Noxious stimulation can trigger persistent sensitization of somatosensory systems that involves memory-like mechanisms. Noxious stimulation of the mollusc Aplysia produces transcription-dependent, long-term hyperexcitability (LTH) of nociceptive sensory neurons that requires a nitric oxide (NO)-cyclic GMP-protein kinase G (PKG) pathway. Injection of cGMP induces LTH, whereas antagonists of the NO-cGMP-PKG pathway prevent pinch-induced LTH. Co-injection of calcium/cAMP-responsive-element (CRE) blocks both pinch-induced LTH and cAMP-induced LTH, but antagonists of protein kinase A (PKA) fail to block pinch-induced LTH. Thus the NO-cGMP-PKG pathway and at least one other pathway, but not the cAMP-PKA pathway, are critical for inducing LTH after brief, noxious stimulation (Lewin, 1999).
Genes can affect natural behavioral variation in different ways. Allelic variation causes alternative behavioral phenotypes, whereas changes in gene expression can influence the initiation of behavior at different ages. The age-related transition by honey bees from hive work to foraging is associated with an increase in the expression of the foraging (for) gene, which encodes a guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (PKG). cGMP treatment elevates PKG activity and causes foraging behavior. Previous research has shown that allelic differences in PKG expression result in two Drosophila foraging variants. The same gene can thus exert different types of influence on a behavior (Ban-Shahar, 2002).
Division of labor in honey bee colonies is influenced by the foraging gene (Amfor), which encodes a cGMP-dependent protein kinase (PKG). Amfor upregulation in the bee brain is associated with the age-related transition from working in the hive to foraging for food outside, and cGMP treatment (which increases PKG activity) causes precocious foraging. Two lines of evidence are presented in support of the hypothesis that Amfor affects division of labor by modulating phototaxis. (1) A subset of worker bees involved in the removal of corpses from the hive had forager-like brain levels of Amfor brain expression despite being middle aged; age-matched food-handlers, who do not leave the hive to perform their job, had low levels of Amfor expression. This finding suggests that occupations that involve working outside the hive are associated with high levels of Amfor in brain. (2) Foragers were much more positively phototactic than hive bees in a laboratory assay, and cGMP treatment (bees were treated orally with a 50% sucrose solution containing 8-Br-cGMP) caused a precocious onset of positive phototaxis. The cGMP effect was not due to a general increase in behavioral activity; cGMP treatment had no effect on locomotor activity under either constant darkness or a light:dark regime. The cGMP effect also was not due to changes in circadian rhythmicity; neither age at onset of locomotor circadian rhythmicity nor the period of rhythmicity was affected by cGMP treatment. The effects of Amfor on phototaxis are not related to peripheral processing; electroretinogram analysis revealed no effect of cGMP treatment on photoreceptor activity and no differences between untreated hive bees and foragers. The cAMP/PKA pathway does not appear to be playing a similar role to cGMP/PKG; cAMP treatment (oral administration) did not affect phototaxis and gene expression analysis revealed task-related differences only for the gene encoding the regulatory subunit, but not the catalytic subunit, of PKA. These findings implicate one neural process associated with honey bee division of labor that can be affected by naturally occurring changes in the expression of Amfor (Ben-Shahar, 2003).
The dynamic regulation of nitric oxide synthase (NOS) activity and cGMP levels suggests a functional role in the development of nervous systems. NO/cGMP signalling cascade plays a key role in regulating migration of postmitotic neurons in the enteric nervous system of the embryonic grasshopper. During embryonic development, a population of enteric neurons migrates several hundred micrometers on the surface of the midgut. These midgut neurons (MG neurons) exhibit nitric oxide-induced cGMP-immunoreactivity coinciding with the migratory phase. Using a histochemical marker for NOS, potential sources were identified of NO in subsets of the midgut cells below the migrating MG neurons. Pharmacological inhibition of endogenous NOS, soluble guanylyl cyclase (sGC) and protein kinase G (PKG) activity in whole embryo culture significantly blocks MG neuron migration. This pharmacological inhibition can be rescued by supplementing with protoporphyrin IX free acid, an activator of sGC, and membrane-permeant cGMP, indicating that NO/cGMP signalling is essential for MG neuron migration. Conversely, the stimulation of the cAMP/protein kinase A signalling cascade results in an inhibition of cell migration. Activation of either the cGMP or the cAMP cascade influences the cellular distribution of F-actin in neuronal somata in a complementary fashion. The cytochemical stainings and experimental manipulations of cyclic nucleotide levels provide clear evidence that NO/cGMP/PKG signalling is permissive for MG neuron migration, whereas the cAMP/PKA cascade may be a negative regulator. These findings reveal an accessible invertebrate model in which the role of the NO and cyclic nucleotide signalling in neuronal migration can be analyzed in a natural setting (Haase, 2003).
The induction of a long-term hyperexcitability (LTH) in vertebrate nociceptive sensory neurons (SNs) after nerve injury is an important contributor to neuropathic pain in humans, but the signaling cascades that induce this LTH have not been identified. In particular, it is not known how injuring an axon far from the cell soma elicits changes in gene expression in the nucleus that underlie LTH. The nociceptive SNs of Aplysia (ap) develop an LTH with electrophysiological properties after axotomy similar to those of mammalian neurons and are an experimentally useful model to examine these issues. An Aplysia PKG (cGMP-dependent protein kinase; protein kinase G) has been cloned that is homologous to vertebrate type-I PKGs; apPKG is activated at the site of injury in the axon after peripheral nerve crush. The active apPKG is subsequently retrogradely transported to the somata of the SNs, but apPKG activity does not appear in other neurons whose axons are injured. In the soma, apPKG phosphorylates apMAPK (Aplysia mitogen-activated protein kinase), resulting in its entry into the nucleus. Surprisingly, studies using recombinant proteins in vivo and in vitro indicate that apPKG directly phosphorylates the threonine moiety in the T-E-Y activation site of apMAPK when the -Y- site contains a phosphate. Inhibitors of nitric oxide synthase, soluble guanyl cyclase, or PKG were used after nerve injury; each prevents the appearance of the LTH. Moreover, blocking apPKG activation prevents the nuclear import of apMAPK. Consequently, the nitric oxide-PKG-MAPK pathway is a potential target for treatment of neuropathic pain (Sung, 2004).
