Agonists and hydrogen peroxide mediate hyperoxidation of ß2-adrenergic receptor in airway epithelial cells: Implications for tachyphylaxis to ß2-agonists in constrictive airway disorders.

Asthma and other airway obstructive disorders are characterized by heightened inflammation and excessive airway epithelial cell reactive oxygen species (ROS), which give rise to a highly oxidative environment. After decades of use, ß2-adrenergic receptor (ß2AR) agonists remain at the forefront of treatment options for asthma, however, chronic use of ß2-agonists leads to tachyphylaxis to the bronchorelaxant effects, a phenomenon that remains mechanistically unexplained. We have previously demonstrated that ß2AR agonism increases ROS generation in airway epithelial cells, which upholds proper receptor function via feedback oxidation of ß2AR cysteine thiolates to Cys-S-sulfenic acids (Cys-SOH). Our previous results also demonstrate that prevention of normal redox cycling of this post-translational oxi-modification back to the thiol prevents proper receptor function. Given that Cys-S-sulfenic acids can be irreversibly overoxidized to Cys-S-sulfinic (Cys-SO2H) or S-sulfonic (Cys-SO3H) acids, which are incapable of further participation in redox reactions, we hypothesized that ß2-agonist tachyphylaxis may be explained by hyperoxidation of ß2AR to S-sulfinic acids. Here, using airway epithelial cell lines and primary small airway epithelial cells from healthy and asthma-diseased donors, we show that ß2AR agonism generates H2O2 in a receptor and NAPDH oxidase-dependent manner. We also demonstrate that acute and chronic receptor agonism can facilitate ß2AR S-sulfination, and that millimolar H2O2 concentrations are deleterious to ß2AR-mediated cAMP formation, an effect that can be rescued to a degree in the presence of the cysteine-donating antioxidant N-acetyl-L-cysteine. Our results reveal that the oxidative state of ß2AR may contribute to receptor functionality and may, at least in part, explain ß2-agonist tachyphylaxis.

Free-fatty acid receptor-1 (FFA1/GPR40) promotes papillary RCC proliferation and tumor growth via Src/PI3K/AKT/NF-κB but suppresses migration by inhibition of EGFR, ERK1/2, STAT3 and EMT.

BACKGROUND: Papillary renal cell carcinoma (pRCC) is a highly metastatic genitourinary cancer and is generally irresponsive to common treatments used for the more prevalent clear-cell (ccRCC) subtype. The goal of this study was to examine the novel role of the free fatty-acid receptor-1 (FFA1/GPR40), a cell-surface expressed G protein-coupled receptor that is activated by medium-to-long chained dietary fats, in modulation of pRCC cell migration invasion, proliferation and tumor growth. METHODS: We assessed the expression of FFA1 in human pRCC and ccRCC tumor tissues compared to patient-matched non-cancerous controls, as well as in RCC cell lines. Using the selective FFA1 agonist AS2034178 and the selective FFA1 antagonist GW1100, we examined the role of FFA1 in modulating cell migration, invasion, proliferation and tumor growth and assessed the FFA1-associated intracellular signaling mechanisms via immunoblotting. RESULTS: We reveal for the first time that FFA1 is upregulated in pRCC tissue compared to patient-matched non-cancerous adjacent tissue and that its expression increases with pRCC cancer pathology, while the inverse is seen in ccRCC tissue. We also show that FFA1 is expressed in the pRCC cell line ACHN, but not in ccRCC cell lines, suggesting a unique role in pRCC pathology. Our results demonstrate that FFA1 agonism promotes tumor growth and cell proliferation via c-Src/PI3K/AKT/NF-κB and COX-2 signaling. At the same time, agonism of FFA1 strongly inhibits migration and invasion, which are mechanistically mediated via inhibition of EGFR, ERK1/2 and regulators of epithelial-mesenchymal transition. CONCLUSIONS: Our data suggest that FFA1 plays oppositional growth and migratory roles in pRCC and identifies this receptor as a potential target for modulation of pathogenesis of this aggressive cancer.

Free-Fatty Acid Receptor-4 (FFA4/GPR120) differentially regulates migration, invasion, proliferation and tumor growth of papillary renal cell carcinoma cells.

