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10 pages, 1981 KiB

Open AccessArticle

Direct Interaction between Calmodulin and the Grb7 RA-PH Domain

by Gabrielle M. Watson and Jacqueline A. Wilce

Int. J. Mol. Sci. 2020, 21(4), 1336; https://doi.org/10.3390/ijms21041336 - 17 Feb 2020

Cited by 5 | Viewed by 2147

Abstract

Grb7 is a signalling adapter protein that engages activated receptor tyrosine kinases at cellular membranes to effect downstream pathways of cell migration, proliferation and survival. Grb7’s cellular location was shown to be regulated by the small calcium binding protein calmodulin (CaM). While evidence [...] Read more.

Grb7 is a signalling adapter protein that engages activated receptor tyrosine kinases at cellular membranes to effect downstream pathways of cell migration, proliferation and survival. Grb7’s cellular location was shown to be regulated by the small calcium binding protein calmodulin (CaM). While evidence for a Grb7/CaM interaction is compelling, a direct interaction between CaM and purified Grb7 has not been demonstrated and quantitated. In this study we sought to determine this, and prepared pure full-length Grb7, as well as its RA-PH and SH2 subdomains, and tested for CaM binding using surface plasmon resonance. We report a direct interaction between full-length Grb7 and CaM that occurs in a calcium dependent manner. While no binding was observed to the SH2 domain alone, we observed a high micromolar affinity interaction between the Grb7 RA-PH domain and CaM, suggesting that the Grb7/CaM interaction is mediated through this region of Grb7. Together, our data support the model of a CaM interaction with Grb7 via its RA-PH domain. Full article

(This article belongs to the Special Issue Calmodulin Function in Health and Disease)

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Review

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17 pages, 1888 KiB

Open AccessReview

A Non-Canonical Calmodulin Target Motif Comprising a Polybasic Region and Lipidated Terminal Residue Regulates Localization

by Benjamin M. M. Grant, Masahiro Enomoto, Mitsuhiko Ikura and Christopher B. Marshall

Int. J. Mol. Sci. 2020, 21(8), 2751; https://doi.org/10.3390/ijms21082751 - 15 Apr 2020

Cited by 16 | Viewed by 3483

Abstract

Calmodulin (CaM) is a Ca²⁺-sensor that regulates a wide variety of target proteins, many of which interact through short basic helical motifs bearing two hydrophobic ‘anchor’ residues. CaM comprises two globular lobes, each containing a pair of EF-hand Ca²⁺-binding [...] Read more.

Calmodulin (CaM) is a Ca²⁺-sensor that regulates a wide variety of target proteins, many of which interact through short basic helical motifs bearing two hydrophobic ‘anchor’ residues. CaM comprises two globular lobes, each containing a pair of EF-hand Ca²⁺-binding motifs that form a Ca²⁺-induced hydrophobic pocket that binds an anchor residue. A central flexible linker allows CaM to accommodate diverse targets. Several reported CaM interactors lack these anchors but contain Lys/Arg-rich polybasic sequences adjacent to a lipidated N- or C-terminus. Ca²⁺-CaM binds the myristoylated N-terminus of CAP23/NAP22 with intimate interactions between the lipid and a surface comprised of the hydrophobic pockets of both lobes, while the basic residues make electrostatic interactions with the negatively charged surface of CaM. Ca²⁺-CaM binds farnesylcysteine, derived from the farnesylated polybasic C-terminus of KRAS4b, with the lipid inserted into the C-terminal lobe hydrophobic pocket. CaM sequestration of the KRAS4b farnesyl moiety disrupts KRAS4b membrane association and downstream signaling. Phosphorylation of basic regions of N-/C-terminal lipidated CaM targets can reduce affinity for both CaM and the membrane. Since both N-terminal myristoylated and C-terminal prenylated proteins use a Singly Lipidated Polybasic Terminus (SLIPT) for CaM binding, we propose these polybasic lipopeptide elements comprise a non-canonical CaM-binding motif. Full article