cDNA clones (PKG Ia and PKG Ib) for medaka fish cGMP-dependent protein kinase (PKG) Ia and Ib were isolated and characterized, and both were demonstrated to be expressed in the embryos after late gastrula stage. The transcripts of the PKG Ia gene are present in the spinal cord and gill arch, while those of the PKG Ib gene are only weakly expressed in these organs, but highly expressed in the otic vesicles. Injection of PKG Ia-specific morpholino antisense oligonucleotides (Ia-MO) into two-cell stage medaka fish embryos caused severe abnormalities in the developing embryos, such as the development of a hammer-like head, fusion of the developing eyes, and degeneration of cells around the eyes, while injection of PKG Ib-specific morpholino antisense oligonucleotides (Ib-MO) caused fewer abnormalities in the embryos, even when injected at higher concentrations than Ib-MO. The PKG I-overexpressing embryos exhibited smaller eyes and enlargement of the forebrain, a phenotype similar to that observed in the cAMP-dependent protein kinase (PKA)-depressed embryos. In the PKG-deficient embryos, a shh-target gene, HNF-3b was expressed weakly; this phenotype is similar to that observed in the PKA-overexpressing embryos suggesting that the cGMP/PKG signaling pathway is involved in some steps of shh signaling. Gli proteins, shh-downstream molecules, are phosphorylated by the NO/cGMP signaling pathway, probably by PKG in NG108-15 neuroblastoma cells. These results imply that PKG and PKA share common substrates and work in an opposite manner during the early embryogenesis of medaka fish (Yamamoto, 2005).
All mammalian cGMP-dependent protein kinases (PKGs) are dimeric. Dimerization of PKGs involves sequences located near the amino termini, which contain a conserved, extended leucine zipper motif. In PKG Ibeta this includes eight Leu/Ile heptad repeats, and in the present study, deletion and site-directed mutagenesis have been used to systematically delete these repeats or substitute individual Leu/Ile. The enzymatic properties and quaternary structures of these purified PKG mutants have been determined. All had specific enzyme activities comparable to wild type PKG. Simultaneous substitution of alanine at four or more of the Leu/Ile heptad repeats of the motif produces a monomeric PKG Ibeta. Mutation of two Leu/Ile heptad repeats can produce either a dimeric or monomeric PKG. Point mutation of Leu-17 or Ile-24 does not disrupt dimerization. These results suggest that all eight Leu/Ile heptad repeats are involved in dimerization of PKG Ibeta. Six of the eight repeats are sufficient to mediate dimerization, but substitutions at some positions appear to have greater impact than others on dimerization. The Ka of cGMP for activation of monomeric mutants is 2- to 3-fold greater than that for wild type dimeric PKG Ibeta, and there is a corresponding 2- to 3-fold increase in cGMP-dissociation rate of the high affinity cGMP-binding site of these monomers. These results indicate that dimerization increases sensitivity for cGMP activation of the enzyme (Richie-Jannetta, 2003).
The cGMP-dependent protein kinase (PKG) is the main mediator of nitric oxide-induced relaxation of smooth muscle. Although this pathway is well established, the cellular action of PKG, nitric oxide, and cGMP is complex and not fully understood. A cross-talk between the cGMP-PKG and other pathways (e.g. cAMP-protein kinase A) seems to exist. cGMP- and cAMP-dependent relaxation of smooth muscle has been examined using PKG-deficient mice (cGKI-/-). In intact ileum strips of wild type mice (cGKI+/+), 8-Br-cGMP inhibited the sustained phase of carbachol contractions by approximately 80%. The initial peak was less inhibited (approximately 30%). This relaxation was associated with a reduction in intracellular [Ca2+] and decreased Ca2+ sensitivity. Contractions of cGKI-/- ileum were not influenced by 8-Br-cGMP. EC50 for 8-Br-cGMP for PKG was estimated to be 10 nm. PKG-independent relaxation by 8-Br-cGMP had an EC50 of 10 microm. Relaxation by cAMP (approximately 50% at 100 microm), Ca2+ sensitivity of force, and force potentiation by GTPgammaS were similar in cGKI+/+ and cGKI-/- tissues. The results show that PKG is the main target for cGMP-induced relaxation in intestinal smooth muscle. cGMP desensitize the contractile system to Ca2+ via PKG. PKG-independent pathways are activated at 1000-fold higher cGMP concentrations. Relaxation by cAMP can occur independently of PKG. Long term deficiency of PKG does not lead to an apparent up-regulation of the cAMP-dependent pathways or changes in Ca2+ sensitivity (Bonnevier, 2004).
cGMP-dependent protein kinase I (PKG-I) has been suggested to contribute to the facilitation of nociceptive transmission in the spinal cord presumably by acting as a downstream target of nitric oxide. However, PKG-I activators cause conflicting effects on nociceptive behavior. In the present study PKG-I-/- mice were used to further assess the role of PKG-I in nociception. PKG-I deficiency is associated with reduced nociceptive behavior in the formalin assay and zymosan-induced paw inflammation. However, acute thermal nociception in the hot-plate test was unaltered. After spinal delivery of the PKG inhibitor, Rp-8-Br-cGMPS, nociceptive behavior of PKG-I(+/+) mice was indistinguishable from that of PKG-I-/- mice. In contrast, the PKG activator, 8-Br-cGMP (250 nmol intrathecally) caused mechanical allodynia (I.F. editor's note: condition in which pain results from a non-injurious stimulus to the skin) only in PKG-I(+/+) mice, indicating that the presence of PKG-I is essential for this effect. Immunofluorescence studies of the spinal cord reveal additional morphological differences. In the dorsal horn of 3- to 4-week-old PKG-I-/- mice, laminae I-III are smaller and contain fewer neurons than controls. Furthermore, the density of substance P-positive neurons and fibers is significantly reduced. The paucity of substance P in laminae I-III may contribute to the reduction of nociception in PKG-I-/- mice and suggests a role of PKG-I in substance P synthesis (Tegeder, 2004).
Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study, the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e., bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the a1 and b1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 hours results in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC, but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC, and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro (Browner, 2004).
The suprachiasmatic nucleus (SCN) circadian clock exhibits a recurrent series of dynamic cellular states, characterized by the ability of exogenous signals to activate defined kinases that alter clock time. To explore potential relationships between kinase activation by exogenous signals and endogenous control mechanisms, clock-controlled protein kinase G (PKG) regulation in the mammalian SCN were examined. Signaling via the cGMP-PKG pathway is required for light- or glutamate (GLU)-induced phase advance in late night. Spontaneous cGMP-PKG activation occura at the end of subjective night in free-running SCN in vitro. Phasing of the SCN rhythm in vitro is delayed by approximately 3 hr after treatment with guanylyl cyclase (GC) inhibitors, PKG inhibition, or antisense oligodeoxynucleotide (alphaODN) specific for PKG, but not PKA inhibitor or mismatched ODN. This sensitivity to GC-PKG inhibition was limited to the same 2 hr time window demarcated by clock-controlled activation of cGMP-PKG. Inhibition of the cGMP-PKG pathway at this time caused delays in the phasing of four endogenous rhythms: wheel-running activity, neuronal activity, cGMP, and Per1. Timing of the cGMP-PKG-necessary window in both rat and mouse depends on clock phase, established by the antecedent light/dark cycle rather than solar time. Because behavioral, neurophysiological, biochemical, and molecular rhythms show the same temporal sensitivities and qualitative responses, it is predicted that clock-regulated GC-cGMP-PKG activation may provide a necessary cue as to clock state at the end of the nocturnal domain. Because sensitivity to phase advance by light-GLU-activated GC-cGMP-PKG occurs in juxtaposition, these signals may induce a premature shift to this PKG-necessary clock state (Tischkau, 2003).
Circadian clocks comprise a cyclic series of dynamic cellular states, characterized by the changing availability of substrates that alter clock time when activated. To determine whether circadian clocks, like the cell cycle, exhibit regulation by key phosphorylation events, endogenous kinase regulation of timekeeping was examined in the mammalian suprachiasmatic nucleus (SCN). Short-term inhibition of PKG-II but not PKG-Ibeta using antisense oligodeoxynucleotides delayed rhythms of electrical activity and Bmal1 mRNA. Phase resetting was rapid and dynamic; inhibition of PKG-II forced repetition of the last 3.5 hr of the cycle. Chronic inhibition of PKG-II disrupted electrical activity rhythms and tonically increased Bmal1 mRNA. PKG-II-like immunoreactivity was detected after coimmunoprecipitation with CLOCK, and CLOCK is phosphorylated in the presence of active PKG-II. PKG-II activation may define a critical control point for temporal progression into the daytime domain by acting on the positive arm of the transcriptional/translational feedback loop (Tischkau, 2004).
Native large conductance, voltage-dependent, and Ca2+-sensitive K+ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: (1) direct phosphorylation of the channel and (2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, the human pore-forming alpha-subunit of the Ca2+-sensitive K+ channel, Hslo, and the alpha-isoform of cGMP-dependent protein kinase I were used. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by approximately 20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates gamma-33P into the immunopurified Hslo band of approximately 125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the alpha-subunit of large conductance Ca2+-sensitive K+ channels is substrate for G-Ialpha kinase in vivo and support direct phosphorylation as a mechanism for PKG-Ialpha-induced activation of maxi-K channels (Alioua, 1998).
Nitric oxide (NO) is thought to play an essential role in neuronal processing, but the downstream mechanisms of its action remain unclear. NO analogs reduce GABA-gated currents in cultured retinal amacrine cells via two distinct, but convergent, cGMP-dependent pathways. Either extracellular application of the NO-mimetic S-nitroso-N-acetyl-penicillamine (SNAP) or intracellular perfusion with cGMP depresses GABA currents. This depression is partially blocked by a pseudosubstrate peptide inhibitor of cGMP-dependent protein kinase (PKG), suggesting both PKG-dependent and independent actions of cGMP. cAMP-dependent protein kinase (PKA) is known to enhance retinal GABA responses. The membrane permiable 8-Bromoinosine 3',5'-cyclic monophosphate (8Br-cIMP), which activates a type of cGMP-stimulated phosphodiesterase that hydrolyzes cAMP, also significantly reduces GABA currents. 1-Methyl-3-isobutylxanthine (IBMX), a nonspecific phosphodiesterase (PDE) inhibitor, blocks both the action of 8Br-cIMP and the portion of SNAP-induced depression that is not blocked by PKG inhibition. These results suggest that NO depresses retinal GABAA receptor function by simultaneously upregulating PKG and downregulating PKA (Wexler, 1998).
A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, has been implicated as the mediator of cGMP-provoked intestinal Cl- secretion, based on cGK localization in the apical membrane of enterocytes and on the cGK capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast, the soluble type I cGK is unable to activate CFTR in intact cells, although both cGK I and cGK II can phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, cGK II or cGK I mutant protein variants, possessing different membrane binding properties, were expressed using adenoviral vectors in a CFTR-transfected intestinal cell line, and the ability of cGMP to phosphorylate and activate the Cl- channel was examined. Mutation of the cGK II N-terminal myristoylation site (Gly2 --> Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Ibeta, acquired the ability to associate with the membrane and activate the CFTR Cl- channel. The potency order of cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I chimer >> nonmyristoylated cGK II > cGK Ibeta) correlated with the extent of 32P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl- channel activation in intact cells (Vaandrager, 1998).
