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The cGMP-dependent protein kinases (cGK), which belong to the family of serine/threonine kinases, exhibit their diverse functions in cells through interaction with a variety of substrate proteins. Several substrates were identified and the interactions studied using different methods inter alia co-immunoprecipitation (Co-IP) and cGMP-agarose affinity purification. In the following chapter, we will describe the preparation of cell or tissue lysates, the procedures of cGMP-agarose affinity purification and co-immunoprecipitation, and finally the separation and analysis of the protein complexes by SDS-PAGE or mass spectrometry.

The cGMP-dependent protein kinases (cGK), which belong to the family of serine/threonine kinases, exhibit their diverse functions in cells through interaction with a variety of substrate proteins. Several substrates were identified and the interactions studied using different methods inter alia co-immunoprecipitation (Co-IP) and cGMP-agarose affinity purification. In the following chapter, we will describe the preparation of cell or tissue lysates, the procedures of cGMP-agarose affinity purification and co-immunoprecipitation, and finally the separation and analysis of the protein complexes by SDS-PAGE or mass spectrometry.

Cyclic guanosine 3′,5′-monophosphate (cGMP) serves as a second messenger molecule, which regulates pleiotropic cellular functions in health and disease. cGMP is generated by particulate or soluble guanylyl cyclases upon stimulation with natriuretic peptides or nitric oxide, respectively. Furthermore, the cGMP concentration is modulated by cGMP-degrading phosphodiesterases. Several targets of cGMP are utilized to effect its various cellular functions. These effector molecules comprise cGMP-dependent protein kinases, ion channels, and phosphodiesterases. During the last decade, it emerged that cGMP is a novel drug target for the treatment of pulmonary and cardiovascular disorders. In this respect, several drugs were developed, which are now in clinical phase studies for, e.g., pulmonary hypertension or cardiovascular diseases. These new drugs act NO-independently with/without heme on soluble guanylyl cyclases or induce subtypes of particular guanylyl cyclases and thereby lead to new therapeutic concepts and horizons. In this regard, the fifth cGMP meeting held in June 2011 in Halle, Germany, comprised the new therapeutic challenges with the novel functional and structural concepts of cGMP generating and effector molecules. This report summarizes the new data on molecular mechanisms, (patho)physiological relevance, and therapeutic potentials of the cGMP signaling system that were presented at this meeting.

Electrophysiological and behavioral experiments in mice reveal that a cGMP-dependent kinase amplifies neurotransmitter release from peripheral pain sensors, potentiates spinal synapses, and leads to exaggerated pain., Synaptic long-term potentiation (LTP) at spinal neurons directly communicating pain-specific inputs from the periphery to the brain has been proposed to serve as a trigger for pain hypersensitivity in pathological states. Previous studies have functionally implicated the NMDA receptor-NO pathway and the downstream second messenger, cGMP, in these processes. Because cGMP can broadly influence diverse ion-channels, kinases, and phosphodiesterases, pre- as well as post-synaptically, the precise identity of cGMP targets mediating spinal LTP, their mechanisms of action, and their locus in the spinal circuitry are still unclear. Here, we found that Protein Kinase G1 (PKG-I) localized presynaptically in nociceptor terminals plays an essential role in the expression of spinal LTP. Using the Cre-lox P system, we generated nociceptor-specific knockout mice lacking PKG-I specifically in presynaptic terminals of nociceptors in the spinal cord, but not in post-synaptic neurons or elsewhere (SNS-PKG-I−/− mice). Patch clamp recordings showed that activity-induced LTP at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) was completely abolished in SNS-PKG-I−/− mice, although basal synaptic transmission was not affected. Analyses of synaptic failure rates and paired-pulse ratios indicated a role for presynaptic PKG-I in regulating the probability of neurotransmitter release. Inositol 1,4,5-triphosphate receptor 1 and myosin light chain kinase were recruited as key phosphorylation targets of presynaptic PKG-I in nociceptive neurons. Finally, behavioural analyses in vivo showed marked defects in SNS-PKG-I−/− mice in several models of activity-induced nociceptive hypersensitivity, and pharmacological studies identified a clear contribution of PKG-I expressed in spinal terminals of nociceptors. Our results thus indicate that presynaptic mechanisms involving an increase in release probability from nociceptors are operational in the expression of synaptic LTP on spinal-PAG projection neurons and that PKG-I localized in presynaptic nociceptor terminals plays an essential role in this process to regulate pain sensitivity., Author Summary Pain is an important physiological function that protects our body from harm. Pain-sensing neurons, called nociceptors, transduce harmful stimuli into electrical signals and transmit this information to the brain via the spinal cord. When nociceptors are persistently activated, such as after injury, the connections they make with neurons in the spinal cord are altered in a process called synaptic long-term potentiation (LTP). In this study, we examine the molecular and cellular mechanisms of LTP at synapses from nociceptors onto spinal neurons. We use multiple experimental approaches in mice, from genetic to behavioural, to show that this form of LTP involves presynaptic events that unfold in nociceptors when they are repetitively activated. In particular, an enzyme activated by the second messenger cGMP, referred to as Protein Kinase G-I, phosphorylates presynaptic proteins and increases the release of neurotransmitters from nociceptor endings in the spinal cord. When we genetically silence Protein Kinase G-I or block its activation in nociceptors, inflammatory pain is markedly reduced at the behavioural level. These results clarify basic mechanisms of pathological pain and pave the way for new therapeutic approaches.

