14-3-3 proteins: a family of versatile molecular regulators.

The 14-3-3 proteins are a family of acidic regulatory molecules found in all eukaryotes. 14-3-3 proteins function as molecular scaffolds by modulating the conformation of their binding partners. Through the functional modulation of a wide range of binding partners, 14-3-3 proteins are involved in many processes, including cell cycle regulation, metabolism control, apoptosis, and control of gene transcription. This minireview includes a short overview of 14-3-3 proteins and then focuses on their role in the regulation of two important binding partners: FOXO forkhead transcription factors and an enzyme tyrosine hydroxylase.

Subcellular redistribution of trimeric G-proteins--potential mechanism of desensitization of hormone response: internalization, solubilization, down-regulation.

Agonist-induced subcellular redistribution of G-protein coupled receptors (GPCR) and of trimeric guanine-nucleotide binding regulatory proteins (G-proteins) represent mechanisms of desensitization of hormone response, which have been studied in our laboratory since 1989. This review brings a short summary of these results and also presents information about related literature data covering at least small part of research carried out in this area. We have also mentioned sodium plus potassium dependent adenosine triphosphatase (Na, K-ATPase) and 3H-ouabain binding as useful reference standard of plasma membrane purity in the brain.

Functional changes in the vanilloid receptor subtype 1 channel during and after acute desensitization.

Agonist-induced desensitization of the transient receptor potential vanilloid receptor-1 (TRPV1) is one of the key strategies that offer a way to alleviate neuropathic and inflammatory pain. This process is initiated by TRPV1 receptor activation and the subsequent entry of extracellular Ca(2+) through the channel into sensory neurones. One of the prominent mechanisms responsible for TRPV1 desensitization is dephosphorylation of the TRPV1 protein by the Ca(2+)/calmodulin-dependent enzyme, phosphatase 2B (calcineurin). Of several consensus phosphorylation sites identified so far, the most notable are two sites for Ca(2+)/calmodulin dependent kinase II (CaMKII) at which the dynamic equilibrium between the phosphorylated and dephosphorylated states presumably regulates agonist binding. We examined the mechanisms of acute Ca(2+)-dependent desensitization using whole-cell patch-clamp techniques in human embryonic kidney (HEK) 293T cells expressing the wild type or CaMKII phosphorylation site mutants of rat TRPV1. The nonphosphorylatable mutant S502A/T704I was capsaicin-insensitive but the S502A/T704A construct was fully functional, indicating a requirement for a specific residue at position 704. A point mutation at the nearby conserved residue R701 strongly affected the heat, capsaicin and pH-evoked currents. As this residue constitutes a stringent CaMKII consensus site but is also predicted to be involved in the interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), these data suggest that in addition to dephosphorylation, or as its consequence, a short C-terminal juxtamembrane segment adjacent to the transient receptor potential box composed of R701 and T704 might be involved in the decelerated gating kinetics of the desensitized TRPV1 channel.

Oxidizing reagent copper-o-phenanthroline is an open channel blocker of the vanilloid receptor TRPV1.

The TRPV1 channel plays an important role in generating nociceptive signals in mammalian primary sensory neurons. It consists of 838 amino acids with six transmembrane segments (TM1-TM6), a pore-forming loop between TM5 and TM6 and N- and C- terminals located intracellularly. It is a homotetramer and forms a nonselective cationic channel that can be opened by capsaicin, weak acids and noxious heat. There are 18 cysteines (Cys), three of which are located on the extracellular side of the receptor in and around the region of the pore-forming loop. We report that the TRPV1 channel in transfected HEK293T cells and in cultured rat DRG neurons is blocked in the open state by an oxidizing agent Cu-o-phenanthroline complex (Cu:Phe). The effects of Cu:Phe are concentration dependent ( IC50 = 5.2 : 20.8 microm ) and fully reversible. Cu:Phe applied immediately before exposure to an acidic solution, capsaicin or noxious heat is without effect. Substitutions of the extracellular Cys residues (616, 621, 634) by glycine individually or together do not alter the blocking effects of Cu:Phe suggesting that disulfide cross-linking does not represent the underlying mechanism. It is suggested that the complex Cu:Phe, a bulky, positively charged molecule, represents a very effective and reversible open channel blocker of TRPV1.

Biochemistry of transmembrane signaling mediated by trimeric G proteins.

Many extracellular signals are at the cell surface received by specific receptors, which upon activation transduce information to the appropriate cellular effector molecules via trimeric G proteins. The G protein-mediated cascades ultimately lead to the highly refined regulation of systems such as sensory perception, cell growth, and hormonal regulation. Transmembrane signaling may be seriously deranged in various pathophysiological conditions. Over the last two decades the major experimental effort of our group has been devoted to better understanding the molecular mechanisms underlying transmembrane signaling regulated by G proteins and to the closely related process of desensitization of hormone response. This review provides general information about the basic principles of G protein-regulated transmembrane signaling as well as about our contribution to the current progress in the field.

