Agonist-stimulated Adenylate Cyclase Activity Research Articles

Allosteric sodium binding cavity in GPR3: a novel player in modulation of A\u03b2 production

The orphan G-protein coupled receptor 3 (GPR3) belongs to class A G-protein coupled receptors (GPCRs) and is highly expressed in central nervous system neurons. Among other functions, it is likely associated with neuron differentiation and maturation. Recently, GPR3 has also been linked to the production of Aβ peptides in neurons. Unfortunately, the lack of experimental structural information for this receptor hampers a deep characterization of its function. Here, using an in-silico and in-vitro combined approach, we describe, for the first time, structural characteristics of GPR3 receptor underlying its function: the agonist binding site and the allosteric sodium binding cavity. We identified and validated by alanine-scanning mutagenesis the role of three functionally relevant residues: Cys2676.55, Phe1203.36 and Asp2.50. The latter, when mutated into alanine, completely abolished the constitutive and agonist-stimulated adenylate cyclase activity of GPR3 receptor by disrupting its sodium binding cavity. Interestingly, this is correlated with a decrease in Aβ production in a model cell line. Taken together, these results suggest an important role of the allosteric sodium binding site for GPR3 activity and open a possible avenue for the modulation of Aβ production in the Alzheimer’s Disease.

Expression and function of adenosine receptors in human dendritic cells.

Dendritic cells (DCs) are specialized antigen-presenting cells characterized by their ability to migrate into target sites, process antigens, and activate naive T cells. In this study, we analyzed the biological activity and intracellular signaling of adenosine by using reverse transcriptase-polymerase chain reaction assays to investigate mRNA expression of A(1), A(2a) and A(3) adenosine receptors in immature and mature human DCs. Functional experiments on adenosine stimulation showed chemotaxis, intracellular calcium transients, and actin polymerization, but no activation of adenylate cyclase in immature DCs. Experiments with receptor isotype-selective agonists and antagonists as well as pertussis toxin revealed that chemotaxis, calcium transients, and actin polymerization were mediated via G(i-) or G(0-)protein-coupled A(1) and A(3) receptors. Maturation of DCs induced by lipopolysaccharide (LPS) resulted in down-regulation of A(1) and A(3) receptor mRNAs, although A(2a) receptor mRNA was still expressed. However, in LPS-differentiated DCs, adenosine and an A(2a) receptor agonist stimulated adenylate cyclase activity, enhanced intracellular cAMP levels, and inhibited interleukin 12 (IL-12) production. These effects could be completely prevented by pretreatment with A(2) receptor antagonist. These findings strongly suggest that adenosine has important but distinct biological effects in DCs activity as a chemotaxin for immature DCs and as a modulator of IL-12 production in mature DCs. These effects can be explained by differential expression of adenosine receptor subtypes.

Neuronal signaling systems and ethanol dependence.

In recent years there have been remarkable developments toward the understanding of the molecular and/or cellular changes in the neuronal second-messenger pathways during ethanol dependence. In general, it is believed that the cyclic adenosine 3',5'-monophosphate (cAMP) and the phosphoinositide (PI) signal-transduction pathways may be the intracellular targets that mediate the action of ethanol and ultimately contribute to the molecular events involved in the development of ethanol tolerance and dependence. Several laboratories have demonstrated that acute ethanol exposure increases, whereas protracted ethanol exposure decreases, agonist-stimulated adenylate cyclase activity in a variety of cell systems, including the rodent brain. Recent studies indicate that various postreceptor events of the cAMP signal transduction cascade (i.e., Gs protein, protein kinase A [PKA], and cAMP-responsive element binding protein [CREB]) in the rodent brain are also modulated by chronic ethanol exposure. The PI signal-transduction cascade represents another important second-messenger system that is modulated by both acute and chronic ethanol exposure in a variety of cell systems. It has been shown that protracted ethanol exposure significantly decreases phospholipase C (PLC) activity in the cerebral cortex of mice and rats. The decreased PLC activity during chronic ethanol exposure may be caused by a decrease in the protein levels of the PLC-beta 1 isozyme but not of PLC-delta 1 or PLC-gamma 1 isozymes in the rat cerebral cortex. Protein kinase C (PKC), which is a key step in the PI-signaling cascade, has been shown to be altered in a variety of cell systems by acute or chronic ethanol exposure. It appears from the literature that PKC plays an important role in the modulation of the function of various neurotransmitter receptors (e.g., gamma-aminobutyrate type A [GABAA], N-methyl-D-aspartate [NMDA], serotonin2A [5-HT2A], and 5-HT2C, and muscarinic [m1] receptors) resulting from ethanol exposure. The findings described in this review article indicate that neuronal-signaling proteins represent a molecular locus for the action of ethanol and are possibly involved in the neuro-adaptational mechanisms to protracted ethanol exposure. These findings support the notion that alterations in the cAMP and the PI-signaling cascades during chronic ethanol exposure could be the critical molecular events associated with the development of ethanol dependence.