Canonical transient receptor potential (TRPC) channels are Ca2+-permeable nonselective cation channels that are widely expressed in numerous cell types. Seven different members of TRPC channels have been isolated. The activity of these channels is regulated by the filling state of intracellular Ca2+ stores and/or diacylglycerol and/or Ca2+/calmodulin. However, no evidence is available as to whether TRPC channels are regulated by direct phosphorylation on the channels. In the present study, TRPC isoform 3 (TRPC3) gene was overexpressed in HEK293 cells that were stably transfected with protein kinase G (PKG). The overexpressed TRPC3 mediates store-operated Ca2+ influx and this type of Ca2+ influx is inhibited by cGMP. The inhibitory effect of cGMP was abolished by KT5823 or H8. Point mutations at two consensus PKG phosphorylation sites (T11A and S263Q) of TRPC3 channel markedly reduced the inhibitory effect of cGMP. In addition, TRPC3 proteins were purified from HEK293 cells that were transfected with either wild-type or mutant TRPC3 constructs, and an in vitro PKG phosphorylation assay was carried out. It was found that wild-type TRPC3 could be directly phosphorylated by PKG in vitro and that the phosphorylation was abolished in the presence of KT5823. The phosphorylation signal was greatly reduced in mutant protein T11A or S263Q. Taken together, TRPC3 channels can be directly phosphorylated by PKG at position T11 and S263, and this phosphorylation abolishes the store-operated Ca2+ influx mediated by TRPC3 channels in HEK293 cells (Kwan, 2004).
Cyclic-GMP-dependent protein kinase (PKG) is widely appreciated as having diverse roles in a variety of cell types. Many reports have indicated that PKG might regulate cell function by activating members of the mitogen-activated protein kinase (MAPK) family of signaling proteins. Stimulation of HEK-293 cells with nitric oxide (NO) was found to induce a rapid accumulation of phosphorylated p38 MAPK. The involvement of PKG in this process was confirmed by cotransfection of a dominant negative PKG construct (G1alphaR-GFP), which was able to block cGMP-induced p38 MAPK activation. Transfection of cells to express dominant negative Rac1(T17N) is also able to dose-dependently block cGMP-stimulated activation of p38 MAPK, thus indicating the importance of this pathway downstream of PKG. GST-PDB affinity-precipitation experiments have revealed that stimulation of HEK293 cells with either nitric oxide or 8-Br-cGMP results in a rapid and transient activation of Rac1 with kinetics similar to p38 MAPK phosphorylation. Moreover, using in vitro kinase assays it was found that cGMP also stimulates the activity of the Rac1 effector Pak1. The activation of both Rac1 and Pak1 by 8-Br-cGMP is completely abolished by transfection of the cells with G1alphaR-GFP. Expression of the Rac1(T17N) mutant inhibits PKG-dependent activation of PAK1 indicating that Rac1 functions upstream of PAK1 in this pathway. Immunofluorescence experiments demonstrate clear colocalization of PKG and Rac1 in membrane ruffles and dynamic membrane regions, supporting a functional interaction. However, in vitro kinase assays demonstrate that Rac1 is not a substrate for PKG, suggesting an indirect activation mechanism. Taken together these data demonstrate a novel PKG-dependent pathway by which the Rac1/Pak1 pathway is activated. Furthermore, this pathway is central to the activation of p38 MAPK by PKG in these cells (Hou, 2004).
Agrin acting through nitric oxide induces the formation of AChR aggregates on myotubes in culture. Soluble guanylyl cyclase (sGC), which is present at the neuromuscular junction, is a common target of NO. Therefore, it was hypothesized that sGC and cGMP are involved in the agrin signaling cascade. Inhibition of sGC hinders AChR aggregation in both agrin- and NO donor-treated cultured myotubes; whereas, a cGMP analog is able to induce the formation of AChR aggregates on naive muscle cells. Due to the presence of cyclic GMP-dependent protein kinase (PKG) at the neuromuscular junction, the ability of a PKG inhibitor to alter the agrin signaling cascade was tested. PKG inhibition does not prevent nascent AChR aggregate formation; however, these aggregates were diffuse and composed of numerous microaggregates consistent with incomplete maturation. Thus, it is concluded that cGMP is important for the initiation of AChR aggregation, while PKG is involved in the maturation of AChR aggregates (Jones, 2004).
Platelet secretion (exocytosis) is critical in amplifying platelet activation, stabilizing thrombus, and in arteriosclerosis and vascular remodeling. The signaling mechanisms leading to secretion have not been well defined. PKG plays a stimulatory role in platelet activation via the glycoprotein Ib-IX pathway. PKG also plays an important stimulatory role in mediating aggregation-dependent platelet secretion and secretion-dependent second wave platelet aggregation, particularly those induced via Gq-coupled agonist receptors, the thromboxane A2 (TXA2) receptor and protease-activated receptors (PAR). PKG I knockout mouse platelets and PKG inhibitor-treated human platelets show diminished aggregation-dependent secretion, and also show diminished secondary wave of platelet aggregation induced by a TXA2 analog and thrombin-receptor activating peptides that is rescued by the granule content ADP. Low dose collagen-induced platelet secretion and aggregation are also reduced by PKG inhibitors. Furthermore PKG I knockout and PKG inhibitors significantly attenuate activation of the Gi pathway that is mediated by secreted ADP. These data unveil a novel PKG-dependent platelet secretion pathway and a mechanism by which PKG promotes platelet activation (Li, 2004).