We analysed the function and intracellular signalling of the cyclic pyrimidinic nucleotide cCMP. The membrane-permeable cCMP analogue dibutyryl-cCMP mediated mouse aorta relaxation. cCMP activated purified cGMP-dependent protein kinase (cGK) Iα and Iβ and stimulated cGK in aorta lysates. cCMP-induced relaxation was abolished in cGKI-knockout tissue. Additionally, deletion of inositol–trisphosphate receptor associated cGKI substrate (IRAG) suppressed cCMP-mediated relaxation. Signalling of cCMP via cGKI/IRAG appears to be of broader physiological importance because cCMP-mediated inhibition of platelet aggregation was absent in cGKI- and IRAG-deficient platelets. These results demonstrate that cCMP acts as intracellular messenger molecule, most unexpectedly utilizing the cGMP signal transduction pathway.

MITOCHONDRIA contain a complex machinery for the import of nuclear-encoded proteins1,2. Receptor proteins exposed on the outer membrane surface are required for the specific binding of precursor proteins to mitochondria, either by binding of cytosolic signal recognition factors or by direct recognition of the precursor polypeptides1–5. Subsequently, the precursors are inserted into the outer membrane at the general insertion site GIP (general insertion protein)6–10. Here we report the analysis of receptors and GIP by crosslinking of translocation intermediates and by coimmunoprecipitation. Surface-accumulated precursors were cross-linked to the receptors MOM19 and MOM72, suggesting a direct interaction of preproteins with surface receptors. We identified three novel mitochondrial outer membrane proteins, MOM7, MOMS, and MOM30 that, together with the previously identified MOM38, seem to form the GIP site and are present in the mitochondrial receptor complex.

Stimulation of cGMP signalling cascade in smooth muscle results in decreased intracellular Ca2+ levels and thus muscle relaxation. Inositoltrisphosphate Receptor 1 (IP3R1) – a Ca2+ release channel located in the membrane of sarcoplasmatic reticulum (SR) – is inhibited by the phosphorylation of IP3R1 associated cGMP kinase dependent substrate (IRAG) via cGMPkinase1β (cGK1β). On the other hand, Ca2+ is pumped back into SR by the Sarco/Endoplasmatic Reticulum Ca2+ ATPase (SERCA), which is inhibited by Phospholamban (PLB). Phosphorylation of PLB by cGK1 suppresses its inhibitory effect on SERCA. To characterize the subcellular distribution of these cGMP signalling proteins upon deletion of IRAG, aortic vascular smooth muscle cells (VSMCs) of IRAG KO mice were immunohistochemically analyzed and compared to VSMCs of littermate wild type animals. Furthermore we investigated the effects of treatment with 8Br-cGMP on the distribution of these proteins in IRAG KO VSMCs versus WT VSMCs.

Results cGKI-/and the RM have a severe anemia and splenomegalie which is associated with a strongly reduced iron content and expression of the ferritin light chain in the spleen. Furthermore, the mRNA levels of transferrin receptor (TfRc) and divalent metal ion transporter (DMT1) are increased in the spleen. Oral or i.m. administration of iron restores partially the iron levels in the spleen of genetargeted cGKI mice, but affects the anemia only moderately. Examination of the intestinal tract showed a massive ulceration in the duodenum that caused intestinal bleeding in cGKI-/and at the later age in the RM as well.

Results Gastric acid production was similar in control and mutant mice. The basal secretion rate of bicarbonate was strongly reduced in the different gene-targeted cGKI mice. Protons induced bicarbonate secretion in controls but not in mutant mice. The dysfunction of the duodenal bicarbonate secretion of RM and cGKI-KO animals was associated with severe gastrointestinal bleedings, which were caused by the age-dependent aggravation of an epithelial ulceration that localized to the papilla Vateri. In conclusion, the analysis of cGKI-KO and RM indicates that a cGMP-cGKI-dependent pathway is present in non-smooth muscle cells of the duodenum, that is involved in the basal and acid-induced secretion of bicarbonate. We suppose, that cGKI might be expressed in the enteric nervous system. Furthermore we hypothesize a crucial role for cGKI signaling via the N. vagus. In contrast to the widespread assumption, that cGKI is expressed in duodenal cells of Cajal we were not able to detect any cGKI in these cells. Conclusion The inability to secrete adequate amounts of bicarbonate ultimately leads to duodenal ulceration. We postulate that the continuous blood loss accounts for the chronic anemia of cGKI mutant mice and causes the premature death of the cGKI-KOs and RM. To administer a therapy against the ulceration and their effects, cGKI knockout mice were treated with a proton pump inhibitor. Chronical treatment of conventional cGKI knockout mice with the proton pump inhibitor esomeprazol prolonged their life expectancy significantly in contrast to the untreated cGKI-knockout mice.

