Adenylyl Cyclase Activity In Response Research Articles

A Novel CRISPR/Cas9‐Based Cellular Model to Explore Adenylyl Cyclase and Cyclic AMP Signaling

Functional characterization of adenylyl cyclase (AC) isoforms has proven to be challenging in mammalian cell systems due to the endogenous expression of multiple AC isoforms, and the high background cyclic AMP (cAMP) responses mediated by AC activators such Gαs or forskolin. Hence, the objective of this study was to generate an isolated HEK293 cell line with low cAMP levels by knocking out the two abundantly expressed AC isoforms using CRISPR/Cas9 technology. Based on qPCR studies examining AC isoform gene expression in HEK293 cells, we chose single guide RNA (sgRNA) sequences that target AC3 and AC6, and generated stable HEK293 cell lines lacking either AC6 (HEK‐AC6 KO) or both AC3 and AC6 (HEK‐AC3/AC6 KO). Loss of function of AC3 and AC6 was evaluated genetically and by comparing the forskolin‐ and Gαs‐coupled receptor‐stimulated cAMP responses in the knockout cells and the parental HEK293 cell line. AC activity in response to 50 μM forskolin was reduced by more than 80% in the HEK‐AC6 KO cells, and by an additional 10% when AC3 was also knocked out resulting in a total reduction of cAMP levels by 95% in the HEK‐AC3/AC6 KO cells compared to the HEK293 parental cell line. For Gαs‐coupled receptor‐mediated AC activation, cAMP accumulation was evaluated for the endogenous β2‐adrenergic receptors (β2AR) and prostaglandin E2 receptors (PGE2R). HEK‐AC6 KO cells produced 50% less cAMP at maximal β2‐adrenergic receptor stimulation (10 μM isoproterenol), whereas the HEK‐AC3/AC6 KO cells displayed >75% lower cAMP accumulation than the HEK293 parental cell line. The cAMP response induced by the PGE2R agonist, PGE‐2, was >85% lower in the HEK‐AC6 KO than that observed in the parental cell line, but no additional decrease in cAMP accumulation was detected in the HEK‐AC3/AC6 KO cells. We subsequently used the HEK‐AC3/AC6 KO cell line to pharmacologically characterize the individual membrane‐bound AC isoform responses to forskolin, β2AR, and PGE2R by reintroducing each individual isoform by transient transfection. Forskolin‐mediated cAMP accumulation for AC1‐6 and AC8 revealed 2‐ to 50‐fold increases over the cAMP levels of the control/venus‐transfected cells. In contrast, AC7 and AC9 showed little or no forskolin response, respectively. The cAMP responses for the nine AC isoforms following β2AR and PGE2R activation were also significantly higher than that observed for the control‐transfected cells. Isoform specific AC regulation by other regulatory mechanisms such as Gαi‐coupled receptors, protein kinases, and Ca+2/calmodulin was also recapitulated in the HEK‐AC3/AC6 KO cell line. We have used CRISPR/Cas9 to develop a HEK293 cell line deficient of endogenous AC3 and AC6 that exhibits very low basal and drug‐stimulated cAMP background levels. These HEK‐AC3/AC6 KO cells will facilitate the selective characterization of AC isoform activity and regulation in a mammalian cellular background.Support or Funding InformationNIH MH101673, Department of Medicinal Chemistry and Molecular Pharmacology (MCMP), and Purdue UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differences in the Expression of VPAC2 Receptors and Adenylyl Cyclase V/VI and VIP‐Induced cAMP Generation in Phasic and Tonic Smooth Muscles