Cyclic GMP-dependent protein kinase I (PKGI) mediates vascular relaxation by nitric oxide and related nitrovasodilators and inhibits vascular smooth muscle cell (VSMC) migration. To identify VSMC proteins that interact with PKGI, the N-terminal protein interaction domain of PKGIalpha was used to screen a yeast two-hybrid human aortic cDNA library. The formin homology (FH) domain-containing protein, FHOD1, was found to interact with PKGIalpha in this screen. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics, cell migration, and gene expression. Antisera to FHOD1 were raised and used to characterize FHOD1 expression and distribution in vascular cells. FHOD1 is highly expressed in human coronary artery, aortic smooth muscle cells, and in human arterial and venous endothelial cells. In glutathione S-transferase pull-down experiments, the FHOD1 C terminus (amino acids 964-1165) binds full-length PKGI. Both in vitro and intact cell studies demonstrate that the interaction between FHOD1 and PKGI is decreased 3- to 5-fold in the presence of the membrane permiable PKG activator, 8Br-cGMP. Immunofluorescence studies of human VSMC show that FHOD1 is cytoplasmic and is concentrated in the perinuclear region. PKGI also directly phosphorylates FHOD1, and studies with wild-type and mutant FHOD1-derived peptides identify Ser-1131 in the FHOD1 C terminus as the unique PKGI phosphorylation site in FHOD1. These studies demonstrate that FHOD1 is a PKGI-interacting protein and substrate in VSMCs and show that cyclic GMP negatively regulates the FHOD1-PKGI interaction. Based on the known functions of FHOD1, the data are consistent with a role for FHOD1 in cyclic GMP-dependent inhibition of VSMC stress fiber formation and/or migration (Y. Wang, 2004).
Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. Baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, were used to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NONOate [ethanamine-2,2'-(hydroxynitrosohydrazone)bis-] has no effect on a chloramphenicol acetyltransferase (CAT) reporter gene under control of the fos promoter in BHK cells transfected with an empty vector or in cells transfected with a G-kinase Ibeta expression vector. In BHK cells transfected with expression vectors for guanylate cyclase, Deta-NONOate markedly increases the intracellular cGMP concentration and causes a small (2-fold) increase in CAT activity; the increased CAT activity appears to be from cGMP activation of cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the presence of Deta-NONOate. Stimulation of CAT activity in the absence of Deta-NONOate appears to be largely from endogenous NO since it was found that: (1) BHK cells produced high amounts of NO; (2) CAT activity was partially inhibited by a NO synthase inhibitor; and (3) the inhibition by the NO synthase inhibitor is reversed by exogenous NO. In CS-54 cells, NO was found to increase fos promoter activity and the increase was prevented by a guanylate cyclase inhibitor. In summary, NO was found to activate the fos promoter by a guanylate cyclase- and G-kinase-dependent mechanism (Idriss, 1999).
Transcriptional regulation of the fos promoter by nitric oxide and cGMP can occur by nuclear translocation of cGMP-dependent protein kinase I. To identify nuclear targets of G-kinase I, a yeast two-hybrid screen was performed with G-kinase I beta as bait. G-kinase I beta was found to interact specifically with TFII-I, an unusual transcriptional regulator that associates with multiple proteins to modulate both basal and signal-induced transcription. By using purified recombinant proteins, the interaction was mapped to the N-terminal 93 amino acids of G-kinase I beta and one of six 95-amino acid repeats found in TFII-I. In baby hamster kidney cells, cGMP analogs enhanced co-immunoprecipitation of G-kinase I beta and TFII-I by inducing co-localization of both proteins in the nucleus, but in other cell types containing cytoplasmic TFII-I, the G-kinase-TFII-I interaction was largely cGMP-independent. G-kinase phosphorylates TFII-I in vitro and in vivo on Ser(371) and Ser(743) outside of the interaction domain. G-kinase strongly enhances TFII-I transactivation of a serum-response element-containing promoter in COS7 cells, and this effect is lost when Ser(371) and Ser(743) of TFII-I are mutated. TFII-I by itself has little effect on a full-length fos promoter in baby hamster kidney cells, but it synergistically enhances transcriptional activation by G-kinase I beta. Binding of G-kinase to TFII-I may position the kinase to phosphorylate and regulate TFII-I and/or factors that interact with TFII-I at the serum-response element (Casteel, 2002).
Activation of the arterial baroreceptors induces expression of the proto-oncogene c-fos in the nucleus tractus solitarii (NTS), the terminal site of baroreceptor afferents in the medulla oblongata. This induced expression is an intracellular event that is crucial to long-term maintenance of stable blood pressure. Using Sprague-Dawley rats maintained under propofol anesthesia, the role of nitric oxide (NO) in this process was evaluated. Baroreceptor activation induced by 30 min of sustained hypertension significantly and sequentially increased the level of cyclic GMP-dependent protein kinase I (PKG-I), phosphorylated cyclic AMP response element-binding protein (pCREB), c-fos mRNA, and Fos protein in the NTS. All of these up-regulated expressions were significantly attenuated in animals that were pretreated immediately before baroreceptor activation with bilateral microinjection into the NTS of a selective neuronal nitric-oxide synthase (nNOS) inhibitor or a soluble guanylyl cyclase (sGC) inhibitor. Bilateral NTS microinjection of a cell-permeable cGMP analog significantly elevated the level of pCREB or c-fos mRNA in the NTS. In contrast, the up-regulated CREB phosphorylation or c-fos induction evoked in the dorsomedial medulla by baroreceptor activation is significantly antagonized by NTS application of a cell-permeable cGMP antagonist or a PKG inhibitor. It is concluded that NO derived from nNOS in the NTS on baroreceptor activation may participate in c-fos expression via phosphorylation of CREB in a process that engages the sGC/cGMP/PKG-I signaling cascade (Chan, 2004).
