Probing the mechanism of rhodopsin-catalyzed transducin activation

An agonist-bound G protein-coupled receptor (GPCR) induces a GDP/GTP exchange on the G protein alpha-subunit (G alpha) followed by the release of G alpha GTP and G beta gamma which, subsequently, activate their targets. The C-terminal regions of G alpha subunits constitute a major receptor recognition domain. In this study, we tested the hypothesis that the GPCR-induced conformational change is communicated from the G alpha C-terminus, via the alpha 5 helix, to the nucleotide-binding beta 6/alpha 5 loop causing GDP release. Mutants of the visual G protein, transducin, with a modified junction of the C-terminus were generated and analyzed for interaction with photoexcited rhodopsin (R*). A flexible linker composed of five glycine residues or a rigid three-turn alpha-helical segment was inserted between the 11 C-terminal residues and the alpha 5 helix of G alpha(t)-like chimeric G alpha, G alpha(ti). The mutant G alpha subunits with the Gly-loop (G alpha(ti)L) and the extended alpha 5 helix (G alpha(ti)H) retained intact interactions with G beta gamma(t), and displayed modestly reduced binding to R*. G alpha(ti)H was capable of efficient activation by R*. In contrast, R* failed to activate G alpha(ti)L, suggesting that the Gly-loop absorbs a conformational change at the C-terminus and blocks G protein activation. Our results provide evidence for the role of G alpha C-terminus/alpha 5 helix/beta 6/alpha 5 loop route as a dominant channel for transmission of the GPCR-induced conformational change leading to G protein activation.     

Biotinylation of transducin and G0 from bovine brain

A method is described for modifying G proteins with biotin. With transducin, better results were obtained with the amino group-specific derivative BXNHS (biotinyl--aminocaproic acid N-hydroxysuccinimide ester) as compared to the -SH-group specific reagent MBB (maleimidobutyrylbiocytin). Modification occurred under conditions preserving functional activity: Interaction of the biotinylated transducin with rod outer segment membranes was shown by its light-dependent association and by a GTPS-binding assay. G₀ from bovine brain was also biotinylated under conditions preserving its activity. Biotinyl-₀ was shown to bind to a streptavidin Sepharose matrix. Biotinyl-G proteins, therefore, are proposed as tools for extracting proteins (receptors and effector systems), which interact under specific conditions with G proteins.     

Calcium-dependent interaction of transducin with calmodulin-sepharose

It has been shown by affinity chromatography on calmodulin-sepharose that transducin, a G protein of bovine retinal rod outer segments interacts with Ca(2+)-calmodulin. This result assumes that the main part of calmodulin in dark retinal rod outer segments is associated with transducin. It has been suggested that photoactivation of retinal rods induces changes in intracellular calmodulin concentration, which may be one of the steps involved in the light adaptation of photoreceptor.     

钙调素及钙调素相关蛋白在植物细胞中的研究进展

植物对一系列生物和非生物刺激所产生的反应都与细胞内Ca2+信号转导有关,而钙调素、钙调素相关蛋白则是Ca2+信号转导的下游靶蛋白。该文介绍了钙调素的结构及其在植物细胞中的分布,钙调素及钙调素相关蛋白在植物细胞中的表达等方面的最近研究进展。     

Nucleoside diphosphate kinase and GTP-binding proteins. Possible mechanisms of coupling

The results of numerous investigations during the last 20 years show that nucleoside diphosphate kinase (NDP kinase) is a multifunctional protein. In this paper, the current data are analyzed indicating that one of the possible mechanisms by which NDP kinase manifests its multifunctional role is its participation in the activation (or regulation) of heterotrimeric GTP-binding proteins (G proteins). We demonstrate that one of the NDP kinase isoforms dynamically interacts with the retinal rod G protein transducin (Gt) and phosphorylates its β-subunit at histidine residue (His 266). It is also shown that it leads to the consecutive transfer of the phosphate group to GDP in the active center of G protein α-subunit and G protein activation. The advantages of this mechanism are considered as compared to the classic G protein activation mechanism, GDP/GTP exchange.     

