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
Calcium-regulated GTPase activity in the calcium-binding protein calexcitin / J. Nelson; A. Quattrone; J. Kim; A. Pacini; V. Cesati; D. Alkon. - In: COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY. PART B, BIOCHEMISTRY & MOLECULAR BIOLOGY. - ISSN 1096-4959. - STAMPA. - 135(2003), pp. 627-638. [10.1016/S1096-4959(03)00144-1]
Titolo: | Calcium-regulated GTPase activity in the calcium-binding protein calexcitin. | |
Autori di Ateneo: | ||
Autori: | J. Nelson; A. Quattrone; J. Kim; PACINI, ALESSANDRA; V. Cesati; D. Alkon | |
Data di pubblicazione: | 2003 | |
Rivista: | ||
Volume: | 135 | |
Pagina iniziale: | 627 | |
Pagina finale: | 638 | |
Abstract: | 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 | |
Handle: | http://hdl.handle.net/2158/7991 | |
Appare nelle tipologie: | 1a - Articolo su rivista |
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