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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three separate processes may contribute to rapid beta-adrenergic receptor desensitization: functional uncoupling from the stimulatory guanine nucleotide-binding protein Gs, mediated by phosphorylation of the receptors by two distinct kinases, the specific beta-adrenergic receptor kinase (beta ARK) and the cyclic AMP-dependent protein kinase A (PKA), as well as a spatial uncoupling via sequestration of the receptors away from the cell surface. To evaluate the relative importance and potential role of the various processes in different physiological situations, a kinetic analysis of these three mechanisms was performed in permeabilized A431 epidermoid carcinoma cells. To allow a separate analysis of each mechanism, inhibitors of the various desensitization mechanisms were used: heparin to inhibit beta ARK, the PKA inhibitor peptide PKI to inhibit PKA, and concanavalin A treatment to prevent sequestration. Isoproterenol-induced phosphorylation of beta 2 receptors in these cells by beta ARK occurred with a t1/2 of less than 20 sec, whereas phosphorylation by PKA had a t1/2 of about 2 min. Similarly, beta ARK-mediated desensitization of the receptors proceeded with a t1/2 of less than 15 sec, and PKA-mediated desensitization with a t1/2 of about 3.5 min. Maximal desensitization mediated by the two kinases corresponded to a reduction of the signal-transduction capacity of the receptor/adenylyl cyclase system by about 60% in the case of beta ARK and by about 40% in the case of PKA. Receptor sequestration was much slower (t1/2 of about 10 min) and involved no more than 30% of the cell surface receptors. It is concluded that beta ARK-mediated phosphorylation is the most rapid and quantitatively most important factor contributing to the rapid desensitization. This rapidity of the beta ARK-mediated mechanism makes it particularly well suited to regulate beta-adrenergic receptor function in rapidly changing environments such as the synaptic cleft.
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PMID:Comparative rates of desensitization of beta-adrenergic receptors by the beta-adrenergic receptor kinase and the cyclic AMP-dependent protein kinase. 164 31

In preparations of synaptic terminals (synaptosomes) isolated from rat brain, the activity of phospholipase A2 (PLA2), a phospholipid hydrolase that serves a central function in signal transduction, was inhibited in a Ca(2+)-dependent manner by incubation with 60 mM K+ or with the Ca(2+)-selective ionophore ionomycin. Reversal by alkaline phosphatase treatment suggested that this inhibitory effect resulted from phosphorylation of a synaptosomal protein substrate. When lysed synaptosomes were incubated with Ca2+/calmodulin (CaM), purified Ca2+/CAM-dependent protein kinase II (Ca2+/CaM-dependent PK II) and ATP, PLA2 activity in lysates was nearly abolished within 10 min. This effect was accompanied by a marked decrease in the Vmax of the enzyme and little or no change in the Km. Furthermore, Ca2+/CaM with ATP but without exogenous Ca2+/CaM-dependent PK II partially inhibited PLA2 activity, and this effect was prevented by treating the lysates with a selective peptide inhibitor of Ca2+/CaM-dependent PK II. In contrast, incubation of intact synaptosomes with 4 beta-phorbol 12-myristate 13-acetate or of lysed synaptosomes with purified protein kinase C had little or no effect on PLA2 activity. The results strongly suggest that the Ca(2+)-dependent inhibition of PLA2 activity observed in intact nerve endings was produced by activation of the multifunctional Ca2+/CaM-dependent PK II. A membrane-permeable adenylyl cyclase activator, forskolin, enhanced PLA2 activity in intact synaptosomes, and cAMP-dependent protein kinase potentiated PLA2 activity in lysed synaptosomes. Furthermore, another broad-spectrum protein kinase present in synaptic terminals, casein kinase II, also potentiated PLA2 activity in lysed synaptosomes. The effects of both protein kinases were associated with a decrease in Km and no change in Vmax. The results suggest that PLA2 activity in synaptic terminals is subject to bidirectional control by distinct signal transduction pathways. Moreover, mutually antagonistic effects of the Ca2+/CaM-dependent PK II and PLA2 pathways provide a possible molecular mechanism for bidirectional modulation of neurotransmitter release.
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PMID:Bidirectional control of phospholipase A2 activity by Ca2+/calmodulin-dependent protein kinase II, cAMP-dependent protein kinase, and casein kinase II. 165 Apr 81