Kidney cancer is among the 10 most common cancers, and renal cell carcinoma (RCC), which represent 90% of all kidney cancers, has the highest mortality rate of all genitourinary cancers. Papillary RCC (pRCC) is the second most frequent subtype of RCC and demonstrates distinct characteristics compared to other subtypes, including a high degree of metastasis and resistance to treatments against the more common clear cell RCC (ccRCC) subtype. Here, we demonstrate that the Free-Fatty Acid Receptor-4 (FFA4), a G protein-coupled receptor that is endogenously activated by medium-to-long chain free-fatty acids, is upregulated in pRCC compared to patient-matched normal kidney tissue, and that the expression of FFA4 increases with the degree of pathological grading of pRCC. Our data also show that FFA4 transcript is not expressed in ccRCC cell lines, but is expressed in the well-characterized metastatic pRCC cell line ACHN. Furthermore, we show that agonism of FFA4 with the selective agonist cpdA positively regulates ACHN cell migration and invasion in a manner dependent on PI3K/AKT/NF-κB signaling to COX-2 and MMP-9, with partial-dependence on EGFR transactivation. Our results also demonstrate that FFA4 agonism induces STAT-3-driven epithelial-mesenchymal transition, suggesting a significant role for FFA4 in pRCC metastasis. On the contrary, FFA4 agonism significantly reduces cell proliferation and tumor growth, suggesting that the receptor may have opposing effects on pRCC cell growth and migration. Together, our data demonstrate that FFA4 has significant functional roles in pRCC cells and may be an attractive target for study of pRCC and development of RCC pharmacotherapeutics.

Oncogenic signaling of the free-fatty acid receptors FFA1 and FFA4 in human breast carcinoma cells.

Globally, breast cancer is the most frequent type of cancer in women, and most breast cancer-associated deaths are due to metastasis and recurrence of the disease. Dietary habits, specifically dietary fat intake is a crucial risk factor involved in breast cancer development and progression. Decades of research has revealed that free-fatty acids (FFA) modulate carcinogenic processes through fatty acid metabolism and lipid peroxidation. The ground-breaking discovery of free-fatty acid receptors, which are members of the G-protein coupled receptor (GPCR) superfamily, has led to the realization that FFA can also act via these receptors to modulate carcinogenic effects. The long-chain free-fatty acid receptors FFA1 (previously termed GPR40) and FFA4 (previously termed GPR120) are activated by mono- and polyunsaturated fatty acids including ω-3, 6, and 9 fatty acids. Initial enthusiasm towards the study of these receptors focused on their insulin secretagogue and sensitization effects, and the downstream associated metabolic regulation. However, recent studies have demonstrated that abnormal expression and/or aberrant FFA1/FFA4 signaling are evident in human breast carcinomas, suggesting that FFA receptors could be a promising target in the treatment of breast cancer. The current review discusses the diverse roles of FFA1 and FFA4 in the regulation of cell proliferation, migration, invasion, and chemotherapy resistance in human breast carcinoma cells and tissue.

Short-chain free-fatty acid G protein-coupled receptors in colon cancer.

The dietary role of macronutrients and their metabolites in cancer has been evident for many decades. Dietary ingestion of fat, carbohydrates, protein, and fiber, as well as probiotics that influence gut microbiota, have all been linked to gastrointestinal (GI) tract health and disease, particularly in the colon, where it has long been known that fat and fiber can regulate inflammation and carcinogenesis. Short-chained fatty acids (SCFA), including acetate, propionate, and butyrate, which are biosynthesized by microbiota-mediated metabolism of dietary fiber, have previously been shown to play important roles in colorectal health, including decreasing inflammation and oxidative stress. Since the 1980s, a growing number of studies have also demonstrated a link between SCFA and colon epithelial cell carcinogenesis and prevention of colorectal cancers (CRC). While the effects of SCFA have historically been associated with their intracellular metabolism and function, the discovery of a family of G protein-coupled free-fatty acid receptors in the early 2000s suggests that many effects of SCFA are cell-surface receptor mediated. Indeed, the SCFA GPCRs FFA2 (previously termed GPR43), FFA3 (previously termed GPR41), and GPR109A are now well established to be expressed within the GI tract, where they modulate a variety of functions in response to luminal SCFA. While the role of SCFA in cancers, including CRC, has been reviewed in detail elsewhere, the goal of this report is to provide a review on the current body of evidence in regard to the effects of SCFA on FFA2, FFA3, and GPR109A in colon cancers.

