Receptor-mediated Activation Of Adenylate Cyclase Research Articles

Mechanisms of synthesis and action of 17α,20β-dihydroxy-4-pregnen-3-one, a teleost maturation-inducing substance

This article briefly reviews the current status of investigations, mainly based on the amago salmon,Oncorhynchus rhodurus, on the mechanisms of synthesis and action of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog). Pituitary gonadotropin is of primary importance in triggering meiotic maturation in teleost oocytes. However, the maturational action of gonadotropin is not direct, but is mediated by the follicular production of maturation-inducing substance (MIS). It is now well established that 17α,20β-diOHprog is the major MIS of salmonids. Production of this steroid occursvia the interaction of two distinct cell layers, the thecal and granulosa cell layers (2-cell type model). The first step of the stimulating effect of gonadotropin in both layers is the receptor-mediated activation of adenylate cyclase and formation of cAMP. Our findings suggest that the major stimulating action of gonadotropin on 17α,20β-diOHprog biosynthesis is due to the stimulation of 17α-hydroxyprogesterone production by the thecal layer and the selective induction of thede novo synthesis of 20β-hydroxysteroid dehydrogenase in the granulosa layer. 17α,20β-diOHprog acts at the surface of the oocyte. The early steps following 17α,20β-diOHprog action involve the formation of the major cytoplasmic mediator of this steroid, maturation-promoting factor (MPF). It was shown that goldfish MPF induces meiotic maturation inXenopus oocytes andvice versa. The chemical characterization of fish MPF is important for our understanding of the precise mode of maturational action of 17α,20β-diOHprog.

Transiently Elevated Levels of c-fosand c-mycOncogene Messenger Ribonucleic Acids in Cultured Murine Leydig Tumor Cells after Addition of Human Chorionic Gonadotropin

The gonadotropic hormones LH and human CG (hCG) normally function to stimulate steroidogenesis in testicular and ovarian cells through receptor-mediated activation of adenylate cyclase. These hormones are also important in regulating the development and growth of responsive cells. Such regulation requires tightly controlled gene expression. Herein we demonstrate that hCG induces increases in mRNAs encoding the competence oncogenes c-fos and c-myc in a murine Leydig cell tumor line (MA-10). When stimulated by hCG (40 ng/ml), the mRNA levels of both genes increase rapidly, peaking at 30 min for c-fos and 1 h for c-myc. Both mRNAs fall to near control levels by 3-6 h. This response to hCG is dose-dependent with half-maximal stimulation of these genes occurring at a concentration of 3 ng/ml, approximating the level required for 50% occupancy of the LH/hCG receptors and the ED50 for steroidogenesis. (Bu)2 cAMP (2 mM) elicits responses similar to those produced by hCG. The observation of oncogene control by the gonadotropin hCG provides further insight regarding the pathways by which such hormones may regulate steroidogenesis, growth, and differentiation of endocrine and neoplastic cells.

Forskolin sensitizes parathyroid hormone-induced cyclic AMP response, but not the bone resorptive effect, in mouse calvarial bones

