UA BIOC 585 - Calmodulin - a prototypical calcium sensor

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322 0962-8924/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. trends in CELL BIOLOGY (Vol. 10) August 2000PII: S0962-8924(00)01800-6Calcium (as Ca21) is an element that is crucial fornumerous biological functions. In many organisms,the vast majority of Ca21is complexed with phos-phates to form exo- or endoskeletons that not onlyserve as structural scaffolds but also buffer the levelsof Ca21 within extracellular fluids at ~1023M. Bycontrast, the resting concentrations of intracellularfree Ca21(~1027M) is 104times lower than that out-side cells, providing the potential for the ready import of Ca21into cells, where it can act as a secondmessenger.Various extracellular stimuli promote the move-ment of Ca21either from outside the cell (viaplasma-membrane Ca21channels) or from intracellularstores into the intracellular milieu (Fig. 1a). TheCa21is released in elemental aliquots called sparks,puffs or waves depending on the extent of the intra-cellular area covered. This free Ca21is only brieflyavailable to act as a cellular signal, however, becauseCa21-binding proteins and Ca21pumps immediatelycombine to sequester and transport it to intracellularstorage sites or outside the cell.The short pulses of Ca21exert specific changes incellular function depending on their route of entryinto the cell, their local sites of action and, finally, bytheir pattern of modulation. The particular mem-brane channel or intracellular receptor responsiblefor the release of Ca21exerts considerable influenceon the eventual effects of the Ca21signal1. The modeof cellular entry also influences the site of action ofthe Ca21signal. Hence, separate intracellular loci ororganelles are potentially distinct compartments oflocalized Ca21signalling2(Fig. 1a). Therefore, Ca21signals in the nucleus exert different effects fromthose generated in the cytoplasm or near the plasmamembrane of the same cell3. Additionally, themodulation of the amplitude or frequency of Ca21spikes (AM and FM, respectively) encodes important signalling information4. This has recently been illustrated for cases in which an optimal frequencyof intracellular Ca21oscillations is important for theexpression of different genes5.Calcium-regulated proteins: calmodulinHow do Ca21signals produce changes in cell func-tion? The information encoded in transient Ca21signals is deciphered by various intracellular Ca21-binding proteins that convert the signals into a widevariety of biochemical changes. Some of these proteins, such as protein kinase C, bind to Ca21andare directly regulated in a Ca21-dependent manner.Other Ca21-binding proteins, however, are inter-mediaries that couple the Ca21signals to biochemicaland cellular changes (Fig. 1b). Among this lattergroup are a family of proteins that is distinguishedby a structural motif known as the E–F hand. An E–Fhand consists of an N-terminal helix (the E helix)immediately followed by a centrally located, Ca21-coordinating loop and a C-terminal helix (the F helix). The three-dimensional arrangement ofthese domains is reminiscent of the thumb, indexand middle fingers of a hand, hence the name ‘E–Fhand’.These proteins respond to Ca21in one of two ways(Fig. 1b). One group (e.g. parvalbumin and calbindin)do not undergo a significant change in confor-mation on binding Ca21and function as Ca21buffersor Ca21transporters. The second group, the Ca21sensors, undergo a Ca21-induced change in confor-mation6. The most prominent examples of sensorsinclude troponin C (a protein dedicated to regu-lating striated-muscle contraction), the multifunc-tional Ca21transducer calmodulin (CaM), the S100family of proteins and, most recently, the neuronalmyristoylated proteins such as recoverin7.The molecular and cellular mechanisms under-lying the ability of a majority of the Ca21-sensor proteins to integrate Ca21signals into specific cellularresponses are not clearly understood. Much of whatwe do know about the mechanisms that the sensorproteins use to transduce Ca21signals is based on information gained from CaM, probably the mostintensively studied member of the E–F-hand familyof sensors. In the remainder of this article, CaM willtherefore serve as a model or prototype for other potential Ca21transducers. A review of some of themechanisms responsible for regulating CaM at thesubcellular and molecular levels might reveal valuableclues as to how Ca21-sensor proteins convert Ca21signals into cellular function.CaM is expressed in all eukaryotic cells where itparticipates in signalling pathways that regulatemany crucial processes such as growth, proliferationand movement. It is relatively small (vertebrate CaMCalmodulin: aprototypicalcalcium sensorDavid Chin and Anthony R. MeansCalmodulin is the best studied and prototypical example of theE–F-hand family of Ca21-sensing proteins. Changes inintracellular Ca21concentration regulate calmodulin in threedistinct ways. First, at the cellular level, by directing itssubcellular distribution. Second, at the molecular level, bypromoting different modes of association with many targetproteins. Third, by directing a variety of conformational states incalmodulin that result in target-specific activation. Thecalmodulin-dependent regulation of protein kinases illustrates thepotential mechanisms by which Ca21-sensing proteins canrecognize and generate affinity and specificity for effectors in aCa21-dependent manner.The authors are inthe Dept ofPharmacologyand CancerBiology, DukeUniversity MedicalCenter, Durham,NC 27710, USA.E-mail: [email protected];[email protected] 08/00 paste-up 30/6/00 8:54 am Page 322reviewstrends in CELL BIOLOGY (Vol. 10) August 2000 323has 148 residues), evolutionarily highly conservedand comprises four E–F hands. The first two E–Fhands combine to form a globular N-terminal domain that is separated by a short flexible linker froma highly homologous C-terminal domain consisting ofE–F hands 3 and 4 (Fig. 2).Ca21sensors must be able to detect and respond to a biologically relevant range of intracellular freeCa21concentrations. CaM fits this profile as itsaffinity for Ca21(Kd5 5 3 1027M to 5 3 1026M) fallswithin the range of intracellular Ca21concentrationsexhibited by most cells (1027M to 1026M). However,it has additional discrimination for Ca21, as the C-terminal pair of E–F hands has a three- to fivefoldhigher affinity for Ca21than the N-terminal pair ofsites. By contrast, many Ca21-binding proteins witha


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