Functional chimeras of the phosphodiesterase 5 and 10 tandem GAF domains

The tandem GAF domain of hPDE10A uses cAMP as an allosteric ligand (Gross-Langenhoff, M., Hofbauer, K., Weber, J., Schultz, A., and Schultz, J. E. (2006) J. Biol. Chem. 281, 2841-2846). We used a two-pronged approach to study how discrimination of ligand is achieved in human (h)PDE10A and how domain selection in the phosphodiesterase GAF tandems is determined. First, we examined which functional groups of cAMP are responsible for purine ring discrimination. Changes at the C-6 ring position (removal of the amino group; chloride substitution) and at the N-1 ring position reduced stimulation efficacy by 80%, i.e. marking those positions as decisive for nucleotide discrimination. Second, we generated a GAF tandem chimera that consisted of the cGMP-binding GAF-A unit from hPDE5A1, which signals through cGMP in PDE5, and the GAF-B from hPDE10A1, which signals through cAMP in PDE10. Stimulation of the reporter enzyme exclusively was through the GAF-B domain of hPDE10A1 (EC(50) = 7 mum cAMP) as shown by respective point mutations. The PDE5 GAF-A domain in the chimera did not signal, and its function was reduced to a strictly structural role. Signaling was independent of the origin of the N terminus. Generating 10 additional PDE5/10 tandem GAF chimeras surprisingly demonstrated that the length-conserved linker in GAF tandems between GAF-A and GAF-B played an unforeseen decisive role in intramolecular signaling. Swapping the linker sections between PDE5 and PDE10 GAF tandem domains abrogated signaling completely pointing to specific domain interactions within GAF tandems, which are not visible in the available crystal structures with bound ligands.     

Characterization of the tandem GAF domain of human phosphodiesterase 5 using a cyanobacterial adenylyl cyclase as a reporter enzyme

We analyzed cGMP signaling by the human phosphodiesterase 5 (hPDE5) tandem GAF domain based on a functional activation assay. The C-terminal catalytic domain of the cyanobacterial adenylyl cyclase (AC) cyaB1 was used as a reporter enzyme. We demonstrate functional coupling between the hPDE5 GAF ensemble and the AC resulting in a chimera stimulated 10-fold by cGMP. The hPDE5 GAF domain has an inhibitory effect on AC activity, which is released upon cGMP activation. Removal of 109 amino acids from the N terminus resulted in partial disengagement of the GAF domain and AC, i.e. in a 10-fold increase in basal activity, and affected cGMP affinity. The Ser-102 phosphorylation site of hPDE5 increased cGMP affinity, as shown by a 5-fold lower K(D) for cGMP in a S102D mutant, which mimicked complete modification. The function of the NKFDE motif, which is a signature of all GAF domains with known cyclic nucleotide binding capacity, was elucidated by targeted mutations. Data with either single and double mutants in either GAF A or GAF B or a quadruple mutant affecting both subdomains simultaneously indicated that it is impossible to functionally assign cGMP binding and intramolecular signaling to either GAF A or B of hPDE5. Both subdomains are structurally and functionally interdependent and act in concert in regulating cycaB1 AC and, most likely, also hPDE5.     

Changes in adenylate cyclase and phosphodiesterase activities during the growth cycle of adult rat hepatocytes in primary culture

Long-term primary adult rat hepatocyte cultures show growth-state-dependent changes in adenylate cyclase and cAMP phosphodiesterase activities. Cellular adenylate cyclase activity decreases to undetectable levels within 1 day postplating, reappears on Days 4-5, and becomes maximal on Day 9. Membrane adenylate cyclase and cellular cAMP formation are insensitive to glucagon during log phase (Days 4-8) but not during lag (Day 1) or stationary phase (Day 12). Cyclic AMP phosphodiesterase activities (soluble and particulate) fall approximately equal to 70% by Day 2 but recover as proliferation begins. By contrast, the particulate phosphodiesterase assayed at 100 microM cAMP, decreased during Days 0-2. These observations simulate changes seen during liver proliferative transitions in vivo and, therefore, further support the use of these cultures as a developmental model.     

Human glucagon-like peptides 1 and 2 activate rat brain adenylate cyclase

Two human glucagon-like peptides, GLP-1 and GLP-2, which are coencoded with pancreatic glucagon in the preproglucagon gene, do not significantly inhibit [125I]monoiodoglucagon binding to rat liver and brain membranes and do not activate adenylate cyclase in liver plasma membranes. Nevertheless, GLP-1 and GLP-2 were each found to be potent stimulators of both rat hypothalamic and pituitary adenylate cyclase. Only 30-50 pM concentrations of each peptide elicited half-maximal adenylate cyclase stimulation. Our data suggest that GLP-1 and GLP-2 may be neurotransmitters and/or neuroendocrine effectors, which would account for their high degree of sequence conservation through vertebrate evolution.     