(This article belongs to the Special Issue Calmodulin Function in Health and Disease)

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12 pages, 743 KiB

Open AccessReview

Calmodulin Mutations Associated with Heart Arrhythmia: A Status Report

by Walter J. Chazin and Christopher N. Johnson

Int. J. Mol. Sci. 2020, 21(4), 1418; https://doi.org/10.3390/ijms21041418 - 19 Feb 2020

Cited by 26 | Viewed by 4389

Abstract

Calmodulin (CaM) is a ubiquitous intracellular Ca²⁺ sensing protein that modifies gating of numerous ion channels. CaM has an extraordinarily high level of evolutionary conservation, which led to the fundamental assumption that mutation would be lethal. However, in 2012, complete exome sequencing [...] Read more.

Calmodulin (CaM) is a ubiquitous intracellular Ca²⁺ sensing protein that modifies gating of numerous ion channels. CaM has an extraordinarily high level of evolutionary conservation, which led to the fundamental assumption that mutation would be lethal. However, in 2012, complete exome sequencing of infants suffering from recurrent cardiac arrest revealed de novo mutations in the three human CALM genes. The correlation between mutations and pathophysiology suggests defects in CaM-dependent ion channel functions. Here, we review the current state of the field for all reported CaM mutations associated with cardiac arrhythmias, including knowledge of their biochemical and structural characteristics, and progress towards understanding how these mutations affect cardiac ion channel function. Full article

(This article belongs to the Special Issue Calmodulin Function in Health and Disease)

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31 pages, 7253 KiB

Open AccessReview

Atomistic Insights of Calmodulin Gating of Complete Ion Channels

by Eider Núñez, Arantza Muguruza-Montero and Alvaro Villarroel

Int. J. Mol. Sci. 2020, 21(4), 1285; https://doi.org/10.3390/ijms21041285 - 14 Feb 2020

Cited by 12 | Viewed by 4117

Abstract

Intracellular calcium is essential for many physiological processes, from neuronal signaling and exocytosis to muscle contraction and bone formation. Ca²⁺ signaling from the extracellular medium depends both on membrane potential, especially controlled by ion channels selective to K⁺, and direct [...] Read more.

Intracellular calcium is essential for many physiological processes, from neuronal signaling and exocytosis to muscle contraction and bone formation. Ca²⁺ signaling from the extracellular medium depends both on membrane potential, especially controlled by ion channels selective to K⁺, and direct permeation of this cation through specialized channels. Calmodulin (CaM), through direct binding to these proteins, participates in setting the membrane potential and the overall permeability to Ca²⁺. Over the past years many structures of complete channels in complex with CaM at near atomic resolution have been resolved. In combination with mutagenesis-function, structural information of individual domains and functional studies, different mechanisms employed by CaM to control channel gating are starting to be understood at atomic detail. Here, new insights regarding four types of tetrameric channels with six transmembrane (6TM) architecture, Eag1, SK2/SK4, TRPV5/TRPV6 and KCNQ1–5, and its regulation by CaM are described structurally. Different CaM regions, N-lobe, C-lobe and EF3/EF4-linker play prominent signaling roles in different complexes, emerging the realization of crucial non-canonical interactions between CaM and its target that are only evidenced in the full-channel structure. Different mechanisms to control gating are used, including direct and indirect mechanical actuation over the pore, allosteric control, indirect effect through lipid binding, as well as direct plugging of the pore. Although each CaM lobe engages through apparently similar alpha-helices, they do so using different docking strategies. We discuss how this allows selective action of drugs with great therapeutic potential. Full article

(This article belongs to the Special Issue Calmodulin Function in Health and Disease)

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15 pages, 1702 KiB

Open AccessReview

Calmodulin and Calmodulin Binding Proteins in Dictyostelium: A Primer

by Danton H. O’Day, Ryan J. Taylor and Michael A. Myre

Int. J. Mol. Sci. 2020, 21(4), 1210; https://doi.org/10.3390/ijms21041210 - 11 Feb 2020

Cited by 13 | Viewed by 4584

Abstract

Dictyostelium discoideum is gaining increasing attention as a model organism for the study of calcium binding and calmodulin function in basic biological events as well as human diseases. After a short overview of calcium-binding proteins, the structure of Dictyostelium calmodulin and the conformational [...] Read more.

