Adenosine A 2B receptors modulate cAMP levels and induce CREB but not ERK1/2 and p38 phosphorylation in rat skeletal muscle cells

The present study examined the existence of the adenosine A(1),A(2A), and A(2B) receptors and the effect of receptor activation on cAMP accumulation and protein phosphorylation in primary rat skeletal muscle cells. Presence of mRNA and protein for all three receptors was demonstrated in both cultured and adult rat skeletal muscle. NECA (10(-9)-10(-4)M) increased the cAMP concentration in cultured muscle cells with an EC(50) of (95% confidence interval)=15 (5.9-25.1) micro M, whereas CGS 21680 (10(-9)-10(-4)M) had no effect on cAMP accumulation. Concentrations of [R]-PIA below 10(-6)M had no effect on cAMP accumulation induced by either isoproterenol or forskolin. NECA resulted in phosphorylation of CREB with an EC(50) of (95% confidence interval)=1.7 (0.40-7.02) micro M, whereas ERK1/2 and p38 phosphorylation was unchanged. The results show that, although the A(1),A(2A), and A(2B) receptors are all present in skeletal muscle cells, the effect of adenosine on adenylyl cyclase activation and phosphorylation of CREB is mainly mediated via the adenosine A(2B) receptor.     

Pharmacology of functional endogenous IP prostanoid receptors in NCB-20 cells: comparison with binding data from human platelets

The objective of these studies was to characterize the effects of a broad range of prostanoid agonists upon the stimulation of cAMP production in National Cancer Bank (NCB-20; mouse neuroblastoma/hamster brain hybridoma) cells. The pharmacology of these functional responses in NCB-20 cells was compared with that of the classic endogenous IP receptor present on human platelets using [3H]-iloprost binding techniques. In both assay systems, agonists from the IP prostanoid class exhibited the highest affinities and functional potencies. Specific prostanoids exhibited the following rank order of potency (EC50 +/- SEM) in stimulating cAMP production in the NCB-20 cells: carbaprostacyclin (4.3 +/- 0.9 nM) = PGI2 (6.6 +/-1.5 nM) > iloprost (75+/-13 nM) > 11-deoxy PGE, (378+/-138 nM) > misoprostol (1,243+/-48) > PGE2 (3020+/-700 nM) > ZK-118182 (7265+/-455 nM). Iloprost wasthe most potent compound in the human platelet binding assay while prostanoidsfromthe DPand EP receptor classes showed modest affinity. These studies provide functional and binding information for a broad range of both natural and synthetic prostanoid receptor ligands at the endogenous IP receptor in two different cell types.     

Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.     

OX2R activation induces PKC-mediated ERK and CREB phosphorylation

Deficiencies in brain orexins and components of mitogen activated protein kinase (MAPK) signaling pathway have been reported in either human depression or animal model of depression. Brain administration of orexins affects behaviors toward improvement of depressive symptoms. However, the documentation of endogenous linkage between orexin receptor activation and MAPK signaling pathway remains to be insufficient. In this study, we report the effects of orexin 2 receptor (OX2R) activation on cell signaling in CHO cells over-expressing OX2R and in mouse hypothalamus cell line CLU172. Short-term extracellular signal-regulated kinase (ERK) phosphorylation and long-term cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) phosphorylation were subsequently observed in CHO cells that over-express OX2R while 20 min of ERK phosphorylation was significantly detected in mouse adult hypothalamus neuron cell line CLU172. Orexin A, which can also activate OX2R, mediated ERK phosphorylation was as the same as orexin B in CHO cells. A MAPK inhibitor eliminated ERK phosphorylation but not CREB phosphorylation in CHO cells. Also, ERK and CREB phosphorylation was not mediated by protein kinase A (PKA) or calmodulin kinase (CaMK). However, inhibition of protein kinase C (PKC) by GF 109203X eliminated the phosphorylation of ERK and CREB in CHO cells. A significant decrease in ERK and CREB phosphorylation was observed with 1 μM GF 109203X pre-treatment indicating that the conventional and novel isoforms of PKC are responsible for CREB phosphorylation after OX2R activation. In contrast, ERK phosphorylation induced by orexin B in CLU172 cells cannot be inhibited by 1 μM of protein kinase C inhibitor. From above observation we conclude that OX2R activation by orexin B induces ERK and CREB phosphorylation and orexin A played the same role as orexin B. Several isoforms of PKC may be involved in prolonged CREB phosphorylation. Orexin B induced ERK phosphorylation in mouse hypothalamus neuron cells differs from CHO cell line and cannot be inhibited by PKC inhibitor GF 109203X. And hypothalamus neuron cells may use different downsteam pathway for orexin B induced ERK phosphorylation. This result supports findings that orexins might have anti-depressive roles.     