Extracellular calmodulin: A polypeptide signal in plants?

Traditionally, calmodulin (CaM) was thought to be a multi-functional receptor for intracellular Ca²⁺ signals. But in the last ten years, it was found that CaM also exists and acts extracellularly in animal and plant cells to regulate many important physiological functions. Laboratory studies by the authors showed that extracellular CaM in plant cells can stimulate the proliferation of suspension cultured cell and protoplast; regulate pollen germination and pollen tube elongation, and stimulate the light-independent gene expression of Rubisco small subunit (rbcS). Furthermore, we defined the trans-membrane and intracellular signal transduction pathways for extracellular CaM by using a pollen system. The components in this pathway include heterotrimeric G-protein, phospholipase C, IP₃, calcium signal and protein phosphorylation etc. Based on our findings, we suggest that extracellular CaM is a polypeptide signal in plants. This idea strongly argues against the traditional concept that there is no intercellular polypeptide signal in plants.     

A point mutation uncouples transducin-alpha from the photoreceptor RGS and effector proteins

A novel gain-of-function mutation, R243Q, has been recently identified in the Candida elegans Gqalpha protein EGL-30. The position corresponding to Arg243 in EGL-30 is absolutely conserved among heterotrimeric G proteins. This mutation appears to be the first gain-of-function mutation in the switch III region of Galpha subunits. To investigate consequences of the R-->Q mutation we introduced the corresponding R238Q mutation into transducin-like Gtalpha* subunit. The mutant retained intact interactions with Gtbetagamma and rhodopsin but exhibited a twofold reduction in the kcat value for guanosine 5'-triphosphate (GTP) hydrolysis. The GTPase activity of R238Q was not accelerated by the RGS domain of the visual GTPase-activating protein, RGS9-1. In addition, R238Q displayed a significant impairment in the effector function. Our data and the crystal structures of transducin suggest that the major reason for the reduced intrinsic GTPase activity of R238Q and the lack of RGS9 function is the break of the conserved ionic contact between Arg238 and Glu39, which apparently stabilizes the transitional state for GTP hydrolysis. We hypothesize that the R243Q mutation in EGL-30 severs the ionic interaction of Arg243 with Glu43, leading to a defective inactivation of the mutant by the C. elegans RGS protein EAT-16.     

Neurogranin enhances synaptic strength through its interaction with calmodulin

Learning‐correlated plasticity at CA1 hippocampal excitatory synapses is dependent on neuronal activity and NMDA receptor (NMDAR) activation. However, the molecular mechanisms that transduce plasticity stimuli to postsynaptic potentiation are poorly understood. Here, we report that neurogranin (Ng), a neuron‐specific and postsynaptic protein, enhances postsynaptic sensitivity and increases synaptic strength in an activity‐ and NMDAR‐dependent manner. In addition, Ng‐mediated potentiation of synaptic transmission mimics and occludes long‐term potentiation (LTP). Expression of Ng mutants that lack the ability to bind to, or dissociate from, calmodulin (CaM) fails to potentiate synaptic transmission, strongly suggesting that regulated Ng–CaM binding is necessary for Ng‐mediated potentiation. Moreover, knocking‐down Ng blocked LTP induction. Thus, Ng–CaM interaction can provide a mechanistic link between induction and expression of postsynaptic potentiation.     

