Arachidonic acid mediates angiotensin II effects on p21ras in renal proximal tubular cells via the tyrosine kinase-Shc-Grb2-Sos pathway

In kidney epithelial cells, an angiotensin II (Ang II) type 2 receptor subtype (AT2) is linked to a membrane-associated phospholipase A2 (PLA2) and the mitogen-activated protein kinase (MAPK) superfamily. However, the intervening steps in this linkage have not been determined. The aim of this study was to determine whether arachidonic acid mediates Ang II’s effect on p21ras and if so, to ascertain the signaling mechanism(s). We observed that Ang II activated p21ras and that mepacrine, a phospholipase A2 inhibitor, blocked this effect. This activation was also inhibited by PD123319, an AT2 receptor antagonist but not by losartan, an AT1 receptor antagonist. Furthermore, Ang II caused rapid tyrosine phosphorylation of Shc and its association with Grb2. Arachidonic acid and linoleic acid mimicked Ang II-induced tyrosine phosphorylation of Shc and activation of p21ras. Moreover, Ang II and arachidonic acid induced an association between p21ras and Shc. We demonstrate that arachidonic acid mediates linkage of a G protein-coupled receptor to p21ras via Shc tyrosine phosphorylation and association with Grb2/Sos. These observations have important implications for other G protein-coupled receptors linked to a variety of phospholipases.

A physiological role of the adenosine A3 receptor: Sustained cardioprotection

Adenosine released during cardiac ischemia exerts a potent, protective effect in the heart. A newly recognized adenosine receptor, the A3 subtype, is expressed on the cardiac ventricular cell, and its activation protects the ventricular heart cell against injury during a subsequent exposure to ischemia. A cultured chicken ventricular myocyte model was used to investigate the cardioprotective role of a novel adenosine A3 receptor. The protection mediated by prior activation of A3 receptors exhibits a significantly longer duration than that produced by activation of the adenosine A1 receptor. Prior exposure of the myocytes to brief ischemia also protected them against injury sustained during a subsequent exposure to prolonged ischemia. The adenosine A3 receptor-selective antagonist 3-ethyl 5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1191) caused a biphasic inhibition of the protective effect of the brief ischemia. The concomitant presence of the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) converted the MRS1191-induced dose inhibition curve to a monophasic one. The combined presence of both antagonists abolished the protective effect induced by the brief ischemia. Thus, activation of both A1 and A3 receptors is required to mediate the cardioprotective effect of the brief ischemia. Cardiac atrial cells lack native A3 receptors and exhibit a shorter duration of cardioprotection than do ventricular cells. Transfection of atrial cells with cDNA encoding the human adenosine A3 receptor causes a sustained A3 agonist-mediated cardioprotection. The study indicates that cardiac adenosine A3 receptor mediates a sustained cardioprotective function and represents a new cardiac therapeutic target.

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease α-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of α-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH2, RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor

The phytochemical resveratrol, which is found in grapes and wine, has been reported to have a variety of anti-inflammatory, anti-platelet, and anti-carcinogenic effects. Based on its structural similarity to diethylstilbestrol, a synthetic estrogen, we examined whether resveratrol might be a phytoestrogen. At concentrations (≈3–10 μM) comparable to those required for its other biological effects, resveratrol inhibited the binding of labeled estradiol to the estrogen receptor and it activated transcription of estrogen-responsive reporter genes transfected into human breast cancer cells. This transcriptional activation was estrogen receptor-dependent, required an estrogen response element in the reporter gene, and was inhibited by specific estrogen antagonists. In some cell types (e.g., MCF-7 cells), resveratrol functioned as a superagonist (i.e., produced a greater maximal transcriptional response than estradiol) whereas in others it produced activation equal to or less than that of estradiol. Resveratrol also increased the expression of native estrogen-regulated genes, and it stimulated the proliferation of estrogen-dependent T47D breast cancer cells. We conclude that resveratrol is a phytoestrogen and that it exhibits variable degrees of estrogen receptor agonism in different test systems. The estrogenic actions of resveratrol broaden the spectrum of its biological actions and may be relevant to the reported cardiovascular benefits of drinking wine.

β-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking

The two widely coexpressed isoforms of β-arrestin (termed βarrestin 1 and 2) are highly similar in amino acid sequence. The β-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of β-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the β-arrestins (βarr1-KO and βarr2-KO) or both (βarr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the β2-adrenergic receptor (β2-AR) and the angiotensin II type 1A receptor (AT1A-R). Both βarr1-KO and βarr2-KO cells showed similar impairment in agonist-stimulated β2-AR and AT1A-R desensitization, when compared with their WT control cells, and the βarr1/2-KO cells were even further impaired. Sequestration of the β2-AR in the βarr2-KO cells was compromised significantly (87% reduction), whereas in the βarr1-KO cells it was not. Agonist-stimulated internalization of the AT1A-R was only slightly reduced in the βarr1-KO but was unaffected in the βarr2-KO cells. In the βarr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two β-arrestins to sequester the β2-AR revealed β-arrestin 2 to be 100-fold more potent than β-arrestin 1. Down-regulation of the β2-AR was also prevented in the βarr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two β-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.

