共查询到20条相似文献,搜索用时 15 毫秒
1.
Y Nakajima T Yamamoto T Nakayama S Nakanishi 《The Journal of biological chemistry》1999,274(39):27573-27577
Metabotropic glutamate receptor subtype 7 (mGluR7) is coupled to the inhibitory cyclic AMP cascade and is selectively activated by a glutamate analogue, L-2-amino-4-phosphonobutyrate. Among L-2-amino-4-phosphonobutyrate-sensitive mGluR subtypes, mGluR7 is highly concentrated at the presynaptic terminals and is thought to play an important role in modulation of glutamatergic synaptic transmission by presynaptic inhibition of glutamate release. To gain further insight into the intracellular signaling mechanisms of mGluR7, with the aid of glutathione S-transferase fusion affinity chromatography, we attempted to identify proteins that interact with the intracellular carboxyl terminus of mGluR7. Here, we report that calmodulin (CaM) directly binds to the carboxyl terminus of mGluR7 in a Ca(2+)-dependent manner. The CaM-binding domain is located immediately following the 7th transmembrane segment. We also show that the CaM-binding domain of mGluR7 is phosphorylated by protein kinase C (PKC). This phosphorylation is inhibited by the binding of Ca(2+)/CaM to the receptor. Conversely, the Ca(2+)/CaM binding is prevented by PKC phosphorylation. Collectively, these results suggest that mGluR7 serves to cross-link the cyclic AMP, Ca(2+), and PKC phosphorylation signal transduction cascades. 相似文献
2.
Scheschonka A Findlow S Schemm R El Far O Caldwell JH Crump MP Holden-Dye K O'Connor V Betz H Werner JM 《The Journal of biological chemistry》2008,283(9):5577-5588
Calmodulin (CaM) binds in a Ca2+-dependent manner to the intracellular C-terminal domains of most group III metabotropic glutamate receptors (mGluRs). Here we combined mutational and biophysical approaches to define the structural basis of CaM binding to mGluR 7A. Ca2+/CaM was found to interact with mGluR 7A primarily via its C-lobe at a 1:1 CaM:C-tail stoichiometry. Pulldown experiments with mutant CaM and mGluR 7A C-tail constructs and high resolution NMR with peptides corresponding to the CaM binding region of mGluR 7A allowed us to define hydrophobic and ionic interactions required for Ca2+/CaM binding and identified a 1-8-14 CaM-binding motif. The Ca2+/CaM.mGluR 7A peptide complex displays a classical wraparound structure that closely resembles that formed by Ca2+/CaM upon binding to smooth muscle myosin light chain kinase. Our data provide insight into how Ca2+/CaM regulates group III mGluR signaling via competition with intracellular proteins for receptor-binding sites. 相似文献
3.
Dynamic light scattering (DLS) has been used to assess the influence of eleven different synthetic peptides, comprising the calmodulin (CaM)-binding domains of various CaM-binding proteins, on the structure of apo-CaM (calcium-free) and Ca(2+)-CaM. Peptides that bind CaM in a 1:1 and 2:1 peptide-to-protein ratio were studied, as were solutions of CaM bound simultaneously to two different peptides. DLS was also used to investigate the effect of Ca(2+) on the N- and C-terminal CaM fragments TR1C and TR2C, and to determine whether the two lobes of CaM interact in solution. The results obtained in this study were comparable to similar solution studies performed for some of these peptides using small-angle x-ray scattering. The addition of Ca(2+) to apo-CaM increased the hydrodynamic radius from 2.5 to 3.0 nm. The peptides studied induced a collapse of the elongated Ca(2+)-CaM structure to a more globular form, decreasing its hydrodynamic radius by an average of 25%. None of the peptides had an effect on the conformation of apo-CaM, indicating that either most of the peptides did not interact with apo-CaM, or if bound, they did not cause a large conformational change. The hydrodynamic radii of TR1C and TR2C CaM fragments were not significantly affected by the addition of Ca(2+). The addition of a target peptide and Ca(2+) to the two fragments of CaM, suggest that a globular complex is forming, as has been seen in nuclear magnetic resonance solution studies. This work demonstrates that dynamic light scattering is an inexpensive and efficient technique for assessing large-scale conformational changes that take place in calmodulin and related proteins upon binding of Ca(2+) ions and peptides, and provides a qualitative picture of how this occurs. This work also illustrates that DLS provides a rapid screening method for identifying new CaM targets. 相似文献
4.
