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Gene name - Calmodulin Synonyms - Cytological map position - 49A1-49A13 Function - calcium ion binding signaling protein Keyword(s) - regulation of Ca2+-dependent processes, Visual signal transduction |
Symbol - Cam FlyBase ID:FBgn0000253 Genetic map position - 2-[64] Classification - EF-hand calcium-binding domain. Cellular location - cytoplasmic |
Calmodulin is a protein of primary importance in the regulation of cellular processes dependent on Ca2+. When cellular Ca2+ levels rise, CaM sequesters Ca2+ ions, binding specifically to CaM-binding domains on a variety of proteins. CaM is involved in most of the important signaling pathways in the cell.
CaM targets include:
CaM in the Ca2+-bound form is a dumbell-shaped molecule: two lobes (globular domains) connected by a long alpha-helix chain, the central portion of which is highly mobile, acting as a flexible tether for the two domains. Each domain binds two Ca2+ ions. The C-terminal lobe binds Ca2+ with high affinity, while the N-terminal shows lower affinity for Ca2+. Binding of Ca2+ ions induces a large conformational change which makes two hydrophobic patches, one in each half of the molecule, available for target interaction (James, 1995 and references).
Most of the characterized Calmodulin binding domains of target proteins are stretches of 16 to 35 amino acids that show a segregation of basic and polar residues on one side of an alpha-helical configuration, and hydrophobic amino acids on the other. Upon target binding of CaM, the extended CaM dumbbell-like structure collapses into a more compact form: the two globular lobes now face each other and the central helical chain twists into a long flexible loop. In many cases, the CaM-binding domain of target proteins constitutes, or is adjacent to, an autoinhibitory domain. Thus, the CaM-binding site interacts with the active site of the enzyme repressing it until inactivated by binding to CaM. In many cases, the CaM-binding domain of target proteins is subject to phosphorylation, generating a CaM independent target (James, 1995 and references). Additional information about these processes can be found at the CaMKII site.
One of CaM's many roles in fly biology is illustrated by the complex interaction of CaM with the unconventional myosin NINAC. CaM localization in the rhabdomere, a microvillar structure in the photoreceptor cell, is disrupted in ninaC mutants. There are two isoforms of NINAC: the p174 isoform is spatially restricted to the rhabdomere and p132 is restricted to the sub-rhabdomeral cytoplasm. Mutant flies lacking the rhabdomere-specific p174 NINAC protein do not concentrate Calmodulin in the rhabdomere, while flies lacking the sub-rhabdomeral p132 isoform have no detectable cytoplasmic Calmodulin (Porter, 1993). Of the two Calmodulin-binding sites in NINAC (C1 and C2), C1 is common to both p132 and p174, while C2 is unique to p174. Spatial location of CaM depends on binding of CaM to both C1 and C2 (Porter, 1995).
Disruption of C1 or C2 disrupts the normal distribution of CaM. In flies carrying a NINAC deleted for C1, Calmodulin staining is detected almost exclusively in the rhabdomeres, and the staining is less intense than in wild-type. In flies carrying a NINAC deleted for C2 (carrying wild-type p132), the relative intensity of staining in the rhabdomeres is reduced, when compared with wild-type. Deletion of C1 or C2, and the consequent disruption of the normal distribution of CaM, results in an abnormal retinal electrophysiology characterized by a prolonged depolarization after-potential (PDA) (Porter, 1995).