Cysteine redox state regulates human ß2-adrenergic receptor binding and function.

Bronchoconstrictive airway disorders such as asthma are characterized by inflammation and increases in reactive oxygen species (ROS), which produce a highly oxidative environment. ß2-adrenergic receptor (ß2AR) agonists are a mainstay of clinical therapy for asthma and provide bronchorelaxation upon inhalation. We have previously shown that ß2AR agonism generates intracellular ROS, an effect that is required for receptor function, and which post-translationally oxidizes ß2AR cysteine thiols to Cys-S-sulfenic acids (Cys-S-OH). Furthermore, highly oxidative environments can irreversibly oxidize Cys-S-OH to Cys-S-sulfinic (Cys-SO2H) or S-sulfonic (Cys-SO3H) acids, which are incapable of further participating in homeostatic redox reactions (i.e., redox-deficient). The aim of this study was to examine the vitality of ß2AR-ROS interplay and the resultant functional consequences of ß2AR Cys-redox in the receptors native, oxidized, and redox-deficient states. Here, we show for the first time that ß2AR can be oxidized to Cys-S-OH in situ, moreover, using both clonal cells and a human airway epithelial cell line endogenously expressing ß2AR, we show that receptor redox state profoundly influences ß2AR orthosteric ligand binding and downstream function. Specifically, homeostatic ß2AR redox states are vital toward agonist-induced cAMP formation and subsequent CREB and G-protein-dependent ERK1/2 phosphorylation, in addition to ß-arrestin-2 recruitment and downstream arrestin-dependent ERK1/2 phosphorylation and internalization. On the contrary, redox-deficient ß2AR states exhibit decreased ability to signal via either Gαs or ß-arrestin. Together, our results demonstrate a ß2AR-ROS redox axis, which if disturbed, interferes with proper receptor function.

The ß2-adrenergic receptor-ROS signaling axis: An overlooked component of ß2AR function?

ß2-Adrenergic receptor (ß2AR) agonists are clinically used to elicit rapid bronchodilation for the treatment of bronchospasms in pulmonary diseases such as asthma and COPD, both of which exhibit characteristically high levels of reactive oxygen species (ROS); likely secondary to over-expression of ROS generating enzymes and chronically heightened inflammation. Interestingly, ß2AR has long-been linked to ROS, yet the involvement of ROS in ß2AR function has not been as vigorously studied as other aspects of ß2AR signaling. Herein, we discuss the existing body of evidence linking ß2AR activation to intracellular ROS generation and importantly, the role of ROS in regulating ß2AR function. The reciprocal interplay of the ß2AR and ROS appear to endow this receptor with the ability to self-regulate signaling efficacy and ligand binding, hereby unveiling a redox-axis that may be unfavorably altered in pathological states contributing to both disease progression and therapeutic drug responses.

The role of free-fatty acid receptor-4 (FFA4) in human cancers and cancer cell lines.

A dietary influence on cancer progression has been evident for many decades, and dietary fatty acids, particularly long chain mono- and polyunsaturated fatty acids, have been shown to play significant roles in influencing growth of a variety of human cancers. The discovery of the family of cell-surface free-fatty acid receptors, which include the long-chain fatty acid receptors FFA1 and FFA4, suggest that many of the effects of dietary fats could be receptor-mediated. FFA4 is ubiquitously expressed and has recently been shown to modulate a variety of important anti-inflammatory and metabolic processes. Since FFA4 is currently an attractive drug target for treatment of metabolic disorders such as diabetes and obesity, understanding its role in cancer progression is critical towards the drug discovery process. In this research update, the current body of knowledge on the role of this receptor in regulating cancer cell proliferation, migration, and invasion, as well as in vivo tumorigenesis is reviewed.

Free-fatty acid receptor-4 (GPR120): Cellular and molecular function and its role in metabolic disorders.

