GTPases of the Ras-related RGK family are negative regulators of high voltage-activated (HVA) Ca2+ channel activity. In this study, we examined the role of calmodulin (CaM) association in Rem-mediated Ca2+ channel inhibition. We found that the Rem/CaM interaction is Ca2+-dependent, and that truncation of the Rem C-terminus before position 277 prevents CaM binding. Serial mutagenesis of the Rem C-terminus between residues 265 and 276 to alanine generated two mutants (RemL271A and RemL274A) that displayed reduced CaM binding, and a subset of these mutants displayed significantly lower cell periphery localization than RemWT. However, reductions in CaM association or membrane trafficking did not affect function, as all Rem mutants could completely inhibit Ca2+ channels. The Rem1–275 truncation mutant partially inhibited Ca2+ channel activity despite its inability to bind CaM. Taken together, these studies indicate that CaM association is not essential for either Rem-mediated Ca2+ channel inhibition or plasma membrane localization.
This is a preview of subscription content, log in to check access.
We wish to thank Dr. Carole Moncman for her assistance with the confocal imaging studies and statistical analysis, Dr. Thomas Vanaman for his gift of calmodulin-sepharose resin used in preliminary studies, and members of the Andres lab for critical reading of this manuscript. This work was supported by Public Health Service Grants HL072936 (to DAA), HL074091 (to JS), and P20 RR20171 from the National Center for Research Resources, National Institutes of Health (to DAA), and an American Heart Association pre-doctoral fellowship and an NIH Interdisciplinary Cardiovascular Training Grant T32 HL072743 (to RNC).
Finlin BS, Andres DA (1997) Rem is a new member of the Rad- and Gem/Kir Ras-related GTP-binding protein family repressed by lipopolysaccharide stimulation. J Biol Chem 272:21982–21988PubMedCrossRefGoogle Scholar
Finlin BS, Shao H, Kadono-Okuda K et al (2000) Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases. Biochem J 347(Pt 1):223–231PubMedCrossRefGoogle Scholar
Maguire J, Santoro T, Jensen P et al (1994) Gem: an induced, immediate early protein belonging to the Ras family. Science 265:241–244PubMedCrossRefGoogle Scholar
Reynet C, Kahn CR (1993) Rad: a member of the Ras family overexpressed in muscle of type II diabetic humans. Science 262:1441–1444PubMedCrossRefGoogle Scholar
Zhu J, Reynet C, Caldwell JS et al (1995) Characterization of Rad, a new member of Ras/GTPase superfamily, and its regulation by a unique GTPase-activating protein (GAP)-like activity. J Biol Chem 270:4805–4812PubMedCrossRefGoogle Scholar
Beguin P, Nagashima K, Gonoi T et al (2001) Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem. Nature 411:701–706PubMedCrossRefGoogle Scholar
Finlin BS, Crump SM, Satin J et al (2003) Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases. Proc Natl Acad Sci U S A 100:14469–14474PubMedCrossRefGoogle Scholar
Finlin BS, Mosley AL, Crump SM et al (2005) Regulation of L-type Ca2+ channel activity and insulin secretion by the Rem2 GTPase. J Biol Chem 280:41864–41871PubMedCrossRefGoogle Scholar
Fischer R, Wei Y, Anagli J et al (1996) Calmodulin binds to and inhibits GTP binding of the ras-like GTPase Kir/Gem. J Biol Chem 271:25067–25070PubMedCrossRefGoogle Scholar
Moyers JS, Bilan PJ, Zhu J et al (1997) Rad and Rad-related GTPases interact with calmodulin and calmodulin-dependent protein kinase II. J Biol Chem 272:11832–11839PubMedCrossRefGoogle Scholar
Ward Y, Spinelli B, Quon MJ et al (2004) Phosphorylation of critical serine residues in Gem separates cytoskeletal reorganization from down-regulation of calcium channel activity. Mol Cell Biol 24:651–661PubMedCrossRefGoogle Scholar
Beguin P, Mahalakshmi RN, Nagashima K et al (2005) 14-3-3 and calmodulin control subcellular distribution of Kir/Gem and its regulation of cell shape and calcium channel activity. J Cell Sci 118:1923–1934PubMedCrossRefGoogle Scholar
Beguin P, Mahalakshmi RN, Nagashima K et al (2005) Roles of 14-3-3 and calmodulin binding in subcellular localization and function of the small G-protein Rem2. Biochem J 390:67–75PubMedCrossRefGoogle Scholar
Beguin P, Mahalakshmi RN, Nagashima K et al (2006) Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14-3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation. J Mol Biol 355:34–46PubMedCrossRefGoogle Scholar
Finlin BS, Andres DA (1999) Phosphorylation-dependent association of the Ras-related GTP-binding protein Rem with 14-3-3 proteins. Arch Biochem Biophys 368:401–412PubMedCrossRefGoogle Scholar
Shi GX, Han J, Andres DA (2005) Rin GTPase couples nerve growth factor signaling to p38 and b-Raf/ERK pathways to promote neuronal differentiation. J Biol Chem 280:37599–37609PubMedCrossRefGoogle Scholar
Andres DA, Crump SM, Correll RN et al (2005) Analyses of Rem/RGK Signaling and Biological Activity. Methods Enzymol 407:484–498PubMedGoogle Scholar
Crump SM, Correll RN, Schroder EA et al (2006) L-type calcium channel alpha-subunit and protein kinase inhibitors modulate Rem-mediated regulation of current. Am J Physiol Heart Circ Physiol 291:H1959–H1971PubMedCrossRefGoogle Scholar
Lee CH, Della NG, Chew CE et al (1996) Rin, a neuron-specific and calmodulin-binding small G-protein, and Rit define a novel subfamily of ras proteins. J Neurosci 16:6784–6794PubMedGoogle Scholar
Payne ME, Fong YL, Ono T et al (1988) Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains. J Biol Chem 263:7190–7195PubMedGoogle Scholar
Heo WD, Inoue T, Park WS et al (2006) PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science 314:1458–1461PubMedCrossRefGoogle Scholar