Potentiation of Growth Factor Signaling by Insulin-like Growth Factor-binding Protein-3 in Breast Epithelial Cells Requires Sphingosine Kinase Activity

We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 ∼2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P₁) or S1P₃, but not S1P₂, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P₁ and S1P₃. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P₁/S1P₃, suggesting that S1P, the product of SphK activity and ligand for S1P₁ and S1P₃, is the “missing link” mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3.Insulin-like growth factor-binding protein-3 (IGFBP-3)² is one of the family of six IGFBPs that bind the peptide growth factors IGF-I and IGF-II with high affinity and regulate their bioactivity (1). As the predominant carrier of IGFs in the endocrine system, IGFBP-3 regulates the movement of these growth factors from the circulation to target tissues and inhibits their proliferative and antiapoptotic cellular effects by blocking their activation of the type 1 IGF receptor (IGFR1) at the cell surface. In vitro studies in a variety of cell types have revealed that IGFBP-3 may also impact on cell growth and survival independently of modulating IGF bioactivity, inducing cell cycle arrest and apoptosis by regulation of apoptotic effector proteins (2–4) and interaction with nuclear receptors (5–7).There is, however, also evidence of an association between IGFBP-3 and enhanced cell proliferation. Some clinical studies in breast, prostate, pancreatic, renal cell, and non-small cell lung cancers have shown that a high level of tissue expression of IGFBP-3 correlates with increased tumor growth or malignancy (8–13). Although the mechanism linking IGFBP-3 with growth stimulation in vivo remains unclear, we and others have shown that, in vitro, IGFBP-3 can enhance the effects of stimulatory growth factors. Human and bovine skin fibroblasts exposed to low concentrations of exogenous IGFBP-3 exhibit enhanced IGF-stimulated DNA synthesis (14, 15), and similarly, exogenous and endogenous IGFBP-3 enhanced the growth response to IGF-I in the MCF-7 breast cancer cell line (16). We have also shown previously that IGFBP-3 is inhibitory to DNA synthesis in MCF-10A breast epithelial cells in the absence of exogenous growth factors or serum (17), but is growth stimulatory in the presence of EGF in the same cell line (18). There is no evidence that potentiation of EGF or IGF bioactivity by IGFBP-3 requires direct interaction between IGFBP-3 and the growth factor receptors (15, 18), but the mechanism underlying the effects of IGFBP-3 on growth factor signaling has not been elucidated.Recently it was suggested that, in human umbilical vein endothelial cells, an antiapoptotic effect of IGFBP-3 is associated with increased expression and activity of sphingosine kinase 1 (SphK1), and formation of the bioactive sphingolipid sphingosine 1-phosphate (S1P) (19, 20). SphK1 has been shown to have a role in oncogenesis (21), and S1P, acting both as an intracellular second messenger and extracellularly through activation of specific S1P receptors, stimulates cell proliferation and survival (22). In addition to transducing S1P signaling, the G-protein-coupled S1P receptors have been implicated in signal amplification of a variety of growth factors receptors, including the EGF and platelet-derived growth factor receptors, via receptor transactivation (23, 24). In this study we investigated whether the sphingosine kinase system is involved in modulation of growth factor receptor signaling pathways by IGFBP-3 and demonstrate that SphK1 expression is stimulated by IGFBP-3 in MCF-10A cells, and its activity is required for potentiation of EGF and IGF-I signaling by IGFBP-3 in these cells.     

Deorphanization of GPR109B as a Receptor for the ��-Oxidation Intermediate 3-OH-octanoic Acid and Its Role in the Regulation of Lipolysis