Signalling of cGK (cGMP-dependent protein kinases) are mediated through phosphorylation of specific substrates. Several substrates of cGKI and cGKII were identified meanwhile. Some cGKI substrates are specifically regulated by the cGKIalpha or the cGKIbeta isozyme. In various cells and tissues, different cGK substrates exist that are essential for the regulation of diverse functions comprising tissue contractility, cell motility, cell contact, cellular secretion, cell proliferation, and cell differentiation. On the molecular level, cGKI substrates fulfill various cellular functions regulating e.g. the intracellular calcium and potassium concentration, the calcium sensitivity, and the organisation of the intracellular cytoskeleton. cGKII substrates are involved e.g. in chloride transport, sodium/proton transport and transcriptional regulation. The understanding of cGK signalling and function depends strongly on the identification of further specific substrates. In the last years, diverse approaches ranging from biochemistry to genetic deletion lead to the identification and establishment of several substrates, which raised new insights in the molecular mechanisms of cGK functions and elucidated new cellular cGK functions. However, the analysis of the dynamic signalling of cGK in tissues and cells will be necessary to discover new signalling pathways and substrates.

A large body of evidence indicates that the release of nitric oxide (NO) is crucial for the central sensitization of pain pathways during both inflammatory and neuropathic pain. Here, we investigated the distribution of NO-sensitive guanylyl cyclase (NO-GC) in the spinal cord and in dorsal root ganglia, and we characterized the nociceptive behavior of mice deficient in NO-GC (GC-KO mice). We show that NO-GC is distinctly expressed in neurons of the mouse dorsal horn, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. GC-KO mice exhibited a considerably reduced nociceptive behavior in models of inflammatory or neuropathic pain, but their responses to acute pain were not impaired. Moreover, GC-KO mice failed to develop pain sensitization induced by intrathecal administration of drugs releasing NO or carbon monoxide. Surprisingly, during spinal nociceptive processing, cGMP produced by NO-GC may activate signaling pathways different from cGMP-dependent protein kinase I (cGKI), whereas cGKI can be activated by natriuretic peptide receptor-B dependent cGMP production. Together, our results provide evidence that NO-GC is crucially involved in the central sensitization of pain pathways during inflammatory and neuropathic pain.

Signaling by NO/cGMP/cGMP-dependent kinase I (cGKI) is important for a variety of physiological functions comprising relaxation of smooth muscle and inhibition of platelet aggregation. An important pathway of this signaling cascade includes the inositol 1,4,5-trisphosphate receptor I (IP3RI) associated protein cGMP kinase substrate (IRAG). This protein interacts in a trimeric macrocomplex with cGKIβ and the IP3RI. To get insight into the physiological function of IRAG two different mice strains were generated by targeted deletion: (1) IRAGΔ12/Δ12 with an exon 12 deletion disrupting the IRAG/IP3RI interaction. (2) IRAG-/with an exon 3 deletion generating an IRAG knockout mutant.

Atherosclerotic vascular lesions are considered to be the predominant cause of morbidity and mortality in industrialized nations. Rupture of atherosclerotic plaques initiates platelet activation/aggregation with subsequent arterial thrombosis triggering myocardial infarction and stroke. Nitric oxide (NO) and its second messenger cyclic GMP (cGMP) comprise the central regulatory pathway that prevents platelet activation/aggregation under physiological conditions is. A major downstream target of NO is cyclic guanosine 3',5'-monophosphate kinase I (cGKI). In previous work, we have tested the intravascular significance of the NO/cGKI signalling pathway in vivo using cGKI-deficient (cGKI-/-) mice. We show that platelet cGKI but not endothelial or smooth muscle cGKI is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. Correspondingly, loss of cGKI in platelets was associated with an increase in platelet accumulation in the postischemic kidney and with a significant enhancement of both platelet adhesion and aggregation in the postischemic intestinal microvasculature. The defect in platelet cGKI is not compensated by the cAMP/cAMP kinase pathway supporting the essential role of cGKI in prevention of ischemia-induced platelet adhesion and aggregation. Next, we addressed the regulatory cascade downstream of cGKI. We found that the inositol1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) is abundantly expressed in platelets and assembled in a macrocomplex together with cGKI and the inositol-1,4,5-trisphosphate receptor type I (InsP3RI). cGKI phosphorylates IRAG at Ser664 and Ser677 in intact platelets. Targeted deletion of the IRAG-InsP3RI interaction in IRAGDelta12/Delta12 mutant mice leads to a loss of NO/cGMP-dependent inhibition of fibrinogen-receptor activation and platelet aggregation. Intracellular calcium transients were not affected by DEA/NO or cGMP in mutant platelets. Furthermore, intravital microscopy shows that NO fails to prevent arterial thrombosis of the injured carotid artery in IRAGDelta12/Delta12 mutants.

The signaling molecule nitric oxide (NO) exerts both beneficial and deleterious effects on the vasculature; however, the molecular signaling pathways are incompletely understood. In smooth muscle cells (SMCs), the second messenger cyclic guanosine monophosphate (cGMP) mediates many effects of NO via the activation of cGMPdependent protein kinase type I (cGKI). The cGKI gene, prkg1, encodes two isoforms, cGKIα and cGKIβ, both of which are expressed in SMCs. The isoforms differ only in their individual amino-terminal ends, which mediate the interaction with partner proteins and target the kinases to different subcellular compartments. Conventional knockouts carrying a cGKI null mutation (cGKI-/-) show multiple phenotypes. These animals are difficult to analyse, since most die before adulthood. Recently, two mouse lines were generated that express either the cGKIα or cGKIβ isoform exclusively in SMCs of cGKI-/mice. These mouse lines allow an isoform specific of individual smooth muscle functions in adult "healthy" animals. 8Br-cGMP treatment of aortic SMCs from rescued mice reduced norepinephrineand depolarization-induced Ca2+-increases to wild type levels. In addition, the 8-BrcGMP-induced relaxation of hormone contracted aorta and jejunum was intact in the rescued animals. Finally, telemetric blood pressure recordings in awake, freely moving mice revealed no differences between rescued and control (wild type) mice. Taken together, these results suggest that both isoforms can rescue a major part of the cGMP/cGKI signaling in SMCs and either isoform can compensate for the other in the modulation of vascular tone. Irrespective of the reconstituting isozyme, 50% of the rescued cGKIα and cGKIβ mice die within 52 weeks. Abnormalities found in the mouse lines that possibly cause premature death will be discussed. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007