Identification and functional characterization of thromboxane A2 receptors in Schwann cells

Previous reports have demonstrated the presence of functional thromboxane A2 (TP) receptors in astrocytes and oligodendrocytes. In these experiments, the presence and function of TP receptors in primary rat Schwann cells (rSC) and a neurofibrosarcoma-derived human Schwann cell line (T265) was investigated. Immunocytochemical and immunoblot analyses using polyclonal anti-TP receptor antibodies demonstrate that both cell types express TP receptors. Treatment with the stable thromboxane A2 mimetic U46619 (10 microM) did not stimulate intracellular calcium mobilization in rSC, whereas T265 cells demonstrated a calcium response that was inhibited by prior treatment with TP receptor antagonists. U46619 also stimulated CREB phosphorylation on Ser133 in T265 cells and, to a lesser extent, in rSC. To identify potential mechanisms of CREB phosphorylation in rSC, we monitored intracellular cAMP levels following U46619 stimulation. Elevated levels of cAMP were detected in both rSC (20-fold) and T265 (15-fold) cells. These results demonstrate that TP receptor activation specifically stimulates CREB phosphorylation in T265 cells, possibly by a calcium- and/or cAMP-dependent mechanism. In contrast, TP receptor activation in rSC stimulates increases in cAMP and CREB phosphorylation but does not elicit changes in intracellular calcium.     

INSL5 is a high affinity specific agonist for GPCR142 (GPR100)

Insulin-like peptide 5 (INSL5) is a peptide that belongs to the relaxin/insulin family, and its receptor has not been identified. In this report, we demonstrate that INSL5 is a specific agonist for GPCR142. Human INSL5 displaces the binding of (125)I-relaxin-3 to GPCR142 with a high affinity (K(i) = 1.5 nM). In a saturation binding assay, (125)I-INSL5 binds GPCR142 with a K(d) value of 2.5 nM. In functional guanosine (gamma-thio)-triphosphate binding and cAMP accumulation assays, INSL5 potently activates GPCR142 with EC(50) values of 1.3 and 1.2 nM, respectively. In addition, INSL5 stimulates Ca(2+) mobilization in HEK293 cells expressing GPCR142 and G alpha(16). Overall, INSL5 behaves as an agonist for GPCR142 similar to relaxin-3. However, unlike relaxin-3, which is also a potent agonist for GPCR135 and LGR7, INSL5 does not activate either GPCR135 or LGR7. INSL5 inhibits (125)I-relaxin-3 binding to GPCR135 with a low potency (K(i) = 500 nM). A functional assay shows that INSL5 (1 microm) is a weak antagonist for GPCR135. In addition, INSL5 (up to 1 microm) shows no affinity or activity at LGR7 or LGR8 either in a binding assay or a bio-functional assay. Previously, we have demonstrated that GPCR142 mRNA is expressed in peripheral tissues, particularly in the colon. Here we show that INSL5 mRNA is expressed in many peripheral tissues, similar to GPCR142. The high affinity interaction between INSL5 and GPCR142 coupled with their co-evolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for GPCR142.     

