Receptor-mediated Activation Of Adenylyl Cyclase Research Articles

Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system

Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein α-subunit (Gα s), G-protein β 1γ 2 subunit dimer (Gβ 1γ 2), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Gα s, Gβ 1γ 2, and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Gα s-coupled receptors together with Gα s, Gβ 1γ 2, and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Gα s and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.

Detection of G Protein Coupled Receptor Mediated Adenylyl Cyclase Activity by Capillary Electrophoresis Using Fluorescently Labeled ATP

A capillary electrophoresis (CE) laser-induced fluorescence (LIF) assay was developed for the detection of G protein coupled receptor mediated adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, cell membranes coexpressing the stimulatory G protein fused to the beta2 adrenergic receptor (beta2AR) and AC were incubated with BATP, the resultant mixture injected, and BATP separated from product BODIPY FL cAMP (BcAMP) by CE. AC activity was quantified by measuring the rate of BcAMP formation. beta2AR agonists isoproterenol and terbutaline increased basal AC activity with EC50s of 2.4 +/- 0.2 and 60 +/- 9 nM, respectively. The antagonist propranolol competed with terbutaline for beta2AR binding sites and expectedly right-shifted the terbutaline dose-response curve to 8 +/- 3 microM. The high sensitivity of the assay was demonstrated by detection of small changes in AC activity, with the partial agonist alprenolol increasing (22 +/- 1%) and the inverse agonist ICI 118,551 decreasing (19 +/- 2%) basal activity. The simplicity and automation of the CE-LIF assay offers advantages over the more traditional assay using radiochemical ATP and column chromatography.

Agonist and Antagonist Effects of 15R-Prostaglandin (PG) D2and 11-Methylene-PGD2on Human Eosinophils and Basophils

Prostaglandin (PG) D2 acts through both the DP(1) receptor, which is coupled to adenylyl cyclase, and the DP2 receptor (chemoattractant receptor-homologous molecule expressed on Th2 cells), which is present on eosinophils, basophils, and Th2 cells and results in cell activation and migration. The most potent prostanoid DP2 agonist so far reported is 15R-methyl-PGD2, in which the hydroxyl group has the unnatural R configuration. In contrast, the corresponding analog possessing the natural 15S configuration is approximately 75 times less potent. This raised the question of whether the isoprostane 15R-PGD2 might have potent DP2 receptor-mediated biological activity. We therefore chemically synthesized 15R-PGD2 and investigated its biological activity. This compound elicited DP2 receptor-mediated CD11b expression in human basophils and eosinophils and induced actin polymerization and migration in eosinophils with a potency about the same as that of PGD2. In contrast, it had only a weak effect on DP1 receptor-mediated adenylyl cyclase activity in human platelets. We also investigated the effects of modification of the 9-hydroxyl and 11-oxo groups of PGD2. Both PGK2, in which the 9-hydroxyl group is replaced by an oxo group, and 11-deoxy-11-methylene PGD2, in which the 11-oxo group is replaced by a CH2 group, have little or no DP1 or DP2 agonist activity. However, the 11-methylene analog is a DP2 antagonist (IC50, approximately 2 microM). We conclude that 15R-PGD2, which may be generated by oxidative stress, is a potent and selective DP2 agonist and that modification of the 11-oxo group of PGD2 can result in DP2 antagonist activity.

Optimized conditions for measuring lipolysis in murine primary adipocytes

The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000-15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells.

The Small Antitumoral Immune Response Modifier Imiquimod Interacts with Adenosine Receptor Signaling in a TLR7- and TLR8-Independent Fashion

