Long-term effect of forskolin on the activation of adenylyl cyclase in astrocytes

Long-term (48-h) forskolin treatment of rat astroglial cells led to a slight decrease (30-40%) in the response to isoproterenol, vasoactive-intestinal peptide, guanyl 5'-(beta gamma-imido)diphosphate, guanosine 5'-O-(3-thiotriphosphate) [GTP(S)], and AIF4- in crude membrane fractions. In contrast, the acute stimulatory effect of forskolin was increased by 1.25-1.5-fold. These two opposite effects of forskolin were mediated by a cyclic AMP-dependent mechanism. No changes in Gs alpha, Gi alpha, or G beta protein levels could be determined by immunoblotting using specific antisera. No significant differences were observed in the ability of G proteins extracted from control and forskolin-treated cells to reconstitute a full adenylyl cyclase activity in membranes from S49 cyc- cells, lacking Gs alpha protein. Gs alpha proteins were detected in two pools of membranes, one in the heavy sucrose fractions and the other in light sucrose fractions. Forskolin treatment of the cells shifted Gs alpha protein toward the light-density membranes. We did not find any significant change in the distribution of adenylyl cyclase. In contrast to the decreased stimulation of adenylyl cyclase activity by agonists acting via Gs alpha, observed in the crude membrane fraction, the responses of adenylyl cyclase to forskolin as well as to GTP(S) were increased in the purified plasma membrane fractions. These results may indicate that sensitization of the catalyst appears to be the dominant component in the astroglial cell response to long-term treatment by forskolin.     

Sodium saccharin inhibits adenylyl cyclase activity in non-taste cells

We have studied the in vitro effect of sodium saccharin (NaSacch) on the rat adipocyte adenylyl cyclase complex. NaSacch (2.5-50 mM) inhibited significantly in a dose-dependent manner basal and isoproterenol-stimulated cAMP accumulation on isolated rat adipocytes. Similarly, NaSacch (2.5-50 mM) inhibited forskolin-stimulated adenylyl cyclase activity measured in the presence of Mg(2+)-ATP on adipocyte, astrocyte and thyrocyte membrane fractions. In contrast, NaSacch did not inhibit but slightly increased the forskolin-stimulated adenylyl cyclase activity measured in the presence of Mn(2+)-ATP and GDP beta S, a stable GDP analogue. The effect of NaSacch was not mediated through either the A1-adenosine receptor (A1R) or the alpha 2-adrenergic receptor (alpha 2AR). The inhibitory effect of NaSacch was additive to that of A1R agonist and was not blocked by the addition of the alpha 2AR antagonist RX 821002. Pretreatment of adipocytes with pertussis toxin slightly attenuated but did not abolish the inhibitory effect of NaSacch on forskolin-stimulated adenylyl cyclase activity on membrane fractions. These data suggest that the inhibitory effect of NaSacch on forskolin stimulated-adenylyl cyclase in adipocytes does not imply only Gi protein but also other direct or indirect inhibitory pathway(s) which remain to be determined.     

Enhanced stimulatory adenylyl cyclase signaling during opioid dependence is associated with a reduction in palmitoylated Gs alpha

Chronic opioid treatment of stably mu-opioid receptor transfected human mammary epidermoid A431 carcinoma cells (clone A431/mu 13) results in sensitization of adenylyl cyclase (AC), a cellular adaptation associated with drug dependence. Up-regulation of AC is characterized by significantly increased levels of both basal and post-receptor-stimulated effector activities, which develop without any apparent change in the quantity of stimulatory G proteins and the maximum catalytic activity of AC. Here, we report that detergent extracts from membranes of chronically morphine-treated (10 microM; 2 days) A431/mu 13 cells display higher stimulatory AC activities as assessed in the S49cyc- reconstitution assay. This finding is most likely due to an increased functional activity of Gs alpha because the addition of exogenous G beta gamma subunits, which per se stimulate AC in S49cyc- membranes, failed to affect the difference in reconstitutive AC activity. Moreover, both chemical depalmitoylation by hydroxylamine and inhibition of palmitoyl-CoA transferase in vivo by tunicamycin treatment incresed the reconstitutive activity of detergent extracts and eliminated the differences between native and opioid-dependent cells, indicating that the increase in stimulatory activity is due to depalmitoylation of Gs alpha. Indeed, metabolic labelling studies with [3H]palmitic acid revealed that chronic opioid treatment reduces considerably the fraction of palmitoylated Gs alpha in the plasma membrane. Furthermore, high affinity [3H]forskolin binding experiments demonstrated that depalmitoylated Gs alpha is able to associated directly with AC during the state of opioid dependence even without preceding receptor activation. These results suggest that post-translational palmitoylation of Gs alpha provides a potential regulator of transmembrane signaling. Moreover, accumulation of the depalmitoylated form of Gs alpha in the plasma membrane as reported herein may contribute to the increase in stimulatory AC signaling, as is characteristic for the state of opioid dependence.     