Developmental regulation of neuronal survival by adenosine in the in vitro and in vivo avian retina depends on a shift of signaling pathways leading to CREB phosphorylation or dephosphorylation

Previous studies have shown a cAMP/protein kinase A-dependent neuroprotective effect of adenosine on glutamate or re-feeding-induced apoptosis in chick retina neuronal cultures. In the present work, we have studied the effect of adenosine on the survival of retinal progenitor cells. Cultures obtained from 6-day-old (E6) or from 8-day-old (E8) chick embryos were challenged 2 h (C0) or 1 day (C1) after seeding and analyzed after 3-4 days in vitro. Surprisingly, treatment with the selective A2a adenosine receptor agonists N(6) -[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) or 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid (CGS21680) promoted cell death when added at E6C0 but not at E6C1 or E8C0. DPMA-induced cell death involved activation of A2a receptors and the phospholipase C/protein kinase C but not the cAMP/protein kinase A pathway, and was not correlated with early modulation of precursor cells proliferation. Regarding cyclic nucleotide responsive element binding protein (CREB) phosphorylation, cultures from E6 embryos behave in an opposite manner from that from E8 embryos, both in vitro and in vivo. While the phospho-CREB level was high at E6C0 cultures and could be diminished by DPMA, it was lower at E8C0 and could be increased by DPMA. Similar to what was observed in cell survival studies, CREB dephosphorylation induced by DPMA in E6C0 cultures was dependent on the Phospholipase C/protein kinase C pathway. Accordingly, cell death induced by DPMA was inhibited by okadaic acid, a phosphatase blocker. Moreover, DPMA as well as the adenosine uptake blocker nitrobenzyl mercaptopurine riboside (NBMPR) modulate cell survival and CREB phosphorylation in a population of cells in the ganglion cell layer in vivo. These data suggest that A2a adenosine receptors as well as CREB may display a novel and important function by controlling the repertoire of developing retinal neurons.     

Arrestin specificity for G protein-coupled receptors in human airway smooth muscle.

Despite a widely accepted role of arrestins as "uncouplers" of G protein-coupled receptor (GPCR) signaling, few studies have demonstrated the ability of arrestins to affect second messenger generation by endogenously expressed receptors in intact cells. In this study we demonstrate arrestin specificity for endogenous GPCRs in primary cultures of human airway smooth muscle (HASM). Expression of arrestin-green fluorescent protein (ARR2-GFP or ARR3-GFP) chimeras in HASM significantly attenuated isoproterenol (beta(2)-adrenergic receptor (beta(2)AR)-mediated)- and 5'-(N-ethylcarboxamido)adenosine (A2b adenosine receptor-mediated)-stimulated cAMP production, with fluorescent microscopy demonstrating agonist-promoted redistribution of cellular ARR2-GFP into a punctate formation. Conversely, prostaglandin E(2) (PGE(2))-mediated cAMP production was unaffected by arrestin-GFP, and PGE(2) had little effect on arrestin-GFP distribution. The pharmacological profile of various selective EP receptor ligands suggested a predominantly EP2 receptor population in HASM. Further analysis in COS-1 cells revealed that ARR2-GFP expression increased agonist-promoted internalization of wild type beta(2)AR and EP4 receptors, whereas EP2 receptors remained resistant to internalization. However, expression of an arrestin whose binding to GPCRs is largely independent of receptor phosphorylation (ARR2(R169E)-GFP) enabled substantial agonist-promoted EP2 receptor internalization, increased beta(2)AR internalization to a greater extent than did ARR2-GFP, yet promoted EP4 receptor internalization to the same degree as did ARR2-GFP. Signaling via endogenous EP4 receptors in CHO-K1 cells was attenuated by ARR2-GFP expression, whereas ARR2(R169E)-GFP expression in HASM inhibited EP2 receptor-mediated cAMP production. These findings demonstrate differential effects of arrestins in altering endogenous GPCR signaling in a physiologically relevant cell type and reveal a variable dependence on receptor phosphorylation in dictating arrestin-receptor interaction.     