Involvement of receptor reserve in D 1 agonistic action of (−)-stepholidine in lesioned rats

(−)-Stepholidine (SPD) is a natural product. Previous studies had demonstrated that SPD displayed D 1 agonism in unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats and D 1 antagonism in reserpinized rats and normal rats. The aim of the present study was to explain this peculiar pharmacological action based on behavioral and biochemical experiments. In the unilaterally 6-OHDA-lesioned rats, SPD (4 mg/kg, s.c.) induced contralateral rotation as did apomorphine (APO), but the rotation response to SPD was 60% lower than that to APO (0.5 mg/kg, i.p.). Coadministration with APO (0.5 mg/kg, i.p.) and SPD (0.5 to 10 mg/kg, s.c.) produced a biphasic action curve. At low doses (0.5 or 1 mg/kg), SPD potentiated APO action; at high doses (4 or 10 mg/kg), however, SPD suppressed APO. In striatal homogenate of the unilaterally lesioned rats, SPD stimulated cyclic AMP (cAMP) formation and produced a maximal response comparable to that of dopamine (DA) in the denervated striatum, but 70% lower than that of DA in the intact striatum. Coadministration of 10 μM DA with various concentrations of SPD yielded different results, with a biphasic response in the intact side and a synergistic effect in the denervated side. Furthermore, based on the determination of receptor-mediated cAMP formation, the D 1 receptor reserve was analyzed in both denervated and intact striatum by using the DA receptor inactivator N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The results showed that following EEDQ administration, the receptor density [revealed by [ 3H]R(+)-7- chloro-8- hydioxy-3- methyl-1- phenyl-2,3,4,5- tetrahydro-1H-3- benzazepine ([ 3H]SCH-23390) binding] and the agonist-stimulated adenylate cyclase (AC) activity (revealed by cAMP formation) were reduced concurrently. In the intact striatum, the reduction in SPD-stimulated AC activity paralleled the receptor loss, indicating the absence of receptor reserve, while in the denervated striatum the reduction in AC activity was less than the receptor loss, indicating a significant level of receptor reserve (estimated 16.4%). By comparison, receptor reserve for DA was 45.7 and 25.3% in the denervated and intact striatum, respectively, representing an 80% increase of receptor reserve. In conclusion, SPD is a D 1 partial agonist, and receptor reserve permits SPD to display its D 1 agonistic action in the unilaterally 6-OHDA-lesioned rats.

Lack of Effect of Repeated Administration of Milnacipran, a Double Noradrenaline and Serotonin Reuptake Inhibitor, on the β-adrenoceptor-linked Adenylate Cyclase System in the Rat Cerebral Cortex

The effects of subacute administration of the double noradrenaline and serotonin uptake inhibitor antidepressant, milnacipran, and the tricyclic antidepressant, imipramine, on radioligand binding to β-adrenergic receptors and on β-adrenergic agonist-stimulated adenylate cyclase activity, in the rat cerebral cortex, have been determined. Rats were injected intraperitoneally for 21 days with milnacipran (3, 10 or 30 mg/kg/day) or imipramine (10 mg/kg/day). The treatment with milnacipran up to 30 mg/kg/day did not modify either the maximum number of [ 3H]CGP-12177 binding sites ( B max) or the equilibrium dissociation constant ( K d). On the other hand, treatment of the rats with 10 mg/kg/day imipramine induced a decrease (27%) in B max [ 3H]CGP-12177 binding sites without affecting the K d value. Furthermore, milnacipran did not affect the stimulation of cAMP production induced by either 30 μM isoprenaline, 10 μM GTPγS or 10 μM forskolin. Under similar conditions, treatment with imipramine reduced by 70% the isoprenaline-induced stimulation of cAMP production without affecting that induced by either GTPγS or forskolin. These results demonstrate that, unlike imipramine, subacute administration of milnacipran does not produce any change in β-adrenoceptor sensitivity in the rat brain cortex. Copyright © 1996 Elsevier Science Ltd.