Defective regulation of platelet activation/aggregation is a predominant cause for arterial thrombosis, the major complication of atherosclerosis triggering myocardial infarction and stroke. A central regulatory pathway conveying inhibition of platelet activation/aggregation is nitric oxide (NO)/cyclic GMP (cGMP) signaling by cGMP-dependent protein kinase I (cGKI). However, the regulatory cascade downstream of cGKI mediating platelet inhibition is still unclear. Here, we show that the inositol-1,4,5-trisphosphate receptor–associated cGMP kinase substrate (IRAG) is abundantly expressed in platelets and assembled in a macrocomplex together with cGKIβ and the inositol-1,4,5-trisphosphate receptor type I (InsP3RI). cGKI phosphorylates IRAG at Ser664 and Ser677 in intact platelets. Targeted deletion of the IRAG-InsP3RI interaction in IRAGΔ12/Δ12 mutant mice leads to a loss of NO/cGMP-dependent inhibition of fibrinogen-receptor activation and platelet aggregation. Intracellular calcium transients were not affected by DEA/NO or cGMP in mutant platelets. Furthermore, intravital microscopy shows that NO fails to prevent arterial thrombosis of the injured carotid artery in IRAGΔ12/Δ12 mutants. These findings reveal that interaction between IRAG and InsP3RI has a central role in NO/cGMP-dependent inhibition of platelet aggregation and in vivo thrombosis.

Over the past few years, a wealth of biochemical and functional data have been gathered on mammalian cGMP-dependent protein kinases (cGKs). In mammals, three different kinases are encoded by two genes. Mutant and chimeric cGK proteins generated by molecular biology techniques yielded important biochemical knowledge, such as the function of the NH2-terminal domains of cGKI and cGKII, the identity of the cGMP-binding sites of cGKI, and the substrate specificity of the enzymes. Genetic approaches have proven especially useful for the analysis of the biological functions of cGKs. Recently, some of the in vivo targets and mechanisms leading to changes in neuronal adaptation, smooth muscle relaxation and growth, intestinal water secretion, bone growth, renin secretion, and other important functions have been identified. These data show that cGKs are signaling molecules involved in many biological functions.

The regulation of neutrophil functions by Type I cGMP-dependent protein kinase (cGKI) was investigated in wild-type (WT) and cGKI-deficient (cGKI−/−) mice. We demonstrate that murine neutrophils expressed cGKIα. Similar to the regulation of Ca2+ by cGKI in other cells, there was a cGMP-dependent decrease in Ca2+ transients in response to C5a in WT, but not cGKI−/− bone marrow neutrophils. In vitro chemotaxis of bone marrow neutrophils to C5a or IL-8 was significantly greater in cGKI−/− than in WT. Enhanced chemotaxis was also observed with cGKI−/− peritoneal exudate neutrophils (PE-N). In vivo chemotaxis with an arachidonic acid-induced inflammatory ear model revealed an increase in both ear weight and myeloperoxidase (MPO) activity in ear punches of cGKI−/− vs WT mice. These changes were attributable to enhanced vascular permeability and increased neutrophil infiltration. The total extractable content of MPO, but not lysozyme, was significantly greater in cGKI−/− than in WT PE-N. Furthermore, the percentage of MPO released in response to fMLP from cGKI−/− (69%) was greater than that from WT PE-N (36%). PMA failed to induce MPO release from PE-N of either genotype. In contrast, fMLP and PMA released equivalent amounts of lysozyme from PE-N. However, the percentage released was less in cGKI−/− (∼60%) than in WT (∼90%) PE-N. Superoxide release (maximum velocity) revealed no genotype differences in responses to PMA or fMLP stimulation. In summary, these results show that cGKIα down-regulates Ca2+ transients and chemotaxis in murine neutrophils. The regulatory influences of cGKIα on the secretagogue responses are complex, depending on the granule subtype.

Intracellular signalling by NO/cGMP/cGMP-dependent protein kinase type I (cGKI) regulates various physiological processes including smooth muscle contractility and platelet aggregation. An important mediator of this signalling cascade is the inositol 1,4,5-trisphosphate receptor I (IP3RI) associated protein cGMP kinase substrate (IRAG). This protein forms a trimeric complex together with the cGMP kinase Iβ (cGKIβ) and the IP3RI. Targeted deletion of exon 12 of IRAG coding for the N-terminal part of the coiled-coil domain disrupted in vivo the IRAG-IP3RI interaction. The resulting IRAG∆12/∆12 mice showed an increased mortality and a severely reduced gastrointestinal motility. The relaxation of hormone-contracted aortic and longitudinal colonic smooth muscle by cGMP was abolished in IRAG∆12/∆12 mice, whereas cAMP-mediated relaxation was not altered. In contrast to WT mice, norepinephrine-induced increases in [Ca]i were not reduced by cGMP in aortic smooth muscle cells from IRAG∆12/∆12 mice. These data suggest, that IRAG is involved in the cGMP-dependent decrease of [Ca]i in vivo and is essential for cGMP-dependent relaxation of hormone-induced vascular and colonic muscle contraction. However, cGMP-mediated relaxation of small intestinal smooth muscles was only partially affected in IRAG∆12/∆12 mice suggesting tissue specific selectivity of cGKI mechanisms.