Beta-adrenergic signal transduction in the failing and hypertrophied myocardium

A strong sympathetic activation has been observed in heart failure and is the cause of beta-adrenergic desensitization in this condition. On the receptor level there is downregulation of beta1-adrenergic receptors and uncoupling of beta2-adrenoceptors. The latter mechanism has been related to an increased activity and gene expression of beta-adrenoceptor kinase in failing myocardium, leading to phosphorylation and uncoupling of receptors. beta3-Adrenoceptors mediate negative inotropic effects, but alterations in these receptors are not known. In addition, an increase in inhibitory G protein alpha subunits (Gi alpha) has been suggested to be causally linked to adenylyl cyclase desensitization in heart failure. In contrast, the catalytic subunit of adenylyl cyclase, stimulatory G protein alpha and betagamma subunits, have been observed to be unchanged. Recent evidence shows that increases in Gi alpha also depress adenylyl cyclase in compensated cardiac hypertrophy both in monogenic and polygenic and in secondary hypertension. These increases of Gi alpha can suppress adenylyl cyclase in the absence of beta-adrenergic receptor downregulation. Since cardiac hypertrophy in pressure overload is a strong predictor of cardiac failure, these observations indicate that adenylyl cyclase desensitization by Gi alpha may be a pathophysiologically relevant mechanism contributing to the progression from compensated cardiac hypertrophy to heart failure.     

Tocolytic therapy with fenoterol induces selective down-regulation of beta-adrenergic receptors in human myometrium

Tocolytic therapy with beta-adrenergic receptor agonists is a standard regimen to prevent preterm birth. Agonists exposure of beta-adrenergic receptors causes receptor desensitization in other organs, and this may limit the therapeutic value of beta-adrenergic receptor agonists. To study the effects of prolonged beta-adrenergic agonist treatment in human myometrium, we obtained biopsies during Caesarean section of 14 pregnant patients who had received fenoterol for at least 5 days and 14 untreated pregnant controls. The densities of total beta-adrenergic receptors, which are mainly of the beta 2-subtype as assessed by [125I]iodo-cyanopindolol binding in crude membrane fractions, were more than 50% smaller in women receiving fenoterol, whereas alpha 2-adrenergic receptor densities were similar. Gs and Gi G-protein alpha-subunit densities were unaltered as assessed by Western blotting and pertussis toxin-catalyzed [32P]ADP-ribosylation. beta-Adrenergic receptor kinase (beta ARK) activity, as determined using bovine rhodopsin as the substrate, was the same in the two groups. Adenylyl cyclase activities in the presence of guanine nucleotides, NaF, forskolin, or Mn+2 were also not altered by fenoterol treatment. The messenger RNA (mRNA) concentrations of beta 2-adrenergic receptors, beta ARK-I and glyceraldehyde-3-phosphate dehydrogenase (as a reference), as determined by quantitative PCR, were unaffected by fenoterol treatment. We conclude that tocolysis with fenoterol results in a selective down-regulation of myometrial beta-adrenergic receptors, which is not associated with a reduction in the respective mRNA concentrations or alterations of alpha 2-adrenergic receptors, Gs and Gi alpha-subunits, or beta ARK activity or mRNA.     

