Kinetics and concentration dependency of cAMP-induced desensitization of a subpopulation of surface cAMP receptors in Dictyostelium discoideum

Extracellular cAMP induces the rapid activation of guanylate cyclase, which adapts within 10 s to constant cAMP concentrations. A new response can be induced either by a higher cAMP concentration or by the same cAMP concentration at some time (t1/2 = 90 s) after removal of the previous stimulus. Stimulation of guanylate cyclase is supposed to be mediated by a subpopulation of cell surface cAMP receptors (B-sites). These sites can exist in three states, BF, BS, and BSS, which interconvert in a cAMP and guanine nucleotide dependent manner. It has been proposed that the transition of BS to BSS represents the activation of a guanine nucleotide regulatory protein [Van Haastert, P.J.M., De Wit, R.J.W., Janssens, P.M.W., Kesbeke, F., & DeGoede, J. (1986) J. Biol. Chem. 261, 9604-9611]. Binding of [3H]cAMP to these sites was measured after a short preincubation with an identical concentration of nonradioactive cAMP. [3H]cAMP could still bind to BF and BS, but not to BSS, indicating that the transition of BS to BSS is blocked by the preincubation with cAMP. This blockade was rapid and showed first-order kinetics with t1/2 = 4 s. A half-maximal blockade was induced by 0.7 nM cAMP; at this concentration only 5% of the B-sites are occupied with cAMP. The blockade of the transition of BS to BSS was released by two conditions: (i) When the concentration of cAMP was increased, the blockade was released within a few seconds. (ii) When cAMP was removed, the blockade was released slowly with t1/2 = 90 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental regulation of the pathways of folate-receptor-mediated stimulation of cAMP and cGMP synthesis in Dictyostelium discoideum

Recently, we demonstrated the presence of multiple folate-binding sites on the cell surface of Dictyostelium discoideum. These sites were divided into two major classes, with different ligand specificities (A and B). Each major class consists of several interconvertible subtypes. In the present report, the ability of 13 folate analogs to activate both adenylate and guanylate cyclase in pre- as well as postaggregative cells is examined. The patterns of correlation between binding and activation data indicate that guanylate cyclase activation is mediated by the B-sites in both developmental stages (P less than 0.001). In postaggregative cells, adenylate cyclase also seems to be activated by the B-sites (P less than 0.001). In contrast, adenylate cyclase activation in preaggregative cells was well correlated with the specificity of A-sites (P less than 0.01). Remarkably, the potencies of activation were less affected by molecular modifications than the binding affinities were, as suggested by a slope of 0.4 in a plot of K0.5 values of activation vs. binding. This observation argues against the existence of a transduction mechanism in which the response is proportional to receptor occupancy. For the B-receptor, however, the degree of receptor occupancy appears to determine the response. The existence of folic acid antagonists is demonstrated, some of which are specific for either A-sites coupled to adenylate cyclase or for B-sites coupled to guanylate cyclase.     

Modulation of Anp Receptor-Mediated cGMP Accumulation by Atrial Natriuretic Peptides and Vasopressin in A10 Vascular Smooth Muscle Cells

Abstract

ANP receptor binding and desensitization were demonstrated in the A10 vascular smooth muscle cell (VSMC) line. Concomitantly, the ANP receptor coupled guanylate cyclase activity was reduced by the receptor down-regulation with ANP. The ANP stimulated cGMP accumulation is modulated by arginine-vasopressin, while the arginine-vasopressin mediated cAMP system remained unaffected by ANP. Results suggest negative coupling of arginine-vaso-pressin receptors to the guanylate cyclase activity, and indicate that the vasorelaxant activity of ANP might be regulated in part by arginine-vasopressin via specific receptor sites.     