Imiquimod, a small-molecule immune response modifier of the imidazoquinoline family, has shown profound antitumoral and antiviral efficacy both in vitro and in clinical applications in vivo. It has been demonstrated that this activity is mediated through the Toll-like receptor (TLR)7- and TLR8-signaling cascade resulting in the secretion of proinflammatory cytokines and, consecutively, induction of a tumor-directed cellular immune response. In addition, imiquimod exerts a direct proapoptotic activity in tumor cells. We demonstrate here that imiquimod induces activation of the transcription factor NF-kappaB and the downstream production of proinflammatory cytokines in the absence of TLR7 and TLR8. In Chinese hamster ovary cells stably transfected with the human adenosine receptor subtypes, we then show in radioligand-binding competition experiments that imiquimod binds to adenosine receptors at concentrations relevant in clinical settings, with highest affinities to the A(1) and A(2A) subtypes. The effect on the receptor-mediated activation of adenylyl cyclase was also studied, and these experiments revealed that imiquimod acts as an adenosine receptor antagonist. In addition, imiquimod had an inhibitory effect on adenylyl cyclase activity downstream from the receptor. Finally, using transformed human keratinocytes, we provide experimental evidence that imiquimod and A(2A) adenosine receptor-specific compounds similarly induce proinflammatory cytokines in the absence of immune cells. Thus, imiquimod appears to suppress an important feedback mechanism of inflammation by antagonism of adenosine receptor-dependent increase of cAMP and a concomitant receptor-independent inhibition of cAMP production. These novel mechanisms presumably act synergistic with the positive induction of proinflammatory cytokines and can, at least in part, explain the profound inflammation observed in some patients in vivo.

Heterologous desensitization is evoked by both agonist and antagonist stimulation of the human 5-HT 7 serotonin receptor

Previously, we demonstrated that human serotonin (5-HT) 5-HT 7 receptors display marked constitutive activity. Here, we tested if the constitutive activation of adenylyl cyclase by 5-HT 7 receptors influenced both the desensitization properties of transfected 5-HT 7 receptors and the ability of endogenous G s-coupled receptors to activate adenylyl cyclase. Using membranes from stably transfected HEK293 cells expressing the recombinant human 5-HT 7 receptor splice variants (5-HT 7(a), 5-HT 7(b) and 5-HT 7(d)), we compared the effects of 1-h or 24-h preincubation of the agonist 5-HT, partial inverse agonists mesulergine and SB269970, and full inverse agonists clozapine and methiothepin on subsequent activation of adenylyl cyclase by both 5-HT through transfected 5-HT 7 receptors and the endogenous G s-coupled β-adrenoceptors and prostaglandin receptors of HEK293 cells. The data show that stable expression of 5-HT 7 receptors is sufficient to attenuate adenylyl cyclase activation by endogenous G s-coupled receptors. Interestingly, preincubation with inverse agonists not only failed to result in the predicted resensitization of all receptor mediated adenylyl cyclase activation, but some inverse agonists further attenuated (desensitized) β-adrenoceptor and prostaglandin-stimulated adenylyl cyclase activation similar to long-term agonist exposure by 5-HT. These effects were not correlated with inverse agonist efficacy, were not accompanied by receptor down-regulation and appear to be mediated by a protein kinase A (PKA) independent mechanism. It is concluded that the human 5-HT 7 receptor mediates heterologous desensitization of endogenous G s-coupled receptors through an unknown and potentially novel mechanism.

Identification of a dysfunctional missense single nucleotide variant of human adenylyl cyclase VI

Genetic variants have been described for a range of G protein-coupled receptors (as well as for G proteins) linked to adenylyl cyclase. Furthermore, expression of these variants resulted in alterations in receptor-mediated activation of adenylyl cyclase, as well as alterations in more "downstream" effector pathways mediated by cyclic adenosine monophosphate. However, the identification of dysfunctional variants of adenylyl cyclase has been far more limited. Screening a region of the molecule that we recently demonstrated to be critical in regulation of enzyme activity, we have identified a missense single-nucleotide variant at amino acid 674 of human adenylyl cyclase isoform VI. In a population of 286 healthy white subjects, this variant has an allelic frequency of 3.1% (although 0/90 nonwhite subjects had this variant). Expression of this variant of adenylyl cyclase VI (whether expressed as the S674 human adenylyl cyclase VI [ADCY6] or the S686 ADCY6 rat analog) is characterized by a significant decrease in stimulated adenylyl cyclase activity (forskolin-stimulated activity of the S674 human ADCY6 variant was decreased to 56% +/- 6% of the activity of the A674 variant [mean +/- SEM]; n = 9; P = .004). Furthermore, subjects with the S674 variant demonstrated a significantly higher lymphocyte count (2.68 +/- 4.13 x 10(3)/mm3 versus 1.90 +/- 0.72 x 10(3)/mm3, P = .019). Paralleling this phenotype, expression of the variant was associated with attenuation of the forskolin-mediated reduction in cell growth rate to 64% +/- 5% of the effect seen with expression of the wild-type ADCY6 (n = 4; P = .001). In summary, these data demonstrate an unappreciated variant of adenylyl cyclase isoform VI that has a functional impact on both enzyme activity and cyclic adenosine monophosphate-mediated regulation of cell growth.