Reconstitution of beta2-adrenoceptor-GTP-binding-protein interaction in Sf9 cells--high coupling efficiency in a beta2-adrenoceptor-G(s alpha) fusion protein

In most studies, coupling of the beta2-adrenoceptor (beta2AR) to the stimulatory, heterotrimeric GTP-binding protein of adenylyl cyclase the (Gs) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which beta2AR-Gs interactions can be studied directly at the level of the G-protein. We expressed the beta2AR alone, in combination with the alpha-subunit of Gs (G(s alpha)), and as fusion protein with G(s alpha) (beta2AR-G(s alpha)) in Sf9 insect cells. The beta2AR expressed alone couples poorly to the endogenous G(s alpha)-like G-protein of Sf9 cells since no high-affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high-affinity GTPase and guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding were small. Beta2AR-G(s alpha) reconstituted high-affinity agonist binding and regulated adenylyl cyclase more effectively than the beta2AR co-expressed with a large excess of G(s alpha). In membranes expressing beta2AR-G(s alpha), highly effective agonist- and inverse agonist regulation of high-affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing beta2AR and G(s alpha) as separate proteins was difficult to detect. Our data show that the beta2AR interacts with G(s alpha) more efficiently when expressed as a fusion protein than when expressed with an excess of non-fused G(s alpha). The beta2AR-G(s alpha) fusion protein provides a very sensitive model system to study the regulation of Gs function by beta2AR agonists and inverse agonists directly at the level of the G-protein.     

The delta-opioid receptor in SK-N-BE human neuroblastoma cell line undergoes heterologous desensitization

The human neuroblastoma cell line SK-N-BE expresses delta-opioid receptors negatively coupled to adenylyl cyclase. Prolonged treatment (2 h) of the cells with 100 nM etorphine leads to an almost complete desensitization (8.2 +/- 5.9 vs. 45.8 +/- 8.7% for the control). Other receptors negatively coupled to adenylyl cyclase, namely, D2-dopaminergic, alpha 2-adrenergic, and m2/m4-muscarinic, were identified by screening of these cells, and it was shown that prolonged treatment (2 h) with 1 microM 2-bromo-alpha-ergocryptine or 1 microM arterenol resulted in a marked desensitization of D2-dopaminergic and alpha 2-adrenergic receptors, respectively. Cross-desensitization experiments revealed that pretreatment with etorphine desensitized with the same efficiency the delta-opioid receptor and the D2-dopaminergic receptor, and pretreatment with 2-brorno-alpha-ergocryptine also desensitized both receptors. In contrast, pretreatment with etorphine desensitized only partly the alpha 2-adrenergic receptor response, whereas pretreatment with 1 microM arterenol partly desensitized the delta-opioid receptor response. It is concluded that the delta-opioid receptor-mediated inhibitory response of adenylyl cyclase undergoes heterolgous desensitization, and it is suggested that delta-opioid and D2-dopaminergic receptors are coupled to adenylyl cyclase via a G12 protein, whereas alpha 2-adrenergic receptor could be coupled to the enzyme via two G proteins, G12 and another member of the G1/G0 family.     

Glucocorticoid regulation of G-protein subunits in neonatal liver

The effect of dexamethasone administration in vivo on the steady-state levels of G-protein subunits in liver of neonatal rabbits was investigated using specific antibodies to each subunit as well as bacterial toxin-mediated ADP-ribosylation assays. Parallel measurements were also made of the activity of adenylyl cyclase, as influenced by a variety of activators. Dexamethasone administration modulated the levels of G-protein subunits in liver in an age-dependent and subunit-specific manner but not in 24-h-old newborns. The inductive effect of dexamethasone was observed in animals older than 24 h, the greatest effect being on 2- to 3-day-old neonates. In 48-h-old animals the alpha-subunits Gs alpha-1, Gs alpha-2, Gi alpha and the beta-subunit G beta increased 2.0-, 2.1-, 4.3- and 2.8-fold, respectively, compared to the control. The increases were much less for older animals. Dexamethasone treatment also modulated effector-mediated stimulation of adenylyl cyclase activity in vitro and mimicked its effects on G-protein levels; the greatest increase (approximately 2-fold) in the activation of adenylyl cyclase occurred in membranes isolated from 2- to 3-day-old animals. In older animals there was either no effect of dexamethasone or a decrease in activity. The degree of change in enzyme activity paralleled the change in the amount of Gs alpha rather than of Gi alpha or G beta. These results suggest development-dependent regulation of hepatic G-proteins by glucocorticoids.     