A calmodulin dependent protein kinase activity associated with rabbit heart sarcolemma

Summary Sarcolemmal vesicles prepared from rabbit heart muscle by differential and discontinuous sucrose density gradient centrifugation, exhibited high marker enzyme activities: Na⁺ + K⁺ ATPase 22 µMol Pi × mg^–1 × h^–1. Adenylate cyclase 500 pmole CAMP × mg^–1 × min^–1, calcium antagonist receptors 0.7 pmoles × mg^–1. Calmodulin in the presence of calcium and -ATP³² stimulated rapidly and specifically the ³²P incorporation into two membrane proteins of 54 and 44 kDa. Calmodulin stimulated the phosphorylation of the 44 kDa to a greater extent (17.9 pmol ³²P × mg^–1 protein) than the 54 kDa protein (1.3 pmoles ³²P x mg^–1 protein). Removal of endogenous calmodulin from the membrane by EGTA extraction resulted in a 2.5 fold increase in calmodulin dependent ³²P incorporation into the two proteins in the presence of exogenous calmodulin. It is suggested that the calmodulin dependent protein kinase activity in heart sarcolemma may mediate the effects of calmodulin in the regulation of Ca²⁺ transport across the membrane.     

Distribution of calmodulin and calmodulin-binding proteins in membranes from bovine epididymal spermatozoa

Reproducible concentrations of calmodulin representing approximately 0.1% of the membrane protein were detected in purified plasma membranes from bovine epididymal spermatozoa. When membranes were isolated in the presence of 1 mM EGTA, the amount of calmodulin associated with the plasma membranes was not reduced. Calmodulin-binding proteins were detected in both purified plasma membranes and in a mixed membrane fraction containing both plasma membranes and cytoplasmic droplet membranes. A calcium-dependent, calmodulin-binding protein of apparent molecular weight 123,000 was detected in both fractions. In the presence of 1 mM EDTA, putative calcium-independent calmodulin-binding proteins of apparent molecular weights 93,000, 32,000, 18,000, and 15,000 were detected in the plasma membrane fraction. The 15,000 Mr polypeptide was also present in the mixed membrane fraction but the three proteins of higher molecular weight were reduced or absent in this fraction.     

Systematic delineation of a calmodulin peptide interaction

We present a comprehensive profile of amino acid side-chain constraints in a calmodulin (CaM) peptide complex. These data were obtained from the analysis of calmodulin binding to an array of all single substitution analogues as well as N- and C-terminal truncations of the skMLCK derived M13 peptide ligand. The experimentally derived binding data were evaluated with respect to the known 3D-structure of the CaM/M13 complex. Besides an almost perfect agreement between the measured affinities and the structural data, the unexpected high-affine Asn5Ala variant of the M13(*) peptide described by Montigiani et al. could be verified. In contrast to other reports our data clearly support the postulate of the minor and major hydrophobic anchors of this calcium dependent interaction.     

NMR and molecular dynamics studies of the interaction of melatonin with calmodulin

Pineal hormone melatonin (N-acetyl-5-methoxytryptamine) is thought to modulate the calcium/calmodulin signaling pathway either by changing intracellular Ca(2+) concentration via activation of its G-protein-coupled membrane receptors, or through a direct interaction with calmodulin (CaM). The present work studies the direct interaction of melatonin with intact calcium-saturated CaM both experimentally, by fluorescence and nuclear magnetic resonance spectroscopies, and theoretically, by molecular dynamics simulations. The analysis of the experimental data shows that the interaction is calcium-dependent. The affinity, as obtained from monitoring (15)N and (1)H chemical shift changes for a melatonin titration, is weak (in the millimolar range) and comparable for the N- and C-terminal domains. Partial replacement of diamagnetic Ca(2+) by paramagnetic Tb(3+) allowed the measurement of interdomain NMR pseudocontact shifts and residual dipolar couplings, indicating that each domain movement in the complex is not correlated with the other one. Molecular dynamics simulations allow us to follow the dynamics of melatonin in the binding pocket of CaM. Overall, this study provides an example of how a combination of experimental and theoretical approaches can shed light on a weakly interacting system of biological and pharmacological significance.     

