Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have characterized the opsin phosphatase activities in extracts of rod outer segments and determined their relationship to known protein phosphatases. The opsin phosphatase activity in the extracts was not due to protein phosphatases 1, 2B, or 2C because it was neither stimulated by Mg2+ or Ca2+/calmodulin nor inhibited by protein phosphatase inhibitors-1 or -2. Opsin phosphatase activity in rod outer segment extracts was potently inhibited by okadaic acid (IC50 approximately 10 nM), a preferential inhibitor of protein phosphatase 2A. Moreover, during chromatography on DEAE-Sepharose, the opsin phosphatase activity co-eluted with three peaks of protein phosphatase 2A activity, termed protein phosphatases 2A0, 2A1, and 2A2. The opsin phosphatase activity of each peak was stimulated by polylysine, a known activator of protein phosphatase 2A. Finally, treatment of rod outer segment extracts with 80% ethanol at room temperature converted the activity from a high molecular weight form characteristic of the protein phosphatase 2A0, 2A1, and 2A2 species to a low molecular weight form characteristic of the protein phosphatase 2A catalytic subunit. We conclude that protein phosphatase 2A is likely to be the physiologically relevant rhodopsin phosphatase. The 48-kDa rod outer segment protein arrestin (S-antigen) was found to inhibit the dephosphorylation of freshly photolyzed rhodopsin by protein phosphatase 2A but did not inhibit the dephosphorylation of unbleached rhodopsin. Arrestin has no effect on the dephosphorylation of phorphorylase a, indicating that the effect was substrate-directed. It appears that dephosphorylation of the photoreceptor protein phosphorhodopsin occurs only after decay of the photoactivated protein and that this may be regulated in vivo by arrestin. The binding of arrestin to photolyzed phosphorylated rhodopsin, i.e. the binding of a regulatory protein to a protein phosphatase substrate to form a complex resistant to dephosphorylation represents a novel mechanism for the regulation of protein phosphatase 2A.
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PMID:Regulation of rhodopsin dephosphorylation by arrestin. 255 Apr 22

Arrestins have emerged as one family of proteins that mediate the inactivation of G-protein-coupled receptors. We have isolated cDNA clones encoding two arrestin isoforms of the dipteran visual system, Calliphora arrestin 1 (Arr1) and arrestin 2 (Arr2). Microsequencing established that the arr2 gene encodes the Calliphora 49-kDa protein characterized previously as a photoreceptor-specific protein that undergoes reversible binding to light-activated rhodopsin and thereby activates the phosphorylation of metarhodopsin. Ultrastructural localization of Arr2 to the rhabdomeral part of the photoreceptor cell and quantitation of the amount of Arr2 bound suggest that Arr2 directly interacts with light-activated rhodopsin. In a reconstituted system containing affinity purified Arr2 and isolated rhabdomeric membranes, Arr2 binds to non-phosphorylated and phosphorylated metarhodopsin with comparable affinity. Reaction time courses reveal that Arr2 binding precedes phosphorylation of metarhodopsin, contrary to what has been reported so far for vertebrate photoreceptors. The phosphorylation-independent binding of Arr2 to metarhodopsin provides a mechanism for the rapid inactivation of the long-lived activated rhodopsin state which is characteristic for invertebrate photoreceptors. The dephosphorylation of rhodopsin is catalyzed by a Ca(2+)-dependent protein phosphatase which is shown here for the first time to exist in a membrane-associated form. Only metarhodopsin molecules with bound Arr2 are resistant to dephosphorylation. Thus, in fly photoreceptors, Arr2 acts as a regulatory protein that controls the phosphorylation as well as the dephosphorylation of the light-activated visual pigment.
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PMID:Mechanism of arrestin 2 function in rhabdomeric photoreceptors. 792 36

Calcium (Ca2+) plays an integral role in the light response of the photoreceptors in both vertebrate and invertebrate organisms. In the ventral eye of the horseshoe crab, Limulus polyphemus, a flash of light delivered to a dark-adapted photoreceptor stimulates a rapid rise in intracellular free calcium concentration ([Ca2+]i), which in turn mediates light adaptation. It has previously been demonstrated that in Limulus photoreceptors light, via Ca2+, activates a calcium/calmodulin (Ca2+/CaM)-dependent protein kinase which increases the phosphorylation of arrestin. We now have identified biochemically, a calcium/calmodulin-dependent protein phosphatase (Ca2+/CaM PP) in homogenates of the Limulus lateral and ventral eye, brain, and lateral optic nerve using as a substrate, a 32P-labeled peptide fragment of the regulatory subunit of cAMP-dependent protein kinase (RII). This protein phosphatase shares biochemical properties with calcineurin, a Ca2+/CaM-dependent protein phosphatase (type-2B). Its activity is enhanced by Ca2+, calmodulin and Mn2+; and is inhibited by mastoparan, a calmodulin antagonist, and a synthetic peptide corresponding to the autoinhibitory domain of mammalian calcineurin. Most importantly, light regulates the Ca2+/CaM PP activity in the lateral eye. While there is no difference in basal activity in long-term dark- or light-adapted preparations, Ca2+ enhances Ca2+/CaM PP activity only in long-term light-adapted eyes.
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PMID:Characterization of a calcium/calmodulin-dependent protein phosphatase in the Limulus nervous tissue and its light regulation in the lateral eye. 794 99