Functional activity and expression of the myocardial postreceptor adenylyl cyclase system in pressure overload hypertrophy in rat

OBJECTIVE: The aim of the present study was to investigate the functional regulation of the myocardial postreceptor adenylyl cyclase (AC) system in compensated left ventricular hypertrophy (LVH) and the effect of long-term angiotensin converting enzyme (ACE) inhibition. METHODS: Pressure overload LVH was induced in rats by supravalvular aortic banding for 12 weeks. At 12 weeks left ventricular function and inner diameters were analyzed by echocardiography of anesthetized animals, and responsiveness to forskolin (systolic developed pressure) was determined in isolated perfused hearts. Functional activities of AC and the stimulatory G protein Gs were measured as well as mRNA expression (quantitative slot blot analyses) of AC type V, isoforms of Gs alpha and Gi alpha 2. G protein alpha-subunits were also quantified by immunoblotting. Rats were treated with ramipril (Ram, 10 mg/kg per day p.o.) during weeks 7 to 12 to induce regression of LVH or with vehicle (Veh, tap water). RESULTS: Pressure overload induced severe LVH (3.2 +/- 0.09 g/kg in Veh vs. 1.8 +/- 0.03 in sham; P < 0.05) which was significantly reduced by ramipril (2.7 +/- 0.09; P < 0.05 vs. Veh). In-vivo left ventricular function and diameters were unchanged in LVH. In contrast, in hearts with LVH, responsiveness of left ventricles to forskolin was attenuated and basal, GTP gamma S and forskolin as well as manganese chloride-stimulated adenylyl cyclase activity was significantly downregulated by approximately 40% (basal 20.8 +/- 1.9 pmol cAMP/mg per min vs. 34.0 +/- 2.2 in sham; P < 0.01). However, no significant changes of AC type V mRNA were found in hypertrophied left ventricles. Functional activity of the stimulatory G protein Gs was reduced in LVH (48 +/- 7 pmol cAMP/mg per min in Veh vs. 68 +/- 3 in sham), whereas mRNA expression of long and short Gs alpha-isoforms was not altered and that of Gi alpha 2 was only slightly increased in ramipril-treated animals. Western analysis showed no significant differences of Gs alpha or Gi alpha 2 subunits. Long-term blockade of the renin-angiotensin system had no effect on the activity of the adenylyl cyclase system. CONCLUSIONS: Functional desensitization of adenylyl cyclase and stimulatory G protein occurred in rat adaptive LVH prior to the onset of severe left ventricular dysfunction which was not restored by ACE-inhibitor treatment. The desensitization seems not to be mediated by significant changes of mRNA expression of AC type V or abundance of regulatory G proteins.     

Atypical beta-adrenergic receptors in rat liver: evidence for transient expression during aging

This study was designed to determine whether functional beta 3-adrenergic receptors exist in the rat liver and whether there are alterations in the beta 3-adrenergic response with age in a manner similar to that which occurs for beta 2-adrenergic receptors. The beta 3 stimulation of adenylyl cyclase activity was assessed using the novel beta 3-specific agonist, CGP-12177A. Adenylyl cyclase activation by CGP-12177A was seen only in the adults. Isoproterenol-stimulated adenylyl cyclase activity was high in the neonates, declined by 45% in the adults, and was high again in the senescent rats. ICI 118551, a beta 2-selective antagonist, attenuated the isoproterenol-stimulated activity by two-thirds but had no effect on the CGP-12177A-stimulated activity. These data demonstrated the presence of the beta 3-adrenergic receptor in the rat liver, although only at the adult stage of development. In addition, these data confirm earlier findings that beta 2-adrenergic activation of adenylyl cyclase is biphasic with age and indicate that the emergence of the beta 3-stimulated activity coincides with the attenuation of beta 2-stimulated activity.     