The proximal stomach is characterized by its ability to maintain tone as compared to the distal stomach, which is phasic in nature and contracts transiently. In gastric muscle, the inhibitory transmitters VIP, PACAP and NO mediate relaxation. VIP and PACAP interact with VPAC2 receptors to stimulate adenylyl cyclase (AC) activity and cAMP formation. AIM To determine whether differences in the VPAC2/AC/cAMP pathway are related to tonic and phasic phenotype of smooth muscles in proximal and distal regions of the stomach, respectively. METHODS Expression of VPAC2 receptor and ACV/VI, the main isoforms(s) expressed in smooth muscle, are determined in muscle cells isolated separately from proximal and distal stomach by qRT-PCR and western blot. Stimulation of AC activity in response to VIP was measured as increase in cAMP formation in the presence of IBMX. RESULTS Expression of VPAC2 receptors and ACV/VI was significantly higher in muscle from distal stomach than from proximal stomach. Consistent with the higher expression of VPAC2 receptors and ACV/VI, VIP induced cAMP levels were significantly higher in muscle cells from distal stomach than muscle cells from proximal stomach. CONCLUSION Higher expression of VPAC2 receptors and ACV/VI, and greater generation of cAMP that mediate relaxation in muscle from distal stomach correlate with the phasic phenotype of muscle in distal stomach relative to proximal stomach.

Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum

The adenosine A(2A) receptor (A(2A)R) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α(olf) subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ(7) subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ(7) protein controls the hierarchial assembly of a specific G-protein α(olf)β(2)γ(7) heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A(2A)R activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ(7) subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A(2A)R G-protein α(olf)β(2)γ(7) interface as a possible therapeutic target for Parkinson disease.

Self-Limitation of Intravenous Tocolysis with β2-Adrenergic Agonists Is Mediated through Receptor G Protein Uncoupling

Tocolysis with a beta-adrenergic receptor agonist is the most common approach to premature labor management after the 25th wk of pregnancy. However, prolonged treatment is associated with a marked loss of efficacy. The biochemical mechanisms involved remain unclear. This study was undertaken to investigate the effect of fenoterol on beta-adrenergic receptor signal transduction in human myometrium. Myometrial biopsy specimens were obtained from 40 women at cesarean section between the 25th and 34th wk of pregnancy. Nineteen patients had received no tocolysis (controls, group I) and 21 had been treated with fenoterol (<48 h in 10, group II; > or = 48 h in 11, group III). As methods we used membrane preparation, adenylyl cyclase assay and cAMP RIA. Adenylyl cyclase activity was determined by the measurement of cAMP levels to evaluate signal transduction after stimulation of beta-adrenergic receptors with isoproterenol, G protein with GTP, and adenylyl cyclase with forskolin. The functional activity of GTP-binding regulatory proteins (G(s)) and adenylyl cyclase was not altered by fenoterol treatment. In the control group, the increase in adenylyl cyclase activity in response to GTP plus isoproterenol was greater than in response to GTP alone. The increase was reduced by 50% in group II and was insignificant in group III. There was no correlation between gestational age and basal adenylyl cyclase activity. Intravenous tocolysis with the beta2-adrenergic receptor agonist fenoterol leads to complete desensitization of the beta-adrenergic receptor system. In addition to the known reduction in receptor number (down-regulation) as underlying mechanism, uncoupling of the receptor from the stimulatory G protein G(s) was identified.

Is cyclic AMP formation desensitized in patients with end‐stage renal failure?

1 Cyclic AMP formation has consistently been reported to be desensitized in various tissues including heart of animal models of end-stage renal failure (ESRF). In contrast, reports on desensitization of cAMP formation in ESRF patients remain contradictory. Whether this discrepancy results from a difference between human ESRF and its animal models or from the use of circulating blood cells in the human and various solid tissues in the animal studies, remains unclear. Therefore, we performed three studies with heart and platelets of ESRF patients undergoing haemodialysis or continuous ambulatory peritoneal dialysis and age- and gender-matched controls with normal renal function (n = 11-13 each). 2 In platelets from haemodialysis patients adenylyl cyclase activity in response to receptor-dependent and -independent agonists was reduced by approximately 30%, and this could be explained by an alteration at the level of adenylyl cyclase itself. However, no such desensitization was seen in platelets from peritoneal dialysis patients. 3 In hearts from ESRF patients undergoing haemodialysis, beta-adrenoceptor density and subtype distribution, cAMP formation in response to the beta-adrenoceptor agonist isoprenaline or various receptor-independent stimuli, were very similar to those in control patients but activity of G-protein-coupled receptor kinase was increased by approximately 20%. 4 We conclude that conflicting reports on the desensitization of cAMP formation between ESRF patients and ESRF animal models are not explained by the use of solid tissues in animal studies vs. circulating blood cells in patient studies. Rather desensitization of cAMP formation seems to be a less consistent feature of human ESRF than of its animal models.

