Sonication as a tool for the study of adenylyl cyclase activity.

The technique of sonication was applied in studying adenylyl cyclase activity of cultured fibroblasts. Exposure of BHK 21 c/13 to brief periods of low power sonication gives cell preparations with greater basal, fluoride and hormone sensitive adenylyl cyclase activites than those of broken cell preparations of homogenized cells. The sonicated cells provide a convenient method to study adenylyl cyclase since they are added directly to the adenylyl cyclase reaction vessels without further processing. Maximal epinephrine stimulated activity in sonicated cells is nearly equivalent to that activated by sodium fluoride, but the apparent affinity of the enzyme system is similar to that of broken cell preparations. Furthermore, broken cell preparations of sonicated cells possess greater adenylyl cyclase activity than broken cell preparations of unsonicated cells. This procedure may provide a useful tool for the analysis of the hormonal regulation of adenylyl cyclase activity of isolated cells.     

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

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

The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins.

In the yeast Saccharomyces cerevisiae, adenylyl cyclase is regulated by RAS proteins. We show here that the yeast adenylyl cyclase forms at least two high-molecular-weight complexes, one with the RAS protein-dependent adenylyl cyclase activity and the other with the Mn(2+)-dependent activity, which are separable by their size difference. The 70-kDa adenylyl cyclase-associated protein (CAP) existed in the former complex but not in the latter. Missense mutations in conserved motifs of the leucine-rich repeats of the catalytic subunit of adenylyl cyclase abolished the RAS-dependent activity, which was accompanied by formation of a very high molecular weight complex having the Mn(2+)-dependent activity. Contrary to previous results, disruption of the gene encoding CAP did not alter the extent of RAS protein-dependent activation of adenylyl cyclase, while a concomitant decrease in the size of the RAS-responsive complex was observed. These results indicate that CAP is not essential for interaction of the yeast adenylyl cyclase with RAS proteins even though it is an inherent component of the RAS-responsive adenylyl cyclase complex.     

The intracellular location of adenylyl cyclase in the cellular slime molds Dictyostelium discoideum and Polysphondylium pallidum

Adenylyl cyclase activity was low or not detectable on intact cells and in isolated plasma membranes, phagocytic vacuoles and nuclei of the two slime mold species examined. The entire activity of homogenates was sedimentable and concentrated in a light membrane fraction. When this fraction was centrifugated through sucrose density gradients the adenylyl cyclase activity sedimented differently from all other enzymes measured. The gradient fractions with the highest specific activity of adenylyl cyclase consisted mainly of small vesicles. No changes in adenylyl cyclase distribution were associated with development. The possibility that cellular slime mold adenylyl cyclase activity is associated with vesicles in vivo, as already suggested by Maeda & Gerisch [10], is discussed.     

Characterization of adenylyl cyclase in heart sarcolemma in the absence or presence of alamethicin

Alamethicin is commonly used as an agent for unmasking the latent enzyme activities in vesicular membrane preparations; however, relatively little is known about the effect of this agent on the characteristics of adenylyl cyclase in heart sarcolemma. By employing rat heart sarcolemmal preparation, we observed 5 to 6 fold increase in adenylyl cyclase activity upon treatment with alamethicin. Kinetic experiments using various concentrations of MgATP revealed that the increase in adenylyl cyclase activity in alamethicin treated membranes was associated with an increase in V_max as well as affinity of the substrate for the enzyme. Dose-responses of the control and alamethicin-treated preparations to various activators of adenylyl cyclase revealed that the sensitivity of the enzyme to forskolin, NaF and GppNHp, was markedly increased upon treating sarcolemma with alamethicin. The activation of adenylyl cyclase by forskolin was also enhanced by increasing the concentration of alamethicin in the incubation medium. Furthermore, there was a greater increase in adenylyl cyclase activity with different concentrations of Mn²⁺ in the presence of alamethicin. These results suggest that alamethicin treatment alters the characteristics of adenylyl cyclase in addition to unmasking the enzyme activity in the purified sarcolemmal vesicular preparation.     