The solution structures of complexes between calcium-saturated calmodulin (Ca (2+)/CaM) and a CaM-binding domain of the HIV-1 matrix protein p17 have been determined by small-angle X-ray scattering with use of synchrotron radiation as an intense and stable X-ray source. We used three synthetic peptides of residues 11-28, 26-47, and 11-47 of p17 to demonstrate the diversity of CaM-binding conformation. Ca (2+)/CaM complexed with residues 11-28 of p17 adopts a dumbbell-like structure at a molar ratio of 1:2, suggesting that the two peptides bind each lobe of CaM, respectively. Ca (2+)/CaM complexed with residues 26-47 of p17 at a molar ratio of 1:1 adopts a globular structure similar to the NMR structure of Ca (2+)/CaM bound to M13, which adopted a compact globular structure. In contrast to these complexes, Ca (2+)/CaM binds directly with both CaM-binding sites of residues 11-47 of p17 at a molar ratio of 1:1, which induces a novel structure different from known structures previously reported between Ca (2+)/CaM and peptide. A tertiary structural model of the novel structure was constructed using the biopolymer module of Insight II 2000 on the basis of the scattering data. The two domains of CaM remain essentially unchanged upon complexation. The hinge motions, however, occur in a highly flexible linker of CaM, in which the electrostatic residues 74Arg, 78Asp, and 82Glu interact with N-terminal electrostatic residues of the peptide (residues 12Glu, 15Arg, and 18Lys). The acidic residues in the N-terminal domain of CaM interact with basic residues in a central part of the peptide, thereby enabling the central part to change the conformations, while an acidic residue in the C-terminal domain interacts with two basic residues in the two helical sites of the peptide. The overall structure of the complex adopts an extended structure with the radius of gyration of 20.5 A and the interdomain distance of 34.2 A. Thus, the complex is principally stabilized by electrostatic interactions. The hydrophobic patches of Ca (2+)/CaM are not responsible for the binding with the hydrophobic residues in the peptide, suggesting that CaM plays a role to sequester the myristic acid moiety of p17. 相似文献
5.
Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases 总被引:3,自引:0,他引:3
The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca(2+)-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca(2+) with mutations in the N-lobe (CaM(12)), the C-lobe (CaM(34)), or both lobes of CaM (CaM(1234)) to determine their effect on the binding and activation of the Ca(2+)-dependent neuronal (nNOS) and Ca(2+)-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein-protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca(2+). Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes. 相似文献
6.
Identification of the calmodulin binding domain of connexin 43 总被引:2,自引:0,他引:2
Zhou Y Yang W Lurtz MM Ye Y Huang Y Lee HW Chen Y Louis CF Yang JJ 《The Journal of biological chemistry》2007,282(48):35005-35017
Calmodulin (CaM) has been implicated in mediating the Ca(2+)-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136-158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca(2+)-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its alpha-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K(+) is in the range of 0.7-1 microM. Upon binding of the peptide to CaM, the apparent K(d) of Ca(2+) for CaM decreased from 2.9 +/- 0.1 to 1.6 +/- 0.1 microM, and the Hill coefficient n(H) increased from 2.1 +/- 0.1 to 3.3 +/- 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca(2+)-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136-158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca(2+)-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca(2+)-dependent manner, providing a molecular basis for the well characterized Ca(2+)-dependent inhibition of Cx43-containing gap junctions. 相似文献
7.