Modulation of cyclic AMP production by signal transduction pathways in preglomerular microvessels and microvascular smooth muscle cells.

Cyclic AMP affects microvascular smooth muscle contraction and growth. Therefore, it is important to elucidate mechanisms regulating cyclic AMP production in microvascular smooth muscle. In this study, we determined whether several signal transduction pathways regulate receptor-induced cyclic AMP in isolated preglomerular microvessels and microvascular smooth muscle cells. Preglomerular microvessels were incubated with isoproterenol (beta-adrenoceptor agonist) and with and without U73122 (phospholipase C inhibitor), GF109203X (protein kinase C inhibitor), 1-butanol (phospholipase D inhibitor), CGP77675 (c-src inhibitor), HA1077 (Rho kinase inhibitor), Y27632 (Rho kinase inhibitor), LY294002 (phosphatidylinositol-3-kinase inhibitor), dipenyleneiodonium (NADPH oxidase inhibitor), or Tempol (superoxide dismutase mimetic). Cultured preglomerular microvascular smooth muscle cells were incubated with isoproterenol or forskolin (direct activator of adenylyl cyclase) and with or without U73122, C(2)-ceramide (phospholipase D inhibitor), or PP1 [src family inhibitor, 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine]. All studies were conducted with 3-isobutyl-1-methylxanthine (broad-spectrum phosphodiesterase inhibitor) to eliminate changes in cyclic AMP degradation. In microvessels isoproterenol-induced cyclic AMP was not affected by Y27632, HA1007, LY294002, dipenylene-iodonium, or Tempol; was increased by U73122 and GF109203X; and was decreased by 1-butanol and CGP77675. In cells, U73122 increased and C(2)-ceramide and PP1 decreased isoproterenol-induced cyclic AMP. Forskolin-induced cyclic AMP was not altered. These results indicate that receptor-mediated activation of adenylyl cyclase is 1) not modulated by Rho kinase, phosphatidylinositol-3-kinase, NADPH oxidase, or superoxide; 2) is attenuated by phospholipase C and protein kinase C; and 3) is augmented by phospholipase D and src. Phospholipase C, phospholipase D, and src modulate receptor-induced cyclic AMP by affecting beta-adrenoreceptor/G protein/adenylyl cyclase coupling rather than by directly affecting adenylyl cyclase activity.

Receptor-mediated adenylyl cyclase activation through XLalpha(s), the extra-large variant of the stimulatory G protein alpha-subunit.

XLalpha(s), the large variant of the stimulatory G protein alpha subunit (Gsalpha), is derived from GNAS1 through the use of an alternative first exon and promoter. Gs(alpha) and XLalpha(s) have distinct amino-terminal domains, but are identical over the carboxyl-terminal portion encoded by exons 2-13. XLalpha(s) can mimic some functions of Gs(alpha), including betagamma interaction and adenylyl cyclase stimulation. However, previous attempts to demonstrate coupling of XLalpha(s) to typically Gs-coupled receptors have not been successful. We now report the generation of murine cell lines that carry homozygous disruption of Gnas exon 2, and are therefore null for endogenous XLalpha(s) and Gs(alpha) (Gnas(E2-/E2-)). Gnas(E2-/E2-) cells transfected with plasmids encoding XLalpha(s) and different heptahelical receptors, including the beta2-adrenergic receptor and receptors for PTH, TSH, and CRF, showed agonist-mediated cAMP accumulation that was indistinguishable from that observed with cells transiently coexpressing Gs(alpha) and these receptors. Our findings thus indicate that XLalpha(s) is capable of functionally coupling to receptors that normally act via Gs(alpha).

