Analogues of parathyroid hormone modified at positions 3 and 6. Effects on receptor binding and activation of adenylyl cyclase in kidney and bone.

Predictive and spectroscopic methods were used to develop a model of the structures of the 1-34 peptides of parathyroid hormone (PTH) and the PTH-related protein (PTHrP). Circular dichroism (CD) studies of bovine PTH-(1-34) and human PTHrP-(1-34)amide in the presence of trifluoroethanol suggest the presence of 24-26 alpha-helical residues. For both peptides, interactions between amino- and carboxyl-region alpha-helices are predicted to result in a hydrophobic core with externally facing hydrophilic residues that include probable determinants of receptor binding and activation. Two such residues, Ser3 and Gln6, are conserved in all known members of the PTH/PTHrP family. We have synthesized 13 novel analogues of bovine PTH-(1-34) monosubstituted at positions 3 and 6 and have determined their biological activities in renal and bone cell radioreceptor and adenylyl cyclase assays. Position 3 analogues displayed biological activity that was reduced in direct proportion to the volume of the substituent side-chain. Position 6 analogues also displayed reduced biological activity, but no simple correlation with side-chain volume or hydrophobicity was evident. The analogues fully displaced labeled PTH from binding sites in renal membranes and bone cells, but [Phe3]bPTH-(1-34), [Tyr3]bPTH-(1-34), [Phe6] bPTH-(1-34), and [Ser6]bPTH-(1-34) were only partial agonists in one or both adenylyl cyclase assays. Of these, [Phe3]bPTH-(1-34) and [Phe6]bPTH-(1-34) were tested for antagonist activity and were found to inhibit the activation of adenylyl cyclase in response to bPTH-(1-34) or hPTHrP-(1-34)amide. These results indicate that positions 3 and 6 contribute important determinants of PTH receptor binding and activation. Modification at these positions represents a novel approach to the development of antagonists of PTH action.     

Role of soluble adenylyl cyclase in the heart

This review discusses the potential place of soluble adenylyl cyclase (sAC) in the framework of signaling in the cardiovascular system. cAMP has been studied as a critical and pleiotropic second messenger in cardiomyocytes, endothelial cells, and smooth muscle vascular cells for many years. It is involved in the transduction of signaling by catecholamines, prostaglandins, adenosine, and glucagon, just to name a few. These hormones can act via cAMP by binding to a G protein-coupled receptor on the plasma membrane with subsequent activation of a heterotrimeric G protein and its downstream effector, transmembrane adenylyl cyclase. This has long been the canonical standard for cAMP production in a cell. However, the relatively recent discovery of a unique source of cAMP, sAC, creates the potential for a shift in this signaling paradigm. In fact, sAC has been shown to play a role in apoptosis in coronary endothelial cells and cardiomyocytes. Additionally, it links nutrient utilization with ATP production in the liver and brain, which suggests one of many potential roles for sAC in cardiac function. The possibility of producing cAMP from a source distal to the plasma membrane provides a critical new building block for reconstructing the cellular signaling infrastructure.     

Gbetagamma activation site in adenylyl cyclase type II. Adenylyl cyclase type III is inhibited by Gbetagamma

The Gbetagamma complex of heterotrimeric G proteins is the most outstanding example for the divergent regulation of mammalian adenylyl cyclases. The heterodimeric Gbetagamma complex inhibits some isoforms, e.g. ACI, and stimulates the isoforms ACII, -IV, and -VII. Although former studies identified the QEHA region located in the C2 domain of ACII as an important interaction site for Gbetagamma, the determinant of the stimulatory effect of Gbetagamma has not been detected. Here, we identified the C1b domain as the stimulatory region using full-length adenylyl cyclase. The relevant Gbetagamma signal transfer motif in IIC1b was determined as MTRYLESWGAAKPFAHL (amino acids 493-509). Amino acids of this PFAHL motif were absolutely necessary for ACII to be stimulated by Gbetagamma, whereas they were dispensable for Galpha(s) or forskolin stimulation. The PFAHL motif is present in all three adenylyl cyclase isoforms that are activated by Gbetagamma but is absent in other adenylyl cyclase isoforms as well as other known effectors of Gbetagamma. The emerging concept of two contact sites on different molecule halves for effective regulation of adenylyl cyclase is discussed.     