Nef of HIV-1 interacts directly with calcium-bound calmodulin 总被引:5,自引:0,他引:5
Hayashi N Matsubara M Jinbo Y Titani K Izumi Y Matsushima N 《Protein science : a publication of the Protein Society》2002,11(3):529-537
It was recently found that the myristoyl group of CAP-23/NAP-22, a neuron-specific protein kinase C substrate, is essential for the interaction between the protein and Ca(2+)-bound calmodulin (Ca(2+)/CaM). Based on the N-terminal amino acid sequence alignment of CAP-23/NAP-22 and other myristoylated proteins, including the Nef protein from human immunodeficiency virus (HIV), we proposed a new hypothesis that the protein myristoylation plays important roles in protein-calmodulin interactions. To investigate the possibility of direct interaction between Nef and calmodulin, we performed structural studies of Ca(2+)/CaM in the presence of a myristoylated peptide corresponding to the N-terminal region of Nef. The dissociation constant between Ca(2+)/CaM and the myristoylated Nef peptide was determined to be 13.7 nM by fluorescence spectroscopy analyses. The NMR experiments indicated that the chemical shifts of some residues on and around the hydrophobic clefts of Ca(2+)/CaM changed markedly in the Ca(2+)/CaM-Nef peptide complex with the molar ratio of 1:2. Correspondingly, the radius of gyration determined by the small angle X-ray scattering measurements is 2-3 A smaller that of Ca(2+)/CaM alone. These results demonstrate clearly that Nef interacts directly with Ca(2+)/CaM. 相似文献
8.
Group III metabotropic glutamate receptors (mGluRs) serve as presynaptic receptors that mediate feedback inhibition of glutamate release via a Ca(2+)/calmodulin (CaM)-dependent mechanism. In vitro phosphorylation of mGluR7A by protein kinase C (PKC) prevents its interaction with Ca(2+)/CaM. In addition, activation of PKC leads to an inhibition of mGluR signaling. Here, we demonstrate that disrupting CaM binding to mGluR7A by PKC in vitro is due to phosphorylation of a highly conserved serine residue, S862. We propose charge neutralization of the CaM binding consensus sequence resulting from phosphorylation to constitute a general mechanism for the regulation of presynaptic mGluR signaling. 相似文献
9.
H Kurokawa M Osawa H Kurihara N Katayama H Tokumitsu M B Swindells M Kainosho M Ikura 《Journal of molecular biology》2001,312(1):59-68
Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM. 相似文献
10.
The type IIb class of plant Ca(2+)-ATPases contains a unique N-terminal extension that encompasses a calmodulin (CaM) binding domain and an auto-inhibitory domain. Binding of Ca(2+)-CaM to this region can release auto-inhibition and activates the calcium pump. Using multidimensional NMR spectroscopy, we have determined the solution structure of the complex of a plant CaM isoform with the CaM-binding domain of the well characterized Ca(2+)-ATPase BCA1 from cauliflower. The complex has a rather elongated structure in which the two lobes of CaM do not contact each other. The anchor residues Trp-23 and Ile-40 form a 1-8-18 interaction motif. Binding of Ca(2+)-CaM gives rise to the induction of two helical parts in this unique target peptide. The two helical portions are connected by a highly positively charged bend region, which represents a relatively fixed angle and positions the two lobes of CaM in an orientation that has not been seen before in any complex structure of calmodulin. The behavior of the complex was further characterized by heteronuclear NMR dynamics measurements of the isotope-labeled protein and peptide. These data suggest a unique calcium-driven activation mechanism for BCA1 and other plant Ca(2+)-ATPases that may also explain the action of calcium-CaM on some other target enzymes. Moreover, CaM activation of plant Ca(2+)-ATPases seems to occur in an organelle-specific manner. 相似文献
11.
Calmodulin (CaM) is the primary Ca(2+)-sensor that regulates a wide variety of cellular processes in eukaryotes. Although many Ca(2+)/CaM-binding proteins have been identified, very few such proteins could be found from the genome-wide protein-protein interaction maps of Caenorhabditis elegans constructed by yeast two-hybrid screening. Using a genotype-phenotype conjugation method called mRNA-display, we performed a selection for Ca(2+)/CaM-binding proteins from a proteome library of C. elegans. The method allowed the identification of 9 known and 47 previously uncharacterized Ca(2+)-dependent CaM-binding proteins from the adult worm proteome. The Ca(2+)/CaM-binding properties of these proteins were characterized and their binding motifs were identified. The availability of such information could facilitate our understanding of the signaling pathways mediated by Ca(2+)/CaM in C. elegans. Due to its simplicity and efficiency, the method could be readily applied to examine the Ca(2+)-dependent binding partners of numerous other Ca(2+)-binding proteins, which may play important roles in many signaling pathways in C. elegans. 相似文献
12.