Distinct interactions of G sα-long, G sα-short, and G αolf with GTP, ITP, and XTP

The G s-proteins G sα-short (G sαS) and G sα-long (G sαL), and the olfactory G s protein (G αolf) mediate activation of adenylyl cyclase by the β 2-adrenoceptor (β 2AR). Early studies showed that the purine nucleotides GTP, ITP, and XTP differentially support receptor-mediated adenylyl cyclase activation in various native membrane systems, but those findings have remained unexplained thus far. We systematically analyzed the effects of GTP, ITP, and XTP on the coupling of the β 2AR to G sαS, G sαL, and G αolf, respectively, using fusion proteins expressed in Sf9 insect cells. Fusion proteins ensure defined receptor/G-protein stoichiometry and efficient coupling. At all three fusion proteins, GTP, ITP, and XTP exhibited unique profiles with respect to their potency and efficacy at disrupting high-affinity agonist binding and supporting adenylyl cyclase activation by partial and full agonists. Our data can be interpreted in two ways: (i) GTP, ITP, and XTP may stabilize different active conformations in various G s-proteins, or (ii) GTP, ITP, and XTP may differ from one another in the kinetics of interaction with various G s-proteins. Regardless of which of the two explanations is correct, our present data demonstrate that GTP, ITP, and XTP are highly efficient regulators of signal transduction mediated through a specific G-protein. Also discussed is the possibility that G-protein activation by ITP and XTP may be of relevance in Lesch–Nyhan syndrome, a defect of the purine salvage pathway associated with abnormalities in various neurotransmitter systems.

Regulation of glutamatergic neurotransmission in the striatum by presynaptic adenylyl cyclase-dependent processes

The aim here was to examine the possible roles of adenylyl cyclase- and protein kinase A (PKA)-dependent processes in ionotropic glutamate receptor (iGluR)-mediated neurotransmission using superfused mouse striatal slices and a non-metabolized l-glutamate analogue, d-[3H]aspartate. The direct and indirect presynaptic modulation of glutamate release and its susceptibility to changes in the intracellular levels of cyclic AMP (cAMP), Ca2+ and calmodulin (CaM) and in protein phosphorylation was characterized by pharmacological manipulations. The agonists of iGluRs, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate, stimulated the basal release of d-[3H]aspartate, while N-methyl-d-aspartate (NMDA) was without effect. Both the AMPA- and kainate-mediated responses were accentuated by the β-adrenoceptor agonist isoproterenol. These facilitatory effects were mimicked by the permeable cAMP analogue dibutyryl-cAMP. The β-adrenoceptor antagonist propranolol, the adenylyl cyclase inhibitor MDL12,330A, the inhibitor of PKA and PKC, H-7, and the PKA inhibitor H-89 abolished the isoproterenol effect on the kainate-evoked release. The dibutyryl-cAMP-induced potentiation was also attenuated by H-7. Isoproterenol, propranolol and MDL12,330A failed to affect the basal release of d-[3H]aspartate, but dibutyryl-cAMP was inhibitory and MDL12,330A activatory. In Ca2+-free medium, the kainate-evoked release was enhanced, being further accentuated by the CaM antagonists calmidazolium and trifluoperazine, though these inhibited the basal release. The potentiating effect of calmidazolium on the kainate-stimulated release was counteracted by both MDL12,330A and H-7.We conclude that AMPA- and kainate-evoked glutamate release from striatal glutamatergic terminals is potentiated by β-adrenergic receptor-mediated adenylyl cyclase activation and cAMP accumulation. Glutamate release is enhanced if the Ca2+- and CaM-dependent, kainate-evoked processes do not prevent the excessive accumulation of intracellular cAMP.

15-Deoxy-delta 12,14-prostaglandins D2 and J2 are potent activators of human eosinophils.

