Yijuang Chern

Protein Kinase C Inhibits Adenylyl Cyclase Type VI Activity during Desensitization of the A2a-Adenosine Receptor-mediated cAMP Response

We have previously reported that phosphorylation of adenylyl cyclase type VI (AC6) may result in the suppression of adenylyl cyclase activity during desensitization of the A2a-adenosine receptor-mediated cAMP response (A2a desensitization) in rat pheochromocytoma PC12 cells. In the present study, we demonstrate that protein kinase C (PKC) is responsible for the phosphorylation and inhibition of AC6 during A2a desensitization. Inhibition of PKC by several independent methods markedly blocked the suppression of AC6 during A2a desensitization. Purified PKC from rat brain directly phosphorylated and inhibited recombinant AC6 expressed in Sf21 cells. Substantially lower AC6 activities were also observed in PC12 cells overexpressing PKCδ or PKCε. Stimulation of A2a-R in PC12 cells under the same conditions as those required for A2a desensitization resulted in an increase in Ca2+-independent PKC activity. Most importantly, exogenous PKC did not further suppress AC6 activity in A2a-desensitized membranes. In vitro PKC phosphorylation of AC6 isolated from A2a-desensitized cells was also profoundly lower than that from control cells, suggesting a specific role for PKC in regulating AC6 during A2a desensitization in PC12 cells. Taken together, our data demonstrate that a calcium-independent, novel PKC inhibits AC6 activity during A2a desensitization in PC12 cells. Independent regulation of AC6 by calcium-independent PKC and by Ca2+ provides an exquisite mechanism for integrating signaling pathways to fine-tune cAMP synthesis.

Regulation of adenylyl cyclase type VI activity during desensitization of the A2a adenosine receptor-mediated cyclic AMP response: role for protein phosphatase 2A.

We reported earlier that inhibition of adenylyl cyclase activity is a mechanism involved in desensitization of the A2a adenosine receptor-mediated cAMP response (A2a desensitization) in rat pheochromocytoma PC-12 cells. Here, we investigated the molecular mechanism that modulates adenylyl cyclase activity during A2a desensitization. Reversible inhibition of forskolin-evoked adenylyl cyclase activity in desensitized cells occurred after incubation with an A2a-selective adenosine agonist (CGS21680). However, when okadaic acid (a relatively protein phosphatase 2A-specific phosphatase inhibitor) was added after agonist removal, adenylyl cyclase activity did not recover. Okadaic acid caused significant dose-dependent inhibition of adenylyl cyclase activity in intact PC-12 cells. Prolonged exposure of okadaic acid-treated PC-12 cells to adenosine agonists did not evoke further inhibition, suggesting that the inhibition of adenylyl cyclase activity during A2a desensitization may operate through a pathway that overlaps with the increased phosphorylation caused by okadaic acid. Inclusion of calcium in the adenylyl cyclase assay significantly inhibited cyclase activity. indicating that PC-12 cells contain Ca(2+)-inhibitable type VI adenylyl cyclase (AC6). This was confirmed by polymerase chain reaction-based detection of AC6 cDNA. Furthermore, incubation of PC-12 cell membrane fractions with purified protein phosphatase 2A or coexpression of protein phosphatase 2A with AC6 in COS-1 cells significantly increased AC6 activity. To reduce the possible influence of Gs alpha protein, we substituted guanosine-5-O-(2-thio)diphosphate and MnCl2 for GTP and MgCl2, respectively, in some cyclase assays and found that the suppression of AC6 during A2a desensitization and okadaic acid treatment remained largely unchanged. Taken together, these data suggest that phosphorylation of AC6 might account for the inhibition of adenylyl cyclase activity during A2a desensitization in PC-12 cells.

Molecular cloning of a novel adenosine receptor gene from rat brain.

We have isolated an adenosine receptor gene (RA2) from a rat brain cDNA library. This novel rat adenosine receptor has 410 amino acids, as deduced from its base sequence, and shows 82% amino acid identity with the dog A2 receptor. Amino acid sequence analysis indicates that RA2 protein contains seven transmembrane domains and belongs to the G protein-coupled receptor family. The variations in amino acid sequences between RA2 protein and the dog A2 receptor are largely confined to the extracellular second loop and the carboxyl terminus.