Adenylate cyclase activity in human pancreatic adenocarcinoma cell lines

BxPC-3, Hs 766T, Capan-2, Panc-1, and Capan-1 cells possess receptors for VIP and beta-adrenergic agonists that are functionally coupled to adenylate cyclase. In this respect, they resemble pancreatic duct cells. However, we speculate that the process of neoplastic transformation has either downregulated the expression of secretin receptors or led to a defect in the receptor itself, placing a question mark over the usefulness of these adenocarcinoma cell lines as models of the pancreatic ductal epithelium Because of the importance of ducts in pancreatic disease, we wished to establish which duct cells receptors are functional on adenocarcinoma cell lines. We investigated the expression of agonist-stimulated adenylate cyclase activity in six human pancreatic adenocarcinoma cell lines. Known stimulants of pancreatic ductal secretion, VIP, PHI, secretin, beta-adrenergic, and dopamine, were tested. For responsive cell lines, VIP was the most effective stimulant followed by adrenaline, isoprenaline, PHI, and secretin. Dopamine was without effect. Since high concentrations of PHI and secretin were required to stimulate cyclase activity, their effect is probably mediated by VIP receptors. Based on the degree of stimulation observed with the individual agonist, Hs 766T and BxPC-3 were the most responsive cell lines, followed by Capan-2 and Capan-1, and finally Panc-1. MIAPaCa-2 cells did not respond to any of the agonists tested.

Transforming growth factor-beta 1 regulation of signal transduction in two renal epithelial cell lines

The present studies examine the effect of transforming growth factor-beta 1 (TGF-beta 1) on signal transduction pathways in two cultured renal epithelial cell lines. TGF-beta 1 promotes basal and agonist-stimulated adenylate cyclase activity in LLC-PK1 but not MDCK cell membranes. TGF-beta 1 stimulation of LLC-PK1 membrane adenylate cyclase activity occurs quickly and can be attenuated by pertussis toxin pretreatment. Both TGF-beta 1 and adenosine 3',5'-cyclic monophosphate (cAMP) exert comparable effects on [3H]thymidine uptake in LLC-PK1 cells, suggesting that TGF-beta 1 regulation of adenylate cyclase activity potentially plays a role in mediating biological responses to TGF-beta 1. The activities of protein kinase C and phospholipase A are not affected by TGF-beta 1 in either LLC-PK1 or MDCK cells. Both TGF-beta 1 and epidermal growth factor (EGF) increase expression and induce the appearance of new forms of the cAMP response element binding protein (CREB) in LLC-PK1 cells. These effects of TGF-beta 1 and EGF on CREB appear to be specific since neither TGF-beta 1 nor EGF alters expression of an activating transcription factor in LLC-PK1 cells. The effect of TGF-beta 1 and EGF to alter expression of CREB does not affect CREB binding to its regulatory element in LLC-PK1 cell lysates. These results suggest that some of the biological effects of TGF-beta 1 may be attributed to stimulation of adenylate cyclase activity and cAMP formation as well as to enhanced expression and/or modification of the CREB transcription factor in LLC-PK1 cells.

Evidence for beta-adrenoceptors in rat proximal convoluted tubules.

It is well documented that renal catecholamines, acting as paracrine or autocrine substances, modulate reabsorption of sodium and other substances in renal tubules. However, beta-receptors or the resulting stimulation of adenylate cyclase (AC) have not been measured directly in the proximal convoluted tubules (PCT) of mammalian kidney. In this study, the beta adrenergic receptors in the PCT from rat kidney were characterized pharmacologically by measuring agonist stimulated adenylate cyclase activity. Isoproterenol (ISO) (10 microM) significantly increased AC activity in the microdissected PCT (ISO, 193.0 +/- 23.6 vs basal, 118.7 +/- 14.3 fmol/mm/20; n = 8, p = 0.004). The stimulation of AC in PCT by ISO was blocked by 10 microM propranolol (p < 0.01); it was also blocked by either the beta-1-adrenergic blocker, 10 microM atenolol (p < 0.05), or the beta-2-adrenergic blocker, 10 microM ICI-118551 (p < 0.05). The beta adrenergic receptor binding sites were localized by autoradiography; specific [125I] iodocyanopindolol ([125I] ICYP) binding could be measured in the microdissected rat PCT. In conclusion, these findings provide evidence for beta adrenergic receptors in the PCT in rat kidney by radioligand binding and beta adrenergic agonist stimulated AC activity.