Interaction between brain natriuretic peptide and atrial natriuretic peptide in caecal circular smooth muscle cells

Guinea pig caecal circular smooth muscle cells were used to determine whether brain natriuretic peptide (BNP) can inhibit the contractile response produced by cholecystokinin-octapeptide (CCK-8). In addition, we examined the effect of an inhibitor of cAMP-dependent protein kinase, an inhibitor of particulate or soluble guanylate cyclase, an atrial natriuretic peptide (ANP) antagonist (ANP 1-11), and selective receptor protection on the BNP-induced relaxation of these muscle cells. The effect of BNP on cAMP formation was also examined. BNP inhibited the contractile response produced by CCK-8 in a dose-response manner, with an IC50 value of 8.5 nM, and stimulated the production of cAMP. The inhibitor of cAMP-dependent protein kinase and the inhibitor of soluble guanylate cyclase significantly inhibited the relaxation produced by BNP. In contrast, the inhibitor of particulate guanylate cyclase did not have any significant effect on the relaxation produced by BNP. ANP 1-11 significantly but partially inhibited the relaxation produced by BNP. The muscle cells where CCK-8 and ANP binding sites were protected completely preserved the inhibitory response to ANP, but partially preserved the inhibitory response to BNP. The muscle cells where CCK-8 and BNP binding sites were protected completely preserved the inhibitory response to both ANP and BNP. This study demonstrates that BNP induces relaxation of these muscle cells via both ANP binding sites coupled to soluble guanylate cyclase and distinct BNP binding sites coupled to adenylate cyclase.     

Activation of particulate guanylate cyclase by adrenomedullin in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells

We investigated the effects of adrenomedullin (ADM) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ADM increased cGMP accumulation in a time- and concentration- dependent manner. The peptide increased cGMP formation in the transformed cells by 405-fold as compared to 1. 6-fold in primary cultured CISM cells. The basal cGMP concentrations in both cell types were comparable. In addition, ADM increased cAMP accumulation in SV-CISM-2 cells and in primary cultured cells by 18. 9- and 5.8-fold, respectively. The ADM receptor antagonist, ADM(26-52), but not the atrial natriuretic peptide (ANP) receptor antagonist, anantin, inhibited ADM-induced cGMP formation. The phorbol ester, phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylate cyclases in smooth muscle, blocked ADM-stimulated cGMP accumulation. In contrast, inhibitors of the soluble guanylate cyclases, such as LY83583 and ODQ, and inhibitors of the nitric oxide cascade had little effect on ADM-stimulated cGMP production. The stimulatory effect of ADM on cGMP formation is due to activation of the guanylate cyclase system and not to a much reduced phosphodiesterase activity. ADM stimulated guanylate cyclase activity in membrane fractions isolated from SV-CISM-2 cells in a concentration-dependent manner with EC(50) value of 72 nM. Pertussis toxin, an activator of the G-protein, Gi, inhibited ADM-stimulated cGMP accumulation, whereas cholera toxin, a stimulator of the Gs G-protein and subsequently cAMP accumulation, had little effect. Pretreatment of the plasma membrane fraction with Gialpha antibody attenuated ADM-stimulated guanylate cyclase activity by 75%. We conclude that ADM increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ADM receptor and subsequent stimulation of a Gi-mediated membrane-bound guanylate cyclase.     

Demonstration of receptor heterogeneity and affinity modulation by nonequilibrium binding experiments. The cell surface cAMP receptor of Dictyostelium discoideum

The binding of [3H]cAMP to Dictyostelium discoideum cells was analyzed on a seconds time scale under both equilibrium and nonequilibrium conditions. The binding of [3H]cAMP increases rapidly to a maximum obtained at about 6 s, which is followed by a decrease to an equilibrium value reached at about 45 s. This decrease of [3H]cAMP binding is not the result of ligand degradation or isotope dilution by cAMP secretion but is due to a transition of high-affinity binding to low-affinity binding. Analysis of the dissociation rate of [3H]cAMP from the binding sites indicates that these high- and low-affinity binding sites are both fast dissociating with a half-life of about 1 s. In addition, these dissociation experiments reveal a third binding type which is slowly dissociating with a half-life of about 15 s. The number and affinity of these slowly dissociating sites does not change during the incubation with [3H]cAMP. The drugs caffeine and chlorpromazine do not change the total number of binding sites, but they change the ratio of the three binding types. In the presence of 10 mM caffeine almost all binding sites are in the low affinity conformation, while in the presence of 0.1 mM chlorpromazine the ratio is shifted to both the high-affinity type and slowly dissociating type. The results indicate that the cAMP-binding activity of D. discoideum cells is heterogeneous. In the absence of cAMP about 4% of the sites are slowly dissociating with Kd = 12.5 nM, about 40% are fast dissociating with high affinity (Kd = 60 nM), and about 60% are fast dissociating with low affinity (Kd = 450 nM). During the binding reaction the number of slowly dissociating sites does not change. The number of high-affinity sites decreases to a minimum of about 10% with a concomitant increase of low-affinity sites to about 90%. This transition of binding types shows first-order kinetics with a half-life of about 9 s. A half-maximal transition is induced by 12.5 nM cAMP.     