The orphan G-protein-coupled receptor GPR109B is the result of a recent gene duplication of the nicotinic acid and ketone body receptor GPR109A being found in humans but not in rodents. Like GPR109A, GPR109B is predominantly expressed in adipocytes and is supposed to mediate antilipolytic effects. Here we show that GPR109B serves as a receptor for the β-oxidation intermediate 3-OH-octanoic acid, which has antilipolytic activity on human but not on murine adipocytes. GPR109B is coupled to G_i-type G-proteins and is activated by 2- and 3-OH-octanoic acid with EC₅₀ values of about 4 and 8 μm, respectively. Interestingly, 3-OH-octanoic acid plasma concentrations reach micromolar concentrations under conditions of increased β-oxidation rates, like in diabetic ketoacidosis or under a ketogenic diet. These data suggest that the ligand receptor pair 3-OH-octanoic acid/GPR109B mediates in humans a negative feedback regulation of adipocyte lipolysis to counteract prolipolytic influences under conditions of physiological or pathological increases in β-oxidation rates.Triacylglycerols stored in the white adipose tissue serve as the major energy reserve in higher eukaryotes (1). Although they are constantly turned over by lipolysis and re-esterification, their mobilization and storage are precisely balanced by various hormones and other factors depending on the nutritional state (2). The net rate of lipolysis is increased during fasting or periods of increased energy demand. Fatty acids generated via lipolysis undergo β-oxidation in the muscle and liver to serve directly as a source of energy or as a precursor for ketone bodies (3). The major intracellular regulator of lipolysis is cyclic AMP, which stimulates cAMP-dependent kinase to activate lipolytic enzymes (2, 4–6). This lipolytic pathway is induced, for example, via β-adrenergic receptors that couple to the G-protein G_s and thereby stimulate adenylyl cyclase (7, 8). To adjust lipolysis at the appropriate rate, the effects of prolipolytic stimuli are balanced by various antilipolytic influences. Besides insulin, which promotes the degradation of cAMP via activation of phosphodiesterase 3B (2, 5, 7), several antilipolytic stimuli decrease cAMP levels by activation of G_i-coupled receptors, which mediate an inhibition of adenylyl cyclase (5, 8). One of these receptors, GPR109A, has recently been shown to mediate the anti-lipolytic effects of high concentrations of the ketone body 3-OH-butyrate thereby providing a negative feedback mechanism during fasting (9, 10). GPR109A also binds nicotinic acid (11–13) and mediates the anti-lipolytic effects of this anti-dyslipidemic drug (12).GPR109B, a close relative of GPR109A, is the result of a recent gene duplication being present in humans but not in rodents and most other mammals (14). GPR109B differs from GPR109A in an extended C-terminal tail as well as in 16 amino acids (11, 13). Despite its high homology to GPR109A, GPR109B does not bind nicotinic acid or 3-OH-butyrate with reasonable affinity (10, 11, 13). Because GPR109A and GPR109B have very similar expression patterns (11, 13, 15) and are likely to have the same basic signaling properties, agonists of GPR109B are expected to have physiological and pharmacological effects comparable with those of the GPR109A agonist 3-OH-butyrate and nicotinic acid, respectively. Recently, several synthetic compounds as well as various aromatic d-amino acids have been shown to be selective agonists at GPR109B (16–18). However, endogenous physiological anti-lipolytic ligands of GPR109B are unknown.In this study we tested endogenous carboxylic acids for their ability to activate GPR109B. We found that the fatty acid β-oxidation intermediate 3-OH-octanoic acid is a highly specific agonist of GPR109B. 3-OH-octanoic acid has anti-lipolytic activity, and its plasma concentration in humans reflects the β-oxidation flux. Our data suggest that 3-OH-octanoic acid and GPR109B mediate a negative feedback regulation of adipocyte lipolysis.     

Ubiquitination Regulates Proteolytic Processing of G Protein-coupled Receptors after Their Sorting to Lysosomes

Ubiquitination is essential for the endocytic sorting of various G protein-coupled receptors to lysosomes. Here we identify a distinct function of this covalent modification in controlling the later proteolytic processing of receptors. Mutation of all cytoplasmic lysine residues in the murine δ-opioid receptor blocked receptor ubiquitination without preventing ligand-induced endocytosis of receptors or their subsequent delivery to lysosomes, as verified by proteolysis of extramembrane epitope tags and down-regulation of radioligand binding to the transmembrane helices. Surprisingly, a functional screen revealed that the E3 ubiquitin ligase AIP4 specifically controls down-regulation of wild type receptors measured by radioligand binding without detectably affecting receptor delivery to lysosomes defined both immunochemically and biochemically. This specific AIP4-dependent regulation required direct ubiquitination of receptors and was also regulated by two deubiquitinating enzymes, AMSH and UBPY, which localized to late endosome/lysosome membranes containing internalized δ-opioid receptor. These results identify a distinct function of AIP4-dependent ubiquitination in controlling the later