15-Deoxy-Delta(12,14)-PDJ(2) (15d-PGJ(2)) is a degradation product of PGD(2) that has been proposed as an anti-inflammatory compound because of its various inhibitory effects, some of which are mediated by peroxisome proliferator-activated receptor-gamma. In contrast to its reported inhibitory effects on macrophages and other cells, we found that this compound is a potent activator of eosinophils, inducing calcium mobilization, actin polymerization, and CD11b expression. It is selective for eosinophils, having little or no effect on neutrophils or monocytes. 15d-PGJ(2) has an EC(50) of approximately 10 nM, similar to that of its precursor, PGD(2). The concentrations of 15d-PGJ(2) required to activate eosinophils are thus much lower than those required for its anti-inflammatory effects (usually micromolar). 15-Deoxy-Delta(12,14)-prostaglandin D(2) (15d-PGD(2)) is also a potent activator of eosinophils, with an EC(50) about the same as that of PGD(2), whereas Delta(12)-PGJ(2) is slightly less potent. Eosinophils pretreated with PGD(2) no longer respond to 15d-PGJ(2), and vice versa, but in both cases the cells still respond to another eicosanoid proinflammatory mediator, 5-oxo-6,8,11,14-eicosatetraenoic acid. This indicates that the effects of 15d-PGJ(2) are mediated by the DP(2)/chemoattractant receptor-homologous molecule expressed on Th2 cells that has recently been identified in eosinophils. 15d-PGJ(2) is selective for the DP(2) receptor, in that it has no effect on DP(1) receptor-mediated adenylyl cyclase activity in platelets. We conclude that 15d-PGJ(2) and 15d-PGD(2) are selective DP(2) receptor agonists that activate human eosinophils with potencies at least 100 times greater than those for the proposed anti-inflammatory effects of 15d-PGJ(2) on other cells.

The cytoplasmic tail of the D1A receptor subtype: identification of specific domains controlling dopamine cellular responsiveness.

In this study the rat D1A receptor (wild-type, WT) and truncation mutants thereof, are utilized to delineate specific cytoplasmic tail (CT) domains responsible for regulating ligand binding and receptor-mediated adenylyl cyclase activation. In human embryonic kidney (HEK) cells, all truncation mutants of the D1A receptor (Delta425, Delta379, Delta351) display cell surface localization and express at high but different receptor numbers. Binding studies suggest that residues located between Cys(351) and Asp(425) may serve to restrain the agonist binding conformation of the D1A receptor. This contention is supported by the observation that the constitutive activation of Delta351 is significantly increased in comparison with WT, Delta425 and Delta379. Furthermore, we demonstrate that the extent of dopamine-mediated maximal activation of adenylyl cyclase is significantly augmented in cells expressing Delta351 when compared with WT or mutants harboring shorter truncations. These results suggest that in addition to restraining receptor conformation, determinants located downstream of Cys(351) may act as negative regulators of the G protein coupling efficiency and adenylyl cyclase activation. Interestingly, all truncated receptors used in the present study display a decrease in dopamine potency when compared with WT. We show that inhibition of protein kinase A (PKA) activity leads also to a reduction in dopamine potency in cells expressing WT but not Delta351 receptors. These results hint at a potential previously unanticipated role for PKA in facilitating D1A receptor coupling efficiency in HEK cells. Overall, the present study has uncovered specific CT domains involved in regulating discrete aspects of the D1A receptor signaling.

Beta(2)-adrenergic and several other G protein-coupled receptors in human atrial membranes activate both G(s) and G(i).

Cardiac G protein-coupled receptors that couple to Galpha(s) and stimulate cAMP formation (eg, beta-adrenergic, histamine, serotonin, and glucagon receptors) play a key role in cardiac inotropy. Recent studies in rodent cardiac myocytes and transfected cells have revealed that one of these receptors, the beta(2)-adrenergic receptor (AR), also couples to the inhibitory G protein Galpha(i) (activation of which inhibits cAMP formation). If beta(2)ARs could be shown to couple to Galpha(i) in the human heart, it would have important ramifications, because levels of Galpha(i) increase with age and in failing human heart. Therefore, we investigated whether beta(2)ARs in the human heart activate Galpha(i). By photoaffinity labeling human atrial membranes with [(32)P]azidoanilido-GTP, followed by immunoprecipitation with antibodies specific for Galpha(i), we found that Galpha(i) is activated by stimulation of beta(2)ARs but not of beta(1)ARs. In addition, we found that other Galpha(s)-coupled receptors also couple to Galpha(i), including histamine, serotonin, and glucagon. When coupling of these receptors to Galpha(i) is disrupted by pertussis toxin, their ability to stimulate adenylyl cyclase is enhanced. These data provide the first evidence that beta(2)AR and many other Galpha(s)-coupled receptors in human atrium also couple to Galpha(i) and that abolishing the coupling of these receptors to Galpha(i) increases the receptor-mediated adenylyl cyclase activity.