Differential effects of temperature on cAMP-induced excitation, adaptation, and deadaptation of adenylate and guanylate cyclase in Dictyostelium discoideum

Extracellular cAMP induces excitation of adenylate and guanylate cyclase in Dictyostelium discoideum. Continuous stimulation with cAMP leads to adaptation, while cells deadapt upon removal of the cAMP stimulus. Excitation of guanylate cyclase by cAMP has a lag time of approximately 1 s; excitation of adenylate cyclase is much slower with a lag time of 30 s. Excitation of both enzyme activities is less than twofold slower at 0 degrees C than at 20 degrees C. Adaptation of guanylate cyclase is very fast (t1/2 = 2.4 s at 20 degrees C), and virtually absent at 0 degrees C. Adaptation of adenylate cyclase is much slower (t1/2 = 110 s at 20 degrees C) but not very temperature sensitive (t1/2 = 290 s at 0 degrees C). At 20 degrees C, deadaptation of adenylate cyclase is about twofold slower than deadaptation of guanylate cyclase (t1/2 = 190 and 95 s, respectively). Deadaptation of adenylate cyclase is absent at 0 degrees C, while that of guanylate cyclase proceeds slowly (t1/2 = 975 s). The results show that excitation, adaptation, and deadaptation of guanylate cyclase have different kinetics and temperature sensitivities than those of adenylate cyclase, and therefore are probably independent processes.     

Ca2+- or phorbol ester- dependent effect of ATP on a subpopulation of cAMP cell-surface receptors in membranes from D. discoideum. A role for protein kinase C

D. discoideum cells contain surface receptors for the chemoattractant cAMP which are composed of fast and slowly dissociating binding sites with half-lifes of respectively about 1 s and 15 s (Van Haastert and De Wit, J. Biol. Chem. 259, 13321-13328). In membranes prepared by shearing the cells through a Nucleopore filter, ATP has no effect on cAMP-binding at equilibrium, but the number of slowly dissociating sites is increased about 2-fold by ATP while their apparent affinity and off-rate are not altered by ATP. The effect of ATP is stimulated about 3-fold by Ca2+ with a half maximal effect at 100 microM Ca2+. The tumor promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), increases this Ca2+-sensitivity of the ATP effect to about 0.2 microM Ca2+. These data suggest that a specific subpopulation of cAMP receptors in membranes from D. discoideum is altered by the action of protein kinase C.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Homologous desensitization of the human guanylate cyclase C receptor. Cell-specific regulation of catalytic activity.

Guanylate Cyclase C (GCC) serves as a receptor for the endogenous ligands, guanylin and uroguanylin, as well as the family of bacterial heat-stable enterotoxins (ST), which are one of the major causes of diarrhoea the world over. We had earlier provided evidence that GCC, present in the human colonic T84 cell line, is desensitized on prolonged exposure to ST, and this desensitization was reflected in a reduced ST-stimulated guanylate cyclase activity of GCC [Bakre, M.M. & Visweswariah, S.S. (1997) FEBS Lett. 408, 345-349]. In this study, we have investigated the mechanisms that underlie this cellular desensitization process. Desensitization of T84 cells was not a result of reduction in GCC present in membranes prepared from desensitized T84 cells, nor due to increased cGMP-phosphodiesterase activity associated with the membrane fraction. The decrease in ST-stimulatable guanylate cyclase activity of GCC was due to a dramatic reduction in the Vmax of the cyclase, which was also seen when MnGTP was used as the substrate. GCC undergoes ligand-induced inactivation in vitro, which is alleviated in the presence of ATP. In vivo desensitized GCC could be further inactivated in vitro when preincubated with ST, indicating that the two mechanisms of GCC inactivation are distinct. Cellular refractoriness as reflected by a reduced responsiveness to further ST-stimulation following prior exposure to IST, coupled with GCC desensitization was also observed in another colonic cell line, Caco2. However, HEK293 cells, stably transfected with GCC cDNA, when exposed to ST for prolonged periods, did not result in GCC desensitization, indicating that desensitization of GCC appeared to be a cell specific phenomenon. GCC expressed in HEK293-GCC cells, however, showed in vitro ligand induced inactivation, suggesting that there are two independent means of ligand-induced desensitization of GCC, perhaps distinct from the mechanisms that have been described earlier for other members of the guanylate cyclase receptor family.     