GPCR screening via ERK 1/2: a novel platform for screening G protein-coupled receptors

Discovery of novel agonists and antagonists for G protein-coupled receptors (GPCRs) relies heavily on cell-based assays because determination of functional consequences of receptor engagement is often desirable. Currently, there are several key parameters measured to achieve this, including mobilization of intracellular Ca2+ and formation of cyclic adenosine monophosphate or inositol triphosphate. However, no single assay platform is suitable for all situations, and all of the assays have limitations. The authors have developed a new high-throughput homogeneous assay platform for GPCR discovery as an alternative to current assays, which employs detection of phosphorylation of the key signaling molecule p42/44 MAP kinase (ERK 1/2). The authors show that ERK 1/2 is consistently activated in cells stimulated by Gq-coupled GPCRs and provides a new high-throughput platform for screening GPCR drug candidates. The activation of ERK 1/2 in Gq-coupled GPCR systems generates comparable pharmacological data for receptor agonist and antagonist data obtained by other GPCR activation measurement techniques.     

Discovery of novel quinolinone adenosine A2B antagonists

A novel series of quinolinone-based adenosine A(2B) receptor antagonists was identified via high throughput screening of an encoded combinatorial compound collection. Synthesis and assay of a series of analogs highlighted essential structural features of the initial hit. Optimization resulted in an A(2B) antagonist (2i) which exhibited potent activity in a cAMP accumulation assay (5.1 nM) and an IL-8 release assay (0.4 nM).     

Immature osteoblastic MG63 cells possess two calcitonin gene-related peptide receptor subtypes that respond differently to [Cys(Acm)(2,7)] calcitonin gene-related peptide and CGRP(8-37)

Calcitonin gene-related peptide (CGRP) is clearly an anabolic factor in skeletal tissue, but the distribution of CGRP receptor (CGRPR) subtypes in osteoblastic cells is poorly understood. We previously demonstrated that the CGRPR expressed in osteoblastic MG63 cells does not match exactly the known characteristics of the classic subtype 1 receptor (CGRPR1). The aim of the present study was to further characterize the MG63 CGRPR using a selective agonist of the putative CGRPR2, [Cys(Acm)(2,7)]CGRP, and a relatively specific antagonist of CGRPR1, CGRP(8-37). [Cys(Acm)(2,7)]CGRP acted as a significant agonist only upon ERK dephosphorylation, whereas this analog effectively antagonized CGRP-induced cAMP production and phosphorylation of cAMP response element-binding protein (CREB) and p38 MAPK. Although it had no agonistic action when used alone, CGRP(8-37) potently blocked CGRP actions on cAMP, CREB, and p38 MAPK but had less of an effect on ERK. Schild plot analysis of the latter data revealed that the apparent pA2 value for ERK is clearly distinguishable from those of the other three plots as judged using the 95% confidence intervals. Additional assays using 3-isobutyl-1-methylxanthine or the PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89) indicated that the cAMP-dependent pathway was predominantly responsible for CREB phosphorylation, partially involved in ERK dephosphorylation, and not involved in p38 MAPK phosphorylation. Considering previous data from Scatchard analysis of [125I]CGRP binding in connection with these results, these findings suggest that MG63 cells possess two functionally distinct CGRPR subtypes that show almost identical affinity for CGRP but different sensitivity to CGRP analogs: one is best characterized as a variation of CGRPR1, and the second may be a novel variant of CGRPR2.     

Differential expression of functional adrenocorticotropic hormone receptors by subpopulations of lymphocytes

In an effort to investigate the presence of adrenocorticotropic hormone (ACTH) receptors on rat lymphocytes, cells were separated by a panning procedure into T and B cell populations. By using the radiolabeled ACTH agonist, (125I-Tyr23) phenylalanine2-norleucine4-ACTH1-24, substantial numbers of ACTH binding sites were detected on T and B lymphocytes, but not on thymocytes. Scatchard analysis revealed two types of binding sites on each cell population, one with Kd1 = 0.088 +/- 0.025 nM and one with Kd2 = 4.2 +/- 0.6 nM; however, the absolute number of binding sites per cell was different. B lymphocytes expressed approximately three times the number of Kd1 binding sites per cell when compared with T lymphocytes. However, ACTH receptor expression by these cell populations was not static as suggested by the ability to induce receptor expression via mitogens. B or T cells and thymocytes stimulated with the mitogens LPS or Con A, respectively, substantially increased their number of Kd1 binding sites per cell (approximately three-fold). Even more dramatic increases in Kd1 receptor expression (approximately 100-fold) were observed when comparing "normal" and stimulated thymocytes. To demonstrate that these ACTH binding sites were in fact functional, cAMP levels were measured in lymphocytes 10 min after exposure to varying concentrations of ACTH. Dose-dependent increases in cAMP levels were observed, with significant stimulation occurring with as little as 0.1 nM ACTH added. Taken together, these studies demonstrate the presence of functional ACTH receptors on normal, rat T and B lymphocytes.     

