cGMP-binding Domains Research Articles

An Essential Aspartic Acid at Each of Two Allosteric cGMP-binding Sites of a cGMP-specific Phosphodiesterase

The amino acid sequences of all known cGMP-binding phosphodiesterases (PDEs) contain internally homologous repeats (a and b) that are 80-90 residues in length and are arranged in tandem within the putative cGMP-binding domains. In the bovine lung cGMP-binding, cGMP-specific PDE (cGB-PDE or PDE5A), these repeats span residues 228-311 (a) and 410-500 (b). An aspartic acid (residue 289 or 478) that is invariant in repeats a and b of all known cGMP-binding PDEs was changed to alanine by site-directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE displayed identical cGMP-binding kinetics, with approximately 1.8 microM cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (Kd > 10 microM) and the D478A mutant showed increased affinity for cGMP (Kd approximately 0.5 microM) as compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an identical curvilinear profile of cGMP dissociation which was consistent with the presence of distinct slowly dissociating (koff = 0.26 h-1) and rapidly dissociating (koff = 1.00 h-1) sites of cGMP binding. In contrast, the D289A mutant displayed a single koff = 1.24 h-1, which was similar to the calculated koff for the fast site of WT and native cGB-PDE, and the D478A mutant displayed a single koff = 0.29 h-1, which was similar to that calculated for the slow site of WT and native cGB-PDE. These results were consistent with the loss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in repeat b of the D478A mutant, suggesting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for interaction with cGMP at each site.

Identification and partial characterization of a domain in CFTR that may bind cyclic nucleotides directly.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is activated by cAMP-dependent phosphorylation. CFTR channel activity is also stimulated by cGMP-dependent protein kinase and protein kinase C. Here, we show that CFTR channel activation by cGMP may also occur directly. In oocytes from one-third of Xenopus donors, the activation of CFTR by cGMP averaged 87% of the level achieved by cAMP. The currents activated by either cyclic nucleotide displayed similar current-voltage relationships, kinetics, pharmacology and halide selectivity. Sequential stimulation by cAMP and cGMP was not additive, suggesting that both cyclic nucleotides activate the same channel; cGMP was one order of magnitude more potent than cAMP, and its action was insensitive to protein kinase inhibitors. Analysis of the amino-acid sequence of CFTR revealed a domain in the amino-terminal portion of the third cytoplasmic loop that resembles a class of cyclic-nucleotide-binding domains related to that of the catabolite-gene activator protein, CAP. Two CFTR residues in this domain--Val397 and Lys420--were identified which, when changed to alanine, altered the response to cGMP independently of the response to cAMP. We conclude that direct cyclic nucleotide binding may play a role in channel gating of CFTR. The cGMP-binding domain may provide a useful target for pharmacologic intervention in cystic fibrosis.

The cGMP-gated channel of photoreceptor cells: Its structural properties and role in phototransduction

AbstractThe cyclic GMP-gated channel responds to changes in free intracellular cGMP, and as a result, it plays a central role in the phototransduction process in rod and cone photoreceptor cells. Recent biochemical, immunochemical, and molecular biology studies indicate that this channel consists of a complex of two distinct subunits and one or more associated proteins. Primary structural analysis indicates that the a and (3 subunits contain a cGMP-binding domain, an even number of membrane-spanning segments, a voltage sensor motif and a pore region. The latter two features are found in voltage-gated channels and suggest that these two classes of channels have evolved from the same ancestral channel protein. A working model for the membrane topography of the channel subunits is proposed based on immunogold labeling studies and sequence analysis. Recent studies also indicate that calmodulin binds to the 240 kDa protein of the channel complex and modulates the sensitivity of the channel for cGMP in a Ca2+-dependent manner. The molecular properties of the channel complex and the possible role of Ca2+-calmodulin modulation of the channel during photoactivation and photorecovery are discussed in relation to the current mechanism of phototransduction in photoreceptor cells

Cross-activation: overriding cAMP/cGMP selectivities of protein kinases in tissues.

cAMP- and cGMP-dependent protein kinases are homologous proteins and are predicted to exhibit very similar three-dimensional structures. Their cyclic nucleotide binding domains share a high degree of amino acid sequence identity. cAMP- and cGMP-dependent protein kinases are activated relatively specifically by cAMP and cGMP, respectively; and a single alanine-threonine difference between cAMP- and cGMP-binding domains partially accounts for this specificity. Thus, it would be expected that cAMP and cGMP mediate separate physiological effects. However, owing in part to the lack of absolute specificity of either enzyme and to the relatively high level of cAMP or cGMP in certain tissues, it is also possible that either cyclic nucleotide could cross-activate the other kinase. Increases in either cAMP or cGMP cause pig coronary artery relaxation. However, only cGMP-dependent protein kinase specific cyclic nucleotide analogues are very effective in causing relaxation, and cAMP elevation in arteries treated with isoproterenol or forskolin activates cGMP-dependent protein kinase, in addition to cAMP-dependent protein kinase. Conversely, increases in either cAMP or cGMP cause Cl- secretion in T-84 colon carcinoma cells, and the cGMP level in T-84 cells can be elevated sufficiently by bacterial enterotoxin to activate cAMP-dependent protein kinase. These results imply specific regulation of cAMP- and cGMP-dependent protein kinases by the respective cyclic nucleotides, but either cyclic nucleotide is able to cross-activate the other kinase in certain tissues.