Dual role of cAMP and involvement of both G-proteins and ras in regulation of ERK2 in Dictyostelium discoideum.

Dictyostelium discoideum expresses two Extracellular signal Regulated Kinases, ERK1 and ERK2, which are involved in growth, multicellular development and regulation of adenylyl cyclase. Binding of extracellular cAMP to cAMP receptor 1, a G-protein coupled cell surface receptor, transiently stimulates phosphorylation, activation and nuclear translocation of ERK2. Activation of ERK2 by cAMP is dependent on heterotrimeric G-proteins, since activation of ERK2 is absent in cells lacking the Galpha4 subunit. The small G-protein rasD also activates ERK2. In cells overexpressing a mutated, constitutively active rasD, ERK2 activity is elevated prior to cAMP stimulation. Intracellular cAMP and cAMP-dependent protein kinase (PKA) are essential for adaptation of the ERK2 response. This report shows that multiple signalling pathways are involved in regulation of ERK2 activity in D.discoideum.     

Relationship between adaptation of the folic acid and the cAMP mediated cGMP response in Dictyostelium

Chemotactic stimulation of post-vegetative Dictyostelium cells with folic acid or aggregative cells with cAMP results in a fast transient cGMP response which peaks at 10 s; basal levels are recovered in about 30–40 s. Stimulation with folic acid or cAMP rapidly desensitizes the cells for equal or lower concentrated stimuli. However, cells remain responsive for stimuli with higher concentration, which indicates that desensitization is caused by an adaptation process. Removal of the stimulus induces deadaptation, which for both cAMP and folic acid has first order kinetics with a half-life of 1.5 min.Cells were prepared which are simultaneously sensitive to folic acid and to cAMP. The cGMP responses to saturated folic acid and cAMP stimuli are not additive, which suggests that the transduction pathways of these signals meet each other at or before the guanylate cyclase. Cells which are adapted to folic acid are not adapted to cAMP and vice versa. This demonstrates that adaptation of Dictyostelium cells to chemotactic stimuli is localized at a step in the transduction chain before the transduced folic acid and cAMP signals combine in one pathway.     

Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium.

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.     

Desensitization of vasopressin sensitive adenylate cyclase by vasopressin and phorbol esters

Desensitization of vasopressin V2 receptor-mediated adenylate cyclase was studied in canine kidney cell line, MDCK cells. Overnight treatment of MDCK cells with arginine vasopressin (AVP) resulted in a loss of vasopressin receptors and an inhibition of cAMP accumulation in response to AVP. Both the loss of receptor and reduction in cAMP accumulation were time- and AVP concentration-dependent. Desensitization was selective for AVP because cAMP formation in response to isoproterenol, prostaglandin E1 (PGE1) and forskolin was not affected by AVP pre-treatment. Pre-treatment of MDCK cells with phorbol dibutyrate (PDBu) also caused a dose-dependent inhibition of AVP mediated cAMP accumulation, but not of isoproterenol-, PGE1- and forskolin-induced cAMP accumulation. PDBu pre-treatment did not cause loss of vasopressin receptors. Instead, the affinity for vasopressin was changed by PDBu treatment. Pre-treatment of the cells with pertussis toxin (PT) had no effect on the desensitization and downregulation of vasopressin (V2) receptors, suggesting that the desensitization may not be mediated by pertussis toxin sensitive G-protein. Our data suggest that pre-treatment of MDCK cells with AVP or PDBu caused desensitization of AVP-mediated cAMP accumulation and that downregulation of V2 receptors required agonist occupancy of the receptors, whereas the affinity of the receptors was changed by phorbol ester treatment.     