proteolytic processing of G protein-coupled receptors, without detectably affecting their endocytic sorting to lysosomes. We propose that ubiquitination or ubiquitination/deubiquitination cycling specifically regulates later proteolytic processing events required for destruction of the receptor''s hydrophobic core.A fundamental cellular mechanism contributing to homeostatic regulation of receptor-mediated signal transduction involves ligand-induced endocytosis of receptors followed by proteolysis in lysosomes. The importance of such proteolytic down-regulation has been documented extensively for a number of seven-transmembrane or G protein-coupled receptors (GPCRs),³ which comprise the largest known family of signaling receptors expressed in animals, as well as for other important signaling receptors, such as the epidermal growth factor receptor tyrosine kinase (1–5).One GPCR that is well known to undergo endocytic trafficking to lysosomes is the δ-opioid peptide receptor (DOR or DOP-R) (6). Following endocytosis, DOR traffics efficiently to lysosomes in both neural and heterologous cell models (6–8), whereas many membrane proteins, including various GPCRs, recycle rapidly to the plasma membrane (9–12). Such molecular sorting of internalized receptors between divergent recycling and degradative pathways is thought to play a fundamental role in determining the functional consequences of regulated endocytosis (2, 3, 13, 14). The sorting process that directs internalized DOR to lysosomes is remarkably efficient and appears to occur rapidly (within several min) after receptor endocytosis (11). Nevertheless, biochemical mechanisms that control lysosomal trafficking and proteolysis of DOR remain poorly understood.A conserved mechanism that promotes lysosomal trafficking of a number of membrane proteins, including various signaling receptors, is mediated by covalent modification of cytoplasmic lysine residues with ubiquitin (4, 15–17). Ubiquitination was first identified as an endocytic sorting determinant in studies of vacuolar trafficking of the yeast GPCR Ste2p (18). Subsequent studies have established numerous examples of lysyl-ubiquitination being required for sorting endocytic cargo to lysosomes and have identified conserved machinery responsible for the targeting of ubiquitinated cargo to lysosomes (3, 17, 19–22).The CXCR4 chemokine receptor provides a clear example of ubiquitin-dependent lysosomal sorting of a mammalian GPCR. Ubiquitination of the carboxyl-terminal cytoplasmic domain of the CXCR4 receptor, mediated by the E3 ubiquitin ligase AIP4, is specifically required for the HRS- and VPS4-dependent trafficking of internalized receptors to lysosomes. Blocking this ubiquitination event by Lys → Arg mutation of the receptor specifically inhibits trafficking of internalized receptors to lysosomes, resulting in recycling rather than lysosomal proteolysis of receptors after ligand-induced endocytosis (23–25).Lysosomal trafficking of DOR, in contrast, is not prevented by mutation of cytoplasmic lysine residues (26) and can be regulated by ubiquitination-independent protein interaction(s) (27, 28). Nevertheless, both wild type and lysyl-mutant DORs traffic to lysosomes via a similar pathway as ubiquitin-dependent membrane cargo and require both HRS and active VPS4 to do so (29). These observations indicate that DOR engages the same core endocytic mechanism utilized by ubiquitination-directed membrane cargo but leave unresolved whether ubiquitination of DOR plays any role in this important cellular mechanism of receptor down-regulation.There is no doubt that DOR can undergo significant ubiquitination in mammalian cells, including HEK293 cells (30–32), where lysosomal trafficking of lysyl-mutant receptors was first observed (26). Ubiquitination was shown previously to promote proteolysis of DOR by proteasomes and to function in degrading misfolded receptors from the biosynthetic pathway (30, 31). A specific role of ubiquitination in promoting proteasome- but not lysosome-mediated proteolysis of DOR has been emphasized (32) and proposed to contribute to proteolytic down-regulation of receptors also from the plasma membrane (33).To our knowledge, no previous studies have determined if DOR ubiquitination plays any role in controlling receptor proteolysis mediated by lysosomes, although this represents a predominant pathway by which receptors undergo rapid down-regulation following ligand-induced endocytosis in a number of cell types, including HEK293 cells (8). In the present study, we have taken two approaches to addressing this fundamental question. First, we have investigated in greater detail the effects of lysyl-mutation on DOR ubiquitination and trafficking. Second, we have independently investigated the role of ubiquitination in controlling lysosomal proteolysis of wild type DOR. Our results clearly establish the ability of DOR to traffic efficiently to lysosomes in the absence of any detectable ubiquitination. Further, they identify a distinct and unanticipated function of AIP4-dependent ubiquitination in regulating the later proteolytic processing of receptors and show that this distinct ubiquitin-dependent regulatory mechanism operates effectively downstream of the sorting decision that commits internalized receptors for delivery to lysosomes.     