Mutation of tyrosine-350 impairs the coupling of the beta 2-adrenergic receptor to the stimulatory guanine nucleotide binding protein without interfering with receptor down-regulation

Long-term stimulation of the beta 2-adrenergic receptor (beta 2AR) leads to an internalization and degradation of the receptor. This down-regulation of the beta 2AR number contributes to the desensitization of the adenylyl cyclase activity induced by chronic exposure to agonists. It was proposed that two tyrosine residues (Tyr-350 and Tyr-354) located in the cytoplasmic tail of the beta 2AR play a crucial role in agonist-induced down-regulation. In addition to perturbation of the down-regulation, the substitution of these tyrosines for alanines also led to a functional uncoupling of the receptor from Gs [Valiquette et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5089-5093]. To further characterize the relative contribution of Tyr-350 and Tyr-354 to the receptor interaction with Gs and agonist-promoted down-regulation, both tyrosines were individually replaced by alanines and mutant receptors expressed in CHW cells. We show here that mutation of Tyr-350 but not that of Tyr-354 significantly decreased the ability of the beta 2AR to be functionally coupled to Gs and thereby to stimulate the adenylyl cyclase. Moreover, in contrast to the double tyrosine mutation, neither of the single-point mutations affected the agonist-induced down-regulation pattern. These data suggest that the presence of either Tyr-350 or Tyr-354 is sufficient to maintain normal agonist-induced down-regulation whereas the integrity of Tyr-350 is required for an appropriate coupling to Gs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human-Xenopus chimeras of Gs alpha reveal a new region important for its activation of adenylyl cyclase

G proteins are heterotrimeric GTPases that play a key role in signal transduction. The alpha subunit of Gs bound to GTP is capable of activating adenylyl cyclase. The amino acid sequences derived from two X. laevis cDNA clones that apparently code for Gs alpha subunits are 92% identical to those found in the short form of human Gs alpha. Despite this high homology, the X. laevis Gs alpha clones expressed in vitro, yielded a protein that are not able to activate the adenylyl cyclase present in S49 cyc- membranes in contrast with human Gs alpha similarly expressed. This finding suggested that the few amino acid substitutions found in the amphibian subunit are important in defining the functionality of the human Gs alpha. The construction of chimeras composed of different fractions of the cDNAs of the two species was adopted as an approach in determining the regions of the molecule important in its functionality in this assay. Four pairs of chimeras were constructed using reciprocal combinations of the cDNAs coding for human and Xenopus Gs alpha. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTP gamma and isoproterenol. The results obtained here clearly indicate that the G alpha sequence that extends from amino acid 70 to 140, is important for the functionality of human Gs alpha in activating adenylyl cyclase.     

Palmitoylated cysteine 341 modulates phosphorylation of the beta2-adrenergic receptor by the cAMP-dependent protein kinase

We previously showed that substitution of a glycine residue for the palmitoylated cysteine 341 of the human beta2-adrenergic receptor (Gly341beta2AR), increases the basal level of the receptor phosphorylation and reduces its ability to functionally interact with Gs. In the present study, we show that additional mutation of serines 345 and 346 (Ala345,346Gly341beta2AR) restored normal phosphorylation and receptor-Gs coupling, thus suggesting that the increased phosphorylation of this site, rather than the lack of palmitoylation per se, is responsible for the poor coupling of the unpalmitoylated receptor. This is supported by the observation that chemical depalmitoylation of purified beta2AR did not affect the ability of the receptor to stimulate adenylyl cyclase in reconstitution assays. Furthermore, mutation of Ser345,346 in a wild type receptor background (Ala345,346beta2AR) significantly decreased the rate of agonist-promoted desensitization of the receptor-stimulated adenylyl cyclase activity, supporting a role for this phosphorylation site in regulating the functional coupling of the receptor. Since serines 345 and 346 are located in a putative cyclic AMP-dependent protein kinase (PKA) phosphorylation site immediately downstream of the palmitoylated cysteine 341, the hypothesis that the accessibility of this site may be regulated by the receptor palmitoylation state was further assessed in vitro. In membrane phosphorylation assays, Gly341beta2AR was found to be a better substrate for PKA than the wild type receptor, thus supporting the notion that palmitoylation restrains access of the phosphorylation site to the enzyme. Taken together, the data demonstrate that palmitoylation of cysteine 341 controls the phosphorylation state of the PKA site located in the carboxyl tail of the beta2AR and by doing so modulates the responsiveness of the receptor.     