Reactivity of the adenylyl cyclase system in rat tissues to biogenic amines and peptide hormones under starvation condition

Under starvation condition, sensitivity of the adenylyl cyclase system to regulatory action of biogenic amines and peptide hormones in rat tissues are changed. In the myocardium and skeletal muscles, after 2 and 4 days of starvation, the regulatory effects of isoproterenol and relaxin acting via G,-proteins on the adenylyl cyclase activity and the G-protein GTP-binding are significantly increased compared with control. At the same time, regulatory effects ofsomatostatin which are realized via Gi-proteins, on adenylyl cyclase system in the myocardium are decreased. Under prolonged starvation consisting of two consecutive 4-days periods, the effects of hormones acting via Gs-proteins on the adenylyl cyclase activity in muscle tissues are decreased to control value levels. The effects of hormones acting via Gi-proteins are largely reduced. In the brain, intensification of adenylyl cyclase stimulating hormonal effects was late and only observed after a 4-day starvation. Unlike muscle tissues, the increase of adenylyl cyclase stimulating effects in the brain is preserved after two-period starvation. The weakening of adenylyl cyclase inhibiting hormonal signals both in the brain and muscles is observed after a 2-day starvation and then the weakening is intensified. Possible role of glucose level and basal adenylyl cyclase activity in determination of the sensitivity of the adenylyl cyclase system to hormones under study is discussed. It is suggested that one of the key causes of physiological changes in animal organism under starvation involves alteration of hormonal signalling systems sensitivity, in particular that of the adenylyl cyclase system, to hormone regulatory action.     

Motor dysfunction in type 5 adenylyl cyclase-null mice

Various neurotransmitters, such as dopamine, stimulate adenylyl cyclase to produce cAMP, which regulates neuronal functions. Genetic disruption of the type 5 adenylyl cyclase isoform led to a major loss of adenylyl cyclase activity in a striatum-specific manner with a small increase in the expression of a few other adenylyl cyclase isoforms. D1 dopaminergic agonist-stimulated adenylyl cyclase activity was attenuated, and this was accompanied by a decrease in the expression of the D1 dopaminergic receptor and G(s)alpha. D2 dopaminergic agonist-mediated inhibition of adenylyl cyclase activity was also blunted. Type 5 adenylyl cyclase-null mice exhibited Parkinsonian-like motor dysfunction, i.e. abnormal coordination and bradykinesia detected by Rotarod and pole test, respectively, and to a lesser extent locomotor impairment was detected by open field tests. Selective D1 or D2 dopaminergic stimulation improved some of these disorders in this mouse model, suggesting the partial compensation of each dopaminergic receptor signal through the stimulation of remnant adenylyl cyclase isoforms. These findings extend our knowledge of the role of an effector enzyme isoform in regulating receptor signaling and neuronal functions and imply that this isoform provides a site of convergence of both D1 and D2 dopaminergic signals and balances various motor functions.     

Identification of RGS2 and type V adenylyl cyclase interaction sites

The production of cAMP is controlled on many levels, notably at the level of cAMP synthesis by the enzyme adenylyl cyclase. We have recently identified a new regulator of adenylyl cyclase activity, RGS2, which decreases cAMP accumulation when overexpressed in HEK293 cells and inhibits the in vitro activity of types III, V, and VI adenylyl cyclase. In addition, RGS2 blocking antibodies lead to elevated cAMP levels in olfactory neurons. Here we examine the nature of the interaction between RGS2 and type V adenylyl cyclase. In HEK293 cells expressing type V adenylyl cyclase, RGS2 inhibited Galpha(s)-Q227L- or beta(2)-adrenergic receptor-stimulated cAMP accumulation. Deletion of the N-terminal 19 amino acids of RGS2 abolished its ability to inhibit cAMP accumulation and to bind adenylyl cyclase. Further mutational analysis indicated that neither the C terminus, RGS GAP activity, nor the RGS box domain is required for inhibition of adenylyl cyclase. Alanine scanning of the N-terminal amino acids of RGS2 identified three residues responsible for the inhibitory function of RGS2. Furthermore, we show that RGS2 interacts directly with the C(1) but not the C(2) domain of type V adenylyl cyclase and that the inhibition by RGS2 is independent of inhibition by Galpha(i). These results provide clear evidence for functional effects of RGS2 on adenylyl cyclase activity that adds a new dimension to an intricate signaling network.     