The effect of forskolin on parathyroid hormone (PTH) stimulated bone resorption, as assessed in vitro by the release of 45Ca from prelabelled neonatal mouse calvarial bones, and cyclic AMP formation in mouse calvarial bones and osteoblast-like cells was investigated. Much higher concentrations (100–300-times) of PTH were required to stimulate cyclic AMP accumulation than to stimulate mineral mobilization in murine calvarial bones. PTH, in the absence of phosphodiesterase inhibitor, stimulated cyclic AMP formation in mouse calvarial bones at and above concentrations of 3–10 nmol/l with EC 50 at 10–15 nmol/l. In the presence of forskolin ( 1 or 10 μmol/l) the minimal concentration required to obtain a cyclic AMP response to PTH was decreased by a factor of 30–100 and the EC 50 value was decreased to 1–2 nmol/l. Similar results were seen in osteoblast-cnriched cells. In addition, the magnitude of the PTH-induced cyclic AMP response was substantially potentiated by forskolin, both in calvarial bones and in isolated osteoblasts. Forskolin, in the absence of PTH, stimulated cyclic AMP levels in mouse calvaria at and above 1 μmol/l. In the presence of PTH, the response to forskolin was potentiated over the whole dose-response curve with apparent EC 50 value at 1–2 μmol/l of forskolin. Forskolin (1 μmol/l) did not affect the magnitude of the 45Ca release response to PTH in 24 or 48 h cultures. In 96 h cultures, forskolin, in an additive manner, potentiated the effect of PTH on calcium mobilization. These results show that forskolin, in mouse calvarial bones and in isolated osteoblasts, in addition to directly stimulating cyclic AMP, can enhance receptor-mediated activation of adenylate cyclase. The finding that forskolin did not synergistically potentiate PTH-induced bone resorption suggests that there is no simple relationship between PTH-induccd cyclic AMP formation and stimulation of bone resorption.

CAMP regulation of early gene expression in signal transduction mutants of Dictyostelium

We have examined the regulation of three early developmentally regulated genes in Dictyostelium. Two of these genes (D2 and M3) are induced by pulses of cAMP and the other (K5) is repressed. Expression of these genes has been examined in a number of developmental mutants that are specifically blocked in various aspects of the signal transduction/cAMP relay system involved in aggregation and control of early development. The mutant strains include Synag mutants, which are blocked in receptor-mediated activation of adenylate cyclase and do not relay cAMP pulses; FrigidA mutants, which are blocked in receptor-mediated activation of both adenylate cyclase and the putative phosphoinositol bisphosphate (PIP 2) turnover pathway and appear to be mutations in the gene encoding one of the G α protein subunits; and a StreamerF allele, which lacks cGMP-specific cGMP phosphodiesterase. From the analysis of the developmental expression of these genes under a variety of conditions in these mutant strains, we have drawn a number of conclusions concerning the modes of regulation of these genes. Full induction of D2 and M3 genes requires cAMP interaction with the cell surface receptor and an “oscillation” of the receptor between active and adapted forms. Induction of these genes does not require activation of the signal transduction pathway that leads to adenylate cyclase activation and cAMP relay, but does require activation of other receptor-mediated intracellular signal transduction pathways, possibly that involving PIP 2 turnover. Likewise, repression of the K5 gene requires pulses of cAMP. Expression of this gene is insensitive to cAMP pulses in FrigidA mutants, suggesting that a signal transduction pathway is necessary for its repression. Results using the StreamerF mutant suggest that the rise in cGMP in response to cAMP/receptor interactions may not be directly related to control of the pulse-induced genes. In addition, we have examined the effect of caffeine, which M. Brenner and S. D. Thoms (1984, Dev. Biol., 101, 136–146) showed preferentially blocks the cAMP relay system by blocking receptor-mediated activation of adenylate cyclase. We show that in many of the mutants and in an axenic wild-type strain, caffeine causes the induction of pulse-induced gene expression to almost wild-type levels or in some cases to higher than wild-type levels. Our data suggest that caffeine works by activating some step in the signal transduction pathway that must lie downstream from both the receptor and at least one of the G proteins and thus has effects other than simply blocking the receptor-mediated cAMP relay system.

Pituicytes, glia and control of terminal secretion.