Effect of morphine sulfate on adenylate cyclase and phosphodiesterase activities in rat corpus striatum.

The effect of morphine sulfate (MS) on adenylate cyclase (AC) and phosphodiesterase (PDE) activities in the rat striatum was investigated. MS produced a dose-dependent increase in basal AC activity and did not alter sodium fluoride-induced stimulation both $\text{in}$$\text{vivo}$ (7.5–30 mg/kg, 1 hr pretreatment, i.p.) and $\text{in}$$\text{vitro}$ (1–100μM). $\text{in}$$\text{vitro}$, when submaximal effective concentrations of dopamine and MS were combined, there was an additive effect. However, administration of MS $\text{in}$$\text{vivo}$ did not alter dopamine-induced stimulation of AC activity. MS, $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ inhibited PDE activity in a dose-dependent manner only with the high substrate concentration (3.3 × 10⁻³M cyclic AMP). Preliminary results from this study indicate that morphine affects the cyclic AMP system.     

Dopamine-sensitive adenylate cyclase and cAMP phosphodiesterase in substantia nigra and corpus striatum of rat brain.

A dopamine-sensitive adenylate cyclase has been identified in substantia nigra of the rat brain. Low concentrations of dopamine markedly increased the accumulation of cyclic AMP while l-norepinephrine and isoproterenol had little effect at concentrations up to 100 μM. The phenothiazine, trifluoperazine, is a potent inhibitor of the s. nigral adenylate cyclase (I.C.₅₀ of 0.1 μM), while the adrenergic receptor blocking agents propranolol and phentolamine are ineffective. Basal activity of striatal adenylate cyclase and cAMP phosphodiesterase is approximately 3-fold higher than that found in substantia nigra.     

Age-related parallel decline in beta-adrenergic receptors, adenylate cyclase and phosphodiesterase activity in rat erythrocyte membranes.

The activities of three components of the cyclic AMP system were compared in erythrocyte ghost membranes prepared from the blood of rats at various ages from 1.5 to 15 months. The apparent number of β-adrenergic receptor sites, adenylate cyclase activity and cyclic AMP phosphodiesterase activity all declined about 50% in the membranes from the older animals (>5 months) as compared to the 1.5 month ones. The soluble erythrocyte phosphodiesterase also declined with age, but the decline did not parallel that of the membrane-associated activity. In contrast, there was no age-related change in the number of β-adrenergic receptors in membranes from the brains of the same animals. In erythrocyte ghosts, both the ratio of isoproterenol-stimulated adenylate cyclase activity to basal activity and the ratio of sodium fluoride-stimulated activity to basal were constant with age. Neither the dissociation constant for the β-adrenergic receptor nor the Michaelis constant for the phosphodiesterase changed as a function of age. Together with other data in the literature, these results suggest a close functional association of the components of the cyclic AMP system in the mature erythrocyte membrane, and support a physiological role for the cyclic AMP mediated β-adrenergic effects in the red blood cell.     

The size of adenylate cyclase and guanylate cyclase from the rat renal medulla

The size distribution of adenylate cyclase from the rate renal medulla solubilized with the nonionic detergents Triton X-100 and Lubrol PX was determined by gel filtration and by centrifugation in sucrose density gradients made up in H₂O or D₂O. The physical parameters of the predominant from in Triton X-100 are ²20,w, 5.9 S; Stokes radius, 62 A; partial specific volume (v ), 0.74 ml/g; mass, 159,000 daltons; f/f₀, 1.6; axial ratio (prolate ellipsoid), 11. For the minor form the values are : ²20,w, 3.0; Stokes radius, 28 A; mass, 38,000 daltons; f/f₀, 1.2. The corresponding values determined in Lubrol PX are similar. The value of v for the enzyme indicates that it binds less than 0.2 mg detergent/mg protein. Since interactions with detergents probably substitute for interactions with lipids and hydrophobic amino acid side chains, these findings suggest that no more than 5% of the surface of adenylate cyclase is involved in hydrophobic interactions with other membrance components. Thus, most of the mass of the enzyme is not deeply embedded in the lipid bilayer of the plasma membrance. Similar studies have been performed on the soluble guanylate cyclase of the rate renal medulla. In the absence of detergent, the molecular properties of this enzyme are: ^s20,w, 6.3 S; Stokes radius, 54 A, v , 0.75 ml/g; mass, 154,000 daltons f/f₀, 1.4; axial ratio, 7. The addition of 0.1% Lubrol PX to this soluble enzyme increases its activity two- to fourfold and changes the physical properties to : ^s20,w, 5.5 S; Stokes radius, 62 A; v , 0.74 ml/g; mass, 148,000 daltons; f/f₀, 1.6; axial ratio, 11. These results show that Lubrol PX activates the enzyme by causing a conformational change with unfolding on the polypeptide chain. Guanylate cyclase from the particulate cell fraction can be solubilized with Lubrol PX but has properties quite different from those of the enzyme in the soluble cell fraction. It is a heterogeneous aggregrate with ^s20,w, 10 S; Stokes radius, 65 A; mass about 300,000 daltons. The conditions which solubilize guanylate cyclase also solubilize adenylate cyclase and the two activities can be separated on the same sucrose gradient.     

