Dominant negative mutants of ornithine decarboxylase.

Conserved lysines of mouse ornithine decarboxylase were individually mutated to arginines. The mutations at amino acid residues 69, 115, and 169 greatly reduced or abolished enzymatic activity. Lysine 69 is the site of Schiff base formation with the cofactor pyridoxal phosphate; the functional role of the other two lysines essential for activity is not known. Coexpression of wild type ornithine decarboxylase along with the lysine 115 to arginine mutant reduced the activity of the former without diminishing the amount of wild type protein. This form of negative complementation was seen when wild type and mutant protein were coexpressed either by in vitro translation or in bacteria. The data are consistent with the conclusion that a wild type and mutant subunit form a heterodimer that is enzymatically inactive.     

Dominant negative mutants of transducin-alpha that block activated receptor

Mutations counterpart to dominant negative RasSer17Asn in the alpha-subunits of heterotrimeric G-proteins are known to also produce dominant negative effects. The mechanism of these mutations remains poorly understood. Here, we examined the effects and mechanism of the Ser43Cys and Ser43Asn mutants of transducin-like chimeric Gtalpha* in the visual signaling system. Our analysis showed that both mutants have reduced affinity for GDP and are likely to exist in an empty-or partially occupied-pocket state. S43C and S43N retained the ability to interact with Gtbetagamma and, as heterotrimeric proteins, bind to photoexcited rhodopsin (R*). The interaction with R* is unproductive as the mutants failed to bind GTPgammaS and become activated. S43C and S43N inhibited R*-dependent activation of Gtalpha* and Gtalpha, apparently by blocking R*. Finally, both Gtalpha* mutants lacked interaction with the gamma-subunit of PDE6, an effector protein in phototransduction. These results indicate that the S43C and S43N mutants of Gtalpha* are dominant negative inhibitors that bind and block the activated receptor in a mechanism that parallels that of RasSer17Asn. Dominant negative mutants of Gtalpha sequestering R*, such as S43C and S43N, may become useful instruments in probing the mechanisms of visual dysfunctions caused by abnormal phototransduction signaling.     

Molecular mechanisms of action of natural amino acids and serotonin on infusorian adenylyl cyclase and guanylyl cyclase

Earlier, it has been shown that some amino acids and their derivatives are able to regulate activities of adenylyl cyclase (AC) and guanylyl cyclase (GC) in free-living infusoria Dileptus anser and Tetrahymena pyriformis. The goal of this work consisted in studying the molecular mechanisms of action of methionine, tyrosine, alanine, and neurohormone serotonin on the activity of enzyme-cyclases and in identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both infusoria; the effect of serotonin on AC in T. pyriformis took place with participation of the Ca²⁺-dependent form of AC and of the heterotrimeric G-proteins. The AC-stimulating effect of tyrosine and alanine was expressed weakly and was revealed only in D. anser. Serotonin in both infusoria and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine did not affect GC. Methionine and serotonin were bound with a high affinity to the surface receptors of infusoria. The K_D for [methyl-³H]methionine binding to D. anser and T. pyriformis were equal to 7.5 and 35.6 nM, and for [³H]serotonin binding, they were 2.7 and 4.7 nM, respectively. Alanine and tyrosine were bound to infusoria with low affinity. Thus, in the infusoria D. anser and T. pyriformis, there are chemosignal systems regulated by amino acids and their derivatives, including enzymes with cyclase activity. These systems are suggested to be similar to the hormonal signal systems of the higher eukaryotes and to be their predecessors.     