A nonradioactive method for isolating complementary DNAs endocing calmodulin-binding proteins

Calmodulin labeled with¹²⁵I or³⁴S has been used to screen expression libraries to isolate cDNAs encoding calmodulin-binding proteins (CBPs) from several eukaryotic systems. The use of radiolabeled calmodulin has, however, several disadvantages. We have developed a nonradiactive method to isolate cDNAs for CBPs using biotinylated calmodulin. Screening of a cDNA library in an expression vector with biotinylated calmodulin resulted in the isolation of cDNAs encoding CBPs. Avidin and biotin blocking steps, prior to incubation of the filters with biotinylated calmodulin, are found to be essential to eliminate the cDNAs that code for biotin-containing polypeptides. The cDNA clones isolated using this nonradioactive method bound calmodulin in a calcium-dependent manner. The binding of biotinylated calmodulin to these clones was completely abolished by ethylene glycolbis(\-aminoethylether)-N,N′-tetraacetic acid (EGTA), a calcium chelator. Furthermore, the isolated cDNAs were confirmed by probing the clones with³⁵S-labeled calmodulin. All the isolated clones bound to radiolabeled calmodulin in the presence of calcium but not in the presence of EGTA. The method described here is simple, fast, and does not involve preparation and handing of radiolabeled calmodulin. All the materials used in this method are commercially available; hence, this procedure should be widely applicable to isolate cDNAs encoding CBPs from any eukaryotic organism.     

Light and GTP dependence of transducin solubility in retinal rods

The physical origin and functional significance of the near infra-red light scattering changes observable upon flash illumination of diluted suspensions of magnetically oriented, permeabilised frog retinal rods has been reinvestigated with particular attention paid to the degree with which transducin remains attached to the membrane. In the absence of GTP, the so called binding signal is shown to include two components of distinctive origins, widely different kinetics, and whose relative amplitudes depend on the dilution of the suspension and resulting detachment of transducin from the disc membrane. The fast component is a consequence of the fast interaction between photoexcited rhodopsin (R^*) and the transducin remaining on the membrane. Its kinetics monitors a structural modification of the discs caused by a change in electrostatic interaction between closely packed membranes upon the formation of R^*-T complexes. The slow component monitors the slow rebinding to the membrane and possible subsequent interaction with excess R^* of T-GDP which, in spite of its low solubility, had eluted into solution given the high dilution of the permeated rods. In the presence of GTP, the so called dissociation signal includes a fast, anisotropic release component that specifically monitors the release into the interdiscal space of T -GTP formed from the membrane-bound pool, and a slower isotropic loss component monitoring the leakage from the permeated rod of the excess T -GTP which did not interact with the cGMP phosphodiesterase. The amplitudes of both components depend exclusively on the membrane bound T-GDP pool. The kinetics of the loss component is limited by the size and degree of permeation of the rod fragments, rather than by the dissociation rate of T -GTP from the membrane.Abbreviations ROS rod outer segment - R rhodopsin - R^* photoactivated rhodopsin - T, T-GDP, T -GDP, T -GTP, T transducin and its various forms - T _mb, T _sol: T bound to membrane or soluble - PDE cGMP-phosphodiesterase - GTP guanosine 5-triphosphate - GDP guanosine 5-diphosphate - GDP S guanosine 5-O-(2-thiodiphosphate) - cGMP guanosine-3-5 cyclic-monophosphate - DTT dithiothreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - TRIS Tris (hydroxymethyl)aminomethane - SDS sodium dodecyl sulfate     

Arrestin interaction with rhodopsin

It is becoming increasingly apparent that G protein-coupled receptors (GPCRs) can exist and function as oligomers. This notion differs from the classical view of signaling wherein the receptor has been presumed to be monomeric. Despite this shift in views, the interpretation of data related to GPCR function is still largely carried out within the framework of a monomeric receptor. Rhodopsin is a prototypical GPCR that initiates phototransduction. Like other GPCRs, the activity of rhodopsin is regulated by phosphorylation and the binding of arrestin. In the current investigation, we have explored by modeling methods the interaction of rhodopsin and arrestin under the assumption that either one or two rhodopsin molecules bind each arrestin molecule. The dimeric receptor framework may provide a more accurate representation of the system and is therefore likely to lead to a better and more accurate understanding of GPCR signaling.     