The regulatory tandem domains of human phosphodiesterases 1 and 4 regulate a cyanobacterial adenylyl cyclase

Human phosphodiesterase 1 is regulated by a tandem of N-terminal calmodulin/Ca(2+)-binding domains. We grafted the tandems from hPDE1A3 and -B1 onto the cyanobacterial adenylyl cyclase CyaB1 thus replacing an intrinsic tandem GAF-domain. Cyclase activity was stimulated by Ca(2+)/calmodulin 1.9 to 4.4-fold, i.e. similarly as reported for hPDE1 regulation. hPDE4 long isoforms are activated by phosphorylation of a serine located in a conserved RRESF motif in a tandem of N-terminal upstream-conserved regions (UCR). We grafted the UCR tandems from hPDE4A4, -B1, and -D3 onto the CyaB1 cyclase as a reporter enzyme. Activity was enhanced 1.4 to 4.5-fold by respective phosphomimetic (S/D) point mutations. Similarly, cyclase activity was increased 2.5-fold by phosphorylation of the chimera with the PDE4D3 UCR tandem by cAMP-dependent protein kinase. We propose a common mechanism of activation in mammalian phosphodiesterases containing N-terminal tandem regulatory domains. A downstream region is suggested to alternate between random and ordered conformations and to enable switching between inactive, the catalytic domain occluding PDE homodimers and active monomeric PDE catalytic domains.     

Changes in rat testicular adenylate cyclase activities and gonadotrophin binding during unilateral experimental cryptorchidism

Unilaterally cryptorchid rats were examined at 3, 8, 15, 22 and 28 days after operation. There was a selective decrease in the adenylate cyclase (ATP pyrophosphate--lyase (cyclizing), EC 4.6.1.1) responses to gonadotrophin stimulation in the abdominal testis. This was associated with a parallel decrease in specific FSH and LH binding. There was no reduction in the response of testicular adenylate cyclases to prostaglandin (PG) E-1 or fluoride stimulation, indicating that both the GTP binding protein (N-component) and the catalytic subunit of the adenylate cyclase complexes were intact. The reduction in FSH-responsive adenylate cyclase activity in the abdominal testis was not due to a change in the Km for adenylate cyclase activation, but was due to a reduction in maximal velocities. Unilateral cryptorchidism was also associated with a rapid decline in soluble Mn2+-dependent adenylate cyclase activity in germ cells (spermatids). By 3 days after operation there was an 82% decrease in germ cell adenylate cyclase activity. The loss of soluble Mn2+-dependent adenylate cyclase activity was associated with a parallel decrease in Sertoli cell secretion of androgen binding protein, indicating that Sertoli cell factors may be important for the maintenance of germ cell adenylate cyclase activity. The desensitization of the gonadotrophin--responsive adenylate cyclases and the loss of gonadotrophin receptors in Leydig and Sertoli cells were not due to changes in plasma gonadotrophin values because LH concentrations were within normal limits and plasma FSH was only marginally elevated in the cryptorchid rats. No significant alterations of any of these parameters were seen in the scrotal testis of unilaterally cryptorchid rats when compared to values for intact controls.     

Dopamine-inhibited adenylate cyclase in female rat adenohypophysis

A dopamine-inhibited adenylate cyclase has been demonstrated in anterior pituitary gland of adult female rats, lactating and not lactating. This inhibitory effect was completely GTP dependent. In contrast, in the adenohypophysis of male rats, dopamine had no detectable effect on adenylate cyclase activity. In female rats the inhibition of the enzyme appears mediated by specific dopaminergic receptors: the effect of dopamine was mimicked by the dopaminergic agonists apomorphine and the ergot derivative CH 29–717, while norepinephrine was much less potent. On the other hand, the dopaminergic antagonists trifluoperazine and sulpiride competitively antagonized the dopamine inhibition of the adenylate cyclase. The possibility that the dopamine-inhibited enzyme is located in mammotrophs appears supported 1) by its observation in the female rat pituitary, which contains this type of cells in much larger proportion than the male gland (33–38% vs. < 5%); 2) by the pharmacological similarity between the dopaminergic receptors mediating the adenylate cyclase inhibition (this work) and those regulating prolactin release (which have been characterized in previous studies). The well known inhibition of prolactin release brought about by dopamine could therefore be mediated, at least in part, by a decrease in the intracellular level of cAMP.     