The transcription factor NFAT (nuclear factor of activated T-cells) is implicated in cardiac hypertrophy and vasculogenesis. NFAT activation, reflecting dephosphorylation by the calcium-dependent phosphatase, calcineurin, and subsequent nuclear localization, is generally thought to require a sustained increase in intracellular calcium. However, in smooth muscle it was found that elevation of calcium by membrane depolarization fails to induce an increase in nuclear localization of the NFATc3 isoform. Physiological intravascular pressure (100 mm Hg) induces an increase in NFATc3 nuclear localization in mouse cerebral arteries. Pressure-induced NFATc3 nuclear accumulation is abrogated by endothelial denudation and by nitric-oxide synthase, cGMP-dependent kinase (PKG), and voltage-dependent calcium channels inhibition. It is shown that exogenous nitric oxide, in combination with an elevation in calcium, is an effective stimulus for NFATc3 nuclear accumulation. c-Jun terminal kinase 2 (JNK) activity, which has been shown to regulate NFATc3 nuclear export, is also reduced by pressure, an effect that is prevented by pretreatment with a PKG inhibitor. Consistent with this, pressure-induced NFATc3 nuclear accumulation is independent of PKG in arteries from JNK2-/- mice. Collectively, these results indicate that both activation of the NO/PKG pathway and elevation of smooth muscle calcium are required for NFATc3 nuclear accumulation and that PKG inhibits JNK2 to decrease NFAT nuclear export. These findings suggest that at physiological intravascular pressures NFATc3 is localized to the nucleus in smooth muscle cells of intact arteries and indicate a novel and unexpected role for nitric oxide/PKG in NFAT activation (Gonzalez Bosc, 2004).
Thrombospondin 1 (TSP1) transcription is stimulated by glucose, resulting in increased TGF-beta activation and matrix protein synthesis. Inducible expression of the catalytic domain of cGMP-dependent protein kinase (PKG) inhibits glucose-regulated TSP1 transcription and transforming growth factor (TGF)-beta activity in stably transfected rat mesangial cells. However, the molecular mechanisms by which PKG represses glucose-regulated TSP1 transcription are unknown. Using a luciferase-promoter deletion assay, a single region of the human TSP1 promoter (-1172 to -878, relative to the transcription start site) was identified that is responsive to glucose. Further characterization of this region identified an 18-bp sequence that specifically binds nuclear proteins from mesangial cells. Moreover, binding is significantly enhanced by high glucose treatment and is reduced by increased PKG activity. Gel mobility shift and supershift assays show that the nuclear proteins binding to the 18-bp sequence are USF1 and -2. USF1 and USF2 binds to the endogenous TSP1 promoter using a chromatin immunoprecipitation assay. Glucose stimulates nuclear USF2 protein accumulation through protein kinase C, p38 MAPK, and extracellular signal-regulated kinase pathways. Increased PKG activity down-regulates USF2 protein levels and its DNA binding activity under high glucose conditions, resulting in inhibition of glucose-induced TSP1 transcription and TGF-beta activity. Overexpression of USF2 reversed the inhibitory effect of PKG on glucose-induced TSP1 gene transcription and TGF-beta activity. Taken together these data present the first evidence that USF2 mediates glucose-induced TSP1 expression and TSP1-dependent TGF-beta bioactivity in mesangial cells, suggesting that USF2 is an important transcriptional regulator of diabetic complications (S. Wang, 2004).
The second messengers cAMP and inositol-1,4,5-triphosphate have been implicated in olfaction in various species. The odorant-induced cGMP response was investigated using cilia preparations and olfactory primary cultures. Odorants cause a delayed and sustained elevation of cGMP. A component of this cGMP response is attributable to the activation of one of two kinetically distinct cilial receptor guanylyl cyclases by calcium and a guanylyl cyclase-activating protein (GCAP). cGMP thus formed serves to augment the cAMP signal in a cGMP-dependent protein kinase (PKG) manner by direct activation of adenylate cyclase. cAMP, in turn, activates cAMP-dependent protein kinase (PKA) to negatively regulate guanylyl cyclase, limiting the cGMP signal. These data demonstrate the existence of a regulatory loop in which cGMP can augment a cAMP signal, and in turn cAMP negatively regulates cGMP production via PKA. Thus, a small, localized, odorant-induced cAMP response may be amplified to modulate downstream transduction enzymes or transcriptional events (Moon, 1998).
Norepinephrine (NE) induces a sustained potentiation of transmitter release in the chick ciliary ganglion through a mechanism pharmacologically distinct from any known adrenergic receptors. The adrenergic potentiation of transmitter release is enhanced by a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and by zaprinast, an inhibitor of cGMP-selective phosphodiesterase. Exogenous application of the membrane-permeable cGMP, 8-bromo-cGMP (8Br-cGMP), potentiates the quantal transmitter release, and after potentiation, the addition of NE is no longer effective. In contrast, 8Br-cAMP neither potentiates the transmitter release nor occludes the NE-induced potentiation. The NE-induced potentiation is blocked by neither nitric oxide (NO) synthase inhibitor nor NO scavenger. The quantal transmitter release is not potentiated by NO donors, e.g., sodium nitroprusside. The NE-induced potentiation and its enhancement by IBMX is antagonized by two inhibitors of protein kinase G (PKG). As with NE-induced potentiation, the effects of 8Br-cGMP on both the resting intraterminal [Ca2+] ([Ca2+]i) and the action potential-dependent increment of [Ca2+]i (DeltaCa) in the presynaptic terminal are negligible. The reduction of the paired pulse ratio of EPSC is consistent with the notion that the NE- and cGMP-dependent potentiation of transmitter release is attributable mainly to an increase of the exocytotic fusion probability. These results indicate that NE binds to a novel adrenergic receptor that activates guanylyl cyclase and that accumulation of cGMP activates PKG, which may phosphorylate a target protein involved in the exocytosis of synaptic vesicles (Yawo, 1999).
The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolishes phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolishes PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detect phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 is located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons (Xue, 2004).