Selective down-regulation of cell surface cAMP-binding sites and cAMP-induced responses in Dictyostelium discoideum

Extracellular cAMP induces an intracellular accumulation of cAMP and cGMP levels in Dictyostelium discoideum. cAMP is detected by cell-surface receptors which are composed of a class of fast-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1?2}\text{s}$) and a class of slow-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 15?150}\text{s}$). Exposure of D. discoideum cells to 1 mM cAMP for 30 min induces a reduction of cAMP binding (down-regulation; Klein, C. and Juliani, M.H. (1977) Cell 10, 329–335). The number of fast-dissociating sites was reduced by 80–90% in down-regulated cells. These sites are composed of two forms with high and low affinity which interconvert during the binding reaction. In down-regulated cells this transition still occurred in the residual sites. The accumulation of cellular cAMP levels induced by a saturating stimulus decreased by 80–90%. The number of slow-dissociating sites was not significantly reduced in down-regulated cells, but their affinity decreased about 10-fold. The accumulation of cellular cGMP levels induced by a saturating stimulus was not decreased; however, about 20-fold higher cAMP concentrations were required to induce the same response. These results demonstrate that the cAMP transduction pathways to adenylate cyclase and guanylate cyclase are down-regulated differently. Furthermore, the results suggest that the fast-dissociating sites are involved in the activation of adenylate cyclase, while the slow-dissociating sites are coupled to guanylate cyclase.     

Vascular atrial natriuretic factor receptor subtypes are not independently regulated by atrial peptides

The regulation of the atrial natriuretic factor (ANF) receptor system in cultured rat vascular smooth muscle cells (RVSMC) was examined following long term pretreatment of these cells with rANF99-126 or with any one of a series of truncated and ring-deleted analogs. The latter analogs are reported to bind selectively the ANF-C or clearance receptor. Initial competition binding studies revealed that all analogs examined showed comparable apparent receptor binding affinities (Ki values did not differ by more than 10-fold). In contrast, the extent of interaction of the ANF analogs with the receptor pool coupled to particulate guanylate cyclase (the ANF-B receptor) was much more variable, with some ligands failing to stimulate cGMP production or particulate guanylate cyclase over the concentrations tested. Pretreatment of cells for 24 h with rANF99-126 or any of the truncated analogs that interact with the ANF-B receptor caused a dose- and time-dependent decrease in the number of ANF binding sites (99% of which are uncoupled in RVSMC) without any change in affinity. Examination of the binding activity following pretreatment of the cells with ANF suggested that the observed reduction in 125I-rANF99-126 binding capacity was not because of the retention of the peptide on its receptor. Furthermore, this down-regulation was associated with desensitization of particulate guanylate cyclase resulting in a decreased responsiveness of intracellular cGMP accumulation to ANF. In contrast, however, analogs selective for the ANF-C receptor pool failed to cause down-regulation or desensitization. These findings suggest that ANF-C receptors in RVSMC are not independently down-regulated by selective ligands but that nonselective analogs that down-regulate and desensitize the ANF-B receptor system can by some cooperative mechanism reduce the size of the predominant ANF-C receptor pool in these cells.     

Guanylate Cyclase Activity in Dictyostelium discoideum and Its Increase during Cell Development

Following consumption of the food supply, cells of the cellular slime mould Dictyostelium discoideum aggregate and form a multicellular organism. The mechanism for cell aggregation is chemotaxis. The chemotactic signal in D. discoideum is released periodically from aggregation centers and propagated from cell to cell. cAMP mediates cell aggregation by acting as chemotactic attractant and as propagator of the signal. cAMP signals are measured by cell-surface receptors. Recent evidence indicates a role for cGMP during cAMP-mediated cell aggregation in D. discoideum .
During cell differentiation to aggregation competence, cAMP binding sites appear at the cell surface, and the activity of the enzymes adenylate cyclase and phosphodiesterase increases several-fold. In the present work we investigate the synthesis of cGMP in D. discoideum . Conditions for the assay of guanylate cyclase in cell homogenates are described. Guanylate cyclase activity was followed during cell differentiation to aggregation competence and found to increase fourfold. These results indicate that cGMP is involved in cell differentiation of D. discoideum . In contrast to adenylate cyclase, which is activated by cAMP, guanylate cyclase was under our conditions activated neither by cAMP, nor by folic acid.     