Gs alpha is a substrate for mono(ADP-ribosyl)transferase of NG108-15 cells. ADP-ribosylation regulates Gs alpha activity and abundance.

NG108-15 neuroblastoma x glioma somatic hybrid cells were permeabilized in the presence of [32P]NAD+ and then cultured for 18 h. Resolution of the cell proteins on polyacrylamide gels revealed [32P]ADP-ribosylation of five major protein species with molecular mass values of 52 kDa, 44 kDa, 35 kDa, 30 kDa and 25 kDa. A similar pattern of labelling was also seen when NG108-15 cell membranes were incubated with [32P]NAD+ and hydrolysis of the product revealed mono(ADP-ribosyl)ation. Immunoprecipitation of these products with anti-Gs alpha antiserum revealed a single band identical to cholera toxin substrate. Culture of [32P]NAD(+)-loaded cells for 18 h in the presence of 50 mM-nicotinamide inhibited the eukaryotic mono(ADP-ribosyl)transferase activity. Inhibition of the eukaryotic enzyme was also accompanied by an increase in the abundance of Gs alpha, whether measured by Western blotting with anti-Gs alpha antibody (two separate antisera) or by cholera toxin-dependent [32P]ADP-ribosylation. There was no accompanying change in the abundance of G beta. The increase in Gs alpha abundance in nicotinamide-treated NG108-15 cells was accompanied by a 2-fold increase in basal adenylate cyclase activity (measured in the presence of GTP), and by a smaller but significant increase in iloprost-dependent activation of adenylate cyclase. Receptor number or affinity was not affected by nicotinamide, since this treatment did not alter the binding parameters of [3H]iloprost to NG108-15 cell membranes. Short-term exposure of cells to nicotinamide for 1 h revealed no significant difference in either basal or agonist-stimulated adenylate cyclase activity. These results reveal that mono(ADP-ribosyl)ation of Gs alpha by eukaryotic ADP-ribosyltransferase modifies the abundance and activity of Gs alpha in NG108-15 cells, and hence may play a role in the hormonal regulation of cell function.

Chronic nonocclusive coronary artery constriction in rats. Beta-adrenoceptor signal transduction and ventricular failure.

To determine the effects of chronic coronary artery constriction on the relationship between cardiac function and regulation of beta-adrenoceptor signal transduction, the left main coronary artery was narrowed in rats and the animals were killed 5 mo later. An average reduction in coronary luminal diameter of 44% was obtained and this change resulted in an increase in left ventricular end-diastolic pressure and a decrease in positive and negative dP/dt. Significant increases in left and right ventricular weights indicative of global cardiac hypertrophy were observed. Radioligand binding studies of beta-adrenoreceptors, agonist-stimulated adenylate cyclase activity, and ADP ribosylation of 45-kD substrate by cholera toxin were all depressed in the failing left ventricle. In contrast, in the hypertrophic non-failing right ventricle, beta-adrenoreceptor density was preserved and receptor antagonist affinity was increased. In spite of these findings at the receptor level, agonist stimulated cyclic AMP generation was reduced in the right ventricular myocardium. The quantity of the 45-kD substrate was also decreased. In conclusion, longterm nonocclusive coronary artery stenosis of moderate degree has profound detrimental effects on the contractile performance of the heart in association with marked attenuation of adrenergic support mechanisms.

Inhibition of cAMP accumulation by intracellular calcium mobilization in C6-2B cells stably transfected with substance K receptor cDNA.

C6-2B rat glioma cells were stably transfected with substance K receptor cDNA and used to study interactions between cAMP and Ca2+ signaling pathways. Activation of the newly expressed receptors by substance K increased the intracellular free Ca2+ concentration, as monitored by single-cell fura-2 imaging, and markedly inhibited agonist-stimulated cAMP accumulation. Blockade of intracellular Ca2+ mobilization abolished the substance K receptor-mediated inhibition of isoproterenol-induced cAMP production. Phosphodiesterase inhibitors, down-regulation or inhibition of protein kinase C, and pertussis toxin failed to prevent substance K-induced inhibition of agonist-stimulated cAMP accumulation. An increased intracellular Ca2+ concentration caused by either calcium ionophores or activation of endogenous bradykinin receptors was found to markedly reduce cAMP production in wild-type cells. These results demonstrate that elevated intracellular Ca2+ concentration can negatively modulate agonist-stimulated adenylate cyclase activity in C6-2B glioma cells.