Nitric oxide-sensitive guanylyl cyclase: structure and regulation

By the formation of the second messenger cGMP, NO-sensitive guanylyl cyclase (GC) plays a key role within the NO/cGMP signaling cascade which participates in vascular regulation and neurotransmission. The enzyme contains a prosthetic heme group that acts as the acceptor site for NO. High affinity binding of NO to the heme moiety leads to an up to 200-fold activation of the enzyme. Unexpectedly, NO dissociates with a half-life of a few seconds which appears fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogs act as NO sensitizers and led to the discovery of a novel pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme. The two isoforms of the heterodimeric enzyme (alpha1beta1, alpha2beta1) are known that are functionally indistinguishable. The alpha2beta1-isoform mainly occurs in brain whereas the alpha1beta1-enzyme shows a broader distribution and represents the predominantly expressed form of NO-sensitive GC. Until recently, the enzyme has been thought to occur in the cytosol. However, latest evidence suggests that the alpha2-subunit mediates the membrane association of the alpha2beta1-isoform via interaction with a PDZ domain of the post-synaptic scaffold protein PSD-95. Binding to PSD-95 locates this isoform in close proximity to the NO-generating synthases thereby enabling the NO sensor to respond to locally elevated NO concentrations. In sum, the two known isoforms may stand for the neuronal and vascular form of NO-sensitive GC reflecting a possible association to the neuronal and endothelial NO-synthase, respectively.     

Dimerization of guanylyl cyclase-activating protein and a mechanism of photoreceptor guanylyl cyclase activation.

Ca(2+)-binding guanylyl cyclase-activating proteins (GCAPs) stimulate photoreceptor membrane guanylyl cyclase (retGC) in the light when the free Ca(2+) concentrations in photoreceptors decrease from 600 to 50 nM. RetGC activated by GCAPs exhibits tight dimerization revealed by chemical cross-linking (Yu, H., Olshevskaya, E., Duda, T., Seno, K., Hayashi, F., Sharma, R. K., Dizhoor, A. M., and Yamazaki, A. (1999) J. Biol. Chem. 274, 15547-15555). We have found that the Ca(2+)-loaded GCAP-2 monomer undergoes reversible dimerization upon dissociation of Ca(2+). The ability of GCAP-2 and its several mutants to activate retGC in vitro correlates with their ability to dimerize at low free Ca(2+) concentrations. A constitutively active GCAP-2 mutant E80Q/E116Q/D158N that stimulates retGC regardless of the free Ca(2+) concentrations forms dimers both in the absence and in the presence of Ca(2+). Several GCAP-2/neurocalcin chimera proteins that cannot efficiently activate retGC in low Ca(2+) concentrations are also unable to dimerize in the absence of Ca(2+). Additional mutation that restores normal activity of the GCAP-2 chimera mutant also restores its ability to dimerize in the absence of Ca(2+). These results suggest that dimerization of GCAP-2 can be a part of the mechanism by which GCAP-2 regulates the photoreceptor guanylyl cyclase. The Ca(2+)-free GCAP-1 is also capable of dimerization in the absence of Ca(2+), but unlike GCAP-2, dimerization of GCAP-1 is resistant to the presence of Ca(2+).     

A-350619: a novel activator of soluble guanylyl cyclase

Nitric oxide (NO) is a key mediator in many physiological processes and one of the major receptors through which NO exerts its effects is soluble guanylyl cyclase. Guanylyl cyclase converts GTP to cyclic GMP as part of the cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, inhibition of platelet aggregation and immune response. The properties of A-350619, a novel soluble guanylyl cyclase activator, were examined to determine the modulatory effect on the catalytic properties of soluble guanylyl cyclase. A-350619 increased V(max) from 0.1 to 14.5 micromol/min/mg (145 fold increase), and lowered K(m) from 300 to 50 microM (6 fold decrease). When YC-1 (another sGC activator) and A-350619 were combined, a 156 fold increase in V(max) and a 5 fold decrease in Km were observed, indicating that the modulation of the enzyme brought about by YC-1 and A-350619 are not additive, suggesting a common binding site. Activation of soluble guanylyl cyclase by A-350619 was partially inhibited by ODQ, a specific inhibitor of soluble guanylyl cyclase by oxidation of the enzyme heme. YC-1 and A-350619 after pre-treatment with N-omega-nitro-L-arginine, an NO-synthase inhibitor, relaxed cavernosum tissue strips in a dose-dependent manner with EC(50) of 50 microM and 80 microM, respectively. Addition of SNP potentiated the relaxation effect of YC-1 and A-350619, shifting the dose-response curve to the left to 3 microM and 10 microM, respectively. Consistent with its biochemical activity, A-350619 (1 micromol/kg) alone induced penile erection in a conscious rat model. Activation of soluble guanylyl cyclase in cavernosum tissue as an alternate method of enhancing the effect of NO may provide a novel treatment of sexual dysfunction.     