Kinetics and nucleotide specificity of a surface cAMP binding site in Dictyostelium discoideum, which is not down-regulated by cAMP

Dictyostelium cells exhibit four types of kinetically distinct surface cAMP binding sites, the AH, AL, BS, and BSS sites, which are down-regulated during persistent stimulation with cAMP. Although most cAMP-induced responses are subject to desensitization during constant stimulation, some responses, notably the induction of post-aggregative gene expression, require persistent cAMP stimulation. The kinetics and specificity of residual cAMP-binding activity in cells treated for 4 h with micromolar cAMP were investigated. It was found that around 4000 rapidly dissociating binding sites per cell with an affinity of about 300 nM are retained after down-regulation. The nucleotide specificity of the remaining sites was very similar, but not completely identical to the AH, AL and B sites, suggesting that these sites belong to the same class of cell surface cAMP receptors and may be utilized to mediate responses requiring continuous cAMP stimulation.     

Regulation of adenylate cyclase in electropermeabilized Dictyostelium discoideum cells.

In Dictyostelium discoideum cells the enzyme adenylate cyclase is functionally coupled to cell surface receptors for cAMP. Coupling is known to involve one or more G-proteins. Receptor-mediated activation of adenylate cyclase is subject to adaptation. In this study we employ an electropermeabilized cell system to investigate regulation of D. discoideum adenylate cyclase. Conditions for selective permeabilization of the plasma membrane have been described by C.D. Schoen, J. C. Arents, T. Bruin, and R. Van Driel (1989, Exp. Cell Res. 181, 51-62). Only small pores are created in the membrane, allowing exchange of exclusively low molecular weight substances like nucleotides, and preventing the loss of macromolecules. Under these conditions functional protein-protein interactions are likely to remain intact. Adenylate cyclase in permeabilized cells was activated by the cAMP receptor agonist 2'-deoxy cAMP and by the nonhydrolyzable GTP-analogue GTP gamma S, which activates G-proteins. The time course of the adenylate cyclase reaction in permeabilized cells was similar to that of intact cells. Maximal adenylate cyclase activity was observed if cAMP receptor agonist or GTP-analogue was added just before cell permeabilization. If these activators were added after permeabilization adenylate cyclase was stimulated in a suboptimal way. The sensitivity of adenylate cyclase activity for receptor occupation was found to decay more rapidly than that for G-protein activation. Importantly, the adenylate cyclase reaction in permeabilized cells was subject to an adaptation-like process that was characterized by a time course similar to adaptation in vivo. In vitro adaptation was not affected by cAMP receptor agonists or by G-protein activation. Evidently electropermeabilized cells constitute an excellent system for investigating the positive and negative regulation of D. discoideum adenylate cyclase.     

A novel catecholamine-activated adenosine cyclic 3',5'-phosphate independent pathway for beta-adrenergic receptor phosphorylation in wild-type and mutant S49 lymphoma cells: mechanism of homologous desensitization of adenylate cyclase

Virtually all known biological actions stimulated by beta-adrenergic and other adenylate cyclase coupled receptors are mediated by cAMP-dependent protein kinase. Nonetheless, "homologous" or beta-adrenergic agonist-specific desensitization does not require cAMP. Since beta-adrenergic receptor phosphorylation may be involved in desensitization, we studied agonist-promoted receptor phosphorylation during homologous desensitization in wild-type S49 lymphoma cells (WT) and two mutants defective in the cAMP-dependent pathway of beta-agonist-stimulated protein phosphorylation (cyc- cannot generate cAMP in response to beta-adrenergic agonists; kin- lacks cAMP-dependent kinase). All three cell types demonstrate rapid, beta-adrenergic agonist-promoted, stoichiometric phosphorylation of the receptor which is clearly not cAMP mediated. The amino acid residue phosphorylated is solely serine. These data demonstrate, for the first time, that catecholamines can promote phosphorylation of a cellular protein (the beta-adrenergic receptor) via a cAMP-independent pathway. Moreover, the ability of cells with mutations in the adenylate cyclase-cAMP-dependent protein kinase pathway to both homologously desensitize and phosphorylate the beta-adrenergic receptors provides very strong support for the notion that receptor phosphorylation may indeed be central to the molecular mechanism of desensitization.