Loop 2 Structure in Glycine and GABAA Receptors Plays a Key Role in Determining Ethanol Sensitivity

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABA_ARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABA_ARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABA_AR (δL2), but not the γGABA_AR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABA_ARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA_A γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA_ARs. The δL2 mutations did not affect GlyR or GABA_AR sensitivity, respectively, to Zn²⁺ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.Alcohol abuse and dependence are significant problems in our society, with ∼14 million people in the United States being affected (1, 2). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (3–5). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol.Ligand-gated ion channels (LGICs)² have received substantial attention as putative sites of ethanol action that cause its behavioral effects (6–12). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13, 14), whose members include nicotinic acetylcholine, 5-hydroxytryptamine₃, γ-aminobutyric acid type A (GABA_A), γ-aminobutyric acid type C, and glycine receptors (GlyRs) (10, 11, 15–20) and 2) the glutamate superfamily, including N-methyl d-aspartate, α-amino-3-hydroxyisoxazolepropionic acid, and kainate receptors (21, 22). Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of LGICs (23–25).A series of studies that employed chimeric and mutagenic strategies combined with sulfhydryl-specific labeling identified key regions within Cys-loop receptors that appear to be initial targets for ethanol action that also can determine the sensitivity of the receptors to ethanol (7–12, 18, 19, 26–30). This work provides several lines of evidence that position 267 and possibly other sites in the transmembrane (TM) domain of GlyRs and homologous sites in GABA_ARs are targets for ethanol action and that mutations at these sites can influence ethanol sensitivity (8, 9, 26, 31).Growing evidence from GlyRs indicates that ethanol also acts on the extracellular domain. The initial findings came from studies demonstrating that α1GlyRs are more sensitive to ethanol than are α2GlyRs despite the high (∼78%) sequence homology between α1GlyRs and α2GlyRs (32). Further work found that an alanine to serine exchange at position 52 (A52S) in Loop 2 can eliminate the difference in ethanol sensitivity between α1GlyRs and α2GlyRs (18, 20, 33). These studies also demonstrated that mutations at position 52 in α1GlyRS and the homologous position 59 in α2GlyRs controlled the sensitivity of these receptors to a novel mechanistic ethanol antagonist (20). Collectively, these studies suggest that there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM domain (e.g. position 267) and another in the extracellular domain (e.g. position 52).Subsequent studies revealed that the polarity of the residue at position 52 plays a key role in determining the sensitivity of GlyRs to ethanol (20). The findings with polarity in the extracellular domain contrast with the findings at position 267 in the TM domain, where molecular volume, but not polarity, significantly affected ethanol sensitivity (9). Taken together, these findings indicate that the physical-chemical parameters of residues at positions in the extracellular and TM domains that modulate ethanol effects and/or initiate ethanol action in GlyRs are not uniform. Thus, knowledge regarding the physical-chemical properties that control agonist and ethanol sensitivity is key for understanding the relationship between the structure and the actions of ethanol in LGICs (19, 31, 34–40).GlyRs and GABA_ARs, which differ significantly in their sensitivities to ethanol, offer a potential method for identifying the structures that control ethanol sensitivity. For example, α1GlyRs do not reliably respond to ethanol concentrations less than 10 mm (32, 33, 41). Similarly, γ subunit-containing GABA_ARs (e.g. α1β2γ2), the most predominantly expressed GABA_ARs in the central nervous system, are insensitive to ethanol concentrations less than 50 mm (42, 43). In contrast, δ subunit-containing GABA_ARs (e.g. α4β3δ) have been shown to be sensitive to ethanol concentrations as low as 1–3 mm (44–51). Sequence alignment of α1GlyR, γGABA_AR, and δGABA_AR revealed differences between the Loop 2 regions of these receptor subunits. Since prior studies found that mutations of Loop 2 residues can affect ethanol sensitivity (19, 20, 39), the non-conserved residues in Loop 2 of GlyR and GABA_AR subunits could provide the physical-chemical and structural bases underlying the differences in ethanol sensitivity between these receptors.The present study tested the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABA_ARs. To accomplish this, we performed multiple mutations that replaced the Loop 2 region of the α1 subunit in α1GlyRs and the Loop 2 region of the γ subunit of α1β2γ2 GABA_ARs with corresponding non-conserved residues from the δ subunit of GABA_AR and tested the sensitivity of these receptors to ethanol. As predicted, replacing Loop 2 of WT α1GlyRs with the homologous residues from the δGABA_AR subunit (δL2), but not the γGABA_AR subunit (γL2), markedly increased the sensitivity of the receptor to ethanol. Similarly, replacing the non-conserved residues of the γ subunit of α1β2γ2 GABA_ARs with δL2 also markedly increased ethanol sensitivity of GABA_ARs. These findings support the hypothesis and suggest that Loop 2 may play a role in controlling ethanol sensitivity across the Cys-loop superfamily of receptors. The findings also provide the basis for suggesting structure-function relationships in a new molecular model of the GlyR based on the bacterial Gloeobacter violaceus pentameric LGIC homologue (GLIC).     

Interactions between Calmodulin, Adenosine A2A, and Dopamine D2 Receptors

The Ca²⁺-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D₂ receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D₂ receptor, within an Arg-rich epitope that is also involved in the binding to G_i/o proteins and to the adenosine A_2A receptor, with the formation of A_2A-D₂ receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A_2A receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A_2A-D₂ receptor oligomerization. BRET competition experiments indicated that, in the A_2A-D₂ receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A_2A receptor. Furthermore, Ca²⁺ was found to induce conformational changes in the CaM-A_2A-D₂ receptor oligomer and to selectively modulate A_2A and D₂ receptor-mediated MAPK signaling in the A_2A-D₂ receptor heteromer. These results may have implications for basal ganglia disorders, since A_2A-D₂ receptor heteromers are being considered as a target for anti-parkinsonian agents.G-protein-coupled receptors are able to form homo- and hetero-oligomers with unique biochemical and functional characteristics (1–7), and they are easily detected in vitro by using biophysical techniques (8–10). Heteromers of adenosine A_2A and dopamine D₂ receptors were one of the first G-protein-coupled receptor heteromers to be described (11). A close physical interaction between both receptors was shown using co-immunoprecipitation and co-localization assays (11) and fluorescence and bioluminescence resonance energy transfer (FRET² or BRET) techniques (12–14). At the biochemical level, two types of antagonistic A_2A-D₂ receptor interactions have been discovered that may explain the A_2A-D₂ receptor interactions described both at the neuronal and behavioral level (11, 15–18). First, by means of an allosteric interaction in the receptor heteromer, stimulation of A_2A receptor decreases the affinity of D₂ receptor for their agonists (12). Second, the stimulation of the G_i/o-protein-coupled D₂ receptor inhibits the cAMP accumulation induced by the stimulation of the G_s/olf-protein-coupled A_2A receptor (11, 17, 18). In view of the well known role of dopamine in Parkinson disease, schizophrenia, and drug addiction, it has been suggested that the A_2A-D₂ receptor interactions in the central nervous system may provide new therapeutic approaches to combat these disorders (16, 19).An epitope-epitope electrostatic interaction between an Arg-rich epitope of the N terminus of the third intracellular loop (3IL) of the D₂ receptor and an epitope containing a phosphorylated Ser localized in the distal part of the C terminus of the A_2A receptor is involved in A_2A-D₂ receptor heteromer interface (14, 20, 21). The same Arg-rich epitope of the D₂ receptor is able to interact with CaM (22–25). In the absence of phosphorylated residues, adjacent aspartates or glutamates, which are abundant in CaM, may also form non-covalent complexes with Arg-rich epitopes (26). Therefore, CaM can potentially convey a Ca²⁺ signal to the D₂ receptor through direct binding to the 3IL of the D₂ receptor (22). Mass spectrometry data have shown that bovine CaM can form multiple non-covalent complexes with an Arg-rich peptide corresponding to the N-terminal region of the 3IL of the D₂ receptor (VLRRRRKRVN) (24) as well as a peptide from the proximal C terminus of the A_2A receptor (24). This epitope, whose sequence is ²⁹¹RIREFRQTFR³⁰⁰ in the human A_2A receptor, also contains several Arg residues. Since the suspected interaction between the A_2A receptor and CaM was awaiting confirmation by assays using complete proteins, the present study was undertaken to demonstrate the existence of interactions between the A_2A receptor and CaM both in a recombinant protein expression cell system and in the brain. A proteomics approach was used for the discovery of protein-protein interactions between the A_2A receptor and CaM in rat brain, whereas BRET in transfected cells demonstrated a direct interaction between CaM and this receptor. Furthermore, by using BRET and sequential resonance energy transfer (SRET) techniques and analyzing MAPK signaling in transfected cells, evidence was obtained for CaM-A_2A-D₂ receptor oligomerization and a selective Ca²⁺-mediated modulation of A_2A and D₂ receptor function in the A_2A-D₂ receptor heteromer.     