Conformation-dependent activation of type II adenylyl cyclase by protein kinase C

Phorbol ester treatment enhanced the catalytic activity of type II adenylyl cyclase overexpressed in insect cells. In cells coexpressing type II adenylyl cyclase and protein kinase C-α, type II adenylyl cyclase catalytic activity was higher even in the absence of phorbol ester treatment; phorbol ester treatment further and markedly enhanced type II adenylyl cyclase catalytic activity. However, this enhancement, either by phorbol ester treatment or by coexpression of protein kinase C-α, was lost following membrane solubilization with detergents. This attenuation was unaffected by phosphatase inhibitor or salts. In contrast, membrane solubilization did not affect forskolin-stimulated type II adenylyl cyclase catalytic activity. Purified type II adenylyl cyclase was stimulated by forskolin and Gsα, but not by protein kinase C-α. Therefore, a specific mammalian protein kinase C isoenzyme can activate type II adenylyl cyclase, but the mechanism clearly differs from that underlying either Gsα- or forskolin-mediated stimulation. J. Cell. Biochem. 64:492–498. © 1997 Wiley-Liss, Inc.     

Effects of cholera toxin and 5'-guanylylimidodiphosphate on human spermatozoal adenylate cyclase activity

The effects of cholera toxin and 5′-guanylylimidodiphosphate (Gpp(NH)p) on human spermatozoal adenylate cyclase activity were tested. Cholera toxin had no demonstrable effect on adenylate cyclase activity in human spermatozoa at concentrations between 5 and 20 μg/ml, whether the toxin was preincubated with intact spermatozoa between 5 min and 5 h prior the adenylate cyclase assay, or was added to lysed spermatozoa, where the adenylate cyclase would be accessible to the toxin. In contrast, Gpp(NH)p at concentrations between 10 and 100 μM was effective in activating human spermatozoal adenylate cyclase activity.     

Phenolamine-dependent adenylyl cyclase activation in Drosophila Schneider 2 cells

Drosophila Schneider 2 (S2) cells are often employed as host cells for non-lytic, stable expression and functional characterization of mammalian and insect G-protein-coupled receptors (GPCRs), such as biogenic amine receptors. In order to avoid cross-reactions, it is extremely important to know which endogenous receptors are already present in the non-transfected S2 cells. Therefore, we analyzed cellular levels of cyclic AMP and Ca2+, important second messengers for intracellular signal transduction via GPCRs, in response to a variety of naturally occurring biogenic amines, such as octopamine, tyramine, serotonin, histamine, dopamine and melatonin. None of these amines (up to 0.1 mM) was able to reduce forskolin-stimulated cyclic AMP production in S2 cells. Furthermore, no agonist-induced calcium responses were observed. Nevertheless, the phenolamines octopamine (OA) and tyramine (TA) induced a dose-dependent increase of cyclic adenosine monophosphate (AMP) production in S2 cells, while serotonin, histamine, dopamine and melatonin (up to 0.1 mM) did not. The pharmacology of this response was similar to that of the octopamine-2 (OA2) receptor type. In addition, this paper provides evidence for the presence of an endogenous mRNA encoding an octopamine receptor type in these cells, which is identical or very similar to OAMB. This receptor was previously shown to be positively coupled to adenylyl cyclase.     