A critical role for ATP in the stimulation of retinal guanylyl cyclase by guanylyl cyclase-activating proteins

It has been believed that retinal guanylyl cyclase (retGC), a key enzyme in the cGMP recovery to the dark state, is solely activated by guanylyl cyclase-activating proteins (GCAPs) in a Ca2+-sensitive manner. However, a question has arisen as to whether the observed GCAP stimulation of retGC is sufficient to account for the cGMP recovery because the stimulated activity measured in vitro is less than the light/GTP-activated cGMP phosphodiesterase activity. Here we report that the retGC activation by GCAPs is larger than previously reported and that a preincubation with adenine nucleotide is essential for the large activation. Under certain conditions, ATP is two times more effective than adenylyl imidodiphosphate (AMP-PNP), a hydrolysis-resistant ATP analog; however, this study mainly used AMP-PNP to focus on the role of adenine nucleotide binding to retGC. When photoreceptor outer segment homogenates are preincubated with AMP-PNP (EC50 = 0.65 +/- 0.20 mM), GCAP2 enhanced the retGC activity 10-13 times over the control rate. Without AMP-PNP, GCAP2 stimulated the control activity only 3-4-fold as in previous reports. The large activation is due to a GCAP2-dependent increase in Vmax without an alteration of retGC affinity for GCAP2 (EC50 = 47.9 +/- 2.7 nM). GCAP1 stimulated retGC activity in a similar fashion but with lower affinity (EC50 = 308 nM). In the AMP-PNP preincubation, low Ca2+ concentrations are not required, and retGC exists as a monomeric form. This large activation is accomplished through enhanced action of GCAPs as shown by Ca2+ inhibition of the activity (IC50 = 178 nM). We propose that retGC is activated by a two-step mechanism: a conformational change by ATP binding to its kinase homology domain under high Ca2+ concentrations that allows large enhancement of GCAP activation under low Ca2+ concentrations.     

Mapping sites in guanylyl cyclase activating protein-1 required for regulation of photoreceptor membrane guanylyl cyclases

Guanylyl cyclase activating protein (GCAP)-1 regulates photoreceptor membrane guanylyl cyclase, RetGC, in a Ca2+-sensitive manner. It contains four Ca2+-binding motifs, EF-hands, three of which are capable of binding Ca2+. GCAP-1 activates RetGC in low Ca2+ and inhibits it in high Ca2+. In this study we used deletion and substitution analysis to identify regions of GCAP-1 sequence that are specifically required for inhibition and activation. A COOH-terminal sequence within Met157 to Arg182 is required for activation but not for inhibition of RetGC. We localized one essential stretch to 5 residues from Arg178 to Arg182. Another sequence essential for activation is within the N-terminal residues Trp21 to Thr27. The region between EF-hands 1 and 3 of GCAP-1 also contains elements needed for activation of RetGC. Finally, we found that inhibition of RetGC requires the first 9 amino-terminal residues of GCAP-1, but none of the residues from Gln33 to the COOH-terminal Gly205 are specifically required for inhibition. The ability of GCAP-1 mutants to regulate RetGC was tested on total guanylyl cyclase activity present in rod outer segments. In addition, the key mutants were also shown to produce similar effects on recombinant bovine outer segment cyclases GC1 and GC2.     