Rescue of p53 Blockage by the A2A Adenosine Receptor via a Novel Interacting Protein, Translin-Associated Protein X

Blockage of the p53 tumor suppressor has been found to impair nerve growth factor (NGF)-induced neurite outgrowth in PC-12 cells. We report herein that such impairment could be rescued by stimulation of the A2A adenosine receptor (A2A-R), a G protein-coupled receptor implicated in neuronal plasticity. The A2A-R-mediated rescue occurred in the presence of protein kinase C (PKC) inhibitors or protein kinase A (PKA) inhibitors and in a PKA-deficient PC-12 variant. Thus, neither PKA nor PKC was involved. In contrast, expression of a truncated A2A-R mutant harboring the seventh transmembrane domain and its C terminus reduced the rescue effect of A2A-R. Using the cytoplasmic tail of the A2A-R as bait, a novel-A2A-R-interacting protein [translin-associated protein X (TRAX)] was identified in a yeast two-hybrid screen. The authenticity of this interaction was verified by pull-down experiments, coimmunoprecipitation, and colocalization of these two molecules in the brain. It is noteworthy that reduction of TRAX using an antisense construct suppressed the rescue effect of A2A-R, whereas overexpression of TRAX alone caused the same rescue effect as did A2A-R activation. Results of [3H]thymidine and bromodeoxyuridine incorporation suggested that A2A-R stimulation inhibited cell proliferation in a TRAX-dependent manner. Because the antimitotic activity is crucial for NGF function, the A2A-R might exert its rescue effect through a TRAX-mediated antiproliferative signal. This antimitotic activity of the A2A-R also enables a mitogenic factor (epidermal growth factor) to induce neurite outgrowth. We demonstrate that the A2A-R modulates the differentiation ability of trophic factors through a novel interacting protein, TRAX.

Expression of type VI adenylyl cyclase in the central nervous system: Implication for a potential regulator of multiple signals in different neurotransmitter systems

The aim of this study was to investigate the distribution of a calcium‐inhibitable adenylyl cyclase type VI (type VI AC) in the central nervous system using an antiserum directed against the N‐terminus of type VI AC. Our results indicate that type VI AC immunoreactivity is generally expressed in many brain regions with different levels of intensity. Most interestingly, the majority of the detected type VI AC immunoreactivity is present in cells of neuronal phenotype. Double immunostaining of type VI AC and markers of various neurotransmitter systems suggest that type VI AC might participate in regulation of the classical neurotransmitter systems and therefore appeared to play a very important role in the central nervous system.

Activation of Phosphodiesterase IV During Desensitization of the A2A Adenosine Receptor-Mediated Cyclic AMP Response in Rat Pheochromocytoma (PC12) Cells

Abstract: Prolonged activation of an A2A adenosine receptor significantly inhibits the cellular response to subsequent stimulation (A2A desensitization). We have reported previously that activation of phosphodiesterase (PDE) contributes to A2A desensitization in PC12 cells. In the present study, we show that a type IV PDE (PDE4)‐selective inhibitor (Ro 20‐1724) effectively blocks the increase in PDE activity in desensitized cells. Thus, PDE4 appears to be the PDE specifically activated during A2A desensitization in PC12 cells. Prolonged treatment of PC12 cells with an A2A‐selective agonist (CGS21680) leads to increased PDE4 activity in a dose‐dependent manner, which can be blocked by an A2A‐selective antagonist [8‐(3‐chlorostyryl)caffeine]. Using two PDE4 antibodies, we were able to demonstrate that the levels of two PDE4‐immunoreactive bands (72 and 79 kDa) were increased significantly during A2A desensitization. Prolonged treatment with forskolin to elevate intracellular cyclic AMP contents also resulted in increased PDE4 activity. In addition, activation of PDE4 activity during A2A desensitization could be blocked by a protein kinase A (PKA)‐selective inhibitor (H89) and was not observed in a PKA‐deficient PC12 cell line (A123). Taken together, activation of PDE4 via a cyclic AMP/PKA‐dependent pathway plays a critical role in dampening the signal of the A2A receptor.