Targeted beta-adrenergic receptor kinase (betaARK1) inhibition by gene transfer in failing human hearts.

Failing human myocardium is characterized by an attenuated contractile response to beta-adrenergic receptor (betaAR) stimulation due to changes in this signaling cascade, including increased expression and activity of the beta-adrenergic receptor kinase (betaARK1). This leads to desensitization and downregulation of betaARs. Previously, expression of a peptide inhibitor of betaARK1 (betaARKct) has proven beneficial in several animal models of heart failure (HF). To test the hypothesis that inhibition of betaARK1 could improve beta-adrenergic signaling and contractile function in failing human myocytes, the betaARKct was expressed via adenovirus-mediated (AdbetaARKct) gene transfer in ventricular myocytes isolated from hearts explanted from 10 patients with end-stage HF undergoing cardiac transplantation. AdbetaARKct also contained the marker gene, green fluorescent protein, and successful gene transfer was confirmed via fluorescence and immunoblotting. Compared with uninfected failing myocytes (control), the velocities of both contraction and relaxation in the AdbetaARKct-treated cells were increased in response to the beta-agonist isoproterenol (contraction: 57.5+/-6.6% versus 37.0+/-4.2% shortening per second, P<0.05; relaxation: 43.8+/-5.5% versus 27.5+/-3.9% lengthening per second, P<0.05). Fractional shortening was similarly enhanced (12.2+/-1.2% versus 8.0+/-0.9%, P<0.05). Finally, adenylyl cyclase activity in response to isoproterenol was also increased in AdbetaARKct-treated myocytes. These results demonstrate that as in animal models of HF, expression of the betaARKct can improve contractile function and beta-adrenergic responsiveness in failing human myocytes. Thus, betaARK1 inhibition may represent a therapeutic strategy for human HF.

PTH-dependent adenylyl cyclase activation in SaOS-2 cells: passage dependent effects on G protein interactions.

Parathyroid hormone (PTH) sensitive adenylyl cyclase activity (ACA) in SaOS-2 cells varies as a function of cell passage. In early passage (EP) cells (< 6), ACA in response to PTH and forskolin (FOR) was relatively low and equivalent, whereas in late passage (LP) cells (> 22), PTH exceeded FOR dependent ACA. Potential biochemical mechanisms for this passage dependent change in ACA were considered. In EP, prolonged exposure to pertussis toxin (PT) markedly enhanced ACA activity in response to PTH, Isoproterenol and Gpp(NH)p, whereas ACA in response to FOR was decreased. In contrast, the identical treatment of LP with PT diminished all ACA in response to PTH, Gpp(NH)p, and FOR. The dose dependent effects of PT on subsequent [(32)P]ADP-ribosylation of its substrates, GTPase activity, as well as FOR-dependent ACA, were equivalent in EP and LP. The relative amounts of G(alpha)i and G(alpha)s proteins, as determined both by Western blot, PT and cholera toxin (CT) dependent [(32)P]ADP-ribosylation, were quantitatively similar in EP and LP. Western blot levels of G(alpha)s and G(alpha)i proteins were not influenced by prior exposure to PT. Both PT and CT dependent [(32)P]ADP-ribosylation were dose-dependently decreased following exposure to PT. However, the PT-dependent decline in CT-dependent [(32)P]ADP-ribosylation occurred with enhanced sensitivity in LP. The protein synthesis inhibitor cycloheximide partially reversed the PT associated decrease in FOR dependent ACA in EP. In contrast, cycloheximide completely reversed the PT associated decrease in FOR and as well as PTH dependent ACA in LP. G(alpha)s activity, revealed by cyc(-) reconstitution, was not altered either by cell passage or exposure to PT. The results suggest that the coupling between the components of the complex may be pivotally important in the differential responsiveness of early and late passage SaOS-2 cells to PTH.