Role of cysteine sulfhydryl groups in activation of adenylyl cyclase signaling system by biogenic amines in molluscan and rat tissues

It has been shown that in smooth muscles of the freshwater bivalve molluscAnodonta cygnea as well as in skeletal muscles and brain striatum of rats a blocker of SH-groups,para-chlormercury benzoate (ChMB), and an alkylating agent,N-ethylmaleimide, inhibit both the basal adenylyl cyclase (AC) activity and the activity of the enzyme stimulated by non-hormonal agents (NaF, Gpp[NH]p) and by hormonal agents such as serotonin (mollusc muscles, rat brain) or isoproterenol (rat muscles and rat brain). The inhibitory effects of ChMB andN-ethylmaleimide on AC are partly eliminated by an SH-group containing reagent, β-mercaptoethanol (ME, 5 mM). Restoration of the basal and of the stimulated enzyme activity inhibited by ME is better in the case of the ChMB than of theN-ethylmaleimide action. It has also been found that ME stimulates both the basal and the stimulated by non-hormonal agents AC activity. In the presence of ME the hormonal stimulating effects on the enzyme are also preserved, except for the effect of isoproterenol on AC in rat skeletal muscles; this effect is inhibited by ME. Potentiation of the stimulating effect of the hormones on AC by Gpp[NH]p is only preserved in the molluscan smooth muscles (the effect of serotonin—90%). The data obtained indicate that cysteine sulfhydryl groups play a key role in hormonal regulation of the functional activity of the components of the hormone-sensitive adenylyl cyclase signaling system.     

Differences in response to activation of adenylyl cyclase by various stimulants in human myocardium

The response of adenylyl cyclase complex in human atrial tissue removed at corrective surgery of normoxemic and hypoxemic congenital heart defects in children to various stimulants was evaluated and related to the oxygenation state of the myocardium. When comparing response to stimulation in normoxemic and hypoxemic atria a higherbasal as well as stimulated adenylyl cyclase activity was found in hypoxemic atria; an insignificant stimulatory effect of isoprenaline in normoxemic hearts became significant in the atria of hypoxemic patients. Hypoxemic samples also showed two times higher activity when the total catalytic activity was evaluated by the stimulation with forskolin. Higher stimulatory effect of Gpp/NH/p was also observed in hypoxemic than in normoxemic state. Increased adenylyl cyclase activity might represent one of adaptive mechanisms to hypoxemia in patients with congenital heart defects.     

Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system

Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein -subunit (G_s), G-protein β₁γ₂ subunit dimer (Gβ₁γ₂), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, G_s, Gβ₁γ₂, and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other G_s-coupled receptors together with G_s, Gβ₁γ₂, and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, G_s and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.     

Type-specific regulation of adenylyl cyclase. Selective pharmacological stimulation and inhibition of adenylyl cyclase isoforms

Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2'-deoxyadenosine 3'-monophosphate, a classical and 3'-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel non-nucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination.     

Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons

In immortalized GnRH neurons, cAMP production is elevated by increased extracellular Ca2+ and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with nifedipine, consistent with the expression of adenylyl cyclase type I (AC I). Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of G(i)-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to G(i)-related proteins. The stimulation of cAMP production by activation of endogenous LH receptors is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of Ca2+-stimulated AC I. Furthermore, the Ca2+ influx-dependent activation of AC I acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production.     

Regulation of adenylyl cyclase isoforms by N-alkanols

We examined the effect of n-alkanols on adenylyl cyclase isoforms (types II and V) overexpressed in insect cells. Ethanol stimulated the type II isoform but not the type V isoform. Ethanol stimulated type II adenylyl cyclase greater than GTPγS, and the treatment of the membrane with GDPβS or cholera toxin did not affect this stimulation. Other n-alkanols inhibited type V adenylyl cyclase activity in proportion to their lipophilic potency. In contrast, type II adenylyl cyclase was stimulated by weakly lipophilic n-alkanols and inhibited by strongly lipophilic n-alkanols. When solubilized membranes and purified preparations were used, all the n-alkanols inhibited type II adenylyl cyclase. Our data suggest that n-alkanols regulated adenylyl cyclase isoform-dependently. Stimulation of the type II isoform was independent from the interaction with Gsα but required the presence of an intact membrane structure. Our study may provide another step to understanding how membrane protein subtypes are differentially regulated by n-alkanols. J. Cell. Biochem. 66:450–456, 1997. © 1997 Wiley-Liss, Inc.     