Global increases in seizure susceptibility in mice lacking 5-HT2C receptors: a behavioral analysis

OBJECTIVE: Vasodilation by beta-adrenergic receptors of smooth muscle cells appears to be impaired early after the onset of hypercholesteremia. The aim of this study was to analyze the modulation of beta-adrenergic receptor density and adenylyl cyclase activity in the presence of moderately elevated concentrations of LDL. The effects of beta 1- and beta 2-adrenergic receptor antagonists on LDL-induced receptor changes were studied. METHODS AND RESULTS: Media explants of porcine coronary arteries were incubated with moderately elevated LDL concentrations (0.7-3.9 mmol/l). The density of beta-adrenergic receptors was determined in plasma membranes using the radioligand [125I]iodocyanopinodolol. LDL (3.9 mmol/l) resulted in a decrease of beta-adrenergic receptor density (control 137 +/- 5 vs. 89 +/- 7 fmol/mg protein, P < 0.01). After removal of LDL and cultivation for an additional 3 days beta-adrenergic receptors increased to 129 +/- 5 fmol/mg. In the presence of the beta 1- or beta 2-adrenergic receptor antagonists the LDL-mediated decrease was inhibited. Addition of metoprolol after 3 days of LDL incubation caused a restoration of receptor density. The basal, isoproterenol- and forskolin-stimulated adenylyl cyclase activities were increased after LDL incubation by 180, 110 or 80%, respectively. CONCLUSION: Moderately elevated LDL levels decreased beta-adrenergic receptor density while adenylyl cyclase activity was simultaneously increased. beta 1- or beta 2-adrenergic receptor antagonists prevented this receptor decrease and might preserve the beta-adrenergic receptor density in the presence of moderately elevated LDL levels.     

Repeated topical administration of fenoterol in rabbit reverses its initial ocular hypotensive effect and decreases sensitivity of adenylyl cyclase in ciliary processes to stimulatory agents

PURPOSE: The effects of repeated topical administration of the selective beta 2-adrenergic agonist fenoterol on the intraocular pressure and on the adenylyl cyclase activity in ciliary processes in rabbit were examined in order to detect their possible causal relationship. METHODS: Intraocular pressure was measured by pneumatonometry. Adenylyl cyclase activity in homogenates of ciliary processes was assayed ex vivo by measurement of conversion of 32P-alpha-ATP to 32P-cyclic AMP. RESULTS: A single topical dose of 1% solution of fenoterol elicited a clear-cut decrease of the intraocular pressure lasting for several h. Repeated administration of fenoterol for 2-5 days led to a significant increase of intraocular pressure, observable from the second to the fifth day. The stimulation of adenylyl cyclase activity ex vivo by isoproterenol, vasoactive intestinal polypeptide or forskolin was significantly decreased on the fifth day (24 h after the administration of the last dose of fenoterol). CONCLUSIONS: Our data showed that repeated topical administration of the selective beta 2-adrenergic agonist increased intraocular pressure and desensitized adenylyl cyclase in ciliary processes; if these two effects are related then they would support the idea of direct relationship of decreased cAMP production in ciliary processes to the increase of intraocular pressure, and vice versa. However, conclusive evidence of this suggestion and of its possible significance in another animal species or man would require further study.     