Affinity purification of the photoreceptor cGMP-gated cation channel

The cGMP-gated cation channel is a member of a new family of channel proteins that appear to be directly regulated by cyclic nucleotides. A protein with a subunit molecular mass of 78 kDa that exhibits cGMP-gated calcium flux when reconstituted into phospholipid-containing vesicles has been purified using 8-bromo-cGMP-agarose affinity chromatography. This channel activity is sensitive to the inhibitor l-cis-diltiazem. Treatment of the reconstituted channel with trypsin abolishes the l-cis-diltiazem sensitivity. Apparent endogenous proteolysis can also result in smaller molecular weight polypeptides that exhibit cGMP-gated channel activity but are insensitive to l-cis-diltiazem. These results show that the channel can bind cGMP and that it contains a l-cis-diltiazem inhibitory domain that is distinct from the cGMP-binding domain.

Characterization of a purified bovine lung cGMP-binding cGMP phosphodiesterase.

A bovine lung cGMP-binding phosphodiesterase (cG-BPDE) was purified to homogeneity and exhibited specific cGMP hydrolytic (Km = 5.6 microM) and cGMP binding (half-maximum approximately 0.2 microM) activities which comigrated throughout the purification. A chimeric structure was suggested for cG-BPDE since DEAE chromatography of a partial alpha-chymotryptic digest of cG-BPDE separated cGMP-binding fragments from a cGMP hydrolytic fragment. Native cG-BPDE (178 kDa) appeared to be a homodimer comprised of two 93-kDa subunits. The order of potency of inhibitors of cG-BPDE hydrolysis of cGMP was as follows: zaprinast greater than dipyridamole greater than 3-isobutyl-1-methyl-8-methoxymethylxanthine greater than 3-isobutyl-1-methylxanthine greater than cilostamide greater than theophylline greater than rolipram. Minimum [3H]cGMP binding stoichiometry was 0.93 mol of cGMP bound/mol of monomer, but [3H]cGMP dissociation from cG-BPDE in the presence of excess unlabeled cGMP was curvilinear, suggesting multiple cGMP-binding sites. Two chymotryptic cGMP-binding fragments of 35 and 45 kDa were specifically photoaffinity labeled with [32P] cGMP, exhibited [3H]cGMP association and dissociation behavior indistinguishable from native cG-BPDE, and each had the amino-terminal sequence: Thr-Ser-Pro-Arg-Phe-Asp-Asn-Asp-Glu-Gly-. Cochromatography of the two cGMP-binding fragments suggested that both a dimerization domain and a cGMP-binding domain were located in a 35-kDa segment of cG-BPDE. Increased [3H]cGMP binding to or [32P]cGMP photoaffinity labeling of cG-BPDE binding sites in the presence of hydrolytic site-specific cyclic nucleotide analogs suggested communication between hydrolytic and binding sites. The principle of reciprocity thus predicts that cGMP binding to the binding sites may affect the hydrolytic site. In the presence of cGMP, the binding fragments or native cG-BPDE exhibited an electronegative shift on high performance liquid chromatography-DEAE, consistent with a cGMP-induced change in cG-BPDE conformation.

Identification of a noncatalytic cGMP-binding domain conserved in both the cGMP-stimulated and photoreceptor cyclic nucleotide phosphodiesterases.

Partial amino acid sequence has been determined for the cone, alpha' subunit of the bovine photoreceptor cyclic nucleotide phosphodiesterase (PDE) and deduced from nucleotide sequences of a partial cDNA clone. These sequences identify the alpha' subunit as the product of a gene that is distinct from those encoding the alpha or beta subunits of the membrane-associated rod photoreceptor PDE. Comparisons between the recently determined cGMP-stimulated-PDE sequence and those of the alpha and alpha' photoreceptor PDE subunits reveal an unexpected sequence similarity. In addition to the catalytic domain conserved in eukaryotic PDEs, all three PDEs possess a second conserved segment of approximately 340 residues that contains two internally homologous repeats. Limited proteolysis and direct photolabeling studies indicate that the noncatalytic, cGMP-binding site(s) in the cGMP-stimulated PDE is located within this conserved domain, suggesting that it also may serve this function in the photoreceptor PDEs. Moreover, other PDEs that do not bind cGMP at noncatalytic sites do not contain this conserved domain. The function of the conserved segment in the photoreceptor PDEs is not known, but the homology to allosteric sites of the cGMP-stimulated PDE suggests a role in cGMP binding and modulation of enzyme activity.