Changes in isoproterenol-stimulated adenylate cyclase activity in rat testicular tissue during cryptorchidism and after orchidopexy

Cryptorchidism was associated with increased responsiveness of the isoproterenol-sensitive adenylate cyclase in membrane particles from rat testis. Abdominal testes from uni- and bilaterally cryptorchid rats showed the same activities. The change in isoproterenol-responsive adenylate cyclase was independent of the age at which the animals were made cryptorchid. The isoproterenol response was maximal 3-4 weeks after the rats were made cryptorchid. By 2-3 months after orchidopexy the isoproterenol response in the rat testis had decreased to normal control values.     

Active site specificity of the adenylate cyclase catalytic unit from bovine caudate nucleus

ATP analogues were used to study the active site specificity of the catalytic unit (C) of solubilized and partially purified bovine brain caudate nucleus adenylate cyclase. Phenylenediamine ATP (PD-ATP), 8-azido ATP (8-N3ATP), chromium(III) 3'-beta-alanylarylazido ATP (CrATPa), and 2',3'-dialdehyde ATP (oATP) are competitive inhibitors of C in the presence of the substrate MnATP and the activator forskolin. (Km for MnATP is 50 +/- 11 microM, n = 13). The Ki values determined under initial velocity conditions are: PD-ATP, Ki = 695 +/- 60 microM, n = 5; 8-N3ATP, Ki = 155 +/- 23 microM, n = 5; CrATPa, Ki = 7 +/- 3 microM, n = 2; oATP, Ki = 42 +/- 5 microM, n = 3. Irradiation of 100 microM 8-N3ATP by UV light (254 nm) causes the first-order loss of reagent either in the presence or absence of C. Concomitant irreversible inhibition of C in the presence of 8-N3ATP was more complex and asymptotically approached 50% within 4-6 min. Loss of C activity in controls was 10-20%. The fraction of C covalently modified by 8-N3ATP, alpha, was calculated for each time point of irradiation for an increasing initial concentration ([A]o) of 8-N3ATP. Extrapolated to infinite time of photolysis, the value of alpha reached a final level, termed alpha t whose magnitude depended on [A]o. From these data we calculated an apparent KD of 4.5 microM for 8-N3ATP. ATP protected against the irreversible inhibition due to 8-N3ATP. These data are most consistent with a mechanism of photoaffinity labeling involving equilibrium binding and covalent insertion of 8-N3ATP into the active site. These results indicate that the active site binds analogues of ATP which are considerably modified in the adenine, ribose, and gamma-phosphate portions and that the affinity of C for these analogues is within an order of magnitude of the Km for ATP.     

Changes in the properties of adenylate cyclase from guinea pig lungs in tuberculosis

Changes in the properties of adenylate cyclase from the lungs of tuberculotic guinea pigs were revealed. The number of beta-adrenergic receptors in the lungs was found to be reduced by 30% at the second and by 70% at the third stage of the disease. The degree and the value of Ka for adenylate cyclase activation by isoproterenol remained thereby unchanged. The basal activity of adenylate cyclase was increased by 20% against the control level at the second stage and decreased by 20% at the third stage of the disease. At these periods, the stimulating effects of guanylyl imidodiphosphate, NaF and forskolin on lung adenylate cyclase were diminished. The experimental results point to the significant role of the enzymes of cAMP metabolism and reflect the course of the tuberculosis process in experimental animals.     

Multiple localizations of adenylate cyclase in rat hippocampus

Summary A new method for the histochemical demonstration of adenylate cyclase activity, introduced and biochemically tested by Poeggel et al. (1981a), was employed in nervous tissue. Using this method a multiple pattern of activity was detectable. Activity occurs in nervous as well as glial elements. Biochemical results and physiological conclusions could be confirmed by ultrahistochemical visualization of adenylate cyclase activity in nervous tissue. The specificity of the reaction is controlled by a number of variations of the incubation methods.     

Activation of adenylate cyclase from rat striatum and tuberculum olfactorium by adenosine

Adenosine caused a dose-dependent stimulation of adenylate cyclase in homogenates from rat striatum and tuberculum olfactorium (200 and 300% stimulation by 100 muM adenosine). The effect of adenosine was not antagonized by haloperidol. Subcellular fractionation suggested that adenosine stimulates a different adenylate cyclase than dopamine. Basal adenylate cyclase activity in freshly prepared homogenates was reduced by dialysis and by the addition of adenosine deaminase. Basal adenylate cyclase activity was enchanced by papaverine and dipyridamole, but reduced by theophylline and isobutylmethylxanthine. The results are compatible with the opinion that endogenous adenosine is capable of activating adenylate cyclase in these areas of the rat brain.