Cytoplasmic domain of natriuretic peptide receptor C constitutes Gi activator sequences that inhibit adenylyl cyclase activity

We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhibited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) protein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase activity. The peptides used were KKYRITIERRNH (peptide 1), RRNHQEESNIGK (peptide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITIYKKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y have partial G(i) activator sequences with truncated carboxyl or amino terminus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inhibited adenylyl cyclase activity in a concentration-dependent manner with apparent K(i) between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyclase activity with a higher K(i) of about 10 nm, and peptides X, Y, and Z were unable to inhibit adenylyl cyclase activity. The maximal inhibitions observed were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleotides and was completely attenuated by PT treatment. In addition, the stimulatory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These results suggest that the small peptide fragments of the cytoplasmic domain of the NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibit adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptides having complete structural specificity of G(i) activator sequences at both amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus, whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.     

A Quantitative Study of the Ca2+/Calmodulin Sensitivity of Adenylyl Cyclase in Aplysia, Drosophila, and Rat

Studies in Aplysia and Drosophila have suggested that Ca2+/calmodulin-sensitive adenylyl cyclase may act as a site of convergence for the cellular representations of the conditioned stimulus (Ca2+ influx) and unconditioned stimulus (facilitatory transmitter) during elementary associative learning. This hypothesis predicts that the rise in intracellular free Ca2+ concentration produced by spike activity during the conditioned stimulus will cause an increase in the activity of adenylyl cyclase. However, published values for the Ca2+ sensitivity of Ca2+/calmodulin-sensitive adenylyl cyclase in mammals and in Drosophila vary widely. The difficulty in evaluating whether adenylyl cyclase would be activated by physiological elevations in intracellular Ca2+ levels is in part a consequence of the use of Ca2+/EGTA buffers, which are prone to several types of errors. Using a procedure that minimizes these errors, we have quantified the Ca2+ sensitivity of adenylyl cyclase in membranes from Aplysia, Drosophila, and rat brain with purified species-specific calmodulins. In all three species, adenylyl cyclase was activated by an increase in free Ca2+ concentration in the range caused by spike activity. Ca2+ sensitivity was dependent on both calmodulin concentration and Mg2+ concentration. Mg2+ raised the threshold for adenylyl cyclase activation by Ca2+ but also acted synergistically with Ca2+ to activate maximally adenylyl cyclase.     

Benzyloxybenzaldehyde analogues as novel adenylyl cyclase activators.

Several benzyloxybenzaldehyde analogues were prepared and found to have significant inhibitory activity toward neutrophil superoxide formation. Consequently, these compounds were evaluated for cAMP-elevating capability. Among them, benzyloxybenzaldehyde (7), exhibiting activity equivalent to forskolin, was determined as an adenylyl cyclase activator since it elevates cAMP levels by activation of adenylyl cyclase but not by inhibition of phosphodiesterase. Having a chemical structure very different from known adenylyl cyclase activators, compound 7 is recommended by us for use as a new lead compound in the future development of adenylyl cyclase activators.     

Exposure to intermittent high altitude induces different changes in adenylyl cyclase activity in hearts of young and adult Wistar rats

This study investigates changes of adenylyl cyclase activity in the heart of young and adult Wistar rats exposed to experimental conditions simulating high altitude hypoxia as a model for interpretation of some adaptive changes of adenylyl cyclase observed in human. The exposure of rats to intermittent high altitude (IHA) hypoxia (5000 m) showed significant adaptive changes. The right ventricular weight and the ratio of right/left ventricular weights of adult rats exposed to IHA were significantly increased when compared to appropriate controls; adaptive changes of cardiac adenylyl cyclase being dependent on the age of the animals. The isoprenaline-stimulated activity was higher in the left than in the right ventricle, and in both ventricles it was higher in young rats than in adult rats. When compared to controls, isoprenaline stimulation was decreased in the right ventricles of adapted young rats and, by contrast, it was increased in the left ventricles of adapted adult rats. This decrease and increase of adenylyl cyclase activity evoked by isoprenaline was paralleled by forskolin-induced adenylyl cyclase activity in these experimental groups. It seems therefore that the changes in the pattern of total adenylyl cyclase activity observed under IHA hypoxia may at least be partially explained by the changes of beta-adrenergic receptor susceptibility following IHA hypoxia.     