Cyclic guanosine-3', 5'-monophosphate (cGMP)-dependent protein kinases (cGKs) are key enzymes of nitric oxide-cGMP and natriuretic peptide signalling cascades. These kinases mediate most of the effects of cGMP-elevating drugs, such as nitrates and phosphodiesterase inhibitors. cGKs modulate smooth muscle relaxation (e.g. the vasculature, gastrointestinal tract, bladder and penile), platelet aggregation, renin release, intestinal secretion, learning and memory. Furthermore, several cGK substrates have been identified. Isozyme-specific inhibitors and activators of cGK and its downstream substrates might act more specifically than upstream signalling activators, such as organic nitrates and phosphodiesterase inhibitors.

Nitric oxide (NO) modulates a variety of processes in the mammalian brain, but the mechanisms of neuronal NO signaling are poorly understood. In the periphery, many effects of NO are mediated via the generation of the second messenger cyclic guanosine-3′,5′-monophosphate (cGMP) and activation of the cGMP-dependent protein kinase type I (cGKI). However, previous studies suggested that the expression of cGKI in the nervous system is rather restricted, thus, questioning the functional significance of the cGMP/cGKI pathway as a mediator of NO signaling in the brain. Here we have performed a detailed immunohistochemical study to elucidate the distribution of cGKI in the CNS and eye of the mouse. Expression of cGKI protein was detected not only in the previously described areas (cerebellum, hippocampus, dorsomedial hypothalamus) but also in a number of additional regions, such as medulla, subcommissural organ, cerebral cortex, amygdala, habenulae, various hypothalamic regions, olfactory bulb, pituitary gland, and retina. Immunoblotting with isoform-specific antibodies indicated that the cGKIα isoform is prominent in the cerebellum and medulla, whereas the cGKIβ isoform is predominant in the cortex, hippocampus, hypothalamus, and olfactory bulb. Similar levels of the isoforms were detected in the pituitary gland and eye. Thus, it appears that distinct brain regions express distinct cGKI isoforms that signal via distinct pathways. Together, these results improve our understanding of the cellular and molecular mechanisms of NO/cGMP/cGKI signaling and indicate that the distribution and functional relevance of this pathway in the mammalian brain is broader than previously thought.

Signalling by cGMP-dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKI-dependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5-trisphosphate receptor I (IP(3)RI)-associated protein (IRAG), which decreases hormone-induced IP(3)-dependent Ca(2+) release. We show now that the targeted deletion of exon 12 of IRAG coding for the N-terminus of the coiled-coil domain disrupted in vivo the IRAG-IP(3)RI interaction and resulted in hypomorphic IRAG(Delta12/Delta12) mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol- and phenylephrine-contracted smooth muscle strips from colon and aorta, respectively, of IRAG(Delta12/Delta12) mice were not relaxed by cGMP, while cAMP-mediated relaxation was unperturbed. Norepinephrine-induced increases in [Ca(2+)](i) were not decreased by cGMP in aortic smooth muscle cells from IRAG(Delta12/Delta12) mice. In contrast, cGMP-induced relaxation of potassium-induced smooth muscle contraction was not abolished in IRAG(Delta12/Delta12) mice. We conclude that cGMP-dependent relaxation of hormone receptor-triggered smooth muscle contraction essentially depends on the interaction of cGKI-IRAG with IP(3)RI.

cGMP kinase I (cGKI) signaling modulates multiple physiological processes including smooth muscle relaxation. The expression of cGKI and its substrate IRAG (Inositol 1,4,5-trisphosphate receptor associated cGMP kinase substrate) was studied. IRAG and cGKI were colocalized in the smooth muscle of aorta and colon. IRAG was present in the thalamus and in most of the myenteric plexus in the absence of cGKI. Coexpression of IRAG and cGKIbeta or cGKIalpha in COS-7 cells revealed that IRAG recruits cGKIbeta but not cGKIalpha to the endoplasmic reticulum. These results suggest that IRAG may be involved in cGKI-dependent and -independent pathways.

Nitric oxide (NO)-mediated relaxation of colonic smooth muscle is crucial for the maintenance of human gut function. The molecular mechanisms of NO-dependent smooth muscle relaxation involve cyclic GMP-mediated inhibition of store-dependent calcium signaling. Recently, IRAG (inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate) has been characterized as a novel target molecule of cGMP-dependent protein kinase (cGKI) mediating NO-/cGMP-dependent inhibition of inositol 1,4,5-trisphosphate (InsP3)-dependent calcium release in transfected COS cells. The aim of the present study was to characterize IRAG expression and its functional role in NO-dependent signaling in human colonic smooth muscle. Reverse transcriptase-PCR revealed IRAG mRNA expression in human colon, rectum, and cultured colonic smooth muscle cells. In cultured human colonic smooth muscle cells, bradykinin (BK) elicited InsP3-dependent calcium transients that were repeatable and independent of extracellular calcium. The NO donor sodium nitroprusside and the specific cGK activator 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic-monophosphate (8-pCPT-cGMP) significantly inhibited BK-induced increase in intracellular calcium. Cells transfected with antisense oligonucleotides raised against IRAG (IRAG-AS) showed strongly decreased IRAG protein expression. In these cells, sodium nitroprusside and 8-pCPT-cGMP both failed to modulate BK-induced calcium transients. Thus, endogenous IRAG appears to be essentially involved in the NO/cGK-dependent inhibition of InsP3-dependent Ca2+-signaling in colonic smooth muscle.