Reducing agent dithiothreitol facilitates activity of the capsaicin receptor VR-1.

The vanilloid receptor subtype 1 (VR1) is expressed in a sub-population of small dorsal root ganglion (DRG) neurones in mammals and serves as the common transducer of the pain-producing signals, such as noxious heat, acids and capsaicin [Caterina et al., Nature 389 (1997) 816-824; Tominaga et al., Neuron 21 (1998) 531-543]. On the extracellular side, VR1 has three cysteine residues at positions 616, 621 and 634. Here we report that dithiothreitol (DTT) (2-60 mM), an agent that maintains -SH groups of cysteines in a reduced state, greatly facilitates membrane currents induced by noxious heat or capsaicin (1 microM) in cultured DRG neurones from the rat and in VR1-transfected HEK293 cells. The effects of DTT are concentration-dependent and fully reversible. We suggest that the ratio between free sulfhydryl groups and disulfide bonds of the cysteine residues of VR1 pre-sets sensitivity of primary nociceptors to algogens and may represent a new target for treating some pain states in humans.

Molecular and functional properties of synaptically activated NMDA receptors in neonatal motoneurons in rat spinal cord slices.

The functional properties of N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSC) were studied in fluorescence-labelled motoneurons in thin spinal cord slices. The deactivation of NMDA receptor EPSCs in motoneurons voltage-clamped at +40 mV was independent of intensity or location of stimulation and of postnatal age [taufast = 28.5 +/- 4.6 ms (63.6 +/- 8.8%) and tauslow = 165.6 +/- 49.6 ms]. In the presence of 1 mM Mg2+ the amplitude of NMDA receptor EPSCs was voltage-dependent. Boltzmann analysis of the relationship between peak NMDA receptor EPSC amplitude and membrane potential suggested an apparent Kd of Mg2+ (at 0 mV) of 0.87 mM. Nonstationary variance analysis of NMDA receptor EPSCs gave an estimated single-channel conductance of 59 +/- 14 pS. Direct measurement of the NMDA receptor channel openings in outside-out patches isolated from motoneurons indicated the presence of single-channel conductance levels of 21.8 +/- 2.8 pS, 37. 1 +/- 3.2 pS, 49.6 +/- 5.1 pS and 69.6 +/- 4.2 pS. Single-cell RT-PCR analysis of mRNA revealed that NR1, NR2A-D and NR3A transcripts were expressed in motoneurons. These results suggest that specific assembly of NMDA receptor subunits in motoneurons determines the functional and pharmacological properties of the receptors in these cells.

Different cation binding to the I domains of alpha1 and alpha2 integrins: implication of the binding site structure.

In the present work, we studied the interactions of recombinant alpha1 and alpha2 integrin I domains with cations Tb(3+), Mn(2+), Mg(2+) and Ca(2+). We observed that alpha1 and alpha2 I domains bind these cations with significantly different characteristics. The binding of Mg(2+) by the alpha1 I domain was accompanied by significant changes of tryptophan fluorescence which could be interpreted as a conformational change. Comparison of the alpha1 integrin I domain structure obtained by comparative modeling with a known structure of the alpha2 integrin I domain shows distinct differences in the metal ion binding sites which could explain the differences in cation binding.

Chemical modifications of melatonin receptors in chicken brain.

The membrane-bound or solubilized melatonin receptors were treated with protein-modifying agents under specific conditions and then assayed for 125I-melatonin binding in order to obtain information on amino acids present in the ligand binding domain. The reagents specific for sulfhydryl (N-ethylmaleimide and p-chloromercuribenzoate), guanidyl (phenylglyoxal), and amino groups (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1-fluoro-2,4-dinitrobenzene) inhibited 125I-melatonin binding in a dose-dependent manner, and their effects were prevented by pretreatment with cold melatonin. These results suggest the presence of cysteine, arginine, and lysine residues in the melatonin binding domain. Decreased sensitivity of 125I-melatonin binding to guanine nucleotides after N-ethylmaleimide pretreatment suggests the presence of another sulfhydryl group within the coupling domain between the receptor and G protein. Tyrosine reagents tetranitromethane, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, N-acetylimidazole, and p-nitrobenzenesulfonyl fluoride also inhibited 125I-melatonin binding, and their effects were prevented by cold melatonin pretreatment; however, they were effective only at concentrations when cross-reaction with a sulfhydryl group may occur. Histidine reagent diethyl pyrocarbonate inhibited 125I-melatonin binding in a dose-dependent manner, and its action was reversed by cold melatonin. However, diethyl pyrocarbonate had a smaller effect in a solubilized receptor preparation and, therefore, it could have modified a site remote from the ligand binding site. Our data do not suggest the presence of tryptophanyl, aspartic, or glutamic residues at the ligand binding domain.