Myometrial adenylyl cyclase protein decreases on the last day of pregnancy in the rat

To determine whether gestation-related changes in responsiveness of the rat uterus to beta-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12-14) and then late (Days 18-20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the beta-adrenergic receptor (isoproterenol, 10(-4) M), the G protein (GTP, 10(-5) M), or the adenylyl cyclase enzyme (MnCl(2), 20 mM), was similarly altered during gestation. Total adenylyl cyclase protein was quantified by [(3)H]forskolin binding assay in myometrial membranes from nonpregnant and pregnant (Day 14, Day 20, Day 21, and intrapartum Day 22) rats. Adenylyl cyclase protein increased progressively from nonpregnant rats to pregnant rats at mid (Day 14) and late (Day 20) gestation, but it decreased abruptly to nonpregnant levels on Day 21, the day before parturition, and remained at similar levels on Day 22 (intrapartum). The gestation-related increase in expression of myometrial adenylyl cyclase protein may facilitate uterine quiescence during pregnancy, and the abrupt decrease of adenylyl cyclase protein on the last day of pregnancy may be a contributing mechanism for the initiation of labor.     

Tyrosine kinase-mediated serine phosphorylation of adenylyl cyclase

Receptor tyrosine kinase (RTK) activation is associated with modulation of heptahelical receptor-stimulated adenylyl cyclase responses. The mechanisms underlying the RTK-mediated enhancement of adenylyl cyclase function remain unclear. In the present studies, we show that the tyrosine kinase-dependent enhancement of adenylyl cyclase isoform VI function parallels an enhancement in serine phosphorylation of the enzyme. This effect was mediated by both RTK activation, with IGF-1, and by tyrosine phosphatase inhibition, with sodium orthovanadate. This enhancement of adenylyl cyclase function was not attenuated by inhibitors of ERK, PKC, PKA, or PI3 kinase activity but was blunted by inhibition of endogenous p74(raf-1)() activity. To characterize the molecular site of this effect we identified multiple candidate serine residues in and adjacent to the adenylyl cyclase VI C1b catalytic region and performed serine-to-alanine site-directed mutagenesis using adenylyl cyclase VI as a template. Mutation of serine residues 603 and 608 or serine residues 744, 746, 750, and 754 attenuated both the tyrosine kinase-mediated enhancement of enzyme phosphorylation as well as the sensitization of function. Together, these data define a novel tyrosine kinase-mediated mechanism leading to serine phosphorylation of adenylyl cyclase isoform VI and the sensitization of adenylyl cyclase responsiveness.     

The SH3 domain of the S. cerevisiae Cdc25p binds adenylyl cyclase and facilitates Ras regulation of cAMP signalling

Cdc25 and Ras are two proteins required for cAMP signalling in the budding yeast Saccharomyces cerevisiae. Cdc25 is the guanine nucleotide exchange protein that activates Ras. Ras, in turn, activates adenylyl cyclase. Cdc25 has a Src homology 3 (SH3) domain near the N-terminus and a catalytic domain in the C-terminal region. We find that a point mutation in the SH3 domain attenuates cAMP signalling in response to glucose feeding. Furthermore, we demonstrate, by using recombinant adenylyl cyclase and Cdc25, that the SH3 domain of Cdc25 can bind directly to adenylyl cyclase. Binding was specific, because the SH3 domain of Abp1p (actin-binding protein 1), which binds the 70,000 Mr subunit of adenylyl cyclase, CAP/Srv2, failed to bind adenylyl cyclase. A binding site for Cdc25-SH3 localised to the C-terminal catalytic region of adenylyl cyclase. Finally, pre-incubation with Ras enhanced the SH3-bound adenylyl cyclase activity. These studies suggest that a direct interaction between Cdc25 and adenylyl cyclase promotes efficient assembly of the adenylyl cyclase complex.     

Alterations of Tubulin Function Caused by Chronic Antidepressant Treatment in Rat Brain