Heart rate and force can be increased by noradrenaline and adrenaline through an interaction with both beta 1-adrenoceptors (beta 1AR) and beta 2-adrenoceptors (beta 2 AR). Several ionic currents (I) can flow upon beta AR activation: ICa (through either beta 1AR or beta 2AR), INa, IK, and ICl. Calcium currents (ICa) can be increased directly by the alpha s unit of a GTP binding protein, Gs, or by coupling of Gs to adenylyl cyclase with subsequent formation of cyclic AMP, release of the catalytic unit of cyclic AMP-dependent protein kinase, and phosphorylation of calcium channels and other proteins. Chronic exposure (days or months), but not acute exposure (hours), to a catecholamine downregulates human heart beta 1AR. Acute desensitization partially uncouples human heart beta AR from the adenylyl cyclase. Both acute and chronic desensitization reduce positive inotropic responses to catecholamines. In human heart, catecholamine-induced activation of one beta 2AR causes the production of at least four times more cyclic AMP than activation of one beta 1AR. Chronic treatment of patients with beta 1AR-selective blockers paradoxically induces selective inotropic beta 2AR hyperresponsiveness, presumably by increasing coupling of beta 2AR to Gs. Several partial agonists with high affinity for heart beta 1AR and beta 2AR cause stimulant effects that are resistant to blockade of beta 1AR and beta 2AR. Such nonconventional partial agonists could perhaps interact with beta AR that resemble beta 3 adrenoceptors.
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PMID:Some aspects of heart beta adrenoceptor function. 165 75

1. The characteristics have been examined of the high threshold calcium channel current in cultured rat dorsal root ganglion (DRG) neurones recorded in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S; 200 microM in the patch pipette). This current, termed IBa, GTP gamma S, was slowly activating and showed little inactivation over 100 ms. 2. External application of forskolin (10 microM) to elevate internal cyclic AMP levels increased the amplitude of IBa, GTP gamma S whereas it had no effect on the control IBa. This cyclic AMP-dependent protein kinase (PKI; 25 microM). 3. The cyclic AMP-dependent phosphorylation induced enhancement of IBa, GTP gamma S was voltage dependent and either did not occur or was observed only transiently at a holding potential (VH) of -30 mV. The forskolin-stimulated enhancement seen at VH -80 mV was lost with a t1/2 of about 1 min when VH was depolarized to -30 mV. Cholera toxin pre-treatment also increased the amplitude of IBa, GTP gamma S at VH -80 mV but not at VH -30 mV. 4. The calcium channel antagonist (-)-202-791 (5 microM) increased the amplitude of IBa, GTP gamma S when applied at VH -80 mV, but either not, or only transiently, at VH -30 mV, as previously observed. This 'agonist' effect of (-)-202-791 was prevented by PKI and was occluded by prior enhancement of IBa, GTP gamma S with forskolin. (-)-202-791 did not increase cyclic AMP levels in DRG neurones. 5. The 'agonist' response of IBa, GTP gamma S to D600 (10 microM) was also occluded by application of forskolin (10 microM) in the patch pipette. Forskolin alone, applied in this manner, increased IBa, GTP gamma S to a similar extent to D600 applied alone. 6. The agonist effect of (+)-202-791 (5 microM) on IBa, GTP gamma S was not prevented by prior enhancement with forskolin, nor was it prevented by PKI. 7. In conclusion, internal GTP gamma S activates G proteins which may interact directly with calcium channels to influence the kinetics of activation and to reduce steady-state inactivation of the channels. There is also an indirect effect on the generation of second messengers such as cyclic AMP. It is likely that forskolin enhances IBa, GTP gamma S by increasing activated Gs coupling to adenylyl cyclase and increasing cyclic AMP generation. The mechanism of action of (-)-202-791 to enhance IBa, GTP gamma S also involves cyclic AMP-dependent phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Ca2+ channel currents in rat sensory neurones: interaction between guanine nucleotides, cyclic AMP and Ca2+ channel ligands. 165 19

1. Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. The effect of internal perfusion with the hydrolysis-resistant GTP analogue, GppNHp (5'guanylylimidodiphosphate), on basal ICa and ICa stimulated with forskolin or isoprenaline was examined to gain insight into the role of G proteins in ICa regulation. 2. Without added guanine nucleotides, isoprenaline stimulated ICa approximately 14-fold with an EC50 of 0.09 microM. Forskolin stimulated ICa approximately 10-fold with an EC50 of 0.30 microM. 3. Internal 30 microM-GppNHp produced an approximately 80% decrease in ICa elevated by 0.3 microM-isoprenaline or 3 microM-forskolin. The inhibition of isoprenaline stimulation was due to a decrease in the maximal stimulation from approximately 14-fold to approximately 14-fold without a significant change in the EC50. In contrast, the reduction in forskolin stimulation was due to a 22-fold increase in the EC50 to 11.4 microM, with little change in maximal stimulation. 4. The inhibition of stimulated ICa by GppNHp is likely to be mediated by a G protein, because the effects of GppNHp are irreversible, and are blocked by excess GTP. ICa is affected similarly by GppNHp and by ACh. This suggests that GppNHp activates the same G protein that is normally activated by ACh, but activation by GppNHp occurs in the absence of agonist occupation of the muscarinic receptor. 5. The increase in the EC50 for forskolin produced by internal GppNHp was reversed by exposure to isoprenaline, which itself did not affect ICa amplitude. On average, exposure to isoprenaline in the presence of GppNHp caused an irreversible 81-fold decrease in the EC50 for forskolin to 0.14 microM. Stimulation of ICa by forskolin after internal GppNHp and exposure to isoprenaline was completely blocked by the protein kinase A inhibitor PKI(5-22). 6. These effects do not involve the phospholipase C system, because they are not mimicked by phorbol esters or internal inositol 1,4,5-trisphosphate (IP3) and are not blocked by bromophenacyl bromide or neomycin. 7. Direct effects of G proteins on ICa were not evident, because internal perfusion with PKI(5-22) completely inhibited isoprenaline- or forskolin-stimulated increases in ICa, and neither ACh nor internal GppNHp (30-500 microM) affected basal ICa or ICa elevated by internally perfused cyclic AMP. 8. These results suggest that the predominant site of action of the inhibitory G protein activated by either GppNHp or ACh is adenylyl cyclase. Furthermore, the internally perfused frog cardiomyocytes may provide a useful approach for probing the detailed interactions of G proteins, forskolin, and adenylyl cyclase in an intact cell.
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PMID:Regulation of Ca2+ current in frog ventricular cardiomyocytes by 5'-guanylylimidodiphosphate and acetylcholine. 165 25

1. Our objective was to evaluate the mechanism of cyclic AMP-dependent arterial smooth muscle relaxation. Cyclic AMP-dependent relaxation has been proposed to result from either (a) a decrease in intracellular [Ca2+] or (b) a decrease in [Ca2+] sensitivity of myosin light chain kinase by protein kinase A-dependent phosphorylation of myosin kinase. 2. We evaluated these proposed mechanisms by examining forskolin-induced changes in aequorin-estimated myoplasmic [Ca2+], [cyclic AMP], myosin phosphorylation and stress generation in agonist-stimulated or KCl-depolarized swine common carotid media tissues. 3. Forskolin, an activator of adenylyl cyclase, increased [cyclic AMP] and reduced [Ca2+], myosin phosphorylation and stress in tissues pre-contracted with phenylephrine or histamine. This relaxation was not associated with an alteration of the [Ca2+] sensitivity of phosphorylation, nor the dependence of stress on phosphorylation. 4. Forskolin pre-treatment attenuated, but did not abolish, agonist-induced increases in [Ca2+] and stress. 5. These results suggest that cyclic AMP-induced relaxation of the agonist-stimulated swine carotid media is primarily caused by cyclic AMP-mediated decreases in myoplasmic [Ca2+].
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PMID:Cyclic AMP relaxes swine arterial smooth muscle predominantly by decreasing cell Ca2+ concentration. 165 11

Hormone-sensitive adenylyl cyclase is a model system for the study of receptor-mediated signal transduction. It is comprised of three types of components: 1) receptors for hormones that regulate cyclic AMP (cAMP) synthesis, 2) regulatory GTP binding proteins (G proteins), and 3) the family of enzymes, the adenylyl cyclases. Concentrations of cAMP are altered by at least 35 different stimulatory or inhibitory hormones and neurotransmitters. Other signalling pathways may also influence cAMP production through regulation of particular adenylyl cyclase subtypes. The second messenger, cAMP propagates the hormone signal through the effects of cAMP-dependent protein kinase. While structural information on the adenylyl cyclases is limited, a cDNA clone for a calmodulin-sensitive form of bovine brain adenylyl cyclase has been isolated. The amino acid sequence encoded by the Type I cDNA is approximately 40% identical to those specified by three other adenylyl cyclase cDNAs that have been cloned subsequently. This degree of structural variation implies that there must be functional differences between the adenylyl cyclases.
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PMID:The adenylyl cyclase family. 165 97