Decreased adenylyl cyclase protein and function in airway smooth muscle by chronic carbachol pretreatment.

Cellular levels of cAMP are an important determinant of airway smooth muscle tone. We have previously shown that chronic (18 h) but not acute (30 min or 2 h) pretreatment with the muscarinic receptor agonist carbachol resulted in decreased adenylyl cyclase activity in response to GTP, isoproterenol, or forskolin via a pathway blocked by the protein kinase C inhibitor staurosporine. The present study was designed to determine if carbachol-induced decreases in adenylyl cyclase activity were due to regulatory events at the level of either G(s)alpha or adenylyl cyclase. Detergent-solubilized G(s)alpha from control or carbachol-pretreated bovine airway smooth muscle had similar adenylyl cyclase activity in response to either NaF or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) when reconstituted into S49 cyc(-) membranes that lack endogenous G(s)alpha (carbachol pretreated: GTPgammaS, 93 +/- 13% of control; NaF/AlCl(3), 99 +/- 8.6% of control; n = 4). Exogenous G(s)alpha solubilized from red blood cells failed to restore normal adenylyl cyclase activity when reconstituted into carbachol-pretreated bovine airway smooth muscle (carbachol pretreated: GTP, 36 +/- 10% of control; NaF/AlCl(3), 54 +/- 11% of control; n = 4). [(3)H]forskolin radioligand saturation binding assays revealed a decreased quantity of total adenylyl cyclase protein after carbachol pretreatment (maximal binding: 152 +/- 40 and 107 +/- 31 fmol/mg protein in control and carbachol-pretreated airway smooth muscle, respectively). These results suggest that chronic activation of muscarinic receptors downregulates the expression of adenylyl cyclase protein in bovine airway smooth muscle.

The ADP ribosylation factor nucleotide exchange factor ARNO promotes beta-arrestin release necessary for luteinizing hormone/choriogonadotropin receptor desensitization.

Desensitization of guanine nucleotide binding protein-coupled receptors is a ubiquitous phenomenon characterized by declining effector activity upon persistent agonist stimulation. The luteinizing hormone/choriogonadotropin receptor (LH/CGR) in ovarian follicles exhibits desensitization of effector adenylyl cyclase activity in response to the mid-cycle surge of LH. We have previously shown that uncoupling of the agonist-activated LH/CGR from the stimulatory G protein (G(s)) is dependent on GTP and attributable to binding of beta-arrestin present in adenylyl cyclase-rich follicular membrane fraction to the third intracellular (3i) loop of the receptor. Here, we report that LH/CGR-dependent desensitization is mimicked by ADP ribosylation factor nucleotide-binding site opener, a guanine nucleotide exchange factor of the small G proteins ADP ribosylation factors (Arfs) 1 and 6, and blocked by synthetic N-terminal Arf6 peptide, suggesting that the GTP-dependent step of LH/CGR desensitization is receptor-dependent Arf6 activation. Arf activation by GTP and ADP ribosylation factor nucelotide-binding site opener promotes the release of docked beta-arrestin from the membrane, making beta-arrestin available for LH/CGR; Arf6 but not Arf1 peptides block beta-arrestin release from the membrane. Thus, LH/CGR appears to activate two membrane delimited signaling cascades via two types of G proteins: heterotrimeric G(s) and small G protein Arf6. Arf6 activation releases docked beta-arrestin necessary for receptor desensitization, providing a feedback mechanism for receptor self-regulation.

Mutated human beta3-adrenergic receptor (Trp64Arg) lowers the response to beta3-adrenergic agonists in transfected 3T3-L1 preadipocytes.

