PubMed ID: 15366927
Author(s): Subramanian L, Crabb JW, Cox J, Durussel I, Walker TM, van Ginkel PR, Bhattacharya S, Dellaria JM, Palczewski K, Polans AS. Ca2+ binding to EF hands 1 and 3 is essential for the interaction of apoptosis-linked gene-2 with Alix/AIP1 in ocular melanoma. Biochemistry. 2004 Sep 7;43(35):11175-86. PMID 15366927
Journal: Biochemistry, Volume 43, Issue 35, Sep 2004
Apoptosis-linked gene-2 (ALG-2) encodes a 22 kDa Ca(2+)-binding protein of the penta EF-hand family that is required for programmed cell death in response to various apoptotic agents. Here, we demonstrate that ALG-2 mRNA and protein are down-regulated in human uveal melanoma cells compared to their progenitor cells, normal melanocytes. The down regulation of ALG-2 may provide melanoma cells with a selective advantage. ALG-2 and its putative target molecule, Alix/AIP1, are localized primarily in the cytoplasm of melanocytes and melanoma cells independent of the intracellular Ca(2+) concentration or the activation of apoptosis. Cross-linking and analytical centrifugation studies support a single-species dimer conformation of ALG-2, also independent of Ca(2+) concentration. However, binding of Ca(2+) to both EF-1 and EF-3 is necessary for ALG-2 interaction with Alix/AIP1 as demonstrated using surface plasmon resonance spectroscopy. Mutations in EF-5 result in reduced target interaction without alteration in Ca(2+) affinity. The addition of N-terminal ALG-2 peptides, residues 1-22 or residues 7-17, does not alter the interaction of ALG-2 or an N-terminal deletion mutant of ALG-2 with Alix/AIP1, as might be expected from a model derived from the crystal structure of ALG-2. Fluorescence studies of ALG-2 demonstrate that an increase in surface hydrophobicity is primarily due to Ca(2+) binding to EF-3, while Ca(2+) binding to EF-1 has little effect on surface exposure of hydrophobic residues. Together, these data indicate that gross surface hydrophobicity changes are insufficient for target recognition.