title: Ca2+ binding to EF hands 1 and 3 is essential for the interaction of apoptosis-linked gene-2 with Alix/AIP1 in ocular melanoma.
abstract: 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.
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53792, USA.
authors.forename: Lalita
authors.initials: L
authors.lastname: Subramanian
authors.forename: John W
authors.initials: JW
authors.lastname: Crabb
authors.forename: Jos
authors.initials: J
authors.lastname: Cox
authors.forename: Isabelle
authors.initials: I
authors.lastname: Durussel
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Walker
authors.forename: Paul R
authors.initials: PR
authors.lastname: van Ginkel
authors.forename: Saswati
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: Julia M
authors.initials: JM
authors.lastname: Dellaria
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
authors.forename: Arthur S
authors.initials: AS
authors.lastname: Polans
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D019281:Dimerization; D015536:Down-Regulation / Q000235:genetics; D020832:EF Hand Motifs / Q000235:genetics; D056827:Endosomal Sorting Complexes Required for Transport; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D020349:Surface Plasmon Resonance; D014604:Uveal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
chemical_list: D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C097753:PDCD6 protein, human; C117020:PDCD6IP protein, human; D010446:Peptide Fragments; D002118:Calcium