The gaseous molecule nitric oxide (NO) modulates a large variety of physiological functions including vascular tone, intestinal motility, platelet aggregation, proliferation, apoptosis, and neurotransmission. NO initiates diverse cellular signaling cascades which comprise nitrosylation of proteins, adenosine 5'-diphosphate (ADP)-ribosylation, or stimulation of soluble guanylyl cyclases which catalyze intracellular guanosine 3',5'-cyclic monophosphate (cGMP) synthesis. cGMP activates cGMP-dependent protein kinases (cGK) which mediate localized and global signaling. Furthermore, cGMP regulates the activity of phosphodiesterases (PDE) which modulate the duration and amplitude of cyclic nucleotide signaling. Two different types of cGK are expressed in mammals, cGKI and cGKII. Activation of the NO/cGMP/cGKI pathway induces relaxation of smooth muscle by lowering the cytosolic calcium level and/or by calcium desensitization of the contractile elements.

International audience; The cGMP kinase signaling complex identified previously in tracheal smooth muscle membranes contains a number of cGMP kinase substrates termed G0 through G4. G0, G1, and G2 were identified as IP3 receptor I (IP3RI), IRAG, and cGMP kinase I. Sequencing of purified G3 and G4 showed that these proteins were proteolytic cleavage products of IRAG. However, the purified cGMP kinase signaling complex contained following additional proteins: α-actin, calponin H1, and phospholamban (PLB) as verified by MALDI-TOF as well as MS/MS sequencing and immune detection. The complex of these six proteins was immune precipitated by antibodies to each protein. The proteins were phosphorylated by the endogenous cGMP kinase I with the exception of α-actin and calponin H1. The complex did not contain the Ca2+-ATPase SERCA II. PLB, IP3RI, and cGMP kinase Iβ were co-immune precipitated after expression in COS-7 cells. These results suggest that PLB may have additional functions to regulate the activity of SERCA II.

Cyclic GMP-dependent protein kinase I (cGKI) affects the inositol 1,4,5-trisphosphate (InsP(3))-dependent release of intracellular calcium by phosphorylation of IRAG (inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate). IRAG is present in a macromolecular complex with the InsP(3) receptor type I (InsP(3)RI) and cGKIbeta. The specificity of the interaction between these three proteins was investigated by using the yeast two-hybrid system and by co-precipitation of expressed proteins. The amino-terminal region containing the leucine zipper (amino acids 1-53) of cGKIbeta but not that of cGKIalpha or cGKII interacted with the sequence between amino acids 152 and 184 of IRAG in vitro and in vivo most likely through electrostatic interaction. cGKIbeta did not interact with the InsP(3)RI, but co-precipitated the InsP(3)RI in the presence of IRAG indicating that IRAG bound to the InsP(3)RI and to cGKIbeta. cGKIbeta phosphorylated up to four serines in IRAG. Mutation of these four serines to alanine showed that cGKIbeta-dependent phosphorylation of Ser(696) is necessary to decrease calcium release from InsP(3)-sensitive stores. These results show that cGMP induced reduction of cytosolic calcium concentrations requires cGKIbeta and phosphorylation of Ser(696) of IRAG.

Over the past few years, a wealth of biochemical and functional data has been gathered on mammalian cGMP-dependent protein kinases (cGKs). In mammals, three different kinases are encoded by two genes. Mutant and chimeric cGMP kinase proteins generated by molecular biology techniques have yielded important biochemical knowledge, such as the function of the N-terminal domains of cGKI and cGKII, the identity of the cGMP-binding sites of cGKI, the substrate specificity of the enzymes and structural details of the catalytic center. Genetic approaches have proved to be especially useful for the analysis of the biological function of cGKs. Recently, some of the in vivo targets and mechanisms leading to smooth muscle relaxation have been identified. In vivo targets are the myosin-binding subunit of myosin phosphatase (PP1M), a member of the protein phosphatase 1, the calcium-activated maxi K(+) channel and a new protein named IRAG that forms a complex with the inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) receptor and cGKI. Phosphorylation of PP1M by cGKI(alpha) activates myosin phosphatase, whereas phosphorylation of IRAG by cGKI(beta) decreases Ins(1,4, 5)P(3)-induced calcium release. cGKII regulates in vivo intestinal fluid secretion by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), bone growth and renal renin secretion by phosphorylation of unknown proteins.

Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIbeta phosphorylated the IP3R and cGKIbeta, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIbeta, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIbeta in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.