An astroglia-linked dopamine D2-receptor action in prefrontal cortex

Typical neuroleptic drugs elicit their antipsychotic effects mainly by acting as antagonists at dopamine D2 receptors. Much of this activity is thought to occur in the cerebral cortex, where D2 receptors are found largely in inhibitory GABAergic neurons. Here we confirm this localization at the electron microscopic level, but additionally show that a subset of cortical interneurons with low or undetectable expression of D2 receptor isoforms are surrounded by astrocytic processes that strongly express D2 receptors. Ligand binding of isolated astrocyte preparations indicate that cortical astroglia account for approximately one-third of the total D2 receptor binding sites in the cortex, a proportion that we found conserved among rodent, monkey, and human tissues. Further, we show that the D2 receptor-specific agonist, quinpirole, can induce Ca2+ elevation in isolated cortical astrocytes in a pharmacologically reversible manner, thus implicating this receptor in the signaling mechanisms by which astrocytes communicate with each other as well as with neurons. The discovery of D2 receptors in astrocytes with a selective anatomical relationship to interneurons represents a neuron/glia substrate for cortical dopamine action in the adult cerebral cortex and a previously unrecognized site of action for antipsychotic drugs with affinities at the D2 receptor.

Novel Nuclear Signaling Pathway Mediates Activation of Fibroblast Growth Factor-2 Gene by Type 1 and Type 2 Angiotensin II Receptors

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1′,3′-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.

Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release.

Angiotensin (Ang) II and Ang III are two peptide effectors of the brain renin-angiotensin system that participate in the control of blood pressure and increase water consumption and vasopressin release. In an attempt to delineate the respective roles of these peptides in the regulation of vasopressin secretion, their metabolic pathways and their effects on vasopressin release were identified in vivo. For this purpose, we used recently developed selective inhibitors of aminopeptidase A (APA) and aminopeptidase N (APN), two enzymes that are believed to be responsible for the N-terminal cleavage of Ang II and Ang III, respectively. Mice received [3H]Ang II intracerebroventricularly (i.c.v.) in the presence or absence of the APN inhibitor, EC33 (3-amino-4-thio-butyl sulfonate) of the APN inhibitor, EC27 (2-amino-pentan-1,5-dithiol). [3H]Ang II and [3H]Ang III levels were evaluated from hypothalamus homogenates by HPLC. EC33 increased the half-life of [3H]Ang II 2.6-fold and completely blocked the formation of [3H]Ang III, whereas EC27 increased the half-life of [3H]Ang III 2.3-fold. In addition, the effects of EC33 and EC27 on Ang-induced vasopressin release were studied in mice. Ang II was injected i.c.v. in the presence or absence of EC33, and plasma vasopressin levels were estimated by RIA. While vasopressin levels were increased 2-fold by Ang II (5 ng), EC33 inhibited Ang II-induced vasopressin release in a dose-dependent manner. In contrast, EC27 injected alone increased in a dose-dependent manner vasopressin levels. The EC27-induced vasopressin release was completely blocked by the coadministration of the Ang receptor antagonist (Sar1-Ala8) Ang II. These results demonstrate for the first time that (i) APA and APN are involved in vivo in the metabolism of brain Ang II and Ang III, respectively, and that (ii) the action of Ang II on vasopressin release depends upon the prior conversion of Ang II to Ang III. This shows that Ang III behaves as one of the main effector peptides of the brain renin-angiotensin system in the control of vasopressin release.

Local stress, not systemic factors, regulate gene expression of the cardiac renin-angiotensin system in vivo: a comprehensive study of all its components in the dog.