Several lines of evidence suggest that cyclic GMP might be involved in long-term potentiation (LTP) in the hippocampus. Arachidonic acid, nitric oxide and carbon monoxide, three molecules that have been proposed to act as retrograde messengers in LTP, all activate soluble guanylyl cyclase. An inhibitor of guanylyl cyclase blocks the induction of LTP in the CA1 region of hippocampal slices. Conversely, cGMP analogues produce long-lasting enhancement of the excitatory postsynaptic potential if they are applied at the same time as weak tetanic stimulation of the presynaptic fibres. The enhancement is spatially restricted, is not blocked by valeric acid (APV), nifedipine, or picrotoxin, and partially occludes LTP. This synaptic enhancement may be mediated by the cGMP-dependent protein kinase (PKG). Inhibitors of PKG block the induction of LTP, and activators of PKG produce activity-dependent long-lasting enhancement. These results suggest that guanylyl cyclase and PKG contribute to LTP, possibly as activity-dependent presynaptic effectors of retrograde messengers (Zhuo, 1994).
Hippocampal cyclic GMP (cGMP) has been recently postulated to participate in an early phase of memory consolidation of an inhibitory avoidance learning in rats. This study reports the effects of the intrahippocampal infusion of a soluble guanylyl cyclase inhibitor (LY 83583) in the consolidation of one-trial step-down inhibitory avoidance and on the effect of this task on hippocampal cGMP levels and cGMP-dependent protein kinase (PKG) activity. Bilateral intrahippocampal administration of LY 83583 (2.5 micrograms per side) caused full amnesia for inhibitory avoidance when given immediately (0 min) after training, but not 30 min post-training. Rats submitted to the inhibitory avoidance task showed a significant increase in both cGMP levels and in PKG activity in the hippocampus at 0 min after training. No changes were observed 30 min after training. These findings provide further evidence that the hippocampal cGMP/PKG cascade is involved in the early stages of memory formation of an inhibitory avoidance task in rats (Bernabeu, 1997).
The involvement of the cGMP-protein kinase G (PKG) signaling pathway in the induction of long-term depression (LTD) and long-term potentiation (LTP) was investigated in the medial perforant path of the dentate gyrus in vitro. Low-frequency stimulation (LFS)-induced LTD of field EPSPs is inhibited by bath perfusion of a selective soluble guanylyl cyclase inhibitor ODQ. LFS-induced LTD of EPSPs and whole-cell patch-clamped EPSCs is also blocked by bath perfusion and postsynaptic intracellular injection, respectively, of the selective PKG inhibitor KT5823. Elevation of intracellular cGMP by perfusion of the cGMP phosphodiesterase inhibitor zaprinast results in induction of LTD of field EPSPs and EPSCs. Occlusion experiments show mutual inhibition between LFS-induced LTD and zaprinast-induced LTD. The zaprinast-induced LTD of field EPSPs is inhibited by perfusion of ODQ and KT5823. In addition, zaprinast-induced LTD of EPSCs is inhibited by postsynaptic application of KT5823. Glutamate receptor stimulation, especially that of metabotropic glutamate receptors (mGluRs), is required for zaprinast-induced LTD, because cessation of test stimulation or perfusion with the mGluR antagonist MCPG inhibits zaprinast-induced LTD. No inhibitory effect of ODQ or KT5823 on the induction of LTP of EPSPs or EPSCs was found. These data indicate that the cGMP-guanyly cyclase-PKG signaling pathway in the dentate gyrus is essential for induction of LTD, although not of LTP, in the dentate gyrus (Wu, 1998).
Nitric oxide (NO) and the C-type natriuretic peptide (CNP) exert their action on brain via the cGMP signaling pathway. NO, by activating soluble guanylyl cyclase, and CNP, by stimulating membrane-bound guanylyl cyclase, causes intracellular increases of cGMP, activating cGMP-dependent protein kinases (PKGs). Injection of CNP into the rat ventral tegmental area strongly reduces cocaine-induced egr-1 expression in the nucleus accumbens in a dose-dependent manner. The effect of CNP is reversed by the previous injection of a selective PKG inhibitor, KT5823. Activation of PKG by 8-bromo-cGMP reduces, like CNP, cocaine-induced gene transcription in dopaminergic structures. To confirm the involvement of PKG, this was overexpressed in either the mesencephalon or the caudate-putamen. Using the polyethyleneimine delivery system, an active protein was expressed by injecting a plasmid vector containing the human PKG-Ialpha cDNA. PKG was overexpressed in dopaminergic and GABAergic neurons when the plasmid was injected in the ventral tegmental area, whereas overexpression was observed in medium spiny GABAergic neurons and in both cholinergic and GABAergic interneurons when the PKG vector was injected into the caudate-putamen. Activation of the overexpressed PKG reduces cocaine-induced egr-1 expression in dopaminergic structures and affects behavior (i.e., locomotor activity). These effects were again reversed by previous injection of the selective PKG inhibitor. The current data suggest that NO and the neuropeptide CNP are potential regulators of cocaine-related effects on behavior (Jouvert, 2004).
Trafficking of AMPA receptors (AMPARs) is regulated by specific interactions of the subunit intracellular C-terminal domains (CTDs) with other proteins, but the mechanisms involved in this process are still unclear. This study found that the GluR1 CTD binds to cGMP-dependent protein kinase II (cGKII) adjacent to the kinase catalytic site. Binding of GluR1 is increased when cGKII is activated by cGMP. cGKII and GluR1 form a complex in the brain, and cGKII in this complex phosphorylates GluR1 at S845, a site also phosphorylated by PKA. Activation of cGKII by cGMP increases the surface expression of AMPARs at extrasynaptic sites. Inhibition of cGKII activity blocks the surface increase of GluR1 during chemLTP and reduces LTP in the hippocampal slice. This work identifies a pathway, downstream from the NMDA receptor (NMDAR) and nitric oxide (NO), which stimulates GluR1 accumulation in the plasma membrane and plays an important role in synaptic plasticity (Serulle, 2007).