Multiple Developmental Roles for CRAC, a Cytosolic Regulator of Adenylyl Cyclase

Receptor-mediated activation of adenylyl cyclase (ACA) inDictyosteliumrequires CRAC protein. Upon translocation to the membrane, this pleckstrin homology (PH) domain protein stimulates ACA and thereby mediates developmental aggregation. CRAC may also have roles later in development since CRAC-null cells can respond to chemotactic signals and participate in developmental aggregation when admixed with wild-type cells, but they do not complete development within such chimeras. To test whether the role of CRAC in postaggregative development is related to the activation of ACA, chemotactic aggregation was bypassed in CRAC-null cells by activating the cAMP-dependent protein kinase (PKA). While such strains formed mounds, they did not complete fruiting body morphogenesis or form spores. Expression of CRAC in the prespore cells of these strains rescued sporulation and fruiting body formation. This later function of CRAC does not appear to require its PH domain since the C-terminal portion of the protein (CRAC-ΔPH) can substitute for full-length CRAC in promoting spore cell formation and morphogenesis. No detectable ACA activation was observed in any of the CRAC-null strains rescued by PKA activation and expression of CRAC-ΔPH. Finally, we found that the development of CRAC-null ACA-null double mutants could be rescued by the activation of PKA together with the expression of CRAC-ΔPH. Thus, there appears to be a required function for CRAC in postaggregative development that is independent of its previously described function in the ACA activation pathway.

Impaired Vasodilator Function in Hypertension: The Role of Alterations in Receptor–G Protein Coupling

Defects in vascular relaxation mechanisms may have an important role in the pathogenesis and maintenance of the elevation in peripheral vascular resistance characteristic of the hypertensive state. Receptor systems that mediate vasorelaxation via elevation of vascular smooth muscle intracellular cAMP concentrations appear to be globally impaired. Recent studies have indicated that this defect may be due to alterations in the transmembrane signaling processes that link receptor activation with the stimulation of adenylyl cyclase. Reduced G-protein function has been reported. However, increased activity of a member of a family of enzymes, the G protein–receptor kinases (GRK), which reduces the efficiency of coupling between the receptor and G protein, may be the key factor accounting for impaired receptor-mediated adenylyl cyclase activation and impaired vasodilator function in the hypertensive state.

The beta2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange.

Stimulation of beta2-adrenergic receptors on the cell surface by adrenaline or noradrenaline leads to alterations in the metabolism, excitability, differentiation and growth of many cell types. These effects have traditionally been thought to be mediated exclusively by receptor activation of intracellular G proteins. However, certain physiological effects of beta2-adrenergic receptor stimulation, notably the regulation of cellular pH by modulation of Na+/H+ exchanger (NHE) function, do not seem to be entirely dependent on G-protein activation. We report here a direct agonist-promoted association of the beta2-adrenergic receptor with the Na+/H+ exchanger regulatory factor (NHERF), a protein that regulates the activity of the Na+/H+ exchanger type 3 (NHE3). NHERF binds to the beta2-adrenergic receptor by means of a PDZ-domain-mediated interaction with the last few residues of the carboxy-terminal cytoplasmic domain of the receptor. Mutation of the final residue of the beta2-adrenergic receptor from leucine to alanine abolishes the receptor's interaction with NHERF and also markedly alters beta2-adrenergic receptor regulation of NHE3 in cells without altering receptor-mediated activation of adenylyl cyclase. Our findings indicate that agonist-dependent beta2-adrenergic receptor binding of NHERF plays a role in beta2-adrenergic receptor-mediated regulation of Na+/H+ exchange.

Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors.