Instead of binding calcium, one of the EF-hand structures in guanylyl cyclase activating protein-2 is required for targeting photoreceptor guanylyl cyclase

Guanylyl cyclase activator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca(2+)-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca(2+) concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca(2+) and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca(2+)-sensitive dimerization. Point mutations within the EF-1 region can interfere with both the activation of the cyclase by the Ca(2+)-free form of GCAP-2 and the inhibition of retGC basal activity by the Ca(2+)-loaded GCAP-2. Our results strongly indicate that evolutionary conserved and GCAP-specific amino acid residues within the EF-1 can create a contact surface for binding GCAP-2 to the cyclase. Apparently, in the course of evolution GCAP-2 exchanged the ability of its first EF-hand motif to bind Ca(2+) for the ability to interact with the target enzyme.     

Studying the structure and regulation of soluble guanylyl cyclase

Soluble guanylyl cyclase acts as the receptor for the signaling molecule nitric oxide. The enzyme consists of two different subunits. Each subunit shows the cyclase catalytic domain, which is also conserved in the membrane-bound guanylyl cyclases and the adenylyl cyclases. The N-terminal regions of the subunits are responsible for binding of the prosthetic heme group of the enzyme, which is required for the stimulatory effect of nitric oxide (NO). The five-coordinated ferrous heme displays a histidine as the axial ligand; activation of soluble guanylyl cyclase by NO is initiated by binding of NO to the heme iron and proceeds via breaking of the histidine-to-iron bond. Recently, a novel pharmacological and possibly physiological principle of guanylyl cyclase sensitization was demonstrated. The substance YC-1 has been shown to activate the enzyme independent of NO, to potentiate the effect of submaximally effective NO concentrations, and to turn carbon monoxide into an effective activator of soluble guanylyl cyclase.     

Particulate guanylyl cyclases: multiple mechanisms of activation

Cyclic GMP (cGMP), a key messenger in several signal transduction pathways, is synthesized from GTP by a family of enzymes termed guanylyl cyclases, which are found in two forms: cytosolic (soluble) and membrane-bound (particulate). The past decade has brought significant progress in understanding the molecular mechanisms that underlie the regulation of particulate guanylyl cyclases and new members of their family have been identified. It has become more evident that the basic mechanism of catalysis of guanylyl cyclases is analogous to that recognized in adenylyl cyclases. Here we review the known basic mechanisms that contribute to the regulation of particulate guanylyl cyclases.     

Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.     

Internalization and trafficking of guanylyl cyclase/natriuretic peptide receptor-A

One of the principal loci involved in the regulatory action of atrial and brain natriuretic peptides (ANP and BNP) is guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), whose ligand-binding efficiency and GC catalytic activity vary remarkably in different target cells and tissues. In its mature form, NPRA resides in the plasma membrane and contains an extracellular ligand-binding domain, a single transmembrane region, and the intracellular protein kinase-like homology domain (KHD) and guanylyl cyclase (GC) catalytic domain. NPRA is a dynamic cellular macromolecule that traverses through different compartments of the cell through its lifetime. Binding of ligand to NPRA triggers a complex array of signal transduction events and accelerates the endocytosis. The endocytic transport is important in regulating signal transduction, formation of specialized signaling complexes, and modulation of specific components of internalization events. The present review describes the experiments which reveal the internalization of ligand-receptor complexes of NPRA, receptor trafficking and recycling, and delivery of both ligand-receptor molecules into subcellular compartments. The ligand-receptor complexes of NPRA are finally degraded within the lysosomes. The experimental evidence provides a consensus forum, which establishes the endocytosis, cellular trafficking, sequestration, and metabolic processing of ANP/NPRA complexes in the intact cells. The discussion is afforded to address the experimental insights into the mechanisms that cells utilize in modulating the delivery and metabolic processing of ligand-bound NPRA into the cell interior.     