Presence of Na+Ca2+ exchange activity and its role in regulation of intracellular calcium concentration in bovine adrenal chromaffin cells

The presence of a Na+/Ca2+ exchanger in bovine adrenal chromaffin cells was demonstrated by measuring the efflux of 45Ca2+ which had been preloaded into cells by a brief depolarization. The efflux of 45Ca2+ was dependent on extracellular Na+ (Na+o); 45Ca2+ efflux was significantly decreased by replacing Na+o with N-methylglucamine (NMG), or Li+. Replacement of Na+o by NMG increased the resting intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated chromaffin cells. This could be reversed by adding Na+, suggesting that Na+/Ca2+ exchanger activity was involved in maintaining [Ca2+]i at its resting level. The initial rate of Na(+)-dependent [Ca2+]i recovery after Ca2+ loading by depolarization was dependent on the level of [Ca2+]i. There was an apparent linear relationship between the activity of the Na+/Ca2+ exchanger and [Ca2+]i both in the presence and absence of Na+o. When cells were treated with other stimuli, including 10 microM DMPP or 40 mM caffeine, the ability of the stimulated cells to decrease [Ca2+]i was significantly reduced upon replacing Na+o with NMG. Our data show that the Na+/Ca2+ exchanger is one of the major pathways for regulating [Ca2+]i in chromaffin cells in both resting and stimulated states.

The 5' untranslated regions of the rat A2A adenosine receptor gene function as negative translational regulators.

The rat A2A adenosine receptor (A2A-R) gene contains two promoters, P1 and P2, which produce transcript 1 and transcript 2, respectively. These transcripts differ in the lengths of their 5 untranslated regions (5UTR1: 514 bp, initiated from P1; 5UTR2: 221 bp, initiated from P2) but encode the same protein. In the present study, we demonstrate that transcript 2 is present in various tissues at different levels, whereas transcript 1 is found only in the striatum. In the striatum, the level of transcript 2 is approximately 300-fold higher than that of transcript 1. The 5UTR of both transcripts suppresses the expression of A2A-R and a firefly luciferase reporter gene at the translational level; this suppression is not observed after mutational inactivation of an out-of-frame upstream AUG codon. Translational suppression by the 5UTR was also confirmed in cells using a bicistronic strategy. Collectively, these data suggest that P2 is the major promoter of the rat A2A-R gene. The 5UTR of the rat A2A-R gene exerts an inhibitory effect on translation by an upstream open reading frame. Because the 5UTR of the A2A-R gene possesses strong interspecies homology, translational suppression may be a general mechanism by which the expression of the A2A-R gene is regulated.

The Adenosine Analogue N6-L-Phenylisopropyladenosine Inhibits Catecholamine Secretion from Bovine Adrenal Medulla Cells by Inhibiting Calcium Influx

Abstract: We reported earlier that adenine nucleotides and adenosine inhibit acetylcholine‐induced catecholamine secretion from bovine adrenal medulla chromaffin cells. In this article, we used an adenosine analogue, N6‐L‐phenyliso‐propyladenosine (PIA), to study the mechanism underlying inhibition of catecholamine secretion by adenosine. PIA inhibits secretion induced by a nicotinic agonist, 1,1‐di‐methyl‐4‐phenylpiperazinium, or by elevated external K+. The half‐maximal effect on 1,1‐dimethyl‐4‐phenylpiperazinium‐induced secretion occurred at ∼5 ± 10‐‐5M. The inhibition is immediate and reversible. Fura‐2 measurements of cytosolic free Ca2+ indicate that PIA inhibits Ca2+ elevation caused by stimulation; measurements of 45Ca2+ influx show that PIA inhibits uptake of Ca2+. PIA does not inhibit calcium‐evoked secretion from digitonin‐permeabilized cells, nor does PIA cause any significant change in the dependence of catecholamine secretion on calcium concentration. These data suggest that inhibition by PIA occurs at the level of the voltage‐sensitive calcium channel.

Insulin-dependent translocation of the small GTP-binding protein rab3C in cardiac muscle: studies on insulin-resistant Zucker rats

The failure of insulin‐regulated recruitment of the GLUT4 glucose transporter in cardiac muscle of obese Zucker rats is associated with alterations of the subcellular distribution of the small‐molecular‐mass GTP‐binding protein rab4A. Here, we show by subcellular fractionation and Western blotting a translocation of the small‐molecular‐mass GTP‐binding protein rab3C from microsomal membranes to plasma membranes in lean control rats following in vivo insulin stimulation. However, in cardiac muscle of obese animals no significant effect of the hormone on the subcellular distribution of rab3C was observed. In GLUT4‐enriched membrane vesicles, obtained from cardiac microsomes of the obese group as well as of lean controls, rab3C was not detectable. It is suggested that the altered behaviour of rab3C may contribute to an impaired trafficking of GLUT4 in the insulin‐resistant state.