Protein Kinase C�� Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Activation of the 5-hydroxytryptamine receptor 2B (5-HT_2B), a G_q/11 protein-coupled receptor, results in proliferation of various cell types. The 5-HT_2B receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT_2B receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT_2B receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 decreased base-line proliferation but had no effect on 5-HT_2B receptor-mediated proliferation. Proliferation of ICC through the 5-HT_2B receptor was inhibited by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the α, β, γ, and μ protein kinase C (PKC) inhibitor Gö6976, and the α, β, γ, δ, and ζ PKC inhibitor Gö6983 inhibited 5-HT_2B receptor-mediated proliferation, indicating the involvement of PKC α, β, or γ. Of all the PKC isoforms blocked by Gö6976, PKCγ and μ mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT_2B receptor activation in primary cell cultures obtained from PKCγ^−/− mice did not result in a proliferative response, further indicating the requirement for PKCγ in the proliferative response to 5-HT_2B receptor activation. The data demonstrate that the 5-HT_2B receptor-induced proliferative response of ICC is through phospholipase C, [Ca²⁺]_i, and PKCγ, implicating this PKC isoform in the regulation of cellular proliferation.Tight control of cell proliferation is essential to maintain organ size and function. Proliferation needs to be tightly regulated to maintain a critical mass of a particular cell type while preventing dysplasia or malignancy. Cell proliferation is regulated by a complex interaction between extrinsic and intrinsic factors. Extrinsic factors usually signal through cell surface receptors such as various growth factor receptors. 5-Hydroxytryptamine (5-HT,² serotonin) is well established as a neurotransmitter and a paracrine factor with over 90% of 5-HT produced by the gastrointestinal tract (1, 2). There is now substantial evidence that, together with these established functions, 5-HT is involved in the control of cell proliferation through various 5-HT receptors, in particular the 5-hydroxytryptamine receptor 2B (5-HT_2B (3–9)). The 5-HT_2B receptor is G_q/11 protein-coupled. Activation of the 5-HT_2B receptor regulates cardiac function, smooth muscle contractility, vascular physiology, and mood control. Recently it was demonstrated that activation of the 5-HT_2B receptor also induces proliferation of neurons, retinal cells (3, 4), hepatocytes (5), osteoblasts (8), and interstitial cells of Cajal (ICC) (9). ICC express the 5-HT_2B receptor, and activation by 5-HT induces proliferation of ICC (9). ICC are specialized, mesoderm-derived mesenchymal cells in the gastrointestinal tract. Their best known function is the generation of slow waves (10), but they also conduct and amplify neuronal signals (11, 12), release carbon monoxide to set the intestinal smooth muscle membrane potential gradient (13), and act as mechanosensors (14, 15). Loss of ICC has been associated with pathological conditions such as gastroparesis (16–18), infantile pyloric stenosis (19, 20), pseudo-obstruction (21, 22), and slow transit constipation (23), whereas increased proliferation of ICC or their precursors is associated with gastrointestinal stromal tumors (24).The mechanisms by which activation of the 5-HT_2B receptor results in increased proliferation are not well understood. In cultured cardiomyocytes, stimulation of the 5-HT_2B receptor activated both phosphatidylinositol 3-kinase (PI3′-K)/Akt and ERK1/2/mitogen-activated protein kinase (MAPK) signaling pathways to protect cardiomyocytes from apoptosis (25). On the other hand, the 5-HT2 subfamily of receptors are also known to couple to phospholipase C (PLC) (26–28).The objective of this study was to utilize the known expression of the 5-HT_2B receptor on ICC to determine whether proliferation through the 5-HT_2B receptor required PI3′-K or PLC. This study demonstrates that proliferation mediated by the 5-HT_2B receptor requires PLC, intracellular calcium release, and the ERK/MAPK signaling pathway and identifies the PKC isoform activated by the 5-HT_2B receptor in ICC as PKCγ.     