1. Antidepressants have been used clinically for many years; however, the neurochemical mechanism for their therapeutic effect has not been clarified yet. Recent reports indicate that chronic antidepressant treatment directly affects the postsynaptic membrane to increase the coupling between the stimulatory GTP-binding (G) protein, G_s, and adenylyl cyclase. Tubulin, a cytoskeletal element, is involved in the stimulatory and inhibitory regulation of adenylyl cyclase in rat cerebral cortex via direct transfer of GTP to G proteins. In this study, we investigated whether the functional change of the adenylyl cyclase system caused by chronic antidepressant treatment involves an alteration of tubulin function in the regulation of adenylyl cyclase activity.2. Male Sprague–Dawley rats were treated once daily with amitriptyline or saline by intraperitoneal injection (10 mg/kg) for 21 days, and their cerebral cortex membranes and GppNHp-liganded tubulin (tubulin-GppNHp) were prepared for what.3. GppNHp-stimulated adenylyl cyclase activity in cortex membranes from amitriptyline-treated rats was significantly higher than that in control membranes. Furthermore, tubulin–GppNHp prepared from amitriptyline-treated rats was more potent than that from control rats in the stimulation of adenylyl cyclase activity in the cortex membranes of the controls. However, there was no significant difference in manganese-stimulated adenylyl cyclase activity between control and amitriptyline-treated rats.4. The present results suggest that chronic antidepressant treatment enhances not only the coupling between G_s and the catalytic subunit of adenylyl cyclase but also tubulin interaction with G_s in the cerebral cortex of the rat.     

Cannabinoid Receptor Activation Differentially Regulates the Various Adenylyl Cyclase Isozymes

Abstract: Two cannabinoid receptors belonging to the superfamily of G protein-coupled membrane receptors have been identified and cloned: the neuronal cannabinoid receptor (CB1) and the peripheral cannabinoid receptor (CB2). They have been shown to couple directly to the G_i/o subclass of G proteins and to mediate inhibition of adenylyl cyclase upon binding of a cannabinoid agonist. In several cases, however, cannabinoids have been reported to stimulate adenylyl cyclase activity, although the mechanism by which they did so was unclear. With the cloning of nine adenylyl cyclase isozymes with various properties, including different sensitivities to α_s, α_i/o, and βγ subunits, it became important to assess the signaling pattern mediated by each cannabinoid receptor via the different adenylyl cyclase isozymes. In this work, we present the results of cotransfection experiments between the two types of cannabinoid receptors and the nine adenylyl cyclase isoforms. We found that independently of the method used to stimulate specific adenylyl cyclase isozymes (e.g., ionomycin, forskolin, constitutively active α_s, thyroid-stimulating hormone receptor activation), activation of the cannabinoid receptors CB1 and CB2 inhibited the activity of adenylyl cyclase types I, V, VI, and VIII, whereas types II, IV, and VII were stimulated by cannabinoid receptor activation. The inhibition of adenylyl cyclase type III by cannabinoids was observed only when forskolin was used as stimulant. The activity of adenylyl cyclase type IX was inhibited only marginally by cannabinoids.     

Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.     

Thyroliberin receptor binding and adenylyl cyclase activation in cultured prolactin-producing rat pituitary tumor cells (GH cells)

A thyroliberin (TRH)-responsive particulate bound adenylyl cyclase is present in two rat anterior pituitary tumor cell strains (GH4C1 and GH3) which synthesize and secrete prolactin. At a given Mg2+ concentration, ATP and the guanyl nucleotides GTP and guanyl 5'-yl-imidodiphosphate (GMP-P(NH)P) caused a dose-dependent increase in adenylyl cyclase activity. The maximum response to thyroliberin occurred with ATP and GTP at concentrations above 0.30 mM and 2 microM, respectively. The maximal stimulatory effect of thyroliberin on adenylyl cyclase activity was 2-fold in the presence of GTP. GMP-P(NH)P increased the basal enzyme activity 4- to 10-fold over and above that of equimolar concentrations of GTP but supported poorly the TRH-induced response. Mg2+ caused a dose-dependent increase in the basal enzyme activity and reduced TRH and fluoride-induced responses. Also, Mn2+ and Co2+ stimulated the basal adenylyl cyclase activity while Zn2+, Ca2+, and Cu2+ inhibited the enzyme, and neither cations supported the TRH response. Half-maximal stimulation of the adenylyl cyclase by TRH and half-maximum binding of [3H]TRH to membranes at 35 degrees C were 102 and 56 nM, respectively. Pretreatment with TRH decreased the apparent Vmax of the enzyme and the maximal binding of [3H]TRH. Of 6 TRH analogs tested, only one was able to displace [3H]TRH from its receptor and to increase the adenylyl cyclase activity. We suggest that adenylyl cyclase activation is an early event in the stimulus secretion coupling between TRH and prolactin-producing GH cells.