Biochemical and Morphological Analysis of the Interaction of Epidermal Growth Factor and Parathyroid Hormone with UMR 106 Osteosarcoma Cells*

The interaction of epidermal growth factor (EGF) and PTH with UMR 106 osteosarcoma cells was examined biochemically and morphologically. EGF inhibited PTH-stimulated adenylate cyclase activity in association with a reduction in PTH receptors and a decrease in the activity of the stimulatory guanyl nucleotide-binding protein (Gs). Universal inhibition of agonist-stimulated adenylate cyclase activity did not occur, inasmuch as EGF did not reduce prostaglandin E2-enhanced enzyme activity. The influence of EGF on PTH action correlated with its effect in the UMR 106 cell population of promoting entry into the cell cycle. Morphological analysis with radioautography indicated that both EGF and PTH receptors could be colocalized to certain UMR 106 cells, but that each were more abundantly distributed over discrete UMR 106 cell types. Based on the distribution of [3H]thymidine incorporation, EGF receptors were predominantly found over rapidly proliferating cells, whereas PTH receptors were most densely distributed over more quiescent cells. The results indicate that EGF and PTH receptors are localized over specific types within the heterogeneous population of UMR 106 cells and suggest that EGF may reduce PTH action in these cultures by enhancing the proliferation of progenitor cells lacking PTH receptors and reducing differentiation in this cell population, which leads to PTH receptor-enriched target cells. EGF and PTH receptors may, therefore, be useful as probes to examine both functional interactions and differentiation pathways among cells in osteoblast models in vitro and perhaps in vivo.

ATP receptor regulation of adenylate cyclase and protein kinase C activity in cultured renal LLC-PK1 cells.

In cultured intact LLC-PK1 renal epithelial cells, a nonhydrolyzable ATP analogue, ATP gamma S, inhibits AVP-stimulated cAMP formation. In LLC-PK1 membranes, several ATP analogues inhibit basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in a dose-dependent manner. The rank order potency of inhibition by ATP analogues suggests that a P2y type of ATP receptor is involved in this inhibition. The compound ATP gamma S inhibits agonist-stimulated adenylate cyclase activity in solubilized and in isobutylmethylxanthine (IBMX) and quinacrine pretreated membranes, suggesting that ATP gamma S inhibition occurs independent of AVP and A1 adenosine receptors and of phospholipase A2 activity. The ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity is not affected by pertussis toxin but is attenuated by GDP beta S, suggesting a possible role for a pertussis toxin insensitive G protein in the inhibition. Exposure of intact LLC-PK cells to ATP gamma S results in a significant increase in protein kinase C activity. However, neither of two protein kinase C inhibitors (staurosporine and H-7) prevents ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity, suggesting that this inhibition occurs by a protein kinase C independent mechanism. These findings suggest the presence of functional P2y purinoceptors coupled to two signal transduction pathways in cultured renal epithelial cells. The effect of P2y purinoceptors to inhibit AVP-stimulated adenylate cyclase activity may be mediated, at least in part, by a pertussis toxin insensitive G protein.

A Renal Dopamine-1 Receptor Defect in Two Genetic Models of Hypertension

Dopamine (DA), via DA-1 receptors, regulates Na+ transport in the kidneys. Dopamine is synthesized from L-DOPA in the proximal tubule and presumably secreted as an autocrine/paracrine substance to stimulate DA-1 receptors localized on proximal tubular cells. We have previously reported the presence of DA-1 receptors in renal cortical homogenates and on the isolated proximal tubule of the rat and rabbit, consistent with the dopamine autocrine/paracrine model. We have localized DA-1 receptors in the proximal straight tubule of the rabbit, and in the cortical collecting duct of the rabbit and rat, but not in the distal collecting tubule or the cortical thick ascending loop of Henle. The presence of functional DA-1 receptors has been substantiated by the coexistence of DA-1 agonist-stimulated adenylate cyclase activity in the same nephron segments in which DA-r receptors have been found. Increased concentrations of intrarenal dopamine induced by dopamine-beta-hydroxylase inhibition with SKF-102698 caused a down regulation of proximal tubular DA-1 receptors and almost complete ablation of DA-1 agonist stimulated adenylate cyclase activity. Thus, dopamine may play a role in the regulation of DA-1 receptors and their linkage with adenylate cyclase. Since alterations in the renal dopaminergic system have been measured in some forms of experimental hypertension, we studied DA-1 receptors and their coupling to adenylate cyclase in the spontaneously hypertensive rat (SHR).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential activation of the stimulatory and inhibitory guanine nucleotide-binding proteins by fluoroaluminate in cells and in membranes.