Once thought to be hormone-synthesizing cells, the pituicytes are now known to be the resident astroglia of the neurohypophysis (also referred to here as the posterior pituitary). Early investigators interpreted light microscopic observations as demonstrating pituicyte secretion, since pituicytes appeared to contain neuro-secretory material when hormone demand was low and not when it was increased. Ultrastructural studies have shown that pituicytes actually engulf or completely surround neurosecretory axons and axonal endings under basal conditions, and release these neural processes when conditions require increased hormone output. Thus, the pituicytes appeared to the early workers to contain and release hormone when they actually contained and released axons and terminals in which the hormone was, in fact, contained. Dynamic interactions of pituicytes with various of the other elements in the gland have also been demonstrated. When hormone demand is low, the pituicytes not only engulf the neurosecretory processes but also interpose their own processes between the secretory endings and the basal lamina. Since any hormone that is secreted must pass through the basal lamina and into the perivascular spaces in order to enter the fenestrated capillaries, pituicyte interpositions form physical, and perhaps chemical, barriers to hormone entering the circulation. Increasing hormone demand results in retraction of pituicyte processes from the basal lamina, permitting increased neural contact. Studies of isolated neurohypophysis and of cultured adult rat pituicytes have shown that these glia undergo appropriate morphological changes in response to osmotic stimuli or to receptor-mediated activation of adenylate cyclase. Both these events are thought to be effectors of the alterations seen in vivo. Some possible mechanisms by which pituicytes may participate in the control of secretory events are discussed.

Arachidonic acid and cyclic adenosine monophosphate stimulation of c-fos expression by a pathway independent of phorbol ester-sensitive protein kinase C.

The stimulation of cell proliferation by platelet-derived and other growth factors is associated with a rapid increase in the expression of the c-fos protooncogene. We and others have shown that phosphosphoinositide turnover and protein kinase C play a role in the activation of this gene by growth factors, but that a second, kinase C-independent pathway(s) exist. Because cAMP potentiates the actions of a number of growth factors and is elevated in platelet-derived growth factor-stimulated Swiss 3T3 cells, we examined the ability of cAMP to stimulate c-fos expression in this cell type. Forskolin, a direct activator of adenylate cyclase, elicited marked increases in c-fos mRNA levels. Receptor-mediated activation of adenylate cyclase by prostaglandin E1 and stimulation with the cAMP analog 8-bromo-cAMP also enhanced c-fos expression. In cells made protein-kinase C deficient, c-fos induction by phorbol ester was abolished; by contrast, c-fos was still induced by cAMP-elevating agents in protein kinase C-depleted cells. Platelet-derived growth factor causes cAMP accumulation by stimulating arachidonic acid release and the formation of prostaglandins capable of activating adenylate cyclase. The addition of arachidonic acid and the arachidonate metabolite prostaglandin E2 to Swiss 3T3 cultures stimulated c-fos expression. These data suggest the existence of a pathway from growth factor receptor to gene induction that is mediated by cAMP and does not depend on a phorbol ester-sensitive protein kinase C.

Formation and metabolism of inositol phosphates: the inositol tris/tetrakisphosphate pathway.

Many cell functions are modulated by receptor-activated mechanisms that act by increasing the free Ca2+ concentration in the cytosol (Williamson et al, 1981; Rasmussen and Barrett, 1984). Calcium mediates its effects by causing activity changes of a variety of proteins, including protein kinases, either directly or after binding to calmodulin or other Ca2+ -binding proteins (Cohen, 1985). A large number of studies have now established that a wide range of compounds, including hormones, secretagogues, neurotransmitters, chemoattractants and other cell activating subtances that involve Ca2+ mobilization, cause an activation of a phosphodiesterase (phospholipase C), which breaks down inositol lipids in the plasma membrane (Berridge and Irvine, 1984; Williamson et al., 1985; Hokin, 1985; Downes and Michell, 1985). However, unlike receptor-mediated activation of adenylate cyclase, which produces cAMP as the only second messenger, receptor-mediated inositol lipid breakdown serves a dual-signalling role with production of two second messengers having different functions. One of these compounds, myoinositol 1,4,5-trisphosphate (Ins(l,4,5)P3), is responsible for eliciting intracellular Ca2+ mobilization (Berridge and Irvine, 1984; Williamson, 1986), while the second compound, 1,2-diacylglycerol (DAG), has as its primary signalling role the activation of protein kinase C (Nishizuka, 1986). Hence in principle agents that interact with inositol lipid metabolism not only cause Ca2+ release with phosphorylation of proteins by Ca2+ -dependent protein kinases but also phosphorylation of a different set of proteins by activation of protein kinase C. However, most of these latter proteins have not been functionally characterized, and presently it is not clear whether receptor-mediated activation of protein kinase C via DAG production has as its physiological mode of action on cell function a synergistic role with Ca2 or a negative feedback modulatory role (Williamson and Hansen, 1987). Probably both effects occur, depending on the cell type and the extent to which protein kinase C becomes activated (Nishizuka, 1986).