Over the last decade, a subfamily of G protein-coupled receptors that are agonized by endogenous and dietary free-fatty acids (FFA) has been discovered. These free-fatty acid receptors include FFA2 and FFA3, which are agonized by short-chained FFA, as well as FFA1 and FFA4, which are agonized by medium-to-long chained FFA. Ligands for FFA1 and FFA4 comprise the family of long chain polyunsaturated omega-3 fatty acids including α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), suggesting that many of the long-known beneficial effects of these fats may be receptor mediated. In this regard, FFA4 has gathered considerable interest due to its role in ameliorating inflammation, promoting insulin sensitization, and regulating energy metabolism in response to FFA ligands. The goal of this review is to summarize the body of evidence in regard to FFA4 signal transduction, its mechanisms of regulation, and its functional role in a variety of tissues. In addition, recent endeavors toward discovery of small molecules that modulate FFA4 activity are also presented.

Fish oil and flax seed oil supplemented diets increase FFAR4 expression in the rat colon.

BACKGROUND AND OBJECTIVE: Omega-3 fatty acids, such as α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are polyunsaturated fatty acids (PUFA) that have long been associated with anti-inflammatory activity and general benefit toward human health. Over the last decade, the identification of a family of cell-surface G protein-coupled receptors that bind and are activated by free-fatty acids, including omega-3 fatty acids, suggest that many effects of PUFA are receptor-mediated. One such receptor, free-fatty acid receptor-4 (FFAR4), previously described as GPR120, has been shown to modulate anti-inflammatory and insulin-sensitizing effects in response to PUFA such as ALA and DHA. Additionally, FFAR4 stimulates secretion of the insulin secretagogue glucagon-like peptide-1 (GLP-1) from the GI tract and acts as a dietary sensor to regulate energy availability. The aim of the current study was to assess the effects of dietary omega-3 fatty acid supplementation on FFAR4 expression in the rat colon. METHODS: Sprague-Dawley rats were fed control soybean oil diets or alternatively, diets supplemented with either fish oil, which is enriched in DHA and EPA, or flaxseed oil, which is enriched in ALA, for 7 weeks. GLP-1 and blood glucose levels were monitored weekly and at the end of the study period, expression of FFAR4 and the inflammatory marker TNF-α was assessed. RESULTS: Our findings indicate that GLP-1 and blood glucose levels were unaffected by omega-3 fatty acid supplementation, however, animals that were fed fish or flaxseed oil-supplemented diets had significantly heightened colonic FFAR4 and actin expression, and reduced expression of the pro-inflammatory cytokine TNF-α compared to animals fed control diets. CONCLUSIONS: These results suggest that similar to ingestion of other fats, dietary-intake of omega-3 fatty acids can alter FFAR4 expression within the colon.

Clinical effects of once-weekly exenatide for the treatment of type 2 diabetes mellitus.

PURPOSE: The pharmacology, pharmacokinetics, clinical efficacy, adverse effects, administration, dosage, place in therapy, and cost of extended-release exenatide are reviewed. SUMMARY: Regular-release exenatide has a half-life of 2.4 hours and is administered twice daily. In order to allow for once-weekly administration, exenatide was encapsulated in poly(lactic-co-glycolic acid) microspheres, a biodegradable polymer. After subcutaneous injection, the microspheres slowly degrade, and the drug is released. A single injection of extended-release exenatide reaches maximum plasma concentration after 4-8 hours and remains at therapeutic levels for 8-16 hours, depending on the dosage. Based on the pharmacokinetics of a single dose, researchers determined that 0.8- and 2-mg once-weekly doses were likely to maintain therapeutic levels in the serum. Patients who used extended-release exenatide monotherapy had significantly lower glycosylated hemoglobin (HbA1c) levels and lost more weight than those receiving sitagliptin or pioglitazone (p < 0.05). In combination with metformin, extended-release exenatide reduced HbA1c levels more than did insulin glargine. This new formulation reduced HbA1c levels by 1.5-1.9%, fasting blood glucose concentrations by 31-42 mg/dL, and weight by 2.3-3.7 kg. The most common adverse events were injection-site reactions and transient nausea. Postmarketing reports have described acute pancreatitis and acute necrotizing or hemorrhagic pancreatitis in patients treated with exenatide. The published average wholesale price for a one-month supply of extended-release exenatide 2 mg is $388. CONCLUSION: Extended-release exenatide taken once weekly is an effective second-line therapy for patients with type 2 diabetes who have not achieved glycemic goals with metformin alone.