An upstream enhancer regulating brown-fat-specific expression of the mitochondrial uncoupling protein gene.

Previous studies on the regulation of a Ucp minigene in transgenic mice demonstrated that the sequences necessary for brown-fat-specific expression and inducibility by norepinephrine were located in the 5' flanking region between 1 and 2.8 kb from the transcriptional start site. We have investigated this region in more detail in cultured mouse brown adipocyte tumor cells. Deletion analysis of two types of chloramphenicol acetyltransferase reporter gene constructs under control of either the Ucp promoter or a heterologous herpes simplex virus-tk promoter defined an enhancer in a 220-bp HindIII-XbaI fragment which was essential for both brown fat specificity and norepinephrine inducibility. Site-directed mutagenesis of the reporter gene constructs established that independent mutations to a cyclic AMP-responsive element (CRE-2) or one of two TTCC motifs (BRE [brown fat regulatory element]), all within 17 bp, eliminated transient expression. Competitive DNA mobility shift assays with probes of the CRE and BRE motifs indicate that nuclear proteins interact with these motifs in a cooperative, synergistic manner. While these CRE-BRE probes do not show changes in binding which is dependent on norepinephrine treatment, a probe containing a third TTCC motif located 130 bp downstream of BRE-1 does show this dependency. The results indicate that a complex interaction of the CRE and BRE motifs, which cannot be functionally separated, control Ucp expression.

Ouabain binding, ATP hydrolysis, and Na+,K(+)-pump activity during chemical modification of brain and muscle Na+,K(+)-ATPase.

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.

Identification of apamin binding sites in rat intestinal mucosa.

Apamin, a specific blocker of one class of Ca(2+)-activated K+ channes, was used to detect the apamin receptors associated with K+ channels in the mucosa of the rat jejunum and colon. Two receptor sites for 125I-apamin have been identified. These sites differed in their affinity for apamin (jejunum: KD1 = 1.1 nM and KD2 = 170 nM; colon: KD1 = 0.5 nM and KD2 = 1.1 nM and KD2 = 140 nM) and the maximum number of sites (jejunum: B(max1) = 111 and B(max2) = 4030; colon: B(max1) = 187 and B(max2) = 7550 fmol/mg of protein). 125I-apamin binding was stimulated by K+ ions with K0.5 = 1.0 mM and inhibited by the neuromuscular blocker tubocurarine (KI = 50 microM). We interpret these data to demonstrate that the high-affinity, low-capacity binding sites reflect the existence of apamin-sensitive K+ channels in the intestinal mucosa.

Na,K-ATPase and the development of Na+ transport in rat distal colon.

Na,K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na+ transport during early postnatal development. The maximum Na(+)-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (ISCN) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats. ISCN was entirely inhibited by ouabain; the inhibitory constant was 0.1 mM in 10-day-old (young) and 0.4 mM in 90-day-old (adult) rats. The affinity of the Na,K pump for Na+ was higher in young (11 mM) than in adult animals (19 mM). The Na,K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (KI) was not changed during development (0.1-0.3 mM). Specific [3H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (KD) was 8 and 21 microM in young and adult rats, respectively. The Na+ turnover rate per single Na,K pump, which was calculated from ISCN and estimated density of binding sites per cm2 of tissue was 500 in adult and 6400 Na+/min.site in young rats. These data indicate that they very high Na+ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na+ of a single isoform of the Na,K pump. The development of Na+ extrusion across the basolateral membrane is not directly regulated by corticosteroids.

Immobilization atrophy and membrane properties in rat skeletal muscle fibres.

Wet mass, resting membrane potential, frequency of miniature end-plate potentials and the concentration of [3H]ouabain-binding sites were studied after 7 days' immobilization of the rat soleus and extensor digitorum longus (EDL) muscles in the shortened or stretched position and after 3 and 7 days of remobilization. We observed that the loss of muscle mass by 37% in the rat soleus immobilized for 7 days in the shortened position is accompanied by a membrane depolarization of about 5 mV, a decrease in frequency of miniature end-plate potentials by 60% and a decrease of [3H]ouabain binding by 25%. Only minor changes were found in stretched soleus and in shortened and stretched EDL. After 3 days of remobilization of stretched soleus the muscle mass, [3H]ouabain binding and miniature end-plate potential frequency recovered to control values but the resting membrane potential continued to decrease. All changes induced by immobilization disappeared on day 7 of remobilization.