Regulation of gene expression by the intracellular second messengers IP3 and diacylglycerol

In Dictyostelium, extracellular cAMP interacts specifically with cell-surface receptors to promote the accumulation of a variety of intracellular second messengers, such as 3'-5' cyclic adenosine monophosphate (cAMP) and 1,4,5 inositol trisphosphate (IP3). We and others have shown that activation of the cell-surface cAMP receptor can also modulate the expression of the Dictyostelium genome during development. In at least one instance, synthesis of intracellular cAMP is required for appropriate gene regulation. However, the induction of most cAMP-dependent gene expression can occur in the absence of receptor-mediated activation of adenylate cyclase and a consequent accumulation of intracellular cAMP. These results suggest that other intracellular second messengers produced in response to receptor activation may potentially act as signal transducers to modulate gene expression during development. In vertebrate cells, IP3 and diacylglycerol (DAG) are intracellular activators of specific protein kinases; they are produced in equimolar amounts by cleavage of phosphoinositol bisphosphate after a receptor-mediated activation of a membrane-bound phosphodiesterase. IP3 and, thus, by inference, diacyl-glycerol are synthesized in Dictyostelium as a response to cAMP interacting with its cell-surface receptor. Using defined conditions to inhibit the accumulation of extracellular cAMP, we have examined the effects of these compounds on the expression of genes that require cAMP for their maximal expression. Our results suggest that intracellular IP3 and DAG may in part mediate the action of extracellular cAMP on the expression of the Dictyostelium genome.

Cell differentiation in the absence of intracellular and extracellular cyclic AMP pulses in Dictyostelium discoideum

The mutant HSB1 was obtained by colony immunoblotting of mutagenized Dictyostelium discoideum cells with a monoclonal antibody against contact site A, a membrane glycoprotein. HSB1 cells undergo early development in the absence of intracellular and extracellular cAMP pulses. The receptor-mediated activation of adenylate cyclase is defective, but the basal activity increases normally during the first hours of development. Other preaggregative gene products, such as the inhibitor of phosphodiesterase, the contact site A glycoprotein, EDTA-stable contacts, and four developmentally regulated gene transcripts are also expressed in the mutant. Postaggregative gene transcripts fail to accumulate, even after pulsatile addition of cAMP. However, complete development of mutant cells is obtained by complementation with cells of the parent strain AX2.

Alterations in the myocardial β-adrenoceptor system of streptozotocin-diabetic rats

Previous investigations in our laboratory revealed subsensitivity of right ventricular tissue, isolated from one month STZ-diabetic rats, to the inotropic effects of isoproterenol. The present study was concerned with the characterization of this subsensitivity phenomenon. Observations of supersensitivity to methoxamine accompanied by decreased responsiveness to glucagon without a change in responsiveness to forskolin suggested a specific effect of diabetes on pathways involving receptor-mediated activation of adenylate cyclase. Radioligand binding analysis further revealed a specific decrease in the population of the high affinity state of the β-adrenoceptor. Since the high affinity receptor state is a necessary intermediate for adenylate cyclase activation and enhanced myocardial contractility, it is proposed that the specific decrease in the high affinity population of the β-adrenoceptor contributes to myocardial subsensitivity to isoproterenol observed in the diabetic animals. It is further proposed that the decrease in receptor population is related to increases in circulating epinephrine levels which were evident in the diabetic animals.