Activation of an Olfactory Receptor Inhibits Proliferation of Prostate Cancer Cells

Olfactory receptors (ORs) are expressed not only in the sensory neurons of the olfactory epithelium, where they detect volatile substances, but also in various other tissues where their potential functions are largely unknown. Here, we report the physiological characterization of human OR51E2, also named prostate-specific G-protein-coupled receptor (PSGR) due to its reported up-regulation in prostate cancer. We identified androstenone derivatives as ligands for the recombinant receptor. PSGR can also be activated with the odorant β-ionone. Activation of the endogenous receptor in prostate cancer cells by the identified ligands evoked an intracellular Ca²⁺ increase. Exposure to β-ionone resulted in the activation of members of the MAPK family and inhibition of cell proliferation. Our data give support to the hypothesis that because PSGR signaling could reduce growth of prostate cancer cells, specific receptor ligands might therefore be potential candidates for prostate cancer treatment.Excessive signaling by G-protein-coupled receptors (GPCRs)³ such as endothelin A receptor (1), bradykinin 1 receptor (2), follicle-stimulating hormone receptor (3), and thrombin receptor (4, 5) is known to occur in prostate cancers due to strong overexpression of the respective receptors. Activation of some of these GPCRs results in androgen-independent androgen receptor activation, thus promoting the transition of prostate cancer cells from an androgen-dependent to an androgen-independent state (6, 7).The prostate-specific G-protein-coupled receptor (PSGR) is a class A GPCR that was initially identified as a prostate-specific tumor biomarker (8–10). It is specifically expressed in prostate epithelial cells, and its expression increases significantly in human prostate intraepithelial neoplasia and prostate tumors, suggesting that PSGR may play an important role in early prostate cancer development and progression (9, 11). Although expression of the human PSGR was found to be prostate-specific (10, 12), mRNA can also be detected in the olfactory zone and the medulla oblongata of the human brain (12). Human PSGR shares 93% amino acid homology to the respective mouse and rat homologues, which are also expressed in the brain (12). Interestingly, PSGR has numerous sequence motifs in common with the large superfamily of olfactory receptors (ORs), which build the largest class of human GPCRs and allow the recognition of a wide range of structurally diverse molecules in the nasal epithelium (13–15). Recently, also the steroid hormones androstenone and androstadienone were identified as OR ligands (16). In addition to their role in the sensory neurons of the nose, ORs have been found in different tissues throughout the body (17, 18). Their function(s) in these extranasal locations are questionable except for in a few cases where functional studies have been performed in spermatozoa (19, 20) and in enterochromaffin cells of the gastrointestinal tract (21).Here, we report the identification of steroid ligands of heterologously expressed PSGR and investigate the functional relevance of PSGR expression in prostate tissue. Steroid hormones elicited rapid Ca²⁺ responses in the LNCaP prostate cancer cell line and in primary human prostate epithelial cells. Moreover, activated PSGR causes phosphorylation of p38 and stress-activated protein kinase/c-Jun NH₂-terminal kinase (SAPK/JNK) mitogen-activated protein kinases (MAPKs), resulting in reduced proliferation rates in prostate cancer cells.     

Characterization of the ATP-dependent Sphingosine 1-Phosphate Transporter in Rat Erythrocytes

Sphingosine 1-phosphate (S1P) is a bioactive lipid signal transmitter present in blood. Blood plasma S1P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S1P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S1P export from erythrocytes, we performed the enzymatic characterization of S1P transporter in rat erythrocytes. Rat erythrocytes constitutively released S1P without any stimulus. The S1P release was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S1P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S1P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5′-(β,γ-imido)triphosphate. The rate of S1P transport increased depending on S1P concentration, with an apparent K_m value of 21 μm. Two phosphorylated sphingolipids, dihydrosphingosine 1-phosphate and ceramide 1-phosphate, did not inhibit S1P transport. Similar to the intact erythrocytes, the uptake of S1P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S1P is exported from the erythrocytes by a novel ATP-dependent transporter.Sphingosine 1-phosphate (S1P),² a bioactive lipid molecule present in the blood, plays an important role in diverse cellular responses, such as migration, proliferation, and differentiation (1, 2). These processes are triggered by the binding of S1P to its specific receptors (3), of which five subtypes (S1P₁-S1P₅) have been identified in endothelial and immune cells (4). Studies using S1P₁ receptor-deficient mice showed abnormalities in lymphocyte egress from lymph nodes, spleen, and thymus (5, 6). Whereas blood plasma contains a basal level of S1P from the nanomolar to the micromolar range (7–12), lymphoid tissues maintain a low S1P environment through the activity of S1P lyase (13). It has been proposed that a higher concentration of S1P in the blood plasma than in the lymphoid organs establishes an essential gradient along which lymphocytes expressing the S1P₁ receptor on cell surfaces migrate (2, 5, 6, 13–15).The source of plasma S1P remains unclear despite its importance in the cellular responses of endothelial cells and lymphocytes. Unlike most cells, blood cells, astrocytes, and vascular endothelial cells are reported to release S1P (8, 16–18). These cells contain sphingosine kinase, which synthesizes S1P through the phosphorylation of sphingosine (16, 18, 19). Whereas platelets and mast cells release S1P in a stimulus-dependent manner (17, 20), erythrocytes, neutrophils, and mononuclear cells release S1P in a stimulus-independent manner (16). The roles of S1P derived from erythrocytes, the most abundant of these blood cells, have not been elucidated. However, recent reports suggest that S1P released from erythrocytes is a major source of plasma S1P (7, 9) and promotes lymphocyte egress to blood (9).Previously, we showed that S1P is released from rat platelets upon stimulation by thrombin or Ca²⁺ (21). We proposed that an ATP-dependent transporter plays a key role in S1P release from platelets (21). However, the detailed mechanism of S1P release is unclear because there is no way to assay the transport of S1P across the membrane. In this study we compared the properties of S1P release from erythrocytes with that of platelets and showed that S1P release from erythrocytes does not require any stimuli. We then established an assay to measure the ATP-dependent S1P uptake into inside-out membrane vesicles (IOVs) prepared from rat erythrocytes and characterized S1P transport in erythrocytes.     