A MAP kinase necessary for receptor-mediated activation of adenylyl cyclase in Dictyostelium

Analysis of a developmental mutant in Dictyostelium discoideum which is unable to initiate morphogenesis has shown that a protein kinase of the MAP kinase/ERK family affects relay of the cAMP chemotactic signal and cell differentiation. Strains in which the locus encoding ERK2 is disrupted respond to a pulse of cAMP by synthesizing cGMP normally but show little synthesis of cAMP. Since mutant cells lacking ERK2 contain normal levels of both the cytosolic regulator of adenylyl cyclase (CRAC) and manganese-activatable adenylyl cyclase, it appears that this kinase is important for receptor-mediated activation of adenylyl cyclase.     

Conformational changes in the parathyroid hormone receptor associated with activation by agonist

Binding of hormones to their cognate G protein-coupled receptors (GPCRs) induces conformational shifts within the receptor based on evidence from a few hormone-receptor systems. Employing an engineered disulfide bond formation strategy and guided by a previously established model of the PTH-PTH receptor (PTHR)1 bimolecular complex, we set out to document and characterize the nature of agonist-induced changes in this family B GPCR. A mutant PTHR1 was generated which incorporates a Factor Xa cleavage site in the third intracellular loop. Treatment with Factor Xa fragments the receptor. However, if a new disulfide bond was formed before exposure to the enzyme, the fragments remain held together. A set of double cysteine-containing mutants were designed to probe the internal relative movements of transmembrane (TM) helices 2 and TM7. PTH enhanced formation of disulfide bonds in the K240C/F447C and A242C/F447C mutants. For the F238C/F447C mutant, a disulfide bond is formed in the basal state, but is disrupted by interaction with PTH. For the D241C/F447C PTHR1 construct, no disulfide bond formation was observed in either the basal or hormone-bound state. These findings demonstrate that the conformation of PTHR1 is altered from the basal state when PTH is bound. Novel information regarding spatial proximities between TM2 and TM7 of PTHR1 and the nature of relative movements between the two transmembrane regions was revealed. The data confirm and extend the experimentally derived model of the PTH-PTHR1 complex and provide insights at a new level of detail into the early events in PTHR1 activation by PTH.     

Constitutive G(i2)-dependent activation of adenylyl cyclase type II by the 5-HT1A receptor. Inhibition by anxiolytic partial agonists

The 5-HT1A receptor is implicated in depression and anxiety. This receptor couples to G(i) proteins to inhibit adenylyl cyclase (AC) activity but can stimulate AC in tissues (e.g. hippocampus) that express ACII. The role of ACII in receptor-mediated stimulation of cAMP formation was examined in HEK-293 cells transfected with the 5-HT1A receptor, which mediated inhibition of basal and G(s)-induced cAMP formation in the absence of ACII. In cells cotransfected with 5-HT1A receptor and ACII plasmids, 5-HT1A agonists induced a 1. 5-fold increase in cAMP level. Cotransfection of 5-HT1A receptor, ACII, and Galpha(i2), but not Galpha(i1), Galpha(i3), or Galpha(o), resulted in an agonist-independent 6-fold increase in the basal cAMP level, suggesting that G(i2) preferentially coupled the receptor to ACII. The 5-HT1B receptor also constitutively activated ACII. Constitutive activity of the 5-HT1A receptor was blocked by pertussis toxin and the Gbetagamma antagonist, betaCT, suggesting an important role for Gbetagamma-mediated activation of ACII. The Thr-149 --> Ala mutation in the second intracellular domain of the 5-HT1A receptor disrupted Gbetagamma-selective activation of ACII. Spontaneous 5-HT1A receptor activity was partially attenuated by 5-HT1A receptor partial agonists with anxiolytic activity (e.g. buspirone and flesinoxan) but was not altered by full agonists or antagonists. Thus, anxiolytic activity may involve inhibition of spontaneous 5-HT1A receptor activity.