Wild-type or mutated human beta3-adrenergic receptor (Trp64Arg) cDNAs were stably expressed in mouse 3T3-L1 cells. Saturation binding study using a beta-adrenergic ligand revealed that there was no significant difference in the receptor density and the equilibrium dissociation constant between the two cell lines. However, the ability of the mutant beta3-adrenergic receptor to accumulate cyclic AMP (cAMP) in response to isoproterenol was much reduced and Kact for cAMP accumulation was lowered as compared to the wild type receptor. The amount of alpha subunit of stimulatory GTP-binding protein (GSalpha) and adenylyl cyclase activity in response to forskolin were not different in the two cell lines. The responses of the mutant receptor to epinephrine, norepinephrine and L-755,507, a highly specific agonist for human beta3-adrenergic receptor, were also reduced, but the reduction of Kact for L-755,507 was more evident than other agonists tested. The cAMP accumulation in response to some conventional beta3 agonists was less than 10% of that to isoproterenol even in the cells expressing the wild type receptor. These results suggest that the Trp64Arg mutant beta3-adrenergic receptor has less ability to stimulate adenylyl cyclase, and that lipolytic activity through the beta3-adrenergic receptor by catecholamines in subjects carrying this mutation might be suppressed.

A combination assay for simultaneous assessment of multiple signaling pathways

We have developed an assay in which modulation of two or more signaling pathways can be assessed concurrently by combining reporter gene systems with fluorescent probe technology. The validation of this method was achieved by indirect analysis of adenylyl cyclase activation with the use of a cyclic AMP response element (CRE)–luciferase reporter system in combination with the measurement of calcium mobilization by Calcium Green™-1 AM fluorescence on a fluorescent imaging plate reader. To demonstrate the utility of the method in studying the pharmacology of receptors that couple to more than one G protein, Chinese hamster ovary (CHO) cells, which stably expressed both the CRE-luciferase reporter gene and the human pituitary adenylyl cyclase-activating peptide (PACAP) receptor, were treated with PACAP 1-27 and 1-38. Calcium mobilization and the induction of adenylyl cyclase activity in response to each concentration of peptide were assessed in individuals wells. This assay may also be used to screen for ligands of two or more unrelated receptors simultaneously without compromising the assessment of either signaling pathway. To illustrate this point, Rat-1 fibroblasts, which expressed human α 1A receptors, were cocultured with CRE-luciferase CHO cells, which expressed human GLP-1 receptors. Calcium mobilization elicited by phenylephrine agonism of the α 1A receptor was assessed in the same assay as GLP-1-induced activation of adenylyl cyclase. The pEC 50 for each agonist was similar to that observed when the cell lines were not cocultured. The number of different receptors that can be screened per well is limited only by the ability to distinguish different reporter gene signals and fluorescent indicators.

Asymmetric distribution of muscarinic acetylcholine receptors in Madin-Darby canine kidney cells.

We have characterized the muscarinic ACh receptors (mAChRs) expressed in Madin- Darby canine kidney (MDCK) strain II epithelial cells. Binding studies with the membrane-impermeable antagonist N-[(3)H]methylscopolamine demonstrated that mAChRs are approximately 2.5 times more abundant on the basolateral than on the apical surface. Apical, but not basolateral, mAChRs inhibited forskolin-stimulated adenylyl cyclase activity in response to the agonist carbachol. Neither apical nor basolateral mAChRs exhibited detectable carbachol-stimulated phospholipase C activity. Carbachol application to the apical or the basolateral membrane resulted in a threefold increase in intracellular Ca(2+) concentration, which was completely inhibited by pertussis toxin on the apical side and partially inhibited on the basolateral side. RT-PCR analysis showed that MDCK cells express the M(4) and M(5) receptor mRNAs. These data suggest that M(4) receptors reside on the apical and basolateral membranes of polarized MDCK strain II cells and that the M(5) receptor may reside in the basolateral membrane of a subset of cells.

Chronic activation of ORL1 receptor induces supersensitization of adenylyl cyclase.