Characterization of corticotropin-releasing factor receptor-mediated adenylate cyclase activity in the rat central nervous system.

We report here that corticotropin-releasing factor (CRF) stimulates adenylate cyclase activity in the rat central nervous system (CNS). In frontoparietal cortex homogenates, the stimulation by CRF was dependent on time, temperature, tissue protein concentration, and guanine nucleotides. The rank order of potency for CRF analogs and fragments in stimulating adenylate cyclase activity [(Nle21,38) rat CRF greater than rat CRF approximately equal to acetyl ovine CRF (4-41) approximately equal to alpha helical ovine CRF greater than ovine CRF much greater than ovine CRF (1-39) approximately equal to ovine CRF (7-41)] was consistent with their affinities for CRF receptors in the brain and their relative potencies in stimulating pituitary adrenocorticotropic hormone secretion in vitro. The putative CRF receptor antagonist, alpha helical ovine CRF (9-41), did not stimulate adenosine 3',5'-cyclic monophosphate (cAMP) production but was able to attenuate the stimulation by various concentrations of rat CRF. The regional distribution of 125I-Tyr(o)-ovine CRF binding (olfactory bulb greater than frontoparietal cortex approximately equal to cerebellum greater than hypothalamus greater than striatum greater than or equal to midbrain greater than hippocampus greater than or equal to spinal cord) did not correspond with the regional degree of CRF receptor-mediated stimulation of adenylate cyclase (frontoparietal cortex greater than olfactory bulb greater than or equal to cerebellum greater than midbrain greater than or equal to hippocampus greater than striatum greater than or equal to hypothalamus greater than spinal cord). In addition, marked differences were observed in the ability of forskolin to potentiate CRF-stimulated cAMP production in the various brain areas examined. In summary, these data demonstrate that at least one of the second-messenger systems mediating the effects of CRF in the CNS involves stimulation of cAMP production and provides further support for a neurotransmitter role for this neuropeptide in the brain. Significant differences in the regulation of CRF-stimulated cAMP production and the disparity between CRF receptor number and receptor-mediated adenylate cyclase activity in discrete regions of the rat CNS suggest that some populations of CRF receptors in the brain may be functionally coupled to alternative signal transduction mechanisms.

Surface receptor-mediated activation of adenylate cyclase in Dictyostelium. Regulation by guanine nucleotides in wild-type cells and aggregation deficient mutants.

GTP and GTP analogs produced significant (up to 17-fold) and persistent activation of adenylate cyclase in lysates of Dictyostelium discoideum amoeba. The activation was enhanced 2- to 4-fold by cAMP (the agonist for receptor-mediated adenylate cyclase activation), was specific for guanine nucleoside triphosphates, and was inhibited by guanosine 5'-(O-2-thio)diphosphate. The order of potency of guanine nucleotides was guanosine 5'-(O-3-thio)triphosphate greater than guanyl-5'-yl imidodiphosphate greater than GTP; half-maximal activation was observed with 1-10 microM guanine nucleotide. Maximal activation occurred when the guanine nucleotide was added within seconds after cell lysis and the lysate was preincubated for 5 min prior to assay. Under these optimal in vitro conditions, the capacity of guanine nucleotides to activate decreased, closely correlating with adaptation or desensitization induced by exposure of intact cells to cAMP during a period of 10 min. These data strongly support that regulation of adenylate cyclase in Dictyostelium occurs via a receptor-linked GTP/GDP exchange protein. Two mutants, designated synag 7 and 49 were isolated in which cAMP and/or guanine nucleotides were not sufficient to activate adenylate cyclase. The wild-type pattern of guanine nucleotide regulation was restored to synag 7 lysates by the addition of a high-speed supernatant from wild-type cells. Characterization of these mutants demonstrates that activation of adenylate cyclase is not required for growth or cell-type specific differentiation but is essential for cellular aggregation and influences morphogenesis and pattern formation. This suggests that Dictyostelium may provide a model suitable for detailed genetic analysis of surface receptor-guanine nucleotide-binding regulatory protein linked adenylate cyclase systems and for determining the role of these systems in development.