Phosphorylation of the cyclic AMP response element binding protein mediates transforming growth factor beta-induced downregulation of cyclin A in vascular smooth muscle cells

Transforming growth factor beta (TGFbeta), a multifunctional cytokine associated with vascular injury, is a potent inhibitor of cell proliferation. The current results demonstrate that the TGFbeta-induced growth arrest of vascular smooth muscle cells (VSMCs) is associated with cyclin A downregulation. TGFbeta represses the cyclin A gene through a cyclic AMP (cAMP) response element, which complexes with the cAMP response element binding protein (CREB). The CREB-cyclin A promoter interaction is hindered by TGFbeta, preceded by a TGFbeta receptor-dependent CREB phosphorylation. Induction of CREB phosphorylation with forskolin or 6bnz-cAMP mimics TGFbeta's inhibitory effect on cyclin A expression. Conversely, inhibition of CREB phosphorylation with a CREB mutant in which the phosphorylation site at serine 133 was changed to alanine (CREB-S133A) upregulated cyclin A gene expression. Furthermore, the CREB-S133A mutant abolished TGFbeta-induced CREB phosphorylation, cyclin A downregulation, and growth inhibition. Since we have previously shown that the novel PKC isoform protein kinase C delta (PKCdelta) is activated by TGFbeta in VSMCs, we tested the role of this kinase in CREB phosphorylation and cyclin A downregulation. Inhibition of PKCdelta by a dominant-negative mutant or by targeted gene deletion blocked TGFbeta-induced CREB phosphorylation and cyclin A downregulation. Taken together, our data indicate that phosphorylation of CREB stimulated by TGFbeta is a critical step leading to the inhibition of cyclin A expression and, thus, VSMC proliferation.     

Regulation of protein phosphorylation in Dictyostelium discoideum

We have examined the phosphorylation of the cyclic adenosine 3':5' monophosphate (cAMP) cell surface chemotactic receptor and a 36 kDa membrane-associated protein (p36) in Dictyostelium discoideum. The activity of CAR-kinase, the enzyme responsible for the phosphorylation of the cAMP receptor, was studied in plasma membrane preparations. It was found that, as in intact cells, the receptor was rapidly phosphorylated in membranes incubated with [gamma 32P] adenosine triphosphate (ATP) but only in the presence of cAMP. This phosphorylation was not observed in membranes prepared from cells which did not display significant cAMP binding activity. cAMP could induce receptor phosphorylation at low concentrations, while cyclic guanosine 3':5' monophosphate (cGMP) could elicit receptor phosphorylation only at high concentrations. Neither ConA, Ca2+, or guanine nucleotides had an effect on CAR-kinase. It was also observed that 2-deoxy cAMP but not dibutyryl cAMP induced receptor phosphorylation. The data suggest that the ligand occupied form of the cAMP receptor is required for CAR-kinase activity. Although the receptor is rapidly dephosphorylated in vivo, we were unable to observe its dephosphorylation in vitro. In contrast, p36 was rapidly dephosphorylated. Also, unlike the cAMP receptor, the phosphorylation of p36 was found to be regulated by the addition of guanine nucleotides. Guanosine diphosphate (GDP) enhanced the phosphorylation while guanosine triphosphate (GTP) decreased the radiolabeling of p36 indicating that GTP can compete with ATP for the nucleotide triphosphate binding site of p36 kinase. Thus was verified using radiolabeled GTP as the phosphate donor. Competition experiments with GTP gamma S, ATP, GTP, CTP, and uridine triphosphate (UTP) indicated that the phosphate donor site of p36 kinase is relatively non-specific.(ABSTRACT TRUNCATED AT 250 WORDS)