A Functional P2X7 Splice Variant with an Alternative Transmembrane Domain 1 Escapes Gene Inactivation in P2X7 Knock-out Mice

The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target. Here we describe a new P2X7 splice variant (P2X7(k)), containing an alternative intracellular N terminus and first transmembrane domain encoded by a novel exon 1 in the rodent P2rx7 gene. Whole cell patch clamp recordings of the rat isoform expressed in HEK293 cells revealed an 8-fold higher sensitivity to the agonist Bz-ATP and much slower deactivation kinetics when compared with the P2X7(a) receptor. Permeability measurements in Xenopus oocytes show a high permeability for N-methyl-d-glucamine immediately upon activation, suggesting that the P2X7(k) channel is constitutively dilated upon opening. The rates of agonist-induced dye uptake and membrane blebbing in HEK cells were also increased. PCR analyses and biochemical analysis by SDS-PAGE and BN-PAGE indicate that the P2X7(k) variant escapes gene deletion in one of the available P2X7^−/− mice strains and is strongly expressed in the spleen. Taken together, we describe a novel P2X7 isoform with distinct functional properties that contributes to the diversity of P2X7 receptor signaling. Its presence in one of the P2X7^−/− strains has important implications for our understanding of the role of this receptor in health and disease.P2X receptors (P2XRs)³ are ATP-gated cation channels. They consist of three subunits (1, 2) each containing two transmembrane domains (TMDs) linked by an extracellular ligand-binding domain (3). The P2X7 receptor is distinguished from other P2X receptors by its long intracellular C terminus, a low ATP sensitivity (EC₅₀: 100 μm to 1 mm), and its ability to induce “cell permeabilization,” meaning that upon prolonged ATP application the opening of a permeation pathway for large molecules is induced. This process eventually leads to apoptosis, requires the C terminus (3–6), and may be mediated by interaction with pannexin hemichannels (7). In addition, “pore dilation,” which allows the passage of the large cation NMDG, is observed if extracellular sodium is replaced by NMDG (8), a property also displayed by the P2X2 (9) and P2X4 (10) receptors. This pore dilation is assumed to represent an intrinsic property of these P2X receptors (11, 12), although it can be influenced by interaction with intracellular proteins (13). However, both processes are still poorly understood.P2X7 receptors are found on cells of the hematopoietic lineage, in epithelia, and endothelia. Several studies report its expression and/or function in neurons, although its presence here is under debate (14, 15). So far, nine splice variants (P2X7(b) through P2X7(j)) have been described, only one of which was shown to be, at least partially, functional (16, 17). In addition, numerous single nucleotide polymorphisms have been identified in the human P2X7 receptor. Some of these have been found to cause either gain or loss of function and have been associated with chronic lymphocytic leukemia, bone fracture risk, and impaired immune functions (18–20). Recent genetic studies indicate an association between the Gln-460 → Arg polymorphism and familial depressive disorders (21).Two P2X7-deficient mouse lines have been described. In the mouse line generated by Glaxo, the P2rx7 gene was knocked out by insertion of a lacZ transgene into exon 1 (22). In the mouse line generated by Pfizer (23) a neomycin cassette was inserted into exon 13, replacing a region that encodes Cys-506–Pro-532 of the intracellular C terminus of the receptor. The Pfizer P2X7 KO mice demonstrated the involvement of this receptor in bone formation (24), cytokine production, and inflammation (23, 25) while the Glaxo^−/− mice established its role in inflammatory and neuropathic pain (26). All these findings and multiple subsequent studies based on these mouse models defined the P2X7R as a promising target for the development of innovative therapeutic strategies, and selective P2X7 inhibitors are already in clinical trials for the treatment of rheumatoid arthritis (27).Here, we describe a novel P2X7 isoform with an alternative N terminus and TMD 1. Compared with the originally identified P2X7(a) variant, it has increased agonist sensitivity and a higher propensity to form NMDG-permeable pores and permit dye uptake. Due to a novel alternative exon 1 and translation start, this splice variant escapes inactivation in the Glaxo P2X7^−/− mice and thus could account for possible inconsistencies between results obtained with different P2X7^−/− mouse lines (28).     