The opioid receptor-like (ORL1) receptor regulates a variety of effectors shared by its close relatives, the opioid receptors. Supersensitization of adenylyl cyclase (AC) is a hallmark of cellular tolerance induced by chronic activation of opioid receptors. To examine if chronic activation of the ORL1 receptor leads to a similar adaptation, a HEK293 cell line stably expressing the human ORL1 receptor (293/ORL1) was established. Chronic treatment of 293/ORL1 cells with nociceptin/OFQ resulted in enhanced AC activity in response to forskolin stimulation. The AC supersensitivity was blocked by pertussis toxin, indicating the involvement of Gi/Go proteins. Likewise, chronic activation of endogenous ORL1 receptors in the neuroblastoma SK-N-SH cells led to Gi/Go-mediated AC supersensitization.

Regulation of Myocardial β-Adrenergic Receptor Function in Adult and Neonatal Rabbits

β-Adrenergic receptor (βAR) desensitization is the decrease in response following sustained agonist stimulation by catecholamines. While developmental changes in βAR response have been well documented in the mammalian heart, much less is known regarding the regulation of βAR function in immature hearts. The purpose of the present study was to determine whether there are developmental differences in myocardial βAR desensitization. We used an isolated heart preparation to examine the βAR-mediated inotropic response before and after sustained exposure to 1 μM isoproterenol in adult and neonatal rabbits. We also assayed the adenylyl cyclase activity and performed radioligand-binding studies to determine βAR characteristics in adult and neonatal ventricular tissues with and without exposure to isoproterenol. Both adult and neonatal rabbit hearts showed a concentration-dependent increase in systolic function, namely, isovolumic left ventricular developed pressure (LVDP) and maximal positive dP/dt of LVDP (dP/dt_max) in response to isoproterenol. Adults, however, showed a significantly greater response than neonates. After sustained exposure to isoproterenol, the subsequent βAR-mediated responses in LVDP and dP/dt_max were significantly attenuated in adults, but much less so in neonates. The adenylyl cyclase activity in response to isoproterenol was significantly different between adult, but comparable in neonatal tissues both exposed or not to isoproterenol. The total βAR density was higher in neonatal than in adult tissues without isoproterenol exposure, but there was no significant change in βAR density in either group following isoproterenol exposure. In addition, isoproterenol exposure increased the amount of the inhibitory G protein in adult, but not neonatal tissues. Our results suggest that there were developmental differences in myocardial βAR functional responses in βAR desensitization.

Expression of adenylyl cyclase mRNAs in the denervated and in the developing mouse skeletal muscle.

Changes in the activity and in the expression of adenylyl cyclase (AC) were examined in mouse skeletal muscle after denervation and during development. Four isoforms of AC (AC2, AC6, AC7, and AC9) were detected by Northern blot analysis in gastrocnemius muscle, AC9 being the most abundant. After denervation, the levels of AC2 and AC9 mRNA decreased, whereas those of AC6 and AC7 increased. AC activity in response to several neurotransmitters was increased after denervation. During development, AC activity was high in fetus and neonate and declined in the adult; the sensitivity of AC activity to various neurotransmitters was the highest on the third postnatal day. The levels of AC6 and AC7 mRNAs were high on the third postnatal day and then decreased in adult, paralleling the decline in AC activity. All the characteristics of AC expression and activity in fetus and neonate resembled those observed in denervated adult muscle. These results indicate that changes in AC activity and AC mRNAs play an important role in the various physiopathological states of skeletal muscle, especially during muscle atrophy.

Pharmacological and molecular characterisation of β-adrenoceptors in adult rat diaphragm muscle

