reveal for the first time that intracellular membrane binding of an annexin in living cells requires Ca(2+) and is mediated in part through an annexin core domain that is capable of establishing specific interactions.
Rescher
U
U
Center for Molecular Biology of Inflammation, Institute for Medical Biochemistry, von-Esmarchstrasse 56, Germany.
Zobiack
N
N
Gerke
V
V
D000818:Animals; D017305:Annexin A1 / Q000032:analysis / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D013552:Swine; D014162:Transfection
D017305:Annexin A1; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium