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PubMed1992

Calcium-myristoyl protein switch.

Recoverin, a recently discovered member of the EF-hand superfamily of Ca(2+)-binding proteins, serves as a Ca2+ sensor in vision. The amino terminus of the protein from retinal rod cells contains a covalently attached myristoyl or related N-acyl group. We report here studies of unmyristoylated and myristoylated recombinant recoverin designed to delineate the biological role of this hydrophobic unit. Ca2+ induces the binding of both the unmyristoylated and myristoylated proteins to phenyl-agarose, a hydrophobic support. Binding was half-maximal at 1.1 and 1.0 microM Ca2+, respectively. The Hill coefficients of 1.8 and 1.7, respectively, indicate that binding was cooperative. In contrast, Ca2+ induced the binding of myristoylated but not of unmyristoylated recoverin to rod outer segment membranes. Binding to these membranes was half-maximal at 2.1 microM Ca2+, and the Hill coefficient was 2.4. Likewise, myristoylated but not unmyristoylated recoverin exhibited Ca(2+)-induced binding to phosphatidylcholine vesicles. These findings suggest that the binding of Ca2+ to recoverin has two effects: (i) hydrophobic surfaces are exposed, allowing the protein to interact with complementary nonpolar sites, such as the aromatic rings of phenyl-agarose; and (ii) the myristoyl group is extruded, enabling recoverin to insert into a lipid bilayer membrane. The myristoyl group is likely to be an active participant in Ca2+ signaling by recoverin and related EF-hand proteins such as visinin and neurocalcin.

Amino Acid Sequence↗
PubMed1995

Recoverin, a photoreceptor-specific calcium-binding protein, is expressed by the tumor of a patient with cancer-associated retinopathy.

Recoverin is a member of the EF-hand family of calcium-binding proteins involved in the transduction of light by vertebrate photoreceptors. Recoverin also was identified as an autoantigen in the degenerative disease of the retina known as cancer-associated retinopathy (CAR), a paraneoplastic syndrome whereby immunological events lead to the degeneration of photoreceptors in some individuals with cancer. In this study, we demonstrate that recoverin is expressed in the lung tumor of a CAR patient but not in similar tumors obtained from individuals without the associated retinopathy. Recoverin was identified intially by Western blot analysis of the CAR patient's biopsy tissue by using anti-recoverin antibodies generated against different regions of the recoverin molecule. In addition, cultured cells from the biopsy tissue expressed recoverin, as demonstrated by reverse transcription-PCR using RNA extracted from the cells. The immunodominant region of recoverin also was determined in this study by a solid-phase immunoassay employing overlapping heptapeptides encompassing the entire recoverin sequence. Two linear stretches of amino acids (residues 64-70, Lys-Ala-Tyr-Ala-Gln-His-Val; and 48-52, Gln-Phe-Gln-Ser-Ile) made up the major determinants. One of the same regions of the recoverin molecule (residues 64-70) also was uniquely immunopathogenic, causing photoreceptor degeneration upon immunization of Lewis rats with the corresponding peptide. These data demonstrate that the neural antigen recoverin more than likely is responsible for the immunological events associated with vision loss in some patients with cancer. These data also establish CAR as one of the few autoimmune-mediated diseases for which the specific self-antigen is known.

Amino Acid Sequence↗
PubMed1996

Responses of the phototransduction cascade to dim light.

The biochemistry of visual excitation is kinetically explored by measuring the activity of the cGMP phosphodiesterase (PDE) at light levels that activate only a few tens of rhodopsin molecules per rod. At 23 degrees C and in the presence of ATP, the pulse of PDE activity lasts 4 s (full width at half maximum). Complementing the rod outer segments (ROS) with rhodopsin kinase (RK) and arrestin or its splice variant p44 does not significantly shorten the pulse. But when the ROS are washed, the duration of the signal doubles. Adding either arrestin or p44 back to washed ROS approximately restores the pulse width to its initial value, with p44 being 10 times more efficient than arrestin. This supports the idea that, in vivo, capping of phosphorylated R* is mostly done by p44. When myristoylated (14:0) recoverin is added to unwashed ROS, the pulse duration and amplitude increase by about 50% if the free calcium is 500 nM. This effect increases further if the calcium is raised to 1 microM. Whenever R* deactivation is changed--when RK is exogenously enriched or when ATP is omitted from the buffer--there is no impact on the rising slope of the PDE pulse but only on its amplitude and duration. We explain this effect as due to the unequal competition between transducin and RK for R*. The kinetic model issued from this idea fits the data well, and its prediction that enrichment with transducin should lengthen the PDE pulse is successfully validated.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

The effect of recombinant recoverin on the photoresponse of truncated rod photoreceptors.

Recoverin is a heterogeneously acylated calcium-binding protein thought to regulate visual transduction. Its effect on the photoresponse was investigated by dialyzing the recombinant protein into truncated salamander rod outer segments. At high Ca2+ (Ca), myristoylated recoverin (Ca-recoverin) prolonged the recovery phase of the bright flash response but had less effect on the dim flash response. The prolongation of recovery had an apparent Kd for Ca of 13 microM and a Hill coefficient of 2. The prolongation was shown to be mediated by inhibition of rhodopsin deactivation. After a sudden imposed drop in Ca concentration, the effect of recoverin switched off with little lag. The myristoyl (C14:0) modification of recoverin increased its activity 12-fold, and the C12:0 or C14:2 acyl group gave similar effects. These experiments support the notion that recoverin mediates Ca-dependent inhibition of rhodopsin phosphorylation and thereby controls light-triggered phosphodiesterase activity, particularly at high light levels.

Ambystoma↗
PubMed1995

Role of heterogeneous N-terminal acylation of recoverin in rhodopsin phosphorylation.

Recoverin, a new member of the EF-hand superfamily, plays a critical role in the light/dark adaptation of retinal rods by regulating rhodopsin phosphorylation in a Ca(2+)-dependent manner. Recoverin is composed of four isoforms, each of which is modified at its N terminus by myristate (C14:0) or its structurally related fatty acid (C12:0, C14:2, or C14:1). Although the N-fatty acylation is implicated in protein-membrane and protein-protein interactions, the functional difference among the recoverin isoforms and the significance of the heterogeneous acylation have not been defined. Here we separated the heterogeneous recoverin into three fractions, C14:0-recoverin, C14:1-recoverin, and a mixture of C14:2- and C12:0- (C14:2/C12:0-) recoverin to evaluate the individual properties. Recoverin in every fraction bound Ca2+ as assessed by fluorescence spectroscopy and inhibited the light-dependent rhodopsin phosphorylation in the same range of free Ca2+ concentration (0.3-0.8 microM). However, the magnitude of the inhibition at higher Ca2+ concentration was different among the isoforms and ranked in the same order of the hydrophobicity of the N-fatty acyl groups: C14:0 > C14:1 > C14:2/C12:0. These results indicate that the diverged hydrophobicity of the recoverin N terminus plays an important role in the interaction with the membranes and/or its target protein but not with Ca2+.

Acylation↗
PubMed1995

Inhibition of rhodopsin kinase by recoverin. Further evidence for a negative feedback system in phototransduction.

Recoverin is a 23-kDa Ca(2+)-binding protein found predominantly in vertebrate photoreceptor cells. Recent electrophysiological and biochemical studies suggest that recoverin may regulate the photoresponse by inhibiting rhodopsin phosphorylation. We find in both cell homogenates and reconstituted systems that the inhibition of rhodopsin phosphorylation by recoverin occurs over a significantly higher free Ca2+ range than previously reported. Half-maximal inhibition occurs at 1.5-3 microM free Ca2+ and is cooperative with a Hill coefficient of approximately 2. Measurements of transducin activation demonstrate that this inhibition prolongs the lifetime of catalytically active rhodopsin. Ca(2+)-recoverin directly inhibits rhodopsin kinase activity, and Ca(2+)-dependent binding of recoverin to rod outer segment membranes is not required for its action. Extrapolation of the in vitro data to in vivo conditions based on simple mass action calculations places the Ca(2+)-recoverin regulation within the physiological free Ca2+ range in intact rod outer segment. The data are consistent with a model in which the fall in free Ca2+ that accompanies rod excitation exerts negative feedback by relieving inhibition of rhodopsin phosphorylation.

Animals↗
PubMed1995

Rhodopsin kinase inhibition by recoverin. Function of recoverin myristoylation.

Recoverin is a Ca(2+)-binding protein that may play a role in vertebrate photoreceptor light adaptation by imparting Ca2+ sensitivity to rhodopsin kinase. It is heterogeneously acylated (mostly myristoylated) at its amino-terminal glycine. Recent studies have shown that recoverin myristolyation is necessary for its Ca(2+)-dependent membrane association and cooperative Ca2+ binding. We have addressed several issues concerning the role of recoverin myristoylation with respect to inhibition of rhodopsin kinase. We find that 1) myristoylation of recoverin is not necessary for inhibition of rhodopsin kinase, 2) myristoylation of recoverin induces a cooperative Ca(2+)-dependence for rhodopsin kinase inhibition, and 3) each Ca(2+)-binding site on the nonmyristoylated recoverin partially inhibits rhodopsin kinase. The available data suggest that the functions of recoverin myristoylation in the living rod are to induce a sharp Ca2+ dependence of rhodopsin kinase inhibition and to bring this dependence into the rod's physiological Ca2+ concentration range.

Animals↗
PubMed1995

Nuclear magnetic resonance evidence for Ca(2+)-induced extrusion of the myristoyl group of recoverin.

Recoverin, a recently discovered member of the EF-hand protein superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group covalently attached to the amino terminus of recoverin enables it to translocate to retinal disc membranes when the Ca2+ level is elevated. Two-dimensional 1H-13C shift correlation NMR spectra of recoverin containing a 13C-labeled myristoyl group were obtained to selectively probe the effect of Ca2+ on the environment of the attached myristoyl group. In the Ca(2+)-free state, each pair of methylene protons bonded to carbon atoms 2, 3, 11, and 12 of the myristoyl group gives rise to two peaks. The splittings, caused by nonequivalent methylene proton chemical shifts, indicate that the myristoyl group interacts intimately with the protein in the Ca(2+)-free state. By contrast, only one peak is seen for each pair of methylene protons in the Ca(2+)-bound state, indicating that the myristoyl group is located in an isotropic environment in this form. Furthermore, the 1H-13C shift correlation NMR spectrum of Ca(2+)-bound recoverin is very similar to that of myristic acid in solution. 1H-(13)C shift correlation NMR experiments were also performed with 13C-labeled recoverin to selectively probe the resonances of methyl groups in the hydrophobic core of the protein. The spectrum of Ca(2+)-bound myristoylated recoverin is different from that of Ca(2+)-free myristoylated recoverin but similar to that of Ca(2+)-bound unmyristoylated recoverin. Hence, the myristoyl group interacts little with the hydrophobic core of myristoylated recoverin in the Ca(2+)-bound state. Three-dimensional (13C/F1)-edited (13C/F3)-filtered heteronuclear multiple quantum correlation-nuclear Overhauser effect spectroscopy spectra of recoverin containing a 13C-labeled myristoyl group were obtained to selectively probe protein residues located within 5 A of the myristoyl group. The myristoyl group makes close contact with a number of aromatic residues in Ca(2+)-free recoverin, whereas the myristoyl group makes no observable contacts with the protein in the Ca(2+)-bound state. These NMR data demonstrate that the binding of Ca2+ to recoverin induces the extrusion of its myristoyl group into the solvent, which would enable it to interact with a lipid bilayer or a hydrophobic site of a target protein.

Calcium↗
PubMed1995

Amino-terminal myristoylation induces cooperative calcium binding to recoverin.

Recoverin, a new member of the EF-hand protein superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group covalently attached to the amino terminus of recoverin enables it to bind to disc membranes when the Ca2+ level is elevated. Ca(2+)-bound recoverin prolongs the lifetime of photoexcited rhodopsin, most likely by blocking its phosphorylation. We report here Ca2+ binding studies of myristoylated and unmyristoylated recombinant recoverin using flow dialysis, fluorescence, and NMR spectroscopy. Unmyristoylated recoverin exhibits heterogeneous and uncooperative binding of two Ca2+ with dissociation constants of 0.11 and 6.9 microM. In contrast, two Ca2+ bind cooperatively to myristoylated recoverin with a Hill coefficient of 1.75 and an apparent dissociation constant of 17 microM. Thus, the attached myristoyl group lowers the calcium affinity of the protein and induces cooperativity in Ca2+ binding. One-dimensional 1H and two-dimensional 15N-1H shift correlation NMR spectra of myristoylated recoverin measured as a function of Ca2+ concentration show that a concerted conformational change occurs when two Ca2+ are bound. The Ca2+ binding and NMR data can be fit to a concerted allosteric model in which the two Ca2+ binding sites have different affinities in both the T and R states. The T and R conformational states are defined in terms of the Ca(2+)-myristoyl switch; in the T state, the myristoyl group is sequestered inside the protein, whereas in the R state, the myristoyl group is extruded. Ca2+ binds to the R state at least 10,000-fold more tightly than to T. In this model, the dissociation constants of the two sites in the R state of the myristoylated protein are 0.11 and 6.9 microM, as in unmyristoylated recoverin. The ratio of the unliganded form of T to that of R is estimated to be 400 for myristoylated and < 0.05 for unmyristoylated recoverin. Thus, the attached myristoyl group has two related roles: it shifts the T/R ratio of the unliganded protein more than 8000-fold, and serves as a membrane anchor for the fully liganded protein.

Allosteric Site↗
PubMed1996

Photoreceptor protein s26, a cone homologue of S-modulin in frog retina.

A frog retinal protein named s26 is a 26-kDa protein found during purification of S-modulin in frog retina (Kawamura, S. (1992) Photochem. Photobiol. 56, 1173-1180). To identify its role in frog retina, first s26 was purified to nearly homogeneity with three chromatographical steps. Based on the partial amino acid sequences of the proteolysed fragments of s26, we isolated cDNAs that encode s26. The analysis of its amino acid sequence revealed that s26 is an S-modulin-like protein, while it shows higher homology to visinin. Visinin is a Ca2+-binding protein reported to be present in chicken cones, but its localization in the retina had been a subject in dispute. The present study showed that s26 is present in cone photoreceptors. The study also showed that s26 inhibits phosphorylation of rhodopsin after a light flash at high Ca2+ concentrations as S-modulin does. From these results, we concluded that s26 is a cone homologue of S-modulin. The result is consistent with the idea that each type of photoreceptors expresses each cell-type specific version of phototransduction proteins.

Amino Acid Sequence↗
PubMed1998

The role of calcium-binding sites in S-modulin function.

S-modulin controls rhodopsin phosphorylation in a calcium-dependent manner, and it has been suggested that it modulates the light sensitivity of the photoreceptor cell. S-modulin binds to the ROS membrane at high Ca2+ concentration, and N-terminal myristoylation is necessary for this property (the calcium-myristoyl switch). S-modulin has four EF-hand motifs, of which two (EF-2 and -3) are functional. Here, we report on the roles of EF-2 and -3 in S-modulin function (calcium binding, membrane association, and inhibition of rhodopsin phosphorylation) by site-directed mutants (E85M and E121M). Surprisingly, E121M, which has a mutation in EF-3, neither binds Ca2+ nor inhibits phosphorylation. In contrast, E85M binds one Ca2+ and has the same membrane affinity as wild-type S-modulin, but has lost the ability to inhibit rhodopsin phosphorylation. It is suggested that the binding of Ca2+ to EF-3 is probably required for EF-2 to be a functional Ca2+-binding site and to induce exposure of the myristoyl group; and that the binding of Ca2+ to EF-2 is important for the interaction with rhodopsin kinase.

Binding Sites↗
PubMed1998

Neuronal Ca2+ sensor 1, the mammalian homologue of frequenin, is expressed in chromaffin and PC12 cells and regulates neurosecretion from dense-core granules.

Neuronal Ca2+ sensor 1 (NCS-1) is the mammalian homologue of the Ca2+-binding protein frequenin previously implicated in regulation of neurotransmission in Drosophila (Pongs, O., Lindemeier, J., Zhu, X. R., Theil, T., Endelkamp, D., Krah-Jentgens, I., Lambrecht, H.-G., Koch, K. W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J. , Canal, I., Barbas, J. A., and Ferrus, A. (1993) Neuron 11, 15-28). NCS-1 has been considered to be expressed only in neurons, but we show that NCS-1 expression can be detected in bovine adrenal chromaffin and PC12 cells, two widely studied model neuroendocrine cells. NCS-1 was present in both cytosolic and membrane fractions including purified chromaffin granules, and in immunofluorescence, its distribution overlapped with peripheral punctate staining seen with the synaptic-like microvesicle marker synaptophysin in PC12 cells. The possible functional role of NCS-1 in exocytosis of dense-core granules was tested using transient transfection in PC12 cells and assay of co-transfected growth hormone (GH) release. Overexpression of NCS-1 increased evoked GH release in intact cells in response to ATP. No effect of overexpression was seen on GH release because of Ca2+ in permeabilized cells suggesting that NCS-1 may have a regulatory but not direct role in neurosecretion.

Adrenal Medulla↗
PubMed1999

Three-dimensional structure of guanylyl cyclase activating protein-2, a calcium-sensitive modulator of photoreceptor guanylyl cyclases.

Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca2+-sensitive regulator of phototransduction in retinal photoreceptor cells. GCAP-2 activates retinal guanylyl cyclases at low Ca2+ concentration (<100 nM) and inhibits them at high Ca2+ (>500 nM). The light-induced lowering of the Ca2+ level from approximately 500 nM in the dark to approximately 50 nM following illumination is known to play a key role in visual recovery and adaptation. We report here the three-dimensional structure of unmyristoylated GCAP-2 with three bound Ca2+ ions as determined by nuclear magnetic resonance spectroscopy of recombinant, isotopically labeled protein. GCAP-2 contains four EF-hand motifs arranged in a compact tandem array like that seen previously in recoverin. The root mean square deviation of the main chain atoms in the EF-hand regions is 2.2 A in comparing the Ca2+-bound structures of GCAP-2 and recoverin. EF-1, as in recoverin, does not bind calcium because it contains a disabling Cys-Pro sequence. GCAP-2 differs from recoverin in that the calcium ion binds to EF-4 in addition to EF-2 and EF-3. A prominent exposed patch of hydrophobic residues formed by EF-1 and EF-2 (Leu24, Trp27, Phe31, Phe45, Phe48, Phe49, Tyr81, Val82, Leu85, and Leu89) may serve as a target-binding site for the transmission of calcium signals to guanylyl cyclase.

Amino Acid Sequence↗
PubMed2000

Amino acid residues of S-modulin responsible for interaction with rhodopsin kinase.

S-modulin in frog or its bovine homologue, recoverin, is a 23-kDa EF-hand Ca(2+)-binding protein found in rod photoreceptors. The Ca(2+)-bound form of S-modulin binds to rhodopsin kinase (Rk) and inhibits its activity. Through this regulation, S-modulin is thought to modulate the light sensitivity of a rod. In the present study, we tried to identify the interaction site of the Ca(2+)-bound form of S-modulin to Rk. First, we mapped roughly the interaction regions by using partial peptides of S-modulin. The result suggested that a specific region near the amino terminus is the interaction site of S-modulin. We then identified the essential amino acid residues in this region by using S-modulin mutant proteins: four amino acid residues (Phe(22), Glu(26), Phe(55), and Thr(92)) were suggested to interact with Rk. These residues are located in a small closed pocket in the Ca(2+)-free, inactive form of S-modulin, but exposed to the surface of the molecule in the Ca(2+)-bound, active form of S-modulin. Two additional amino acid residues (Tyr(108) and Arg(150)) were found to be crucial for the Ca(2+)-dependent conformational changes of S-modulin.

Amino Acid Sequence↗
PubMed2002

Molecular basis of CIB binding to the integrin alpha IIb cytoplasmic domain.

Integrin adhesion receptors appear to be regulated by molecules that bind to their cytoplasmic domains. We previously identified a 22-kDa, EF-hand-containing protein, CIB, which binds to the alpha(IIb) cytoplasmic tail of the platelet integrin, alpha(IIb)beta(3). Here we describe regions within CIB and alpha(IIb) that interact with one another. CIB binding to alpha(IIb) cytoplasmic tail peptides, as measured by intrinsic tryptophan fluorescence, indicates a CIB-binding site within a hydrophobic, 15-amino acid, membrane-proximal region of alpha(IIb). This region is analogous to the alpha-helical targets of other EF-hand-containing proteins, such as calcineurin B or calmodulin. A homology model of CIB based upon calcineurin B and recoverin indicated a conserved hydrophobic pocket within the C-terminal EF-hand motifs of CIB as a potential integrin-binding site. CIB engineered to contain alanine substitutions in the implicated regions retained wild type secondary structure as determined by circular dichroism, yet failed to bind alpha(IIb) in 11 of 12 cases, whereas CIB mutated within the N terminus retained binding activity. Thus, specific hydrophobic residues in the C terminus of CIB appear necessary for CIB binding to alpha(IIb). The identification of essential interacting regions within alpha(IIb) and CIB provides tools for further probing potential interrelated functions of these proteins.

Amino Acid Motifs↗
PubMed2002

Ca2+-myristoyl switch in the neuronal calcium sensor recoverin requires different functions of Ca2+-binding sites.

Recoverin is an EF-hand Ca(2+)-binding protein that is suggested to control the activity of the G-protein-coupled receptor kinase GRK-1 or rhodopsin kinase in a Ca(2+)-dependent manner. It undergoes a Ca(2+)-myristoyl switch when Ca(2+) binds to EF-hand 2 and 3. We investigated the mechanism of this switch by the use of point mutations in EF-hand 2 (E85Q) and 3 (E121Q) that impair their Ca(2+) binding. EF-hand 2 and 3 display different properties and serve different functions. Binding of Ca(2+) to recoverin is a sequential process, wherein EF-hand 3 is occupied first followed by the filling of EF-hand 2. After EF-hand 3 bound Ca(2+), the subsequent filling of EF-hand 2 triggers the exposition of the myristoyl group and in turn binding of recoverin to membranes. In addition, EF-hand 2 controls the mean residence time of recoverin at membranes by decreasing the dissociation rate of recoverin from membranes by 10-fold. We discuss this mechanism as one critical step for inhibition of rhodopsin kinase by recoverin.

Animals↗
PubMed2003

Impact of N-terminal myristoylation on the Ca2+-dependent conformational transition in recoverin.

Recoverin is a Ca2+-regulated signal transduction modulator found in vertebrate retina that has been shown to undergo dramatic conformational changes upon Ca2+ binding to its two functional EF-hand motifs. To elucidate the differential impact of the N-terminal myristoylation as well as occupation of the two Ca2+ binding sites on recoverin structure and function, we have investigated a non-myristoylated E85Q mutant exhibiting virtually no Ca2+ binding to EF-2. Crystal structures of the mutant protein as well as the non-myristoylated wild-type have been determined. Although the non-myristoylated E85Q mutant does not display any functional activity, its three-dimensional structure in the presence of Ca2+ resembles the myristoylated wild-type with two Ca2+ but is quite dissimilar from the myristoylated E85Q mutant. We conclude that the N-terminal myristoyl modification significantly stabilizes the conformation of the Ca2+-free protein (i.e. the T conformation) during the stepwise transition toward the fully Ca2+-occupied state. On the basis of these observations, a refined model for the role of the myristoyl group as an intrinsic allosteric modulator is proposed.

Amino Acid Substitution↗
PubMed2004

Identification of residues that determine the absence of a Ca(2+)/myristoyl switch in neuronal calcium sensor-1.

The neuronal calcium sensor (NCS) family of Ca(2+)-binding proteins regulates a number of different processes in neurons and photoreceptor cells. The first of these proteins to be characterized, recoverin, was shown to exhibit a Ca(2+)/myristoyl switch whereby its N-terminal myristoyl group is sequestered in the Ca(2+)-free form and is exposed on Ca(2+) binding to allow the protein to become membrane-associated. It has subsequently been shown that certain other family members also exhibit this mechanism in living cells. In contrast, NCS-1 does not show the Ca(2+)/myristoyl switch and is membrane-associated even at low Ca(2+) concentrations. We have used sequence comparison combined with information from structural analyses to attempt to identify candidate residues within the NCS proteins that determine whether or not the Ca(2+)/myristoyl switch operates in cells and have tested their functional significance by mutagenesis. The results show that NCS-1 possesses residues within its N terminus that lock the myristoyl group in an exposed conformation. In addition, other structural aspects within the C-terminal domains are required to allow the switch to operate. We have determined a key role for residues within the motif EELTRK in NCS-1 in keeping the myristoyl group exposed and allowing the protein to be constitutively membrane-associated.

Amino Acid Sequence↗