The cloned BK channel alpha subunit from human myometrium was stably expressed in Chinese hamster ovary cells, either alone (CHOalpha cells) or in combination with the auxiliary beta subunit (CHOalpha+beta cells). We studied basic channel properties and the effects of cGMP- and cAMP-dependent protein kinases on the BK channel activity. Coexpression of alpha and beta subunits enhanced the Ca2+ and voltage sensitivity of the BK channel, and decreased the inhibitory potency of iberiotoxin. Blocking and stimulating effects on BK channel activity by charybdotoxin and nitric oxide, respectively, were independent of the beta subunit. The cGMP kinase Ialpha and cAMP kinase failed to affect BK channel activity in CHOalpha and CHOalpha+beta cells at different [Ca2+]i and voltages. In contrast, BK channels in freshly isolated myometrial cells from postmenopausal women responded to cAMP kinase and cGMP kinase with a fourfold and twofold decrease in their open probability (NPo), respectively. These effects could be reversed by alkaline phosphatase and remained unaffected by the phosphatase inhibitor okadaic acid (100 nM). In 28% of myometrial cells, however, cAMP and cGMP kinases increased NPo 2-fold and 3.5-fold, respectively. This stimulation was enhanced rather than reversed by alkaline phosphatase and was abolished by 100 nM okadaic acid. The results suggest that in stably transfected CHO cells the expressed BK channel is not regulated by cAMP kinase and cGMP kinase. However, in native myometrial cells stimulatory and inhibitory regulation of BK channels by cAMP kinase and cGMP kinase was observed, suggesting that channel regulation by the protein kinases requires factors that are not provided by CHO cells. Alternatively, failure of regulation may have been due to the primary structure of the myometrial BK channel protein used in this study.

Background cAMP and cGMP are well established second messengers and essential for numerous of (patho)physiological processes. These purine cyclic nucleotides activate PKA and PKG, respectively. So far, there was no evidence of further cyclic nucleotides acting as second messengers. Meanwhile the existence of cCMP was described [1]. Formation of the cyclic pyrimidine nucleotide cyclic cytidine 3’,5’-monophosphate (cCMP) by cytidylyl cyclases is debated. cCMP induces relaxation of vascular smooth muscle via cGKI [2]. Furthermore, it was postulated that cCMP is relevant for cell growth [3] and blood cell function [4]. However, functions regulated by cCMP are mostly unknown.

The regulation of Ca2+-activated K+ channels (KCa channels) by cGMP-dependent protein kinase (cGMP kinase) and its molecular mechanism were investigated in Chinese hamster ovary (CHO) and tracheal smooth muscle cells. In CHO wild-type cells (CHO-WT cells) and in CHO cells stably transfected with cGMP kinase Ialpha (CHO-cGK cells), KCa channels with intermediate conductance (approximately 50 picosiemens) were identified. Due to the basal activity of cGMP kinase, Ca2+-activated K+ currents had a higher sensitivity toward the cytosolic Ca2+ concentration in CHO-cGK cells than in CHO-WT cells. Dialysis of the active fragment of cGMP kinase (300 n) into CHO-WT cells or of cGMP into CHO-cGK cells increased the Ca2+-activated K+ current, while the catalytic subunit of cAMP-dependent protein kinase (cAMP kinase) was without effect. In cell-attached patches obtained from freshly isolated bovine tracheal smooth muscle cells, the open state probability (NPo) of maxi-KCa channels (conductance of approximately 260 picosiemens) was enhanced by 300 microM 8-(4-chlorophenylthio)-cGMP, a specific and potent activator of cGMP kinase. In contrast, 1 microM isoprenaline, 20 microM forskolin, and 3 mM 8-bromo-cAMP failed to enhance KCa channel activity. In excised inside-out patches, only the active fragment of cGMP kinase (but not that of cAMP kinase) increased NPo when applied to the cytosolic side of the patch. The enhancement of NPo by cGMP kinase was inhibited in CHO cells as well as in tracheal smooth muscle cells by the cGMP kinase inhibitor KT 5823 (1 microM) and the protein phosphatase (PP) inhibitors microcystin (5 microM) and okadaic acid (10 nM). The catalytic subunit of PP2A (but not that of PP1) mimicked the effect of cGMP kinase on NPo in excised inside-out patches. The results show that cGMP kinase regulates two different KCa channels in two unrelated cell types by the same indirect mechanism, which requires the activity of PP2A. The regulation of the KCa channel is specific for cGMP kinase and is not mimicked by cAMP kinase.

The protein Tom71 is encoded by the open reading frame YHR117w (yeast chromosome VIII) and shares 53% amino acid sequence identity with Tom70, a protein import receptor of the mitochondrial outer membrane. We investigated the cellular function of Tom71 and addressed the question of whether Tom71 and Tom70 fulfill similar functions. Like Tom70, Tom71 is anchored to the mitochondrial outer membrane via its N terminus, thereby exposing a large C-terminal domain to the cytosol. Tom71 is associated with the protein import complex of this membrane and can be cross-linked to a protein with a molecular mass of 30-35 kDa. Disruption of the TOM71 gene does not reduce cell growth, except on nonfermentable carbon sources at elevated temperatures. Deletion of both the TOM71 and TOM70 genes does not acerbate this growth defect. In vitro import studies demonstrated no functional requirement for Tom71 in the import of several preproteins destined for each of the mitochondrial subcompartments. In particular, the import of Tom70-dependent preproteins is minimally affected by the deletion of Tom71, irrespective of the presence or absence of the Tom70 receptor. Thus, despite their strikingly similar biochemical properties, Tom71 and Tom70 do not perform identical functions.