NMDAR stimulation activates nNOS and production of NO, which results in cGMP production and cGKII activation. A major mechanism for expression of NMDAR-dependent LTP involves the synaptic insertion of GluR1. This study reports that, following activation by the NMDAR, cGKII binds to GluR1 and phosphorylates S845, leading to an increase of GluR1 in the plasma membrane. Notably, a cGKII dominant-negative inhibitor peptide blocked the cGMP-dependent increase of GluR1 surface expression, prevented the increase in amplitude and frequency of mEPSCs after chemLTP, and strongly reduced LTP in hippocampal slices. These results demonstrate a mechanism in which the NMDAR regulates AMPAR trafficking during LTP via NO and cGKII (Serulle, 2007).
Because NO is produced at postsynaptic sites and can diffuse through lipid membranes, initial studies of NO-dependent plasticity focused on presynaptic NO function through retrograde mechanisms. Some results were controversial, possibly because different methodologies were employed. Indeed, cGMP derivatives only facilitate LTP maximally if briefly applied when the NMDA receptor is active, and deviating protocols would lead to conflicting results. More recently, the use of new NO donors and NOS antagonists (Bon, 2003; Puzzo, 2005), both in vitro and in vivo (Feil, 2005), has demonstrated a role of the NO cascade in synaptic plasticity. Interestingly, as reported here, both the sGC inhibitor ODQ and the cGK inhibitor KT5823 were found to block LTP. Nonetheless, specific molecular mechanisms underlying the effects of NO, in particular in NO control of AMPAR trafficking in LTP, have been wanting. S-nitrosylation of NSF enhances NSF binding to GluR2 and regulates GluR2 surface expression (Huang, 2005). Also, activation of the NO-cGMP-cGKI pathway increases both GluR1 and synaptophysin puncta and the phosphorylation of VASP in hippocampal neurons (Wang, 2005). However, as yet, a specific pathway for NO control of activity-dependent GluR1 trafficking to synapses, an essential component of LTP, has not been reported. The interaction of cGKII with GluR1 reported here, and its consequent effect on GluR1 surface levels, directly link the actions of NO to LTP via GluR1 trafficking (Serulle, 2007).
A physical association of cGKII with GluR1 enables the kinase to phosphorylate GluR1 at S845. This phosphorylation is required for cGMP-dependent GluR1 surface accumulation, since block of the phosphorylation by the S845A GluR1 mutation blocked the surface increase. Phosphorylation of S845 accompanies increases in GluR1 surface levels and is necessary for GluR1 synaptic insertion during LTP. S845 is dephosphorylated during hippocampal LTD, and S845 phosphorylation on its own is sufficient for increase of GluR1 in the extrasynaptic plasma membrane. Thus far only PKA phosphorylation of S845 has been considered, perhaps because it was the initial kinase shown to phosphorylate this site. The present study demonstrates that cGKII activity also phosphorylates S845 (Serulle, 2007).
Increases of surface GluR1 following both PKA and cGKII phosphorylation are restricted to extrasynaptic sites , and AMPAR synaptic incorporation requires at least one additional step, possibly mediated by S818 phosphorylation. Interestingly, although 8-Br-cGMP on its own did not enhance hippocampal synaptic responses, when paired with a weak tetanus that by itself does not enhance responses, 8-Br-cGMP produced an immediate potentiation. This suggests that cGMP can prime the system for potentiation by a weak tetanic stimulation, possibly by increasing the extrasynaptic surface AMPAR population (Serulle, 2007).
The NMDAR and nNOS mutually interact with PSD-95, and Ca2+ fluxes through the NMDAR activate nNOS in this complex to produce NO, which induces sGC to produce cGMP, which activates cGKII. Ca2+ fluxes also stimulate Ca2+-regulated adenylate cyclases, which produce cAMP, which activates PKA, which also phosphorylates S845. PKA binds the A kinase anchoring protein, AKAP79, which in turn binds the PDZ domain scaffolding protein, SAP97, which binds the GluR1 CTD, thus targeting PKA to the GluR1 CTD and facilitating phosphorylation of S845 (Serulle, 2007).
Unlike the SAP97-AKAP-PKA pathway, the NO-cGMP-cGKII pathway does not rely on a scaffold since the kinase binds the receptor directly. Interestingly, a knockin mouse expressing GluR1 that lacks the last 7 aa of its CTD and does not bind SAP97 exhibited normal hippocampal LTP and GluR1 trafficking. This is explained if the NO-cGMP-cGKII pathway phosphorylates S845 in this mutant (Serulle, 2007).
GluR1 interacts with cGKII via auxiliary and core contact CTD sequences that flank S845. Interestingly, a CTD contact sequence resembles an AI domain sequence of cGKII, suggesting that to bind the catalytic domain, GluR1 mimics the AI domain. Also, this receptor-kinase interaction resembles the well-studied CaMKII binding to the NR2B (Serulle, 2007).
In the absence of cGMP, cGKII is inactive. Following NMDAR stimulation, binding of cGMP to cGKII induces a cGKII conformational change that causes AI domain autophosphorylation, AI domain release from the catalytic domain, and elongation of the kinase. The GluR1 CTD binds the newly exposed cGKII catalytic domain, facilitating GluR1 phosphorylation and the increase of surface GluR1. In one model for this increase, S845 phosphorylation promotes GluR1 trafficking to the plasma membrane, perhaps by releasing of GluR1 from a cytosolic retention factor. Alternatively, GluR1 may cycle into and out of the plasma membrane constitutively, and S845 phosphorylation may stabilize the receptor at the neuron surface. With either model, S845 phosphorylation would regulate the size of an extrasynaptic pool from which receptors may be inserted into the synapse during LTP. Such transport may depend on additional GluR1 phosphorylation. Because a highly selective peptide block of cGKII strongly reduces LTP, such an increase in an extrasynaptic receptor pool is likely to be a requirement for the synaptic potentiation associated with LTP. The present work demonstrates that the NMDAR can control the size of such a receptor pool, acting through nNOS, NO, and cGMP production and the activation of cGKII (Serulle, 2007).
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