Inverse agonism at adrenergic and opioid receptors: studies with wild type and constitutively active mutant receptors

Ligands which display inverse agonism at G protein-coupled receptors do so by decreasing the intrinsic ability of a receptor to active the cellular G protein population in the absence of an agonist ligand. Expression of the murine delta opioid receptor in Rat-1 fibroblasts resulted in the inverse agonist ICI174864 being able to cause inhibition of basal high affinity GTPase activity and of the binding of [35S]GTP gamma S in membranes of a clone (D2) of these cells which expresses high levels of the receptor. These effects were blocked by co-addition of the neutral antagonist TIPP[psi], demonstrating a requirement for the delta opioid receptor, and by pertussis toxin pretreatment of the cells, showing them to be produced via a Gi-like G protein. The inverse agonist properties of ICI174864 could also be demonstrated in whole cells. Stimulation of forskolin-amplified adenylyl cyclase activity was produced by ICI174864 following [3H]adenine prelabelling of the cells. Constitutively activated mutants of receptors should provide a convenient means to detect inverse agonists. Incubation of cells either transiently or stably transfected with a constitutively activated mutant of the human beta 2-adrenoceptor with the beta 2-inverse agonists betaxolol or sotalol, which are both able to inhibit CAM beta 2-adrenoceptor-mediated basal adenylyl cyclase activity, resulted in a strong upregulation of levels of the receptor. In the stable cells lines this effect was prevented by co-incubation with neutral antagonists but could not be reproduced by an adenylyl cyclase P-site ligand which also inhibited basal adenylyl cyclase levels.     

A Gsalpha mutant designed to inhibit receptor signaling through Gs

Hormonal signals activate trimeric G proteins by substituting GTP for GDP bound to the G protein alpha subunit (Galpha), thereby generating two potential signaling molecules, Galpha-GTP and free Gbetagamma. The usefulness of dominant negative mutations for investigating Ras and other monomeric G proteins inspired us to create a functionally analogous dominant negative Galpha mutation. Here we describe a mutant alpha subunit designed to inhibit receptor-mediated hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase. To construct this mutant, we introduced into the alpha subunit (alphas) of Gs three separate mutations chosen because they impair alphas function in complementary ways: the A366S mutant reduces affinity of alphas for binding GDP, whereas the G226A and E268A mutations impair the protein's ability to bind GTP and to assume an active conformation. The triple mutant robustly inhibits (by up to 80%) Gs-dependent hormonal stimulation of adenylyl cyclase in cultured cells. Inhibition is selective in that it does not affect cellular responses to expression of a constitutively active alphas mutant (alphas-R201C) or to agonists for receptors that activate Gq or Gi. This alphas triple mutant and cognate Galpha mutants should provide specific tools for dissection of G protein-mediated signals in cultured cells and transgenic animals.     

Alteration of tubulin-Gi protein interaction in rat cerebral cortex with aging

The ability of the tubulin dimer to interact with and to modulate the Gi function inhibiting adenylyl cyclase was examined in cerebral cortex membranes from 2-month-old and 24-month-old rats. The hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp)-dependent inhibition of adenylyl cyclase was significantly decreased in cerebral cortex membranes from 24-month-old rats. Tubulin, prepared from rat brains by polymerization with GppNHp, caused inhibition of adenylyl cyclase (approximately 28%) in 2-month-old rats. Tubulin-GppNHp-dependent inhibition of adenylyl cyclase in 24-month-old rats was significantly attenuated (approximately 15%). In 2-month-old rats, when tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to Gi alpha. Transfer of AAGTP from tubulin to Gi alpha was reduced in 24-month-old rats. Furthermore, photoaffinity labeling of [32P]AAGTP to Gi alpha in cortex membranes was significantly decreased in 24-month-old rats. No differences were observed in the amounts of Gs alpha, Gi alpha, or G beta subunits and tubulin, estimated by immunoblotting, in cortex membranes from 2-month-old and 24-month-old rats. These results suggest that the ability of tubulin to interact with Gi and thereby modulate the inhibitory regulation of adenylyl cyclase is reduced in the cerebral cortex of 24-month-old rats.