Functional analysis of calcium-binding EF-hand motifs of visinin-like protein-1.
Visinin-like protein-1 (VILIP-1), a myristoylated calcium sensor protein with three EF-hand motifs, modulates adenylyl cyclase activity. It translocates to membranes when a postulated "calcium-myristoyl switch" is triggered by calcium-binding to expose its sequestered myristoyl moiety. We investigated the contributions of the EF-hand motifs to the translocation of VILIP-1 to membranes and to the modulation of adenylyl cyclase activity. Mutation of residues crucial for binding calcium within each one of the EF-hand motifs indicated that they all contributed to binding calcium. Simultaneous mutations of all of the three EF-hand motifs completely abolished VILIP-1's ability to bind calcium, attenuated but did not eliminate its modulation of adenylyl cyclase activity, and abolished its calcium-dependence for association with cellular membranes. These results show that the calcium-binding EF-hand motifs of VILIP-1 do not have an essential role in modulating adenylyl cyclase activity but instead have a structural role in activating the "calcium-myristoyl switch" of VILIP-1.
Neuroscience Center of Excellence, 2020 Gravier Street, Suite D, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Lin
L
Lin
Karl-Heinz
KH
Braunewell
Eckart D
ED
Gundelfinger
Rene
R
Anand
D000262:Adenylyl Cyclases / Q000378:metabolism; D020816:Amino Acid Motifs; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D020832:EF Hand Motifs; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D051596:Neurocalcin; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D044169:Receptors, Calcium-Sensing; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D044169:Receptors, Calcium-Sensing; D011994:Recombinant Proteins; C496688:VSNL1 protein, human; D019814:Myristic Acid; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D002118:Calcium