TCB 08/00 paste-up 30/6/00 8:54 am Page 322 reviews Calmodulin: a the Ca21 signal. Hence, separate intracellular loci or organelles are potentially distinct compartments of prototypical localized Ca21 signalling2 (Fig. 1a). Therefore, Ca21 signals in the nucleus exert different effects from those generated in the cytoplasm or near the plasma calcium sensor membrane of the same cell3. Additionally, the modulation of the amplitude or frequency of Ca21 spikes (AM and FM, respectively) encodes important David Chin and Anthony R. Means signalling information4. This has recently been illustrated for cases in which an optimal frequency of intracellular Ca21 oscillations is important for the Calmodulin is the best studied and prototypical example of the expression of different genes5. 21 E–F-hand family of Ca -sensing proteins. Changes in Calcium-regulated proteins: calmodulin 21 intracellular Ca21 concentration regulate calmodulin in three How do Ca signals produce changes in cell func- tion? The information encoded in transient Ca21 distinct ways. First, at the cellular level, by directing its signals is deciphered by various intracellular Ca21- binding proteins that convert the signals into a wide subcellular distribution. Second, at the molecular level, by variety of biochemical changes. Some of these 21 promoting different modes of association with many target proteins, such as protein kinase C, bind to Ca and are directly regulated in a Ca21-dependent manner. proteins. Third, by directing a variety of conformational states in Other Ca21-binding proteins, however, are inter- mediaries that couple the Ca21 signals to biochemical calmodulin that result in target-specific activation. The and cellular changes (Fig. 1b). Among this latter calmodulin-dependent regulation of protein kinases illustrates the group are a family of proteins that is distinguished by a structural motif known as the E–F hand. An E–F 1 potential mechanisms by which Ca2 -sensing proteins can hand consists of an N-terminal helix (the E helix) 21 recognize and generate affinity and specificity for effectors in a immediately followed by a centrally located, Ca - coordinating loop and a C-terminal helix (the Ca21-dependent manner. F helix). The three-dimensional arrangement of these domains is reminiscent of the thumb, index and middle fingers of a hand, hence the name ‘E–F hand’. These proteins respond to Ca21 in one of two ways Calcium (as Ca21) is an element that is crucial for (Fig. 1b). One group (e.g. parvalbumin and calbindin) numerous biological functions. In many organisms, do not undergo a significant change in confor- the vast majority of Ca21 is complexed with phos- mation on binding Ca21 and function as Ca21 buffers phates to form exo- or endoskeletons that not only or Ca21 transporters. The second group, the Ca21 serve as structural scaffolds but also buffer the levels sensors, undergo a Ca21-induced change in confor- 1 2 of Ca2 within extracellular fluids at ~10 3 M. By mation6. The most prominent examples of sensors contrast, the resting concentrations of intracellular include troponin C (a protein dedicated to regu- 1 2 free Ca2 (~10 7 M) is 104 times lower than that out- lating striated-muscle contraction), the multifunc- side cells, providing the potential for the ready tional Ca21 transducer calmodulin (CaM), the S100 import of Ca21 into cells, where it can act as a second family of proteins and, most recently, the neuronal messenger. myristoylated proteins such as recoverin7. Various extracellular stimuli promote the move- The molecular and cellular mechanisms under- ment of Ca21 either from outside the cell (via lying the ability of a majority of the Ca21-sensor plasma-membrane Ca21 channels) or from intracellular proteins to integrate Ca21 signals into specific cellular stores into the intracellular milieu (Fig. 1a). The responses are not clearly understood. Much of what Ca21 is released in elemental aliquots called sparks, we do know about the mechanisms that the sensor puffs or waves depending on the extent of the intra- proteins use to transduce Ca21 signals is based on cellular area covered. This free Ca21 is only briefly information gained from CaM, probably the most The authors are in available to act as a cellular signal, however, because intensively studied member of the E–F-hand family 1 1 the Dept of Ca2 -binding proteins and Ca2 pumps immediately of sensors. In the remainder of this article, CaM will Pharmacology combine to sequester and transport it to intracellular therefore serve as a model or prototype for other 1 and Cancer storage sites or outside the cell. potential Ca2 transducers. A review of some of the 1 Biology, Duke The short pulses of Ca2 exert specific changes in mechanisms responsible for regulating CaM at the University Medical cellular function depending on their route of entry subcellular and