Agonist- and hydrogen peroxide-mediated oxidation of the ß2 adrenergic receptor: evidence of receptor s-sulfenation as detected by a modified biotin-switch assay.

Reactive oxygen species (ROS), including hydrogen peroxide (H(2)O(2)), have recently been shown to be generated upon agonism of several members of the G protein-coupled receptor (GPCR) superfamily, including ß(2)-adrenergic receptors (ß(2)ARs). Previously, we have demonstrated that inhibition of intracellular ROS generation mitigates ß(2)AR signaling, suggesting that ß(2)AR-mediated ROS generation is capable of feeding back to regulate receptor function. Given that ROS, specifically H(2)O(2), are able to post-translationally oxidize protein cysteine sulfhydryls to cysteine-sulfenic acids, the goal of the current study was to assess whether ROS are capable of S-sulfenating ß(2)AR. Using a modified biotin-switch assay that is selective for cysteine-sulfenic acids, our results demonstrate for the first time that H(2)O(2) treatment facilitates S-sulfenation of transiently overexpressed ß(2)AR in human embryonic kidney 293 cells. It is noteworthy that stimulation of cells with the ß-agonist isoproterenol produces both dose- and time-dependent S-sulfenation of ß(2)AR, an effect that is receptor-dependent, and demonstrates that receptor-generated ROS are also capable of oxidizing the ß(2)AR. Receptor-dependent S-sulfenation was inhibited by the chemoselective sulfenic acid alkylator dimedone and the cysteine antioxidant N-acetyl-l-cysteine. Moreover, our results reveal that receptor oxidation occurs in cells that endogenously express physiologically relevant levels of ß(2)AR, because treatment of human alveolar epithelial A549 cells with either H(2)O(2) or the ß(2)-selective agonist formoterol promoted receptor S-sulfenation. These findings provide the first evidence, to our knowledge, that a mammalian GPCR can be oxidized by S-sulfenation and signify an important first step toward shedding light on the overlooked role of ROS in the regulation of ß(2)AR function.

Dynamin2- and endothelial nitric oxide synthase-regulated invasion of bladder epithelial cells by uropathogenic Escherichia coli.

Invasion of bladder epithelial cells by uropathogenic Escherichia coli (UPEC) contributes to antibiotic-resistant and recurrent urinary tract infections (UTIs), but this process is incompletely understood. In this paper, we provide evidence that the large guanosine triphosphatase dynamin2 and its partner, endothelial nitric oxide (NO) synthase (NOS [eNOS]), mediate bacterial entry. Overexpression of dynamin2 or treatment with the NO donor S-nitrosothiols increases, whereas targeted reduction of endogenous dynamin2 or eNOS expression with ribonucleic acid interference impairs, bacterial invasion. Exposure of mouse bladder to small molecule NOS inhibitors abrogates infection of the uroepithelium by E. coli, and, concordantly, bacteria more efficiently invade uroepithelia isolated from wild-type compared with eNOS(-/-) mice. E. coli internalization promotes rapid phosphorylation of host cell eNOS and NO generation, and dynamin2 S-nitrosylation, a posttranslational modification required for the bacterial entry, also increases during E. coli invasion. These findings suggest that UPEC escape urinary flushing and immune cell surveillance by means of eNOS-dependent dynamin2 S-nitrosylation and invasion of host cells to cause recurrent UTIs.

Androgens transduce the G alphas-mediated activation of protein kinase A in prostate cells.