Interactions between opiate- and dopamine-induced effects on adenylate cyclase in rat striatum

In rat striatal P2 membrane preparations examined under constant experimental conditions, activation of either D2-dopamine or opiate receptors inhibited basal adenylate cyclase with a similar maximal inhibition of approximately 30%. These inhibitions were not additive. D1 dopamine receptor-mediated activation of adenylate cyclase by SKF38393 was inhibited by morphine in an additive fashion, but the opiate had minimal effects on the net activation of adenylate cyclase by dopamine which activates both D1 and D2 receptors. From these results it would appear that opiate and D1 dopamine receptor-mediated activation of adenylate cyclase summate, but that opiate and D2 dopamine receptors regulate a common pool of adenylate cyclase in striatal membranes.

Desensitization by histamine of H 2 receptor-mediated adenylate cyclase activation in the human gastric cancer cell line HGT-1

Short-term treatment of cultured HGT-1 cells with histamine produced a time-dependent (half-life: 20 min) and homologous desensitization of histamine H 2, receptor activity mediating cAMP generation in HGT-1 cells and gastric acid secretion in normal gastric mucosa. Histamine treatment resulted in loss of response of the adenylate cyclase to histamine in purified plasma membranes, but had no effect on basal, vasoactive intestinal peptide (VIP)- or NaF-stimulated enzyme activities. We propose that the desensitization of gastric histamine H 2 receptor by histamine evidenced in cellular or subcellular preparations from HGT-1 cells could be involved in the physiological regulation and pharmacological control of gastric cell function in man. Desensitization Histamine H 2receptor Adenylate cyclase Acid secretion HGT-1 cell

Selective blockage by islet-activating protein, pertussis toxin, of negative signal transduction from receptors to adenylate cyclase.

The adenylate cyclase-cyclic AMP system is one of the signal transduction processes that has been the subject of extensive studies in recent years. Stimulation of some membrane receptors (“activatory” receptors) by particular agonists produces, via activation of adenylate cyclase, cyclic AMP, which acts as an intracellular second messenger of the receptors by activating one of the several kinds of cytosolic protein kinases. The receptor-linked adenylate cyclase system is now known to consist of three components; the guanine nucleotide-binding protein usually referred to as N, G, or G/F, has been identified as the functional communicator between receptors (R) and the catalytic unit of the cyclase (C). A growing body of evidence has accumulated in support of the concept that N plays the pivotal role in receptor-mediated activation of adenylate cyclase, as reviewed by Ross and Gilman (1), Limbird (2) and Spiegel and Downs (3). When no extracellular signal is available to the cell, the species of the guanine nucleotide bound to N is GDP, which allows functional dissociation of the three components, R, N, and C. If information is transmitted by an agonist (A) which occupies R, the ternary complex, A-R-N is formed as the “turn-on” reaction which leads to replacement of GDP by intracellular GTP at the specific binding sites on N. The adenylate cyclase enzyme is then activated as a result of association of the GTP-bound N with C. Synthesis of cyclic AMP continues until GTP is hydrolyzed by the GTPase “turn-off” reaction, leaving GDP on N.

Effect of antibody-induced modulation of measles (SSPE) virus membrane proteins on β-adrenergic receptor-mediated adenylate cyclase activity

The effect of measles virus antiserum on the expression of viral glycoproteins on the membranes of measles (SSPE) persistently infected C6 rat glioma cells was studied. There was a gradual loss of virus membrane antigen from these cells until no antigen was detectable 18 days after initiation of antiserum treatment. At this stage approximately 25% of cells still displayed intracellular virus antigen which was also lost after further cell passages. There was an accompanying recovery of the previously reported disrupted catecholamine-dependent β-adrenergic receptor-stimulated cAMP synthesis in antiserum-treated cells which coincided with the loss of viral antigen from the membrane. This was determined to be due to a recovery of fluoride-stimulated activity of the CAMP synthesising adenylate cyclase enzyme to normal values. Thus we demonstrate that the impairment of this important cell function was due to insertion of viral antigen in the cell membrane rather than its accumulation in the cytoplasm.