The interaction of CaM and NINAC may not be restricted solely to a transport function. Besides being an unconventional myosin, NINAC also possesses a kinase domain. What is the function of this kinase and how is it regulated? Is the myosin function of NINAC regulated by CaM? NinaC myosin activity might be regulated by Calmodulin, since the properties of at least some unconventional myosins are affected by Calmodulin association. As far as the kinase function is concerned, the electrophysiological phenotype (PDA) caused by deletion of C1 and C2 suggests that Calmodulin binding to p174 is required for inactivation of the phototransduction cascade. The only other mutation identified that results in a similar reduction in PDA is arrestin2 . In wild-type organisms, Arrestin2 binds rhodopsin (the photoreceptor of the fly), which is subsequently phosphorylated at multiple serine and threonine residues by rhodopsin kinase. The phosphorylated rhodopsin is then protected by Arrestin2 from dephosphorylation and is no longer able to bind the signal transducing G-protein, causing termination of the photoresponse. Arrestin2 also undergoes serine/threonine phosphorylation, apparently in a Ca2+/CaM dependent fashion. It is intriguing to speculate that NINAC p174 may be the CaM-dependent serine/threonine kinase that phosphorylates Arrestin2 (Porter, 1995).
In addition to the PDA phenotype, the deletion of the C2 CaM interaction site of NINAC, but not C1, results in an abnormal electroretinogram recording, a measurement of the summed response of all retinal cells to light. C2 null flies exhibit an electroretinogram measurement similar to a null allele of ninaC , again suggesting an intimate functional interaction between CaM and NINAC (Porter, 1995).
Calcium levels regulate a multitude of cell functions. Calmodulin is crucial to proper calcium function -- a synergistic relationship both highly evolved and evolutionarily conserved. For example, the following proteins and/or interactions are all conserved in evolution:
There are two transcripts produced as a result of alternative polyadenylation site usage. The two polyadenylation signals are separated by 280 bp in genomic DNA. The shorter-length class of transcripts is associated with a group of five polyadenylation signals; the longer one with three such signals. The gene contains five exons, including a 49 bp exon in the 5' untranslated region, and spans over 16 kb. Homologs of this small, 5' noncoding exon have not been found in other calmodulin genes. Primer extension experiments and RNase protection mapping show that both size classes of Drosophila Calmodulin transcripts initiate at the same site but undergo alternative termination within the final exon (Smith, 1987, Doyle, 1990 and Hanson-Painton, 1992).
Bases in 5' UTR - 140
Exons - 5
Bases in 3' UTR - 1109 (for the longest transcript)
Calmodulin contains four "EF-hand"-type calcium binding sites, and the amino- and carboxy-terminal pairs of sites form two discrete globular domains. The two tyrosine-containing Ca2+ binding sites in the C-terminal domain have a significantly higher affinity for Ca2+ than do the two sites in the amino-terminal domain. There is a high degree of mobility near the middle of the central helix of Calmodulin, from residues K77 through S81. The anisotropy observed in the motion of the two globular Calmodulin domains (N- and C-terminal) is much smaller than expected on the basis of hydrodynamic calculations for a rigid dumbbell type structure. This indicates that the N-terminal (L4-K77) and C-terminal (E82-S147) lobes of Calmodulin are effectively independent of one another with respect to tumbling. A slightly shorter motional correlation time is obtained for the C-terminal domain compared to the N-terminal domain, in agreement with the smaller size of the C-terminal domain. A high degree of mobility is also observed in the loop that connects the first with the second EF-hand type calcium binding domain, and in the loop connecting the third and fourth calcium binding domain as well (Barbato, 1992).
The three-dimensional solution structure of the complex between calcium-bound Calmodulin and a 26-residue synthetic peptide comprising the CaM binding domain of skeletal muscle light chain kinase is described. The two domains of CaM remain essentially unchanged by complexation. The long central helix, however, which connects the two terminal domains is disrupted into two helices connected by a long flexible loop, thereby enabling the two domains to clamp the bound peptide. The helical synthetic peptide is located in a hydrophobic channel that passes through the center of the CaM ellipsoid at an angle of approximately 45 degrees to its long axis. The complex is stabilized mainly by hydrophobic interactions, which, from the CaM side, involve an unusually large number of methionines. Sequence comparisons indicate that a number of proteins that bind CaM with high affinity share common features, either containing aromatic residues or long-chain hydrophobic residues, separated by stretches of 12 residues, suggesting that CaM target proteins interact with CaM in a common manner (Ikura, 1992).
date revised: 1 FEB 97
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