Myristoyl moiety of HIV Nef is involved in regulation of the interaction with calmodulin in vivo

Human immunodeficiency virus Nef is a myristoylated protein expressed early in infection by HIV. In addition to the well known down-regulation of the cell surface receptors CD4 and MHCI, Nef is able to alter T-cell signaling pathways. The ability to alter the cellular signaling pathways suggests that Nef can associate with signaling proteins. In the present report, we show that Nef can interact with calmodulin, the major intracellular receptor for calcium. Coimmunoprecipitation analyses with lysates from the NIH3T3 cell line constitutively expressing the native HIV-1 Nef protein revealed the presence of a stable Nef-calmodulin complex. When lysates from NIH3T3 cells were incubated with calmodulin-agarose beads in the presence of CaCl(2) or EGTA, calcium ion drastically enhanced the interaction between Nef and calmodulin, suggesting that the binding is under the influence of Ca(2+) signaling. Glutathione S-transferase-Nef fusion protein bound directly to calmodulin with high affinity. Using synthetic peptides based on the N-terminal sequence of Nef, we determined that within a 20-amino-acid N-terminal basic domain was sufficient for calmodulin binding. Furthermore, the myristoylated peptide bound to calmodulin with higher affinity than nonmyris-toylated form. Thus, the N-terminal myristoylation domain of Nef plays an important role in interacting with calmodulin. This domain is highly conserved in several HIV-1 Nef variants and resembles the N-terminal domain of NAP-22/CAP23, a myristoylated calmodulin-binder. These results for the interaction between HIV Nef and calmodulin in the cells suggested that the Nef might interfere with intracellular Ca(2+) signaling through calmodulin-mediated interactions in infected cells.     

Calmodulin content,Ca2+-dependent calmodulin binding proteins,and testis growth: Identification of Ca2+-dependent calmodulin binding proteins in primary spermatocytes

In contrast with the transient pre-replicative increase in calmodulin (CaM) level observed in proliferative activated cells, postnatal development of rat testis was paralleled by 3 specific rises in CaM. The first one occurred between 5 and 10 days, coincident with the appearance and proliferation start of spermatogonia and Sertoli cells. Meiosis accomplishment and spermatid differentiation were paralleled by 2 additional rises, at 24 and 32 days, respectively. The plateau phase of testis growth was coincident with the appearance of maturating spermatids and spermatozoa in the germinal epithelium, and with a decrease in CaM content. Testicular DNA:g wet tissue ratio reached the highest level in 15-day-old rats and gradually decreased up to 35 days, when a constant level was reached. A similar level of Ca²⁺-CaMBPs was observed in 5- and 20-day-old rat testis. Although all subcellular fractions showed the ability to bind CaM in a Ca²⁺-dependent manner, CaM was mainly recovered in the nuclear and soluble fractions of adult and immature rat testis. Several Ca²⁺-CaMBPs with an apparent M_r of 82, 75, 64, 19, and 14 kD were purified by affinity chromatography from pachytene primary spermatocyte nuclear matrix. Ca²⁺-CaMBPs showing an M_r of 120, 78, 72, and 66 kD were also purified from the supernatant obtained after DNA and RNA hydrolysis of meiotic nuclei. Major cytosolic Ca²⁺-CaMBPs of primary spermatocytes showed an M_r of 120, 84, 44, and 39 kD. The functions that these Ca²⁺-CaMBPs might have during the first meiotic prophase is discussed. Mol. Reprod. Dev. 48:127–136, 1997. © 1997 Wiley-Liss, Inc.     