Androgens regulate the development and function of male reproductive organs and play a crucial role in the onset and progression of prostate cancer. Androgen action is primarily mediated through the nuclear androgen receptor (AR) which acts as a ligand-dependent transcription factor. This mode of androgen action takes hours to manifest and is called the genomic pathway. The androgen-mediated genomic responses require activity of cyclic AMP (cAMP)-dependent protein kinase (PKA). Androgens also act through nongenomic pathways in certain cell types to evoke rapid responses (manifested in minutes) that are mediated through changes in ion currents and second messengers. Here, we show that androgen causes the rapid and cAMP-dependent activation of PKA in prostate cells. The androgen-induced PKA activation is not inhibited by nuclear AR antagonist bicalutamide and can be observed in cells that do not express nuclear AR gene. Reduction of G alphas expression with siRNA attenuates the androgen-mediated activation of PKA, which is required for the androgen-induced prostate cell proliferation. We conclude that androgen actively evokes a nongenomic signaling pathway to activate PKA that is needed for the genomic functioning of nuclear AR. The inhibition of PKA activation, together with standard AR-targeted therapies, may be more efficacious for treatment of patients with prostate cancer.

Agonist-stimulated reactive oxygen species formation regulates beta2-adrenergic receptor signal transduction.

Generation of reactive oxygen species (ROS) can occur upon agonist stimulation of surface receptors to modulate downstream signaling processes. Here, we show that activation of the beta2 adrenergic receptor (beta2AR) by stimulation with the agonist isoproterenol leads to generation of ROS that is required for beta2AR signal transduction. Specifically, we show that inhibition of NADPH oxidase with diphenyliodonium chloride, inhibition of the small GTPase Rac1 with NSC23766, and inhibition of formed ROS with the antioxidant N-acetyl-L-cysteine decreases beta2AR-mediated cAMP formation, protein kinase A activation, and receptor phosphorylation and internalization, but does not impact ligand binding. The results also show that inhibition of ROS attenuates active beta2AR-mediated binding of GTP to alpha subunits of heterotrimeric G proteins. Based on these results, we propose that agonist-dependent ROS formation is needed for beta2AR signal transduction, perhaps through stabilization of active receptor conformers by redox-mediated modification of receptor and/or Galpha proteins cysteine residues.

Role of PKA and PKC in histamine H1 receptor-mediated activation of catecholamine neurotransmitter synthesis.

Activation of the histamine H1 receptor stimulates tyrosine hydroxylase (TH) to increase catecholamine neurotransmitter synthesis in mammalian brain and adrenal tissues. Histamine non-selectively activates both H1-linked phospholipase (PL) C/inositol phosphates (IP)/diacylglycerol (DAG) signaling and adenylyl cyclase (AC)/adenosine 3',5'-cyclic monophosphate (cAMP) signaling, confounding determination of signaling events involved in H(1)-mediated TH activation. This research uses two new functionally-selective H1 agonists, cis-PAB and trans-PAT, that selectively activate H1/PLC/IP/DAG and H1/AC/cAMP signaling, respectively, to characterize H(1)-mediated activation of TH in rat striatum and bovine adrenal chromaffin (BAC) cells. Histamine, cis-PAB, and trans-PAT produced a two-fold maximal TH activation by an H1 receptor mechanism in rat striatum and BAC cells. Histamine is more potent and efficacious in BAC cells (EC50 approximately 0.2 microM, Emax approximately 200% basal) versus rat striatum (EC50 approximately 0.4 microM; Emax approximately 150%). Cis-PAB and trans-PAT are more potent in rat striatum (EC50 approximately 0.1 microM for both agonists) versus BAC cells (EC50 approximately 1.0 microM for both), with similar efficacy in both preparations (Emax approximately 160% for both agonists). Signaling studies in BAC cells revealed that protein kinase (PK) A but not PKC is involved in H1 -mediated TH activation by trans-PAT and histamine, while, both PKA and PKC are involved for cis-PAB. Results for cis-PAB suggest H1/PLC/IP/DAG/PKC signaling activates PKA, downstream of cAMP formation, indicating apparent direct activation of PKA by PKC.

Expression and function of lysophosphatidic acid LPA1 receptor in prostate cancer cells.