We have localized a G protein activator region of the human beta 2-adrenergic receptor to region beta III-2 (from Arg259 to Lys273). The synthetic beta III-2, corresponding to the C-terminal end of the third cytoplasmic loop, activates Gs at nanomolar concentrations and weakly activates Gi. beta III-2 activates adenylyl cyclase at nanomolar concentrations in wild-type S49 lymphoma membranes, but not in membranes of unc mutant S49 cells, in which Gs is uncoupled from beta-adrenergic stimulation. Phosphorylation of beta III-2 by cAMP-dependent protein kinase A, which is involved in the desensitization of the beta-adrenergic receptor from Gs, drastically reduces the effect of beta III-2 on Gs while potentiating its action on Gi, resulting in a total loss of adenylyl cyclase-stimulating activity. These findings indicate that this receptor sequence is a multipotential G protein activator whose G protein specificity is regulated by protein kinase A.
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PMID:Identification of a Gs activator region of the beta 2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation. 165 4

Heterologous expression of the rat 5-HT1A receptor in stably transfected GH4C1 rat pituitary cells (clone GH4ZD10) and mouse Ltk- fibroblast cells (clone LZD-7) (Albert, P.R., Zhou, Q.-Y., VanTol, H.H.M., Bunzow, J.R., and Civelli, O. (1990) J. Biol. Chem. 265, 5825-5832) was used to characterize the cellular specificity of signal transduction by the 5-HT1A receptor. We demonstrate that the 5-HT1A receptor, acting via pertussis toxin-sensitive G proteins, can change its inhibitory signaling phenotype and become a stimulatory receptor, depending on the cell type, differentiation state, or intracellular milieu of the cell in which it is expressed. When expressed in pituitary GH4ZD10 cells, activation of 5-HT1A receptors decreased both basal and vasoactive intestinal peptide-enhanced cAMP accumulation and blocked (+/-)-Bay K8644-induced influx of calcium, inhibitory responses which are typical of neurons which endogenously express this receptor. Similarly, 5-hydroxytryptamine (5-HT) also inhibited adenylyl cyclase in fibroblast LZD-7 cells, reducing the forskolin-induced enhancement of cAMP levels by 50%, but did not alter basal cAMP levels. In contrast to GH4ZD10 cells, where 5-HT had no effect on basal or thyrotropin-releasing hormone-induced phosphatidylinositol turnover, 5-HT enhanced the accumulation of inositol phosphates and induced a biphasic increase in [Ca2+]i in LZD-7 cells. These dominant stimulatory actions of 5-HT, as well as the inhibitory effects, were absent in untransfected cells and displayed the potency and pharmacological specificity of the 5-HT1A receptor, indicating that the 5-HT1A subtype coupled to both inhibitory and stimulatory pathways in the fibroblast cell. The actions of 5-HT in GH and L cells were blocked by 24-h pretreatment with pertussis toxin, suggesting that inhibitory G proteins (Gi/G(o)) mediate both inhibitory and stimulatory signal transduction of the 5-HT1A receptor. However, the 5-HT-induced stimulatory pathway in fibroblasts was blocked selectively by acute (2-min) pretreatment with TPA, an activator of protein kinase C. This action of protein kinase C was potentiated by activation of protein kinase A, indicating that the expression of the stimulatory pathway of the 5-HT1A receptor in LZD-7 cells is modulated by second messengers.
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PMID:Cell-specific signaling of the 5-HT1A receptor. Modulation by protein kinases C and A. 166 Aug 81

Atrial myocardium of man and pig possess receptors that mediate positive inotropic effects and/or positive chronotropic effects of 5-hydroxytryptamine (5-HT). These 5-HT receptors are blocked with moderate affinity (pKB = 6.7-6.9) by 3 alpha-trophanyl-1H-indole-3-carboxylate (ICS 205930) but not by antagonists of alpha- and beta-adrenoceptors, or 5-HT1 subtypes, 5-HT2 and 5-HT3 receptors. In human and porcine atrium the receptors also mediate 5-HT-induced increases of both cyclic AMP levels and cyclic AMP-dependent protein kinase activity. Human and porcine atrial 5-HT receptors are also partially activated by the benzamides renzapride and cisapride albeit with lower potency and efficacy than 5-HT. The properties of these atrial 5-HT receptors resemble those of "so called" 5-HT4 receptors, positively coupled to the adenylyl cyclase of mouse embyonic colliculi neurons. However, for colliculi 5-HT4 receptors the benzamides have greater efficacy and ICS 205930 lower affinity than for atrial 5-HT receptors. The atrial 5-HT receptors of man and pig are, therefore, designated 5-HT4-like receptors. Guinea-pig night atria appear to have a mixture of 5-HT3- and 5-HT4-like receptors involved in the mediation of positive chronotropic effects of 5-HT.
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PMID:5-HT4-like receptors in mammalian atria. 166 72


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