Dictyostelium discoideum is gaining increasing attention as a model organism for the study of calcium binding and calmodulin function in basic biological events as well as human diseases. After a short overview of calcium-binding proteins, the structure of Dictyostelium calmodulin and the conformational changes effected by calcium ion binding to its four EF hands are compared to its human counterpart, emphasizing the highly conserved nature of this central regulatory protein. The calcium-dependent and -independent motifs involved in calmodulin binding to target proteins are discussed with examples of the diversity of calmodulin binding proteins that have been studied in this amoebozoan. The methods used to identify and characterize calmodulin binding proteins is covered followed by the ways Dictyostelium is currently being used as a system to study several neurodegenerative diseases and how it could serve as a model for studying calmodulinopathies such as those associated with specific types of heart arrythmia. Because of its rapid developmental cycles, its genetic tractability, and a richly endowed stock center, Dictyostelium is in a position to become a leader in the field of calmodulin research. Full article

(This article belongs to the Special Issue Calmodulin Function in Health and Disease)

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12 pages, 2369 KiB

Open AccessReview

Calmodulin-Binding Proteins in Muscle: A Minireview on Nuclear Receptor Interacting Protein, Neurogranin, and Growth-Associated Protein 43

by Fereshteh Moradi, Emily N. Copeland, Ryan W. Baranowski, Aiden E. Scholey, Jeffrey A. Stuart and Val A. Fajardo

Int. J. Mol. Sci. 2020, 21(3), 1016; https://doi.org/10.3390/ijms21031016 - 4 Feb 2020

Cited by 12 | Viewed by 4651

Abstract

Calmodulin (CaM) is an important Ca²⁺-sensing protein with numerous downstream targets that are either CaM-dependant or CaM-regulated. In muscle, CaM-dependent proteins, which are critical regulators of dynamic Ca²⁺ handling and contractility, include calcineurin (CaN), CaM-dependant kinase II (CaMKII), ryanodine receptor [...] Read more.

Calmodulin (CaM) is an important Ca²⁺-sensing protein with numerous downstream targets that are either CaM-dependant or CaM-regulated. In muscle, CaM-dependent proteins, which are critical regulators of dynamic Ca²⁺ handling and contractility, include calcineurin (CaN), CaM-dependant kinase II (CaMKII), ryanodine receptor (RyR), and dihydropyridine receptor (DHPR). CaM-regulated targets include genes associated with oxidative metabolism, muscle plasticity, and repair. Despite its importance in muscle, the regulation of CaM—particularly its availability to bind to and activate downstream targets—is an emerging area of research. In this minireview, we discuss recent studies revealing the importance of small IQ motif proteins that bind to CaM to either facilitate (nuclear receptor interacting protein; NRIP) its activation of downstream targets, or sequester (neurogranin, Ng; and growth-associated protein 43, GAP43) CaM away from their downstream targets. Specifically, we discuss recent studies that have begun uncovering the physiological roles of NRIP, Ng, and GAP43 in skeletal and cardiac muscle, thereby highlighting the importance of endogenously expressed CaM-binding proteins and their regulation of CaM in muscle. Full article

(This article belongs to the Special Issue Calmodulin Function in Health and Disease)

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42 pages, 3844 KiB

Open AccessReview

The Role of Calmodulin in Tumor Cell Migration, Invasiveness, and Metastasis

by Antonio Villalobo and Martin W. Berchtold

Int. J. Mol. Sci. 2020, 21(3), 765; https://doi.org/10.3390/ijms21030765 - 24 Jan 2020

Cited by 58 | Viewed by 8796

Abstract

Calmodulin (CaM) is the principal Ca²⁺ sensor protein in all eukaryotic cells, that upon binding to target proteins transduces signals encoded by global or subcellular-specific changes of Ca²⁺ concentration within the cell. The Ca²⁺/CaM complex as well as Ca [...] Read more.