Using pharmacological and molecular approaches to investigate β-adrenoceptor ( β-AR) subtype expression in adult rat diaphragm, we found that adenylyl cyclase (AC) was potently stimulated by the β 2-AR-selective agonist fenoterol, weakly stimulated by the β 1-AR-selective agonist prenalterol and unaffected by the β 3-AR agonist CGP12177. AC activity in response to a submaximal isoproterenol concentration was potently inhibited by the β 2-AR-selective antagonist ICI118551, whereas the β 1-AR-selective antagonist CGP20712A was effective only in very high concentrations. (−)-[ 125I]-cyanopindolol ([ 125I]-CYP) saturation binding experiments indicated a single affinity component (dissociation constant ( K d)=22±2 pM) for β-AR sites (maximal β-AR density ( B max)=14±2 fmol/mg). Eadie–Hofstee analysis of [ 125I]-CYP displacement curves by β 1-, β 2- or β 3-AR-selective ligands allowed to characterise a homogenous population of β 2-AR sites. Finally, reverse transcriptase–polymerase chain reaction analysis of β-AR subtype mRNAs identified β 2-AR transcripts but no β 1- and β 3-AR mRNAs. Our results demonstrate that β 2-AR is the only β-AR subtype expressed in the diaphragm.

Ribozyme-mediated Suppression of the G Protein γ7Subunit Suggests a Role in Hormone Regulation of Adenylylcyclase Activity

Human HEK 293 cells present a simple and tractable system to directly test the hypothesis that the G protein gamma subunits contribute to the specificity of receptor signaling pathways in vivo. To begin to elucidate the functions of the individual gamma subunits in these cells, a ribozyme strategy was used to specifically inactivate the mRNA encoding the gamma7 subunit. A phosphorothioated DNA-RNA chimeric hammerhead ribozyme was constructed and analyzed for specificity toward the targeted gamma7 subunit. In vitro cleavage analysis of this ribozyme revealed a highly efficient cleavage activity directed exclusively toward the gamma7 RNA transcript. In particular, this ribozyme did not result in cleavage of the gamma12 RNA transcript, which is 75% identical to the gamma7 RNA transcript. Using a transient transfection assay, in vivo analysis of this ribozyme showed a specific reduction in both the mRNA and protein expression of the gamma7 subunit in HEK 293 cells. Coincident with this loss in gamma7 subunit, there was a specific reduction in the protein expression of the beta1 subunit, suggesting that the beta1 and gamma7 subunits may functionally interact to form a betagamma dimer in vivo. Functional analysis of the consequences of ribozyme-mediated suppression of the gamma7 subunit expression indicated that it was associated with significant attenuation of isoproterenol-, but not prostaglandin E1-, stimulated adenylylcyclase activity. Suppression of the gamma7 subunit expression had no effect on carbachol- and ATP-mediated stimulation of phosphatidylinositol turnover. Taken together, these results not only indicate the feasibility of using the ribozyme technology to determine the roles of individual gamma subunits in receptor-G protein-effector pathways in vivo, but they point to a specific role of the gamma7 subunit in the regulation of adenylylcyclase activity in response to isoproterenol.

TNF-alpha inhibits isoproterenol-stimulated adenylyl cyclase activity in cultured airway smooth muscle cells.

Inflammation, increased cytokine production, and decreased responsiveness of airway smooth muscle (ASM) to beta-adrenergic agonists are characteristics of asthma. We questioned whether the cytokine tumor necrosis factor-alpha (TNF-alpha) directly impaired beta-adrenergic signal transduction in cultured canine ASM cells. Confluent ASM cells exposed to TNF-alpha (0.1-10 ng/ml) for 72 h showed lower maximal levels of adenylyl cyclase activity in response to isoproterenol (10 ng/ml; 14 +/- 4.3 vs. 7.5 +/- 1.3 pmol adenosine 3',5'-cyclic monophosphate x well(-1) x 20 min(-1), control vs. treated, respectively), despite no changes in beta-adrenergic receptor numbers (maximum number of binding sites = 4.8 +/- 0.72 vs. 4.5 +/- 0.81 fmol/mg protein, control vs. treated, respectively). Adenylyl cyclase activities in response to prostaglandin E1, NaF, or forskolin were not different in treated and untreated cells. These results demonstrate that a cytokine known to be increased during exacerbation of asthmatic symptoms directly impairs beta-adrenergic function in ASM cells and suggests a mechanism by which inflammation impairs beta-adrenergic receptor signal transduction in asthma.

