Calcium-regulated GTPase activity in the calcium-binding protein calexcitin.
Calexcitin (CE) is a calcium-binding protein, closely related to sarcoplasmic calcium-binding proteins, that is involved in invertebrate learning and memory. Early reports indicated that both Hermissenda and squid CE also could bind GTP; however, the biochemical significance of GTP-binding and its relationship to calcium binding have remained unclear. Here, we report that the GTPase activity of CE is strongly regulated by calcium. CE possessed a P-loop-like structure near the C-terminal similar to the phosphate-binding regions in other GTP-binding proteins. Site-directed mutagenesis of this region showed that Gly(182), Phe(186) and Gly(187) are required for maximum affinity, suggesting that the GTP-binding motif is G-N-x-x-[FM]-G. CE cloned from Drosophila CNS possessed a similar C-terminal sequence and also bound and hydrolyzed GTP. GTPase activity in Drosophila CE was also strongly regulated by Ca(2+), exhibiting over 23-fold higher activity in the presence of 0.3 microM calcium. Analysis of the conserved protein motifs defines a new family of Ca(2+)-binding proteins representing the first example of proteins endowed with both EF-hand calcium binding domains and a C-terminal, P-loop-like GTP-binding motif. These results establish that, in the absence of calcium, both squid and Drosophila CE bind GTP at near-physiological concentrations and hydrolyze GTP at rates comparable to unactivated ras. Calcium functions to increase GTP-binding and GTPase activity in CE, similar to the effect of GTPase activating proteins in other low-MW GTP-binding proteins. CE may, therefore, act as a molecular interface between Ca(2+) cytosolic oscillations and the G protein-coupled signal transduction.
Nelson
Thomas J
TJ
Blanchette Rockefeller Neurosciences Institute, 9601 Medical Center Drive, Rockville, MD 20850, USA. tjnelson@brni-jhu.org
Quattrone
Alessandro
A
Kim
Jeongho
J
Pacini
Alessandra
A
Cesati
Valentina
V
Alkon
Daniel L
DL
D000595:Amino Acid Sequence; D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049832:Decapodiformes / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; D029721:Drosophila Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C102751:cex-2 protein, C elegans; D008274:Magnesium; D002118:Calcium