Calmodulin (CaM) is the principal Ca²⁺ sensor protein in all eukaryotic cells, that upon binding to target proteins transduces signals encoded by global or subcellular-specific changes of Ca²⁺ concentration within the cell. The Ca²⁺/CaM complex as well as Ca²⁺-free CaM modulate the activity of a vast number of enzymes, channels, signaling, adaptor and structural proteins, and hence the functionality of implicated signaling pathways, which control multiple cellular functions. A basic and important cellular function controlled by CaM in various ways is cell motility. Here we discuss the role of CaM-dependent systems involved in cell migration, tumor cell invasiveness, and metastasis development. Emphasis is given to phosphorylation/dephosphorylation events catalyzed by myosin light-chain kinase, CaM-dependent kinase-II, as well as other CaM-dependent kinases, and the CaM-dependent phosphatase calcineurin. In addition, the role of the CaM-regulated small GTPases Rac1 and Cdc42 (cell division cycle protein 42) as well as CaM-binding adaptor/scaffold proteins such as Grb7 (growth factor receptor bound protein 7), IQGAP (IQ motif containing GTPase activating protein) and AKAP12 (A kinase anchoring protein 12) will be reviewed. CaM-regulated mechanisms in cancer cells responsible for their greater migratory capacity compared to non-malignant cells, invasion of adjacent normal tissues and their systemic dissemination will be discussed, including closely linked processes such as the epithelial–mesenchymal transition and the activation of metalloproteases. This review covers as well the role of CaM in establishing metastatic foci in distant organs. Finally, the use of CaM antagonists and other blocking techniques to downregulate CaM-dependent systems aimed at preventing cancer cell invasiveness and metastasis development will be outlined. Full article

(This article belongs to the Special Issue Calmodulin Function in Health and Disease)

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37 pages, 7943 KiB

Open AccessReview

Calmodulin-Mediated Regulation of Gap Junction Channels

by Camillo Peracchia

Int. J. Mol. Sci. 2020, 21(2), 485; https://doi.org/10.3390/ijms21020485 - 12 Jan 2020

Cited by 26 | Viewed by 4791

Abstract

Evidence that neighboring cells uncouple from each other as one dies surfaced in the late 19th century, but it took almost a century for scientists to start understanding the uncoupling mechanism (chemical gating). The role of cytosolic free calcium (Ca²⁺_i) [...] Read more.

Evidence that neighboring cells uncouple from each other as one dies surfaced in the late 19th century, but it took almost a century for scientists to start understanding the uncoupling mechanism (chemical gating). The role of cytosolic free calcium (Ca²⁺_i) in cell–cell channel gating was first reported in the mid-sixties. In these studies, only micromolar [Ca²⁺]_i were believed to affect gating—concentrations reachable only in cell death, which would discard Ca²⁺_i as a fine modulator of cell coupling. More recently, however, numerous researchers, including us, have reported the effectiveness of nanomolar [Ca²⁺]_i. Since connexins do not have high-affinity calcium sites, the effectiveness of nanomolar [Ca²⁺]_i suggests the role of Ca-modulated proteins, with calmodulin (CaM) being most obvious. Indeed, in 1981 we first reported that a CaM-inhibitor prevents chemical gating. Since then, the CaM role in gating has been confirmed by studies that tested it with a variety of approaches such as treatments with CaM-inhibitors, inhibition of CaM expression, expression of CaM mutants, immunofluorescent co-localization of CaM and gap junctions, and binding of CaM to peptides mimicking connexin domains identified as CaM targets. Our gating model envisions Ca²⁺-CaM to directly gate the channels by acting as a plug (“Cork” gating model), and probably also by affecting connexin conformation. Full article

(This article belongs to the Special Issue Calmodulin Function in Health and Disease)

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