The bioactive phospholipid lysophosphatidic acid (LPA) promotes cell proliferation, survival, and migration by acting on cognate G protein-coupled receptors named LPA(1), LPA(2), and LPA(3). We profiled gene expression of LPA receptors in androgen-dependent and androgen-insensitive prostate cancer cells and found that LPA(1) gene is differentially expressed in androgen-insensitive and LPA-responsive but not androgen-dependent and LPA-resistant cells. In human prostate specimens, expression of LPA(1) gene was significantly higher in the cancer compared with the benign tissues. The androgen-dependent LNCaP cells do not express LPA(1) and do not proliferate in response to LPA stimulation, implying LPA(1) transduces cell growth signals. Accordingly, stable expression of LPA(1) in LNCaP cells rendered them responsive to LPA-induced cell proliferation and decreased their doubling time in serum. Implantation of LNCaP-LPA(1) cells resulted in increased rate of tumor growth in animals compared with those tumors that developed from the wild-type cells. Growth of LNCaP cells depends on androgen receptor activation, and we show that LPA(1) transduces Galphai-dependent signals to promote nuclear localization of androgen receptor and cell proliferation. In addition, treatment with bicalutamide inhibited LPA-induced cell cycle progression and proliferation of LNCaP-LPA(1) cells. These results suggest the possible utility of LPA(1) as a drug target to interfere with progression of prostate cancer.

Nitric oxide regulates endocytosis by S-nitrosylation of dynamin.

The GTPase dynamin regulates endocytic vesicle budding from the plasma membrane, but the molecular mechanisms involved remain incompletely understood. We report that dynamin, which interacts with NO synthase, is S-nitrosylated at a single cysteine residue (C607) after stimulation of the beta(2) adrenergic receptor. S-nitrosylation increases dynamin self-assembly and GTPase activity and facilitates its redistribution to the membrane. A mutant protein bearing a C607A substitution does not self-assemble properly or increase its enzymatic activity in response to NO. In NO-generating cells, expression of dynamin C607A, like the GTPase-deficient dominant-negative K44A dynamin, inhibits both beta(2) adrenergic receptor internalization and bacterial invasion. Furthermore, exogenous or endogenously produced NO enhances internalization of both beta(2) adrenergic and epidermal growth factor receptors. Thus, NO regulates endocytic vesicle budding by S-nitrosylation of dynamin. Collectively, our data suggest a general NO-dependent mechanism by which the trafficking of receptors may be regulated and raise the idea that pathogenic microbes and viruses may induce S-nitrosylation of dynamin to facilitate cellular entry.

Ligand-directed functional heterogeneity of histamine H1 receptors: novel dual-function ligands selectively activate and block H1-mediated phospholipase C and adenylyl cyclase signaling.

The autacoid and neurotransmitter histamine activates the H(1) G protein-coupled receptor (GPCR) to stimulate predominantly phospholipase C (PLC)/inositol phosphate (IP) signaling and, to a lesser extent, adenylyl cyclase (AC)/cAMP signaling in a variety of mammalian cells and tissues, as well as H(1)-transfected clonal cell lines. This study reports that two novel H(1) receptor ligands developed in our laboratory, (-)-trans-1-phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (trans-PAT) and (+/-)-cis-5-phenyl-7-dimethylamino-5,6,7,8-tetrahydro-9H-benzocycloheptane (cis-PAB), activate H(1) receptors to selectively stimulate AC/cAMP formation and PLC/IP formation, respectively, in Chinese hamster ovary cells transfected with guinea pig H(1) receptor cDNA. trans-PAT and cis-PAB also are shown to be functionally selective antagonists of H(1)-linked PLC/IP and AC/cAMP signaling, respectively. Whereas cis-PAB H(1) receptor activity is shown to be typically competitive, trans-PAT displays a complex interaction with the H(1) receptor that is not competitive regarding antagonism of saturation binding by the standard H(1) antagonist radioligand [(3)H]mepyramine or H(1)/PLC/IP functional activation by histamine. trans-PAT, however, does competitively block H(1)/PLC/IP functional activation by cis-PAB. Molecular determinants for trans-PAT versus cis-PAB differential binding to H(1) receptors, which presumably leads to differential activation of AC/cAMP versus PLC/IP signaling, likely involves stereochemical factors as well as more subtle steric influences. Results suggest the trans-PAT and cis-PAB probes will be useful to study molecular mechanisms of ligand-directed GPCR multifunctional signaling. Moreover, because most untoward cardiovascular-, respiratory-, and gastrointestinal H(1) receptor-mediated effects proceed via the PLC/IP pathway, PAT-type agonists that selectively enhance H(1)-mediated AC/cAMP signaling provide a mechanistic basis for exploiting H(1) receptor activation for drug design purposes.