Fluoroaluminate had no effect on cAMP levels in cells but inhibited agonist-stimulated cAMP accumulation. In membranes, fluoroaluminate stimulated adenylate cyclase activity between 1 and 10 mM but not at higher concentrations, and it inhibited agonist-stimulated adenylate cyclase activity at concentrations greater than 1 mM. Fluoroaluminate is known to activate Gs and Gi, the guanine nucleotide-binding (G) proteins that stimulate and inhibit adenylate cyclase. G proteins are heterotrimeric, with unique alpha and common beta gamma subunits, and activation involves dissociation of alpha from beta gamma. Pertussis toxin catalyzes ADP-ribosylation of alpha i of heterotrimeric Gi but not free alpha i. Fluoroaluminate prevented pertussis toxin-catalyzed ADP-ribosylation of Gi in cells and membranes, suggesting that Gi is activated by fluoroaluminate in both. Cholera toxin catalyzes ADP-ribosylation of the alpha s subunit of Gs. In cells, agonist often increased cholera toxin-catalyzed ADP-ribosylation of Gs, but fluoroaluminate decreased ADP-ribosylation even in the presence of agonist, suggesting that Gs cannot be activated in the presence of fluoroaluminate. In membranes, both agonist and fluoroaluminate increased cholera toxin-catalyzed ADP-ribosylation, suggesting that Gs is activated by these agents. We conclude that fluoroaluminate activates Gi but not Gs in cells and activates both G proteins in membranes. The value of bacterial toxins in assessing the state of G protein in cells and membranes is demonstrated.

Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled β-adrenergic receptors in avian erythrocyte membranes

Desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes results in a 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of beta-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37 degrees C, pH 8.0. In both preparations alkaline phosphatase treatment significantly decreased desensitization of agonist-stimulated adenylate cyclase activity by 40-75% (P less than 0.05). Similar results were obtained after alkaline phosphatase treatment of membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized duck erythrocytes. Moreover, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with 12-O-tetradecanoylphorbol 13-acetate returned agonist-stimulated adenylate cyclase activity to near control values. In all experiments, inclusion of 20 mM-sodium phosphate to inhibit alkaline phosphatase during treatment of membranes attenuated the enzyme's effect on agonist-stimulated adenylate cyclase activity. In addition, alkaline phosphatase treatment of membranes from control and isoprenaline-desensitized turkey erythrocytes increased the mobility of beta-adrenergic-receptor proteins, specifically photoaffinity-labelled with [125I]iodocyanopindolol-diazirine, on SDS/polyacrylamide-gel electrophoresis. The increased mobility of the beta-adrenergic-receptor proteins after alkaline phosphatase treatment of membranes was again inhibited by 20 mM-phosphate. These results provide additional evidence for a direct role for phosphorylation in desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes.

Catecholamine-induced desensitization of adenylate cyclase coupled beta-adrenergic receptors in turkey erythrocytes: evidence for a two-step mechanism.