The interaction of calmodulin with fluorescent and photoreactive model peptides: evidence for a short interdomain separation

Calmodulin is known to bind target enzymes and basic, amphiphilic peptides in a Ca2(+)-dependent manner. Recently, we introduced a photoaffinity label, p-benzoylphenylalanine (Bpa), into the sequence of a model, alpha-helical, calmodulin-binding peptide. When the Bpa residue was introduced at the third position of the peptide, Met-144 on the C-terminal domain of calmodulin was labeled, whereas when the photolabel was placed at the thirteenth position, Met-71 on the N-terminal domain was labeled. Assuming that both peptides bind in similar orientations, these results are not consistent with the crystal structure of calmodulin, in which the domains are held at a significant distance from one another by a long alpha-helical segment. To test the assumption that both peptides bind in similar orientations, we have synthesized a calmodulin-binding peptide with the photolabel in both the third and the thirteenth positions. Upon photolysis, this peptide forms a cross-link between Met-71 and Met-124 on the N- and C-terminal domains, respectively. Furthermore, a peptide with a Bpa in the thirteenth position and a Trp residue in the third position was also synthesized. After photocross-linking the Bpa residue of this peptide to Met-71 of calmodulin, it could be shown that the fluorescence properties of the Trp residue were consistent with its side chain being buried in a hydrophobic pocket on the C-terminal domain of calmodulin. These data indicate that, when complexed with basic, amphiphilic peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal structure of the uncomplexed protein.     

Calcium-dependent interaction of actin filaments with actin binding protein in the presence of calmodulin and caldesmon

Caldesmon, calmodulin-, and actin-binding protein of chicken gizzard did not affect the process of polymerization of actin induced by 0.1 M KCl. Caldesmon binds to F-actin, thus inhibiting the gelation action of actin binding protein (ABP; filamin). Low shear viscosity and flow birefringence measurements revealed that in a system of calmodulin, caldesmon, ABP, and F-actin, gelation occurs in the presence of micromolar Ca2+ concentrations, but not in the absence of Ca2+. Electron microscopic observations showed the Ca2+-dependent formation of actin bundles in this system. These results were interpreted by the flip-flop mechanism: in the presence of Ca2+, a calmodulin-caldesmon complex is released from actin filaments on which ABP exerts its gelating action. On the other hand, in the absence of Ca2+, caldesmon remains bound to actin filaments, thus preventing the action of ABP.     

alpha-Synuclein exhibits competitive interaction between calmodulin and synthetic membranes

alpha-Synuclein, a pathological component of Parkinson's disease by constituting the Lewy bodies, has been suggested to be involved in membrane biogenesis via induction of amphipathic alpha-helices. Since the amphipathic alpha-helix is also known as a recognition signal of calmodulin for its target proteins, molecular interaction between alpha-synuclein and calmodulin has been investigated. By employing a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, alpha-synuclein has been shown to yield a heterodimeric 1 : 1 complex with calmodulin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and even absence of calcium, whereas beta-synuclein was more dependent upon calcium for its calmodulin interaction. The selective calmodulin interaction of alpha-synuclein in the absence of calcium was also demonstrated with the aggregation kinetics of the synucleins in which only the alpha-synuclein aggregation was affected by calmodulin. A reversible binding assay confirmed that alpha-synuclein interacted with the Ca2+-free as well as the Ca2+-bound calmodulins with almost identical Kds of 0.35 micro m and 0.31 micro m, respectively, while beta-synuclein preferentially recognized the Ca2+-bound form with a Kd of 0.68 micro m. By using a C-terminally truncated alpha-synuclein of alpha-syn97, the calmodulin binding site(s) on alpha-synuclein was(were) shown to be located on the N-terminal region where the amphipathic alpha-helices have been suggested to be induced upon membrane interaction. By employing liposome and calmodulin in a state of being either soluble or immobilized on agarose, actual competition of alpha-synuclein between membranes and calmodulin was demonstrated with the observation that alpha-synuclein previously bound to the liposome was released upon specific interaction with the calmodulins. Taken together, these data may suggest that alpha-synuclein could act not only as a negative regulator for calmodulin in the presence and even absence of calcium, but it could also exert its activity at the interface between calmodulin and membranes.