Paracrine Transactivation of the CB1 Cannabinoid Receptor by AT1 Angiotensin and Other Gq/11 Protein-coupled Receptors

Intracellular signaling systems of G protein-coupled receptors are well established, but their role in paracrine regulation of adjacent cells is generally considered as a tissue-specific mechanism. We have shown previously that AT₁ receptor (AT₁R) stimulation leads to diacylglycerol lipase-mediated transactivation of co-expressed CB₁Rs in Chinese hamster ovary cells. In the present study we detected a paracrine effect of the endocannabinoid release from Chinese hamster ovary, COS7, and HEK293 cells during the stimulation of AT₁ angiotensin receptors by determining CB₁ cannabinoid receptor activity with bioluminescence resonance energy transfer-based sensors of G protein activation expressed in separate cells. The angiotensin II-induced, paracrine activation of CB₁ receptors was visualized by detecting translocation of green fluorescent protein-tagged β-arrestin2. Mass spectrometry analyses have demonstrated angiotensin II-induced stimulation of 2-arachidonoylglycerol production, whereas no increase of anandamide levels was observed. Stimulation of G_q/11-coupled M₁, M₃, M₅ muscarinic, V₁ vasopressin, α_1a adrenergic, B₂ bradykinin receptors, but not G_i/o-coupled M₂ and M₄ muscarinic receptors, also led to paracrine transactivation of CB₁ receptors. These data suggest that, in addition to their retrograde neurotransmitter role, endocannabinoids have much broader paracrine mediator functions during activation of G_q/11-coupled receptors.Hormones, neurotransmitters, and other chemical mediators acting on G protein-coupled receptors (GPCRs)² exert their effects on the target cells by stimulating G protein-dependent and independent intracellular signaling pathways (1–4). Activation of G_q/11 protein-coupled receptors causes phospholipase C activation, which produces inositol-trisphosphate and diacylglycerol from phosphatidylinositol (4,5)-bisphosphate, leading to Ca²⁺-signal generation and protein kinase C activation. However, the concerted response of tissues to chemical mediators frequently also involves the activation of cells adjacent to the target cells, due to the release of paracrine mediators. A well known example is NO, which can be released from activated endothelial cells to cause relaxation of adjacent vascular smooth muscle cells. Lipid mediators can also act as intercellular messengers. For example, endocannabinoids released from postsynaptic neurons after depolarization act as retrograde transmitters by binding to and stimulating presynaptic cannabinoid receptors, which leads to inhibition of γ-aminobutyric acid release (an event termed depolarization-induced suppression of inhibition, DSI) (5–7).Cannabinoid receptors were first identified based on their ability to selectively recognize marijuana analogs. To date, two cannabinoid receptors have been identified by molecular cloning, CB₁ and CB₂ receptors (CB₁R and CB₂R, respectively) (5, 8, 9), although additional GPCRs have also been proposed to function as cannabinoid receptors (10, 11). Cannabinoid receptors also recognize certain lipids present in animal tissues termed endocannabinoids, such as arachidonylethanolamide (anandamide), 2-arachidonoylglycerol (2-AG), and 2-arachidonoylglyceryl ether (noladin ether) (7, 12–16). In adult and fetal neural tissues, the two major endocannabinoids, anandamide and 2-AG, are produced on demand, usually after depolarization of postsynaptic cells or following stimulation of G_q-coupled metabotropic glutamate or muscarinic acetylcholine receptors (7, 12, 17–20). Enzymes responsible for 2-AG production and metabolism in tissues are localized to well defined structures at synapses, near the axon terminals of CB₁R-expressing cells (5, 7). In contrast, in peripheral tissues baseline levels of endocannabinoid production usually manifest as “endocannabinoid tone,” with poorly understood localization of the various components of the endocannabinoid system. 2-AG levels in brain homogenates and in many peripheral tissues are near its K_d for the CB₁R (19), suggesting that function of endocannabinoids may not be limited to localized synaptic signaling.There is mounting evidence that endocannabinoids play important roles in peripheral cardiovascular, inflammatory, intestinal, and metabolic regulation (21–24). 2-AG is produced by diacylglycerol-lipase (DAGL) after cleavage of the fatty-acid in the sn-1 position of diacylglycerol (DAG) (19, 25). Phospholipase C activation by G_q/11 protein-coupled receptors produces DAG, which can serve as a substrate for DAGL. Plasma membrane phosphoinositides are enriched in arachidonic acid in the sn-2 position (26), and DAGL is expressed ubiquitously (27), which suggests that phospholipase C-mediated cleavage of polyphosphoinositides may routinely lead to the formation of 2-AG. In accordance with this hypothesis, we have recently shown that angiotensin II- (Ang II)-mediated activation of the G_q/11-coupled AT₁ angiotensin receptor (AT₁R) leads to DAGL-dependent activation of CB₁Rs expressed in Chinese hamster ovary (CHO) cells (28).Here our aim has been to examine the possibility that 2-AG serves as a common paracrine signal generated via activation of G_q/11 protein-coupled, Ca²⁺-mobilizing receptors. Accordingly, we co-expressed CB₁Rs and BRET-based sensors of G protein activation in CHO cells, and used these cells to detect endocannabinoid release from adjacent cells that express AT₁R or other Ca²⁺-mobilizing GPCRs. We have further shown that activation of AT₁R by Ang II increases 2-AG levels in CHO cells. These findings suggest that 2-AG is commonly released following activation of Ca²⁺-mobilizing GPCRs and serves as a paracrine signal to activate CB₁R in neighboring cells.