The import receptor MOM72 constitutes part of the protein translocation machinery of the outer mitochondrial membrane, the receptor-general insertion pore complex. The protein contains a membrane anchor at the NH2 terminus and a large cytosolic domain. In yeast and Neurospora crassa the cytosolic domain comprises about 570-580 amino acid residues. The cytosolic domain of yeast MOM72 was purified after expression in Escherichia coli as a homogeneous monomeric protein. It can recognize precursor proteins as demonstrated by its ability to compete for binding and import into the mitochondria and to physically interact with preproteins. A subset of preproteins including the ADP/ATP carrier and the phosphate carrier interact with very high affinity, precursors that are known to be targeted via MOM72. Thus, the cytosolic domain of MOM72 plays a critical function in the recognition of preproteins by directly binding to precursor proteins and thereby facilitating their targeting to mitochondria.

Results We investigated the relevance of IRAG and the cGKI stimulated phosphorylation of the calcium channel InsP3R-I for the NO/cGMP-dependent inhibition of platelet aggregation and adhesion. After incubation with different agonists (collagen, thrombin, TxA2) we performed aggregation experiments with platelets of WT and IRAG-KO mice, thereby the IRAG-KO platelets aggregated stronger than the WT platelets. After preincubation with NO/cGMP the inhibition of aggregation was decreased in IRAG-KO platelets compared to WT platelets. Furthermore, GPIIb/IIIamediated adhesion of platelets to fibrinogen could only weakly be inhibited in IRAG-deficient platelets contrary to WT platelets. The cGKI-mediated stimulation of InsP3R-I phosphorylation showed an equal increase in WT and IRAG-KO platelets.

Results For the functional analysis of cGMP signalling, transgenic murine mutants were used. cGKI signalling pathways include interaction of the cGKIb-isozyme with the inositol 1,4,5-trisphosphate receptor I (IP3RI) associated protein cGMP kinase substrate (IRAG). NO/cGMP and ANP/cGMP-dependent relaxation of aortic smooth muscle was strongly affected in IRAG-deficient mice. NO/ ANP-dependent inhibition of intracellular calcium release was suppressed in IRAG-deficient vascular smooth muscle cells (VSMC). Furthermore, reduction of store operated calcium entry by cGMP was affected in IRAG-KO VSMC. Basal mean arterial blood pressure, heart rate and activity were not changed in IRAG knockout mice. However, under pathophysiological conditions like sepsis (induced by E. coli lipopolysaccharide application) IRAG knockout mice were resistant to blood pressure reduction.

We analyzed the submitochondrial location and biogenesis pathway of the phosphate carrier (PiC), also termed p32, of Saccharomyces cerevisiae mitochondria, PiC/p32 was found to behave as an integral membrane protein that cofractionated with the ADP/ATP carrier of the inner membrane. Import of the precursor of PiC/p32 required a membrane potential across the inner membrane, supporting its localization to the inner membrane. This makes it unlikely that the major function of PiC/p32 is that of an import receptor on the surface of the mitochondrial outer membrane. Furthermore, we found that both receptors MOM72 and MOM19 were involved in the import pathway of the precursor of PiC/p32 with MOM72 being responsible for the bulk of import. Yeast PiC/p32 is thus not only structurally homologous to the ADP/ATP carrier, but has a similar targeting mechanism and submitochondrial location, supporting its classification as a member of the inner membrane carrier family.

NO/cGMP signalling via cGMP-dependent kinase I (cGKI) induces a variety of physiological functions comprising relaxation of smooth muscle and inhibition of platelet aggregation. Several signalling pathways of cGKI exist including the interaction of the cGKIβ isozyme with the inositol 1,4,5-trisphosphate receptor I (IP3RI) associated protein cGMP kinase substrate (IRAG). To get insight into the physiological function of IRAG protein a knockout mutant of the IRAG gene was generated by targeted deletion. Expression of other cGKI substrate proteins in mutant smooth muscle tissues and platelets was not affected upon IRAG deletion. Interestingly, the localization of cGKIβ was unchanged in IRAG-deficient vascular smooth muscle cells. Analysis of smooth muscle contractility suggests that signalling via IRAG is essential for endogenous and exogenous NO/cGMP-dependent contractility of aortic smooth muscle. Furthermore, NO/cGMP-dependent relaxation of murine colon is dependent on this signalling cascade. In platelets, IRAG-deficiency abolishes the NO/cGMP-dependent inhibition of platelet aggregation and granule secretion. These results strongly suggest that IRAG signalling is a predominant physiological signalling pathway of NO/cGMP in smooth muscle and platelets.

cGMP signalling plays an important physiological role in diverse organs including the vasculature, the GI-tract and the nervous system. Furthermore, cGMP-elevating substances such as glyceryl trinitrate are important drugs used in cardiovascular diseases. Physiologically, cGMP synthesis is induced by nitric oxide (NO) and natriuretic peptides through the stimulation of guanylyl cyclases. Major mediators of cGMP signalling are the cGMP-dependent protein kinases type I and II (cGKI and cGKII). The functional significance of each kinase type in diverse organs was determined using total and tissue-specific cGKI- and cGKII-deficient mice. These studies established that cGKI plays a major role in the regulation of the cardiovascular and the gastrointestinal system, hippocampal and cerebellar learning and pain perception. cGKII is involved in intestinal water secretion, bone growth and circardian rhythmicity. The cGK mutant mice are important tools to obtain detailed insights into cGMP-mediated signalling pathways in health and disease.