El Far O Bofill-Cardona E Airas JM O'Connor V Boehm S Freissmuth M Nanoff C Betz H 《The Journal of biological chemistry》2001,276(33):30662-30669
Ca(2+)/calmodulin (Ca(2+)/CaM) and the betagamma subunits of heterotrimeric G-proteins (Gbetagamma) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and Gbetagamma binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca(2+)/CaM. Mutational analysis of the Ca(2+)/CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic alpha-helix. Substitutions adjacent to the core CaM target sequence selectively prevent Gbetagamma binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating Gbetagamma recruitment. In addition, we present evidence that Gbetagamma uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although Gbetagamma-mediated signaling is abolished in receptors lacking the core CaM binding sequence, alpha subunit activation, as assayed by agonist-dependent GTPgammaS binding, was not affected. This suggests that Ca(2+)/CaM may alter the mode of group III mGluR signaling from mono- (alpha) to bidirectional (alpha and betagamma) activation of downstream effector cascades. 相似文献
13.
Calmodulin (CaM) is a ubiquitous Ca (2+)-sensor protein that binds and activates the nitric oxide synthase (NOS) enzymes. We have used fluorescence resonance energy transfer (FRET) to examine the conformational transitions of CaM induced by its binding to synthetic nitric oxide synthase (NOS) CaM-binding domain peptides and full length heme-free constitutive NOS (cNOS) enzymes over a range of physiologically relevant free Ca (2+) concentrations. We demonstrate for the first time that the domains of CaM collapse when associated with Ca (2+)-independent inducible NOS CaM-binding domain, similar to the previously solved crystal structures of CaM bound to the Ca (2+)-dependent cNOS peptides. We show that the association of CaM is not detectable with the cNOS peptides at low free Ca (2+) concentrations (<40 nM). In contrast, we demonstrate that CaM associates with the cNOS holo-enzymes in the absence of Ca (2+) and that the Ca (2+)-dependent transition occurs at a lower free Ca (2+) concentration with the cNOS holo-enzymes. Our results suggest that other regions outside of the CaM-binding domain in the cNOS enzymes are involved in the recruitment and binding of CaM. We also demonstrate that CaM binds to the cNOS enzymes in a sequential manner with the Ca (2+)-replete C-lobe binding first followed by the Ca (2+)-replete N-lobe. This novel FRET study helps to clarify some of the observed similarities and differences between the Ca (2+)-dependent/independent interaction between CaM and the NOS isozymes. 相似文献
14.
Sasakura D Nunomura W Takakuwa Y 《Biochemical and biophysical research communications》2012,423(2):360-365
Although the 3D structure of the Ca(2+)-bound CaM (Ca(2+)/CaM) complex with the antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), has been resolved, the dynamic changes in Ca(2+)/CaM structure upon interaction with W-7 are still unknown. We investigated time- and temperature-dependent dynamic changes in Ca(2+)/CaM interaction with W-7 in physiological conditions using one- and two-dimensional Fourier-transformed infrared spectroscopy (2D-IR). We observed changes in the α-helix secondary structure of Ca(2+)/CaM when complexed with W-7 at a molar ratio of 1:2, but not at higher molar ratios (between 1:2 and 1:5). Kinetic studies revealed that, during the initial 125s at 25°C, Ca(2+)/CaM underwent formation of secondary coil and turn structures upon binding to W-7. Variations in temperature that induced significant changes in the structure of the Ca(2+)/CaM complex failed to do so when Ca(2+)/CaM was complexed with W-7. We concluded that W-7 induced stepwise conformational changes in Ca(2+)/CaM that resulted in a rigidification of the complex and its inability to interact with target proteins and/or polypeptides. 相似文献
15.
Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase. 总被引:8,自引:2,他引:6
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We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions. 相似文献
16.
Moon BC Choi MS Kang YH Kim MC Cheong MS Park CY Yoo JH Koo SC Lee SM Lim CO Cho MJ Chung WS 《FEBS letters》2005,579(18):3885-3890
Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants. 相似文献
17.
Nunomura W Takakuwa Y Parra M Conboy JG Mohandas N 《The Journal of biological chemistry》2000,275(9):6360-6367
In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1. 相似文献
18.