Na(+), K(+) ATPase activity in membrane fractions of rat central and peripheral nervous tissues modified by opioids.

The inhibition by ouabain of Na(+), K(+) ATPase activity in lumbar spinal cord membranes followed a sigmoid curve and in sciatic nerve membranes an exponential curve. The degree of inhibition in spinal cord depended on the presence of opioid ligands; fractions became more sensitive to ouabain after removal of bound opioids. The saturation of receptors by specific ligands reversed the effect of preincubation. Opioids had no effect on Na(+), K(+) ATPase in sciatic nerve. Membranes isolated from spinal cord were highly sensitive to oubain inhibition and their Na(+), K(+) ATPase resembled that containing the ? (+) subunit; membranes from peripheral nerve were less sensitive to ouabain and their enzyme reacted as that containing the ? subunit.

The role of carboxyl groups of Na+/K+-ATPase in the interaction with divalent cations.

Mg2+-induced subconformational changes of the E1 conformation of partly purified pig kidney Na+/K+-ATPase were studied by fluorescence techniques. In the enzyme with carboxyl groups modified by carbodiimide in the presence of an exogenous nucleophile the efficiency to pass through conformational substates was substantially lower than in the unmodified enzyme. Magnesium could form bridges between carboxyl groups near the membrane/water interface and negatively charged phospholipid polar heads.

Different sensitivity of ATP + Mg + Na (I) and Pi + Mg (II) dependent types of ouabain binding to phospholipase A2.

The effect of phospholipase A2 and of related agents on ouabain binding and Na, K-ATPase activity were studied in intact and detergent-treated membrane preparations of rat brain cortex and pig kidney medulla. It was found that phospholipase A2 (PLA2) may distinguish or dissociate ouabain binding complexes I (ATP + Mg + Na) and II (Pi + Mg), stimulating the former and inhibiting the latter. Procedures which break the permeability barriers of vesicular membrane preparations, such as repeated freezing-thawing, sonication or hypoosmotic shock failed to mimic the effect of PLA2, indicating that it was not acting primarily by opening the inside-out oriented vesicles. The detergent digitonin exhibited similar effects on ouabain binding in both ATP + Mg + Na and Pi + Mg media. Other detergents were ineffective. The ability of PLA2 to distinguish between ouabain binding type I and II can be manifested even in SDS-treated, purified preparations of Na, K-ATPase. The number of ATP + Mg + Na-dependent sites is unchanged, while the Pi + Mg-dependent sites are decreased in number in a manner similar to that seen in original membranes. This inhibition is completely lost in the reconstituted Na, K-ATPase system, where the ATP- as well as Pi-oriented ouabain sites are inhibited by PLA2.

Inhibition of the electrogenic Na,K pump and Na,K-ATPase activity by tetraethylammonium, tetrabutylammonium, and apamin.

The K+-induced hyperpolarization of Na-loaded mouse diaphragm muscle, enzymatic activity of Na,K-ATPase and 3H-ouabain binding to rat brain microsomes was measured in the presence of K+ channel blockers tetraethylammonium (TEA), tetrabutylammonium (TBA) and apamin. TBA, and to a lesser extent TEA in millimolar concentrations, inhibited the electrogenic effect of the Na,K pump, Na,K-ATPase activity, and 3H-ouabain binding. The inhibition of 3H-ouabain binding by TEA or TBA was more evident in the presence of ATP and Na+ ions. Apamin in nanomolar concentrations inhibited the electrogenic effect of Na,K pump and Na,K-ATPase but not the 3H-ouabain binding. The hyperpolarizing effects of insulin and NADH, but not that of noradrenaline, were also prevented by apamin. The inhibition of Na,K pump by TEA and TBA is apparently due to both competition with K+ for a binding site on the Na,K-ATPase and a reduction in the number of transporting sites. The site of action of apamin on Na,K-ATPase is different from that of tetra-alkylammonium compounds; it apparently decreases the turnover rate of the enzyme.

The changes in conformation of (Na+ K+)-ATPase from rat brain membranes are accompanied by changes of protein segment movements in the nanosecond range.

Differential polarized phase fluorometry of fluorescein-5-isothiocyanate (FITC) showed that the activation of (Na,K)-ATPase in crude plasma membranes from rat brain by 10 mmol.l-1 K+ and 100 mmol.l-1 Na+ significantly increased the rotational relaxational rate (R) of enzyme-bound FITC. This increase was blocked by both ouabain (0.1 mmol.l-1) and vanadate (0.1 mmol.l-1). In the absence of ATP, R was increased less after adding of 10 mmol.l-1 K+ to the membranes. The shifts in the nanosecond movements of the protein segments measured as R during the activation of (Na,K)-ATPase suggest that this type of movement might be of some functional importance.