Mutations of neighboring polar residues on the second transmembrane helix disrupt signaling by the parathyroid hormone receptor.

Site-directed mutagenesis was used to assess the role of transmembrane (TM)-charged amino acids in the expression and function of the G protein-coupled receptor for PTH and PTH-related protein (PTHrP). Charged residues that are conserved in the TM regions of most or all members of the PTH/secretin receptor subfamily were targeted. Four mutants (E296A, R337A, H414A, and E459K) displayed properties similar to the wild type PTH/PTHrP receptor with respect to agonist binding and stimulation of adenylyl cyclase when expressed in COS-7 cells. Several mutations, all in TM II, produced receptors that signaled extremely poorly. Mutation of three residues (227S, 230R, and 233S), predicted to be aligned on one helical face of TM II, displayed a similar phenotype: markedly blunted adenylyl cyclase activity in response to PTH (20-30% of the wild type response) and a lower binding affinity for agonist, with no reduction in cell surface receptor expression. These results suggest that TM II contains a polar face that is involved in TM signaling by the PTH/PTHrP receptor. Two of these mutations were made at the corresponding sites in the secretin receptor, and a similar reduction in secretin-stimulated adenylyl cyclase activity was observed. Thus this region of TM II may participate in a mechanism of TM signal transduction that is shared by the PTH/secretin sub-family of G protein-coupled receptors.

Developmental expression and functional activity of β1- and β3-adrenoceptors in murine 3T3-F442A differentiating adipocytes

β 1- and β 3-adrenoceptor mRNA and protein expression, and contribution of each subtype to the catecholamine-sensitive adenylyl cyclase system were studied during the adipose conversion of the murine 3T3-F442A cell line. Northern and reverse transcriptase-polymerase chain reaction analyses indicated that emergence of β 3-adrenoceptor transcripts was concomittant with that of the gene encoding adipsin, a very late marker of adipose differentiation. Conversely, the induction of the β 1-adrenoceptor mRNA occurred early after cell commitment towards adipose conversion. Changes in β-subtype gene expression were accompanied by parallel modifications in receptor expression and function. 125I-cyanopindolol saturation and competition binding experiments showed a 3-fold increase in β 1-adrenoceptor density in day 3 post-confluent cells. The β 3-subtype population became detectable later and represented ∼ 95% of total β-adrenoceptors in day 8 and day 12 post-confluent cells. Adenylyl cyclase activity in response to the β 3-adrenoceptor-selective agonists CGP12177 (4-(3- t-butylamino-2-hydroxypropoxy)-benzimidazol-2-one), ICI201651 ([( R)-4-(2hydroxy-3-phenoxypropylamino-ethoxy)- N-(2-methoxyethyl)phenoxy-acetamidel]) and cyanopindolol was virtually absent in young adipocytes, but dramatically increased in mature cells. The respective contributions of the β 1- and the β 3-subtypes to the production of cAMP were resolved by an Eadie-Hofstee computer analysis of isoproterenol and norepinephrine concentration-response curve of adenylyl cyclase activity. Agonist response curves in the presence of β 1- and β 2-adrenoceptor antagonists indicated that the β 1-subtype accounted for the totality of β-adrenoceptor-mediated adenylyl cyclase activation in young adipocytes. In mature adipose cells ∼ 90% of this response was due to an activation of the β 3-adrenoceptor. In addition, ∼ 84% of the maximal norepinephrine-stimulated lipolysis was mediated by the β 3-adrenoceptor in fully differentiated adipocytes. The differentiation-dependent expression of β-subtypes in adipocytes is a biphasic process involving an initial and moderate induction of β 1-adrenoceptors followed by the emergence of a prominent β 3-adrenoceptor population. Compared analysis of both receptor occupancy and cAMP production shows that the β 3-subtype is more efficiently coupled to the adenylyl cyclase system than the β 1-adrenoceptor. Thus in mature adipose cells this receptor subtype represents the core of cAMP-dependent regulation of the lipolytic, antilipogenic and thermogenic effects of catecholamines.