The Na+/H+ antiporter of trout erythrocytes is activated by agents raising intracellular cAMP, whereas other Na+/H+ exchangers are insensitive to or inhibited by cAMP. Cloning of the beta agonist-activated exchanger (beta NHE) reveals the presence of two consensus sites for phosphorylation by the cAMP-dependent protein kinase A (cAMP-PKA) on the cytoplasmic loop. Transfected to fibroblasts, beta NHE can no longer be activated by cAMP when these consensus sites are removed, indicating regulation through cAMP-PKA. Moreover, it has been shown that activation of the exchanger is rapidly followed by its desensitization. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase and phosphatase inhibitors on the antiporter activation and desensitization in trout red cells. Na+/H+ exchange was not induced by strong acidification, indicating that beta NHE is normally in a nonfunctional state, whereas cAMP did activate the system by forcing beta NHE into a functional conformation; preincubation of cells with the kinase inhibitor H89 blocked cAMP-activation, confirming the role of cAMP-PKA in the activation process. The protein phosphatase inhibitor okadaic acid (OA) neither activated the exchange when added on unstimulated cells nor prevented deactivation of beta agonist-activated beta NHE by propranolol. Hence, the cAMP-dependent phosphorylation involved in the activating process is controlled by an OA-insensitive phosphatase. beta NHE activated by beta agonist or cAMP shifts rapidly into a refractory state, accounting for the previously described desensitization. Desensitization was blocked and reversed by OA, indicating a control by an OA-sensitive phosphatase of the phosphorylation level of a site critical for the desensitizing process. Phosphorylation of this (site 2) and of the activating site (site 1) is mediated by cAMP-PKA, as demonstrated by the effects of both intracellular cAMP concentration and kinase inhibitor H89 on the Na+/H+ exchange activity. Based on these data, we proposed that beta NHE can exist in three different states (inactive I, activated A, and desensitized D). Conversion of I to A needs the simultaneous phosphorylation by cAMP-PKA of sites 1 and 2. These two sites might constitute the two neighboring cAMP-PKA sites located on the cytoplasmic loop as deduced from the oligonucleotide sequence. Dephosphorylation of site 2 and subsequent binding of an arrestin-like protein are assumed to account for desensitization of the antiport.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of protein phosphorylation and dephosphorylation in activation and desensitization of the cAMP-dependent Na+/H+ antiport. 838 66

Excitation of fly photoreceptor cells is initiated by photoisomerization of rhodopsin to the active form of metarhodopsin. Fly metarhodopsin is thermostable, does not bleach, and does not regenerate spontaneously to rhodopsin. For this reason, the activity of metarhodopsin must be stopped by an effective termination reaction. On the other hand, there is also a need to restore the inactivated photopigment to an excitable state in order to keep a sufficient number of photopigment molecules available for excitation. The following findings reveal how these demands are met. The photopigment undergoes rapid phosphorylation upon photoconversion of rhodopsin to metarhodopsin and an efficient Ca2+ dependent dephosphorylation upon regeneration of metarhodopsin to rhodopsin. Phosphorylation decreases the ability of metarhodopsin to activate the guanine nucleotide-binding protein. Binding of 49-kDa arrestin further quenches the activity of metarhodopsin and protects it from dephosphorylation. Light-dependent binding and release of 49-kDa arrestin from metarhodopsin- and rhodopsin-containing membranes, respectively, directs the dephosphorylation reaction toward rhodopsin. This ensures the return of phosphorylated metarhodopsin to the rhodopsin pool without initiating transduction in the dark. Assays of rhodopsin dephosphorylation in the Drosophila retinal degeneration C (rdgC) mutant, a mutant in a gene previously cloned and predicted to encode a serine/threonine protein phosphatase, reveal that phosphorylated rhodopsin is a major substrate for the rdgC phosphatase. We propose that mutations resulting in either a decrease or an improper regulation of rhodopsin phosphatase activity bring about degeneration of the fly photoreceptor cells.
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PMID:Regulatory arrestin cycle secures the fidelity and maintenance of the fly photoreceptor cell. 844 7