molecular levels might reveal valuable 1 1 Center, Durham, into the cell, their local sites of action and, finally, by clues as to how Ca2 -sensor proteins convert Ca2 NC 27710, USA. their pattern of modulation. The particular mem- signals into cellular function. E-mail: chin0001@ brane channel or intracellular receptor responsible CaM is expressed in all eukaryotic cells where it 1 mc.duke.edu; for the release of Ca2 exerts considerable influence participates in signalling pathways that regulate 1 means001@ on the eventual effects of the Ca2 signal1. The mode many crucial processes such as growth, proliferation mc.duke.edu of cellular entry also influences the site of action of and movement. It is relatively small (vertebrate CaM 322 0962-8924/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. trends in CELL BIOLOGY (Vol. 10) August 2000 PII: S0962-8924(00)01800-6 TCB 08/00 paste-up 30/6/00 8:54 am Page 323 reviews (a) Ca2+ (a) (b) Ca2+ Amplitude Frequency modulated (AM) modulated (FM) Ca2+ Ca2+ Nucleus trends in Cell Biology Ca2+ FIGURE 2 2+ (b) Ca buffers and 1 The Ca2 -regulated conformational change in calmodulin. The transporters 1 main chain structure of Ca2 -free (apo) CaM (a) and 21 Ca 4–CaM (b) are shown in red with their respective N-terminal domains on top. Methionine side chains are shown Ca2+ 2+ Effectors Ca sensors in purple to denote the location of potential hydrophobic 21 trends in Cell Biology pockets in each of the two domains. Ca binding produces large changes in the helices in both domains, resulting in the FIGURE 1 exposure of several hydrophobic residues. (a) Sources of intracellular Ca21 signals. Ca21 enters cells via extracellular plasma-membrane receptors or from intracellular 21 stores, producing transient local or global changes in its (Fig. 2). In the absence of Ca , the N-terminal distribution. The Ca21 oscillations are modulated in their domain of the apo-CaM molecule adopts a ‘closed’ amplitudes (AM) or frequencies (FM) and are therefore capable conformation in which the helices in both E–F of conveying signalling information in complex ways. (b) E–F- hands are packed together. By contrast, still in the 21 hand Ca21-binding proteins are classified as buffers/transporters absence of Ca , the C-terminal domain of apo-CaM and sensors. The Ca21 sensors change conformation on binding adopts a ‘semiopen’ conformation in which a par- Ca21 and transduce changes in cell function by regulating tially exposed hydrophobic patch is accessible to downstream effectors. solvent. This might allow the C-terminal domain of CaM to interact with some target proteins at resting levels of intracellular free Ca21 (Ref. 8). has 148 residues), evolutionarily highly conserved In the event of a transient rise in Ca21, the Ca21 and comprises four E–F hands. The first two E–F ion is coordinated in each Ca21-binding loop of hands combine to form a globular N-terminal Ca21–CaM by seven, primarily carboxylate, ligands. domain that is separated by a short flexible linker from The binding of Ca21 leads to substantial alterations a highly homologous C-terminal domain consisting of in the interhelical angles within the E–F hands in E–F hands 3 and 4 (Fig. 2). each domain and dramatically changes the two Ca21 sensors must be able to detect and respond domains of CaM to produce more ‘open’ confor- to a biologically relevant range of intracellular free mations (Fig. 2). These structural rearrangements in Ca21 concentrations. CaM fits this profile as its CaM result in the concerted exposure of hydrophobic 21 5 3 27 3 26 affinity for Ca (Kd 5 10 M to 5 10 M) falls groups in a methionine-rich crevice of each domain within the range of intracellular Ca21 concentrations that is distinct from the Ca21-binding loops. The ex- 2 2 exhibited by most cells (10 7 M to 10 6 M). However, posure to solvent of these hydrophobic residues is it has additional discrimination for Ca21, as the C- akin to a Ca21-controlled unfolding of CaM and un- terminal pair of E–F hands has a three- to fivefold leashes considerable free energy. It is this capacity to higher affinity for Ca21 than the N-terminal pair of convert the Ca21-binding event into biochemical en- sites. By contrast, many Ca21-binding proteins with ergy that characterizes the Ca21-sensor proteins and , 27 21 a considerably higher affinity (Kd 10 M) act as is the basis of their ability to transduce Ca signals. buffers by sequestering excess free Ca21, whereas Ca21-binding proteins with a considerably lower Calmodulin: location, mobility and translocation . 25 affinity (Kd 10 M) could not act as sensors Is CaM regulated at the subcellular level, and how because they are unable to detect the range of is this related to Ca21 signalling? The concentration changes in intracellular free Ca21 concentrations and location of CaM do appear to play an important that normally occur in cells.