1. The rat CCK(A) and CCK(B) receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon. 2. Spectrofluorophotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCK(A) cells, measured as an increase in Fura-2 fluorescence emission ratio, 1000 fold more potently than its non-sulphated form (CCK-8-NS) (EC50 values of 0.19 nM and 0.18 microM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK(B) cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCK(A) receptor agonist JMV-180 increased [Ca2+]i only in CCK(A) cells. Likewise, pentagastrin increased [Ca2+]i only in CCK(B) cells. Finally, CCK-8-S-induced Ca2+ signalling through the CCK(A) receptor was most potently inhibited by the CCK(A) receptor antagonist L364,718, whereas the CCK(B) receptor antagonist L365,260 was more potent in CCK(B) cells. 3. Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK(A) cells. By contrast, none of these agonists increased cyclicAMP in CCK(B) cells. 4. Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca2+ mobilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK(A) cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-alpha-phorbol 12-myristate 13-acetate. 5. During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKC-alpha, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6. This study demonstrates that following expression in CHO cells (i) both CCK(A) and CCK(B) receptors are coupled to Ca2+ mobilization, (ii) only CCK(A) receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.

Desensitization of β 3-adrenergic receptor- stimulated adenylyl cyclase activity and lipolysis in rats

The β 3-adrenergic receptor is an integral membrane protein consisting of seven transmembrane domains. Unlike the β 1 and β 2 receptors, this subtype lacks the consensus phosphorylation sites required for desensitization by serine kinases. Using the rodent specific β 3 agonist BRL 35135, our initial data indicated that β 3 receptor-mediated glycerol levels progressively decreased following daily oral doses of 5 mg kg . Therefore, we initiated studies designed to delineate the possible mechanism(s) for this decreased response. Within 3 hours following a single oral dose of BRL 35135, serum glycerol levels and UCP (uncoupling protein) RNA levels were significantly increased whereas β 3 RNA levels were significantly decreased. Rats were dosed daily for 5 days with either vehicle or BRL 35135 (5 mg kg , p.o.) and blood samples were collected for glycerol analysis. Adipose tissue was excised for lipolysis and adenyl cyclase measurements. In addition, UCP and β 3 receptor RNA levels were assessed. No effect on adipocyte BRL 37344-stimulated adenylyl cyclase activity was observed 3 hours following the initial dose of BRL 35135. Although a slight decrease (~25%) in adenylyl cyclase activity could be observed 24 hours following the initial dose, it wasn't until day 4 of dosing that a significant decrease (50%) was observed. In contrast, β 3- stimulated lipolysis in adipocytes from BRL 35135-treated rats was decreased 85% within 24 hours and this decrease persisted through four days of treatment. These data indicate that the lipolytic response to β 3 receptor activation is decreased after only a single oral dose of BRL 35135, whereas receptor-mediated adenylyl cyclase activation, although initially unaffected, also desensitizes by day four of treatment.

A Novel Interaction between Adrenergic Receptors and the α-Subunit of Eukaryotic Initiation Factor 2B

The alpha-subunit of eukaryotic initiation factor 2B (eIF-2B), a guanine nucleotide exchange protein that functions in regulation of translation, was observed to associate with the carboxyl-terminal cytoplasmic domains of the alpha2A- and alpha2B-adrenergic receptors in a yeast two-hybrid screen of a cDNA library prepared from 293 cells. This protein association was confirmed in vitro by affinity chromatography and was shown to be specific for a subset of G protein-coupled receptors, including the alpha2A-, alpha2B-, alpha2C-, and beta2-adrenergic receptors, but not the vasopressin (V2) receptor. Association of these proteins in vivo was confirmed by specific co-immunoprecipitation of eIF-2Balpha with full-length beta2-adrenergic receptors expressed in transfected 293 cells and by fluorescence microscopy showing co-localization of these proteins in intact cells. Remarkably, eIF-2Balpha co-localized with receptors exclusively in regions of the plasma membrane that are in contact with the extracellular medium, but failed to associate with membranes making cell-cell contacts. Overexpression of eIF-2Balpha in 293 cells caused a small (approximately 15%) but significant enhancement of beta2-adrenergic receptor-mediated activation of adenylyl cyclase, without affecting forskolin or V2 receptor-mediated activation. These observations suggest a new role for a previously identified guanine nucleotide exchange protein in membrane biology and cell signaling.