Cardiac hypertrophy is associated with altered expression of the components of the cardiac renin-angiotensin system (RAS). While in vitro data suggest that local mechanical stimuli serve as important regulatory modulators of cardiac RAS activity, no in vivo studies have so far corroborated these observations. The aims of this study were to (i) examine the respective influence of local, mechanical versus systemic, soluble factors on the modulation of cardiac RAS gene expression in vivo; (ii) measure gene expression of all known components of the RAS simultaneously; and (iii) establish sequence information and an assay system for the RAS of the dog, one of the most important model organisms in cardiovascular research. We therefore examined a canine model of right ventricular hypertrophy and failure (RVHF) in which the right ventricle (RV) is hemodynamically loaded, the left ventricle (LV) is hemodynamically unloaded, while both are exposed to the same circulating milieu of soluble factors. Using specific competitive PCR assays, we found that RVHF was associated with significant increases in RV mRNA levels of angiotensin converting enzyme and angiotensin II type 2 receptor, and with significant decreases of RV expression of chymase and the angiotensin II type 1 receptor, while RV angiotensinogen and renin remained unchanged. All components remained unchanged in the LV. We conclude that (i) dissociated regional regulation of RAS components in RV and LV indicates modulation by local, mechanical, not soluble, systemic stimuli; (ii) components of the cardiac RAS are independently and differentially regulated; and (iii) opposite changes in the expression of angiotensin converting enzyme and chymase, and of angiotensin II type I and angiotensin II type 2 receptors, may indicate different physiological roles of these RAS components in RVHF.

Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice.

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin II-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype.

Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor.

To investigate the molecular mechanism for stereospecific binding of agonists to beta 2-adrenergic receptors we used receptor models to identify potential binding sites for the beta-OH-group of the ligand, which defines the chiral center. Ser-165, located in transmembrane helix IV, and Asn-293, situated in the upper half of transmembrane helix VI, were identified as potential binding sites. Mutation of Ser-165 to Ala did not change the binding of either isoproterenol isomer as revealed after transient expression in human embryonic kidney (HEK)-293 cells. In contrast, a receptor mutant in which Asn-293 was replaced by Leu showed substantial loss of stereospecific isoproterenol binding. Adenylyl cyclase stimulation by this mutant after stable expression in CHO cells confirmed the substantial loss of stereospecificity for isoproterenol. In a series of agonists the loss of affinity in the Leu-293 mutant receptor was strongly correlated with the intrinsic activity of the compounds. Full agonists showed a 10-30-fold affinity loss, whereas partial agonists had almost the same affinity for both receptors. Stereospecific recognition of antagonists was unaltered in the Leu-293 mutant receptor. These data indicate a relationship between stereospecificity and intrinsic activity of agonists and suggest that Asn-293 is important for both properties of the agonist-receptor interaction.

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Correlation between the effects of bombesin antagonists on cell proliferation and intracellular calcium concentration in Swiss 3T3 and HT-29 cell lines.

Bombesin (BN) acts as an autocrine mitogen in various human cancers. Several pseudononapeptide BN-(6-14) analogs with a reduced peptide bond between positions 13 and 14 have been shown to suppress the mitogenic activity of BN or gastrin-releasing peptide (GRP) when assessed by radioreceptor or proliferation assays and may have significant clinical applications. The search for potent and safe BN antagonists requires the evaluation of a large series of analogs in radioreceptor and proliferation assays. In this paper, we report that the ability of BN analogs to inhibit BN-induced calcium transients in Swiss 3T3 cells shows a high correlation with their inhibitory potency as evaluated by classical proliferation tests. The assay of calcium transients allows a rapid characterization of new BN analogs (in terms of minutes rather than days) and can be adapted as a labor and cost-effective screening step in the selection of potentially relevant BN antagonists for further characterization in cell proliferation systems. We also observed that results from the assay of calcium transients in Swiss 3T3 cells can be correlated with the results of the proliferative response in HT-29 cells, a cell line that does not seem to use the same early transmembrane ionic signal system. This result suggests that the calcium pathway is not mandatory for triggering cell division by the BN receptor.

Binding in the growth hormone receptor complex.

Binding reactions between human growth hormone (hGH) and its receptor provide a detailed account of how a polypeptide hormone activates its receptor and more generally how proteins interact. Through high-resolution structural and functional studies it is seen that hGH uses two different sites (site 1 and site 2) to bind two identical receptor molecules. This sequential dimerization reaction activates the receptor, presumably by bringing the intracellular domains into close proximity so they may activate cytosolic components. As a consequence of this mechanism it is possible to build antagonists to the receptor by introducing mutations in hGH that block binding at site 2 and to build even more potent antagonists by combining these with mutants that enhance binding at site 1. Alanine-scanning mutagenesis of all contact residues at the site 1 interface shows that only a small and complementary set of side chains clustered near the center of the interface affects binding. The most important contacts are hydrophobic, and these are surrounded by polar and charged interactions of lesser importance. Kinetic analysis shows for the most part that the important side chains function to maintain the complex, not to guide the hormone to the receptor. Hormone-induced homodimerization or heterodimerization reactions are turning out to be pervasive mechanisms for signal transduction. Moreover, the molecular recognition principles seen in the hGH-receptor complex are likely to generalize to other protein-protein complexes.