Binding of La3+ to calmodulin and its effects on the interaction between calmodulin and calmodulin binding peptide, polistes mastoparan 总被引:1,自引:0,他引:1
Binding of La(3+) to calmodulin (CaM) and its effects on the complexes of CaM and CaM-binding peptide, polistes mastoparan (Mas), were investigated by nuclear magnetic resonance (NMR) spectroscopy, fluorescence and circular dichroism spectroscopy, and by the fluorescence stopped-flow method. The four binding sites of La(3+) on CaM were identified as the same as the binding sites of Ca(2+) on CaM through NMR titration of La(3+) to uniformly (15)N-labeled CaM. La(3+) showed a slightly higher affinity to the binding sites on the N-terminal domain of CaM than that to the C-terminal. Large differences between the (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of Ca(4)CaM and La(4)CaM suggest conformational differences between the two complexes. Fluorescence and CD spectra also exhibited structural differences. In the presence of Ca(2+) and La(3+), a hybrid complex, Ca(2)La(2)CaM, was formed, and the binding of La(3+) to the N-terminal domain of CaM seemed preferable over binding to the C-terminal domain. Through fluorescence titration, it was shown that La(4)CaM and Ca(2)La(2)CaM had similar affinities to Mas as Ca(4)CaM. Fluorescence stopped-flow experiments showed that the dissociation rate of La(3+) from the C-terminal domain of CaM was higher than that from the N-terminal. However, in the presence of Mas, the dissociation rate of La(3+) decreased and the dissociation processes from both global domains were indistinguishable. In addition, compared with the case of Ca(4)CaM-Mas, the slower dissociations of Mas from La(4)CaM-Mas and Ca(2)La(2)CaM-Mas complexes indicate that in the presence of La(3+), the CaM-Mas complex became kinetically inert. A possible role of La(3+) in the Ca(2+)-CaM-dependent pathway is discussed. 相似文献
19.
Ca(2+)-saturated calmodulin (CaM) directly associates with and activates CaM-dependent protein kinase I (CaMKI) through interactions with a short sequence in its regulatory domain. Using heteronuclear NMR (13)C-(15)N-(1)H correlation experiments, the backbone assignments were determined for CaM bound to a peptide (CaMKIp) corresponding to the CaM-binding sequence of CaMKI. A comparison of chemical shifts for free CaM with those of the CaM.CaMKIp complex indicate large differences throughout the CaM sequence. Using NMR techniques optimized for large proteins, backbone resonance assignments were also determined for CaM bound to the intact CaMKI enzyme. NMR spectra of CaM bound to either the CaMKI enzyme or peptide are virtually identical, indicating that calmodulin is structurally indistinguishable when complexed to the intact kinase or the peptide CaM-binding domain. Chemical shifts of CaM bound to a peptide (smMLCKp) corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase are also compared with the CaM.CaMKI complexes. Chemical shifts can differentiate one complex from another, as well as bound versus free states of CaM. In this context, the observed similarity between CaM.CaMKI enzyme and peptide complexes is striking, indicating that the peptide is an excellent mimetic for interaction of calmodulin with the CaMKI enzyme. 相似文献
20.
Yoshiaki Shinohara Yoshiaki Nakajima Shigetada Nakanishi 《Journal of neurochemistry》2001,78(2):365-373
Group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) stimulate phospholipase C (PLC) and lead to mobilization of intracellular Ca(2+) and activation of protein kinase C (PKC). In this investigation, using heterologous receptor-expressing Chinese hamster ovary (CHO) cells, we showed that stimulation of mGluR1 or mGluR5 with glutamate rapidly increases tyrosine phosphorylation of focal adhesion kinase (FAK) (maximum at 1-3 min) in a dose-dependent manner (half-maximal responses at approximately 2 microM). In mGluR1-expressing cells, the glutamate-induced increase of FAK tyrosine phosphorylation was blocked by not only the PLC inhibitor, U73122, but also depletion of intracellular Ca(2+) and effectively abrogated by calmodulin (CaM) inhibitors, calmidazolium and fluphenazine. However, neither the PKC inhibitor, GF109203X, nor the CaM kinase II inhibitor, KN-62, inhibited glutamate-stimulated FAK tyrosine phosphorylation. Stimulation of mGluR1 caused a marked increase in actin stress fiber formation. Importantly, this actin rearrangement was prevented by the CaM inhibitor, but not by the PKC inhibitor and is thus in a good agreement with the signaling cascade of the mGluR1-FAK pathway. These results suggest that the Ca(2+)/CaM signaling and its downstream FAK tyrosine phosphorylation play an important role in cellular function of mGluR1. 相似文献