Prostaglandin E1 action on adenylate cyclase activity and membrane lipids in platelets.

The effect of prostaglandin E1 (PGE1) on temperature-induced membrane lipid phase transitions of human blood platelets was investigated using excimer forming 1-pyrenedecanoic acid as fluorescent probe. PGE1 and PGF2 alpha at 10 microM concentrations had a small but reproducible effect on the melting curves between 20 degrees and 30 degrees C and lowered the temperature for the onset of the phase transition from 26 degrees to 21 degrees C. The adenylate cyclase of the platelet membrane was half maximally stimulated by PGE1 at 0.8 microM and maximally activated at 10 microM concentrations. PGF2 alpha at 100 microM concentrations produced only 40% of the maximal stimulation achieved with PGE1. Stimulation of adenylate cyclase by 10 microM PGE1 increased steeply with temperature between 20 degrees to 30 degrees C. Binding experiments with [3H]PGE1 indicated a high affinity and a low affinity site with KD 13nM and 2 microM, respectively. Non-radioactive PGE1 inhibited binding of [3H]PGE1 to both sites at concentrations corresponding to their respective dissociation constants. PGF2 alpha inhibited binding of PGE1 only at greater than or equal to 1 microM concentrations. It is assumed that only the high affinity binding sites represent specific PGE1 receptors. Binding of PGE1 to the low affinity sites and the observed effects on membrane structure may result from interactions of PGE1 with membrane lipids. Since these latter effects and also stimulation of adenylate cyclase required similar concentrations, it is proposed that local interactions of PGE1 with membrane lipids are a prerequisite for specific receptor-mediated adenylate cyclase activation.

Cell differentiation in the absence of intracellular cyclic AMP pulses in Dictyostelium discoideum

Repeated additions of cyclic AMP to a morphogenetic mutant of Dictyostelium discoideum, agip 53, induced cell differentiation to the aggregation competent state as previously reported [ Darmon, Brachet, and Pereira da Silva (1975). Proc. Nat. Acad. Sci. USA 72, 3163–3166]. Cyclic AMP additions elicited transient increases of the intracellular cyclic GMP concentration but no significant increases of the intracellular cyclic AMP concentration. These results suggest that transient increases of the intracellular cyclic AMP concentration are not necessary for cell differentiation. Agip 53 seems to be unable to relay cyclic AMP signals. A defect in the receptor-mediated activation of adenylate cyclase could be the biochemical basis of the mutant phenotype of agip 53.

Modulation of β-adrenergic agonist binding by guanylnucleotides in avian erythrocytes

The first adenylate cyclase-linked receptor showing a decreased affinity due to the presence of guanine nucleotides, was the glucagon receptor of rat liver plasma membr~es [ 1). Guanine nucleotidedependent modulation of agonist binding to a /3-adrenergic receptor, was initially shown in plasma membranes from cultured rat glioma cells (C6) [2], and has since been observed in membranes from frog erythrocytes [3f, and other cells [4,5]. Guanine nucleotide modulation has also been found for receptors [6-81, which on interaction with adenylate cyclase results in inhibition. Although, the dependence on guanine nucleotides for /.l receptor-mediated activation of adenylate cyclase [9-l l] in avian erythrocyte membranes is well documented, the bind~g of agonists to the fl receptor has been reported to be unaffected by guanine nucleotides [ 12-141. To explain the absence of guanine nucleotide-mediated receptor modulation, and the lack of agonist-dependent desensitization of adenylate cyclase, it has been proposed that avian erythrocyte membranes may lack an additions GTP binding protein present in other cells [IS]. This study aimed to clarify these controversial points.