Receptor guanylyl cyclase C (GC-C): regulation and signal transduction

Receptor guanylyl cyclase C (GC-C) is the target for the gastrointestinal hormones, guanylin, and uroguanylin as well as the bacterial heat-stable enterotoxins. The major site of expression of GC-C is in the gastrointestinal tract, although this receptor and its ligands play a role in ion secretion in other tissues as well. GC-C shares the domain organization seen in other members of the family of receptor guanylyl cyclases, though subtle differences highlight some of the unique features of GC-C. Gene knock outs in mice for GC-C or its ligands do not lead to embryonic lethality, but modulate responses of these mice to stable toxin peptides, dietary intake of salts, and development and differentiation of intestinal cells. It is clear that there is much to learn in future about the role of this evolutionarily conserved receptor, and its properties in intestinal and extra-intestinal tissues.     

Activation of soluble guanylyl cyclase by four-coordinate metalloporphyrins: evidence for a role for porphyrin conformation

Four-coordinate metalloporphyrins activate soluble guanylyl cyclase. Ni(II)PPIX and Cu(II)PPIX are high affinity activators, with activation constants of 24 and 17 nM, respectively. Both metalloporphyrins remain stably bound to the enzyme, enabling spectroscopic characterization of the Ni(II)- and Cu(II)-reconstituted protein. Electronic absorption and resonance Raman spectroscopy reveal that Ni(II)PPIX remains four coordinate when bound to soluble guanylyl cyclase. Analysis of the vibrational frequencies of the Ni(II)-reconstituted enzyme suggests that the protein imposes a constraining force on the porphyrin, favoring a planar conformation. Spectroscopic data for the Cu(II)-substituted protein are also consistent with four coordination. The intensification of the vibrational modes of the peripheral vinyl groups in both Ni(II)- and Cu(II)-reconstituted soluble guanylyl cyclase are consistent with a substantial influence of the protein on the porphyrin environment. Together these data support a model where activation of soluble guanylyl cyclase correlates with the absence of a metal-to-proximal histidine bond and with decreased conformational freedom for the tetrapyrrole in the activated state.     

The functional genomics of guanylyl cyclase/natriuretic peptide receptor-A: perspectives and paradigms

The cardiac hormones atrial natriuretic peptide and B-type natriuretic peptide (brain natriuretic peptide) activate guanylyl cyclase (GC)-A/natriuretic peptide receptor-A (NPRA) and produce the second messenger cGMP. GC-A/NPRA is a member of the growing family of GC receptors. The recent biochemical, molecular and genomic studies on GC-A/NPRA have provided important insights into the regulation and functional activity of this receptor protein, with a particular emphasis on cardiac and renal protective roles in hypertension and cardiovascular disease states. The progress in this field of research has significantly strengthened and advanced our knowledge about the critical roles of Npr1 (coding for GC-A/NPRA) in the control of fluid volume, blood pressure, cardiac remodeling, and other physiological functions and pathological states. Overall, this review attempts to provide insights and to delineate the current concepts in the field of functional genomics and signaling of GC-A/NPRA in hypertension and cardiovascular disease states at the molecular level.     

Dominant negative mutants of the Cdc2 kinase uncouple cell division from iterative plant development.

Because plant cells do not move and are surrounded by a rigid cell wall, cell division rates and patterns are believed to be directly responsible for generating new structures throughout development. To study the relationship between cell division and morphogenesis, transgenic tobacco and Arabidopsis plants were constructed expressing dominant mutations in a key regulator of the Arabidopsis cell cycle, the Cdc2a kinase. Plants constitutively overproducing the wild-type Cdc2a or the mutant form predicted to accelerate the cell cycle did not exhibit a significantly altered development. In contrast, a mutation expected to arrest the cell cycle abolished cell division when expressed in Arabidopsis, whereas some tobacco plants constitutively producing this mutant protein were recovered. These plants had a reduced histone H1 kinase activity and contained considerably fewer cells. These cells were, however, much larger and underwent normal differentiation. Morphogenesis, histogenesis and developmental timing were unaffected. The results indicate that, in plants, the developmental controls defining shape can act independently from cell division rates.