Opposing effects of phorbol‐12‐myristate‐13‐acetate, an activator of protein kinase C, on the signaling of structurally related human dopamine D1 and D5 receptors

Abstract

The 'cross-talk' between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMPdependent signaling by structurally related human D1-like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, followed by analysis of dopamine-mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist-evoked D1 receptor signaling, whereas constitutive and dopamine-mediated D5 receptor activation were rapidly blunted. RT-PCR and immunoblotting analyses showed that phorbol ester-regulated PKC isozymes (conventional: a, bI, bII, c; novel: d, e, g, h) and protein kinase D (PKCl) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca 2+ -independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö 6976, and Gö 6983. The finding that cross-talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1-like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.

Ethanol Regulation of D1 Dopamine Receptor Signaling is Mediated by Protein Kinase C in an Isozyme-Specific Manner

“…It should be noted that the effect of Gö6983 on D 1 -stimulated signaling was not observed in a recent study by Jackson et al, where Gö6983 had no effect on D 1 receptor accumulation of cAMP. Surprisingly, PMA treatment potentiated D 1 receptor signaling (Jackson et al, 2005). Such discrepancies may be due to differences in experimental procedures and perhaps more importantly differences in the expression/activation profile of PKC isozymes.…”

Dopamine D1 receptor-mediated inhibition of NADPH oxidase activity in human kidney cells occurs via protein kinase A–protein kinase C cross talk

“…We have previously reported that in HEK-293 cells heterologously expressing the human D 5 R, stimulation of D 5 R inhibits NADPH oxidase activity, which is PKA-independent, partly PLD dependent, and partly due to interference with the distribution and assembly of NADPH oxidase components (34). Jackson et al have also reported that PMA increases D 1 R but decreases D 5 R signaling (15). Therefore, it is necessary to use cell lines that heterologously and exclusively express D 1 R or D 5 R to distinguish the mechanisms of involved in D 1 R or D 5 R activation because there are no commercially available D 1 -like receptor agonists or antagonists that can distinguish D 1 R from D 5 R. We tested the hypothesis that a specific PKC isoform may be involved in the D 1 R-mediated inhibition of NADPH oxidase activity.…”

“…Phorbol-12-myristate-13-acetate (PMA), a direct activator of PKC, stimulates adenylyl cyclase activity and enhances agonist-induced cAMP production (15). In addition, activation of PKC with PMA stimulates intracellular cAMP production in human eosinophils, in the absence of any agonist (17).…”

The D5 Dopamine Receptor Mediates Large-Conductance, Calcium- and Voltage-Activated Potassium Channel Activation in Human Coronary Artery Smooth Muscle Cells

“…The D 5 R may also stimulate phospholipase C PLC/PKC activity (Sahu et al, 2009). In contrast, increasing PKC inhibits constitutive and dopamine-mediated stimulation of cAMP production via D 5 R (Jackson et al, 2005). Thus, D 1 R and D 5 R may increase cAMP by unique intracellular signaling pathways.…”