Preincubation of turkey erythrocytes with isoproterenol is associated with (1) 50-60% attenuation of agonist-stimulated adenylate cyclase activity, (2) altered mobility of the beta-adrenergic receptor on sodium dodecyl sulfate-polyacrylamide gels, and (3) increased phosphorylation of the beta-adrenergic receptor. Using a low-cross-linked polyacrylamide gel, the beta-adrenergic receptor protein from isoproterenol-desensitized cells, labeled with 32P or with the photoaffinity label 125I-(p-azidobenzyl)carazolol, can be resolved into a doublet (Mr congruent to 37,000 and Mr congruent to 41,000) as compared to a single Mr congruent to 37,000 beta-adrenergic receptor protein from control erythrocytes. The appearance of the doublet was dependent on the concentration of agonist used to desensitize the cells. Incubation of erythrocytes with dibutyryl-cAMP did not promote formation of the doublet but decreased agonist-stimulated adenylate cyclase activity 40-50%. Limited-digestion peptide maps of 32P-labeled beta-adrenergic receptors using papain revealed a unique phosphopeptide in the larger molecular weight band (Mr congruent to 41,000) of the doublet from the agonist-desensitized preparation that was absent in the peptide maps of the smaller band (Mr congruent to 37,000), as well as control or dibutyryl-cAMP-desensitized receptor. These data provide evidence that maximal agonist-induced desensitization of adenylate cyclase coupled beta-adrenergic receptors in turkey erythrocytes occurs by a two-step mechanism.

Corticotropin releasing factor receptor-mediated stimulation of adenylate cyclase activity in the rat brain

Corticotropin releasing factor (CRF)-stimulated adenylate cyclase activity and receptor binding were examined in the rat brain homogenates using a potent synthetic CRF analog—[ d-Tyr 3, d-Pro 4, Nle 18,21, α-helical]CRF 3–41 (α-hel CRF 3–41). Binding of α-hel CRF 3–41 in the rat brain was saturable, reversible, of high affinity and exhibited relevant peptide specificity. This analog also stimulated adenylate cyclase activity of various brain regions; the greatest magnitude of stimulation was in the cerebral cortex followed by the septum, cerebellum and thalamus. Adenylate cyclase stimulation in the cerebral cortex was concentration-dependent with an ED 50 of2.5±0.4nM for α-hel CRF 3–41 and an ED 50 of16 ± 2nM for ovine and rat CRF. Maximal stimulation was comparable for all peptides. Agonist-stimulated adenylate cyclase activity was competitively blocked by the CRF antagonists. The inactive CRF analog, ovine CRF 1–39, at concentrations less than 1μM, did not significantly stimulate adenylate cyclase. Adrenalectomy, which has been reported to modulate CRF receptor number and CRF-stimulated adenylate cyclase activity in the anterior pituitary, had no effect on CRF receptor binding or CRF-stimulated adenylate cyclase activity in the cerebral cortex. These results suggest that, as in the anterior pituitary, at least some of the physiological responses mediated by CRF receptors in the brain utilize the cyclic nucleotide regulatory pathway as a post-receptor mechanism.

Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes

Incubation of intact frog erythrocytes with 12- O-tetradecanoyl phorbol-13-acetate (TPA), a tumor-promoting phorbol diester which activates protein kinase C, results in an approximate two- to threefold increase in subsequently tested β-adrenergic agonist-stimulated adenylate cyclase activity. This increase is due to an elevation in the V max of the enzyme rather than to a change in affinity for the agonist. TPA treatment of frog erythrocytes does not alter the affinity ( K D ) or the binding capacity ( B max) for the β-adrenergic antagonist [ 125I]cyanopindolol. In addition, agonist/[ 125I]cyanopindolol competition curves are not affected by TPA pretreatment nor is their sensitivity to guanine nucleotides. Incubation of frog erythrocyte membranes alone with TPA does not promote sensitization or activation of adenylate cyclase activity. Pretreatment of intact frog erythrocytes with TPA also produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E 1-, forskolin-, NaF-, and MnCl 2-stimulated adenylate cyclase activities in frog erythrocyte membranes. This enhancement of adenylate cyclase activity by TPA is induced rapidly ( t 1 2 ≅ 5 min ) and with an EC 50 of about 10 −7 to 10 −6 m. Other tumor-promoting phorbol diesters or phorbol diester-like compounds including 4β-phorbol 12,13-dibutyrate, 4β-phorbol 12,13-didecanoate, and mezerein are effective in promoting enhanced adenylate cyclase activity. In contrast, phorbols such as 4β-phorbol, 4α-phorbol 12,13-didecanoate, and 4- O-methylphorbol 12-myristate 13-acetate, which are inactive in tumor promotion and which do not activate protein kinase C, do not affect frog erythrocyte adenylate cyclase activity. These data are suggestive of a protein kinase C-mediated phosphorylation of one of the adenylate cyclase components that is distal to the receptor, i.e., the nucleotide regulatory and/or catalytic components.

Calmodulin activation of adenylate cyclase in the mouse B16 melanoma.

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.