Predicted structures of the cGMP binding domains of the cGMP-dependent protein kinase: a key alanine/threonine difference in evolutionary divergence of cAMP and cGMP binding sites

Mammalian cGMP- and cAMP-dependent protein kinase show considerable similarity in amino acid sequence, although they specifically bind different cyclic nucleotides. Results of cGMP analogue binding experiments, combined with modeling of the cGMP binding sites by analogy to the structure of the homologous catabolite gene activator protein, suggest that a threonine residue forms a hydrogen bond with the 2-NH2 of cGMP. This threonine is invariant in all cGMP binding domains, but the corresponding residue in 23 out of 24 cAMP binding sites of protein kinases is alanine, which cannot form the same hydrogen bond. This alanine/threonine difference has the potential for discriminating between cAMP and cGMP and may be important in the evolutionary divergence of cyclic nucleotide binding sites.

CGMP-dependent Protein Kinase Genes in Drosophila

Two Drosophila genes encoding products related to cGMP-dependent protein kinase have been isolated by cross-hybridization to a Drosophila cAMP-dependent protein kinase catalytic subunit gene. Both genes encode products with putative cGMP binding and kinase domains on the same polypeptide chain, as found for the prototypical bovine lung cGMP-dependent protein kinase. The deduced product of one gene (DG1; cytological position, 21D) is 14% larger than the bovine enzyme and differs substantially in sequence at the amino terminus, the region responsible in the bovine enzyme for dimerization. The second gene (DG2; cytological position, 24A) is transcribed into three major RNA species of different size. The largest (DG2; T1) and smallest (DG2;T3) RNAs encode overlapping polypeptides of similar sequence to the whole length of bovine lung cGMP-dependent protein kinase. The translation product of the third major RNA (DG2;T2) lacks sequences similar to those that constitute the dimerization and kinase inhibitory domains of the bovine enzyme. The percentage amino acid identity between DG1 or DG2 and bovine lung cGMP-dependent protein kinase is 55 and 64%, respectively. A common progenitor of the two cGMP-dependent protein kinase genes, DG1 and DG2, is strongly suggested by the conserved positions of introns in these genes.

Properties of a cGMP-dependent Monomeric Protein Kinase from Bovine Aorta

A form of cGMP-dependent protein kinase (cGK) that was different from previously described cGK was purified from bovine aorta smooth muscle. The partial amino-terminal sequencing of this enzyme indicated that it was derived by endogenous proteolysis of the type I beta isozyme of cGK. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this form migrated as a smaller protein (Mr = 70,000) than the parent cGK (Mr = 80,000), and since the calculated nondenatured Mr was approximately 89,000 compared to Mr = 170,000 for the dimeric native enzyme, it represented a monomeric form of cGK. The monomer bound approximately 2 mol of [3H]cGMP per mol of monomer, although it had only one rapid component in [3H]cGMP dissociation assays as compared to one rapid and one slow component for the native cGK. The specific catalytic activity of the kinase was similar to that of the native enzyme, suggesting that the catalytic domain was essentially intact. The monomeric cGK incorporated significant 32P when incubated with Mg2+ and [gamma-32P]ATP in the presence of cGMP, although the phosphorylation proceeded at a slower rate than that obtained with native cGK. In contrast to previous reports of monomeric forms of cGK, this monomer was highly cGMP-dependent, although it had a slightly higher Ka (0.8 microM) for cGMP than that of the native enzyme (0.4 microM) and a low Hill coefficient of 1.0 (1.6 for the native enzyme). The cGMP dependence of the monomer did not decrease with dilution, implying that the cGMP dependence was not due to monomer-monomer interactions in the assay. The results indicated that the catalytic domain, cGMP binding domain(s), and inhibitory domain of cGK interact primarily within the same subunit rather than between subunits of the dimer as previously hypothesized for dimeric cGK.

Guanosine cyclic 3',5'-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families.

The amino acid sequence of bovine lung cGMP-dependent protein kinase has been determined by degradation and alignment of two primary overlapping sets of peptides generated by cleavage at methionyl or arginyl residues. The protein contains 670 residues in a single N alpha-acetylated chain corresponding to a molecular weight of 76 331. The function of the molecule is considered in six segments of sequence which may correspond to four folding domains. From the amino terminus, the first segment is related to the dimerizing property of the protein. The second and third segments appear to have evolved from an ancestral tandem internal gene duplication, generating twin cGMP-binding domains which are homologous to twin domains in the regulatory subunits of cAMP-dependent protein kinase and to the cAMP-binding domain of the catabolite gene activator of Escherichia coli. The fourth and fifth segments may comprise one domain which is homologous to the catalytic subunits of cAMP-dependent protein kinase, of calcium-dependent phosphorylase b kinase, and of certain oncogenic viral protein tyrosine kinases. The regulatory, amino-terminal half of cGMP-dependent protein kinase appears to be related to a family of smaller proteins that bind cAMP for diverse purposes, whereas the catalytic, carboxyl-terminal half is related to a family of protein kinases of varying specificity and varying sensitivity to regulators. These data suggest that ancestral gene splicing events may have been involved in the fusion of two families of proteins to generate the allosteric character of this chimeric enzyme.