We utilize a computer model derived directly from the phototransductive enzyme cascade involving rhodopsin (R), G-proteins (G) and ultimately the membrane channels. In photoreceptors, single photons activate single rhodopsin molecules (R*), resulting in the production of quantal bumps. A CaM-KII inhibitor may inhibit whereas calcineurin (PP2B) inhibitors may augment arrestin activity. Arrestin is known to inactivate R*. We voltage-clamped and digitized quantal bumps for comparison with the kinetic model and simulations. Inhibitors of CaM-KII and PP2B produce results consistent with altering arrestin activity. In sum, we have succeeded in constructing a mathematical description of the phototransduction cascade that is biochemically derived and useful in interpreting results.
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PMID:Kinetic model for phototransduction and G-protein enzyme cascade: understanding quantal bumps during inhibition of CaM-KII or PP2B. 882 40

The trout red blood cell Na+/H+ antiporter (beta NHE) plays two interesting properties: it is the only NHE own to be activated by cyclic AMP, and the activation process is followed by a desensitisation of the transport system itself. Cloning and expression of beta NHE have provided inificant information about Na+/H+ activation, in particular that activation by cyclic AMP is directly dependent upon the presence of two protein kinase A consensus sites in the cytoplasmic tail of the antiporter. Expression of beta NHE in fibroblasts demonstrates that the protein kinase A (PKA) and protein kinase C (PKC) activation pathways are independent and do not converge a common kinase. Moreover, the hydrophilic C-terminal fragment is essential to the mediation of the various hormonal responses. NHE1 (the human ubiquitous isoform) is not activated by cyclic AMP, but a "NHE1 transmembrane domain/beta NHE cytoplasmic domain' chimera is fully activated by cyclic AMP. In red cells, activation of beta NHE is the result of phosphorylation by PKA of at least two independent sites. Desensitisation, inhibited by the phosphatase inhibitor okadaic acid, may consist of the dephosphorylation of one of these two sites. Furthermore, Calyculin A (CIA), another specific protein phosphatase inhibitor, induces in unstimulated cells a Na+/H+ exchange activity whose exchange properties are very different from those of the adrenergically stimulated antiporter. It is suggested that CIA may be able to revive "sequestered' antiporters. We propose that the molecular events underlying beta NHE desensitisation could be similar to those involved in rhodopsin desensitisation. Antibodies were generated against trout red cell arrestin in order to analyse the binding of arrestin to the activated exchanger. Recombinant trout arrestin was produced in a protease-deficient strain of Escherichia coli and its functionality tested in a reconstituted rhodopsin assay.
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PMID:Regulation of Na+/H+ antiporter in trout red blood cells. 905 Feb 44

Changes in intracellular calcium are involved in phototransduction processes in both vertebrate and invertebrate photoreceptors. During this phototransduction process in the Limulus ventral eye, there is a biochemical change in the protein phosphatase, calcineurin, such that it becomes capable of activation by calcium and calmodulin. Here we show that the calcium/calmodulin-dependent calcineurin-like activity in light-adapted ventral eye was completely inhibited by the CaN autoinhibitory peptide, CaN A457-482 and the Merck analog of the membrane-permeable, immunosuppressant drug, FK 506, L-683, 590, but not an inactive analogue, L-685, 818. Whole-cell, voltage-clamp recordings of spontaneous quantal bump activity present in dark-adapted photoreceptors injected with either CaN A457-482 (500 microM) or superfused with L-683, 590 (20 microM) or L-685, 818 revealed that both CaN A457-482 and L-683, 590, but not L-685, 818, caused rapid decreases in quantal bump amplitude, rise time and fall time, resulting in smaller, sharper bumps. This was correlated with enhanced phosphorylation of arrestin in light-adapted ventral eye photoreceptors exposed to L-683, 590 or less reliably okadaic acid. Both CaN A457-482 and L-683, 590 markedly affected the light-stimulated inward currents recorded from light-adapted ventral photoreceptors, causing a "terracing" of the inward current, and an intensity-dependent delay in the time required to reach peak amplitude. Consequently, inhibition of calcineurin markedly affects two major rhodopsin-dependent electrophysiological processes, and implicates CaN as an integral component in the phototransduction cascade.
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PMID:Inhibition of the calcineurin-like protein phosphatase activity in Limulus ventral eye photoreceptor cells alters the characteristics of the spontaneous quantal bumps and the light-mediated inward currents, and enhances arrestin phosphorylation. 983 68

G protein-coupled receptor kinases (GRKs) and beta-arrestin-2 play a crucial role in the regulation of neurotransmitter receptors in brain. In this study, GRK2, GRK6, beta-arrestin-2 and associated regulatory proteins (Gbeta proteins and protein phosphatase (PP)-2A) were quantitated in human brains (immunodensity with specific antibodies) to assess for postmortem changes (pattern of protein degradation) and to investigate the effect of aging on these regulatory proteins as well as their subcellular distribution (cytosol and membrane fractions). In brain (prefrontal cortex, total homogenate) of healthy subjects (n=14) the immunodensities of GRK2 (r=-0.76), GRK6 (r=-0.64), beta-arrestin-2 (r=-0.57), Gbeta proteins (r=-0.59) and neurofilament (NF)-L (r=-0.64), but not PP-2A, declined markedly with the length of postmortem delay (PMD, 3-81 h). With these linear decay models, the average decreases per 12 h of PMD (from 12 to 72 h) were 7-11% for the various proteins. The immunodensities of GRK2 (r=-0.71), GRK6 (r=-0.61), and beta-arrestin-2 (r=-0.54) in human brain (n=12) also declined with aging (16 to 87 years) and the average decreases per decade (from 20 to 80 years) were 3-5%. In contrast, the immunodensities of PP-2A, Gbeta and NF-L in brain did not correlate significantly with the age of the subject at death (16-87 years). The immunodensities of GRK2/6 and beta-arrestin-2 showed marked individual variations and were strongly reduced after several freeze/thaw cycles. In the prefrontal cortex the subcellular distribution (cytosol/membrane) of the two GRKs differed markedly (GRK2: 60%/40%; GRK6: 5%/95%), and that of beta-arrestin-2 was as expected for a soluble protein (60%/40%). In brains of healthy subjects, the immunodensities of cytosolic GRK2 and beta-arrestin-2 correlated, respectively, with those of membrane-associated GRK2 (r=0.67, P=0.049, n=9) and membrane-associated beta-arrestin-2 (r=0.77, P=0.01, n=9). The results of this study emphasize the importance of examining relevant variables (PMD, age) and potential artifacts (individual variation, freeze-thawing effect) when designing signal transduction studies in neuropsychiatric disorders using the postmortem human brain.
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PMID:G protein-coupled receptor kinases, beta-arrestin-2 and associated regulatory proteins in the human brain: postmortem changes, effect of age and subcellular distribution. 1200 30

G protein-coupled receptor kinases (GRKs) and beta-arrestin-2 play a crucial role in the regulation of neurotransmitter receptors in brain. In this study, GRK 2, GRK 6, beta-arrestin-2 and associated proteins (Gbeta proteins and protein phosphatase (PP)-2A) were quantitated in parallel (immunodensity with specific antibodies) in brains of depressed subjects (drug-free and antidepressant-treated) to investigate the effect of major depression and antidepressant drugs on these receptor regulatory proteins. Specimens of the prefrontal cortex (Brodmann's area 9) were collected from 19 suicide and non-suicide depressed subjects and 13 control subjects. In drug-free (n=9), but not in antidepressant-treated (n=10), depressed subjects an increase in the density of membrane-associated GRK 2 (30%, n=9, P=0.005) was found compared with that in sex-, age-, and PMD-matched controls. Comparison between drug-free and antidepressant-treated depressed subjects showed that GRK 2 was reduced in membrane (39%, n=10, P=0.008) and cytosolic (44%, n=10, P=0.09) preparations after antidepressant drug treatment. In contrast, membrane-associated GRK 6 (drug-free and antidepressant-treated depressed subjects) was found unchanged when compared with that in matched controls. Similarly, the densities of beta-arrestin-2, PP-2A, and Gbeta proteins were not significantly different from those in matched controls. There was a positive correlation between the immunodensities of GRK 2 and beta-arrestin-2 in membrane preparations (r=0.48, n=19, P=0.04), suggesting that both proteins are regulated in a coordinated manner in brains of depressed subjects. The results of this study indicate that major depression is associated with upregulation of brain GRK 2, but not GRK 6, and that antidepressant drug treatment appears to induce downregulation of GRK 2 protein.
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PMID:Regulation of GRK 2 and 6, beta-arrestin-2 and associated proteins in the prefrontal cortex of drug-free and antidepressant drug-treated subjects with major depression. 1265 3


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