Phosphorylation and Functional Desensitization of the α_2A-Adrenergic Receptor by Protein Kinase C

Abstract

We have investigated the potential for protein kinase C (PKC) to phosphorylate and desensitize the ␣ 2A -adrenergic receptor (␣ 2A AR). In whole-cell phosphorylation studies, recombinantly expressed human ␣ 2A AR displayed an increase in phosphorylation after short-term exposure to 100 nM phorbol 12-myristate-13-acetate (PMA) that was blocked by preincubation with a PKC inhibitor. This increase in receptor phosphorylation over basal amounted to 172 Ϯ 40% in COS-7 cells and 201 Ϯ 40% in Chinese hamster ovary cells. In permanently transfected Chinese hamster fibroblast cells, PKC activation by brief exposure of the cells to PMA resulted in a marked desensitization of ␣ 2A AR function, amounting to a 68 Ϯ 4% decrease in the maximal agonist (UK14304)-stimulated intracellular calcium release. Such desensitization was blocked by the PKC inhibitor bisindolylmaleimide I and was not evoked by an inactive phorbol ester. The desensitization of this agonist response was not caused by PKC-mediated augmentation of G protein-coupled receptor kinase activity, because PMA-promoted desensitization of a mutated ␣ 2A AR that lacked G protein-coupled receptor kinase phosphorylation sites was identical to that of wild-type ␣ 2A AR. To test whether PKC phosphorylation is a mechanism by which ␣ 2A AR can be regulated by other receptors, the ␣ 1b AR was co-expressed with the ␣ 2A AR in Chinese hamster ovary cells. Upon selective activation of ␣ 1b AR, the function of ␣ 2A AR underwent a 53 Ϯ 5% desensitization. Thus, cellular events that result in PKC activation promote phosphorylation of the ␣ 2A AR and lead to substantial desensitization of receptor function. This heterologous regulation also represents a mechanism by which rapid crosstalk between the ␣ 2A AR and other receptors can occur.The ␣ 2 ARs regulate several effector systems including adenylyl cyclase, potassium channels, calcium channels, and inositol phosphate-mediated intracellular calcium release (Limbird, 1988;Liggett, 1996;Akerman et al. 1997;. Studies in intact organisms and cell culture systems have indicated that the function of G protein-coupled receptors, including ␣ 2 ARs, can be dynamically regulated under various physiologic and pathophysiologic conditions (Liggett and Lefkowitz, 1993;Liggett, 1997). We have recently delineated one pathway whereby persistent agonist activation results in a dampening of ␣ 2 AR signaling, termed desensitization, which is due to receptor phosphorylation by GRKs (Eason and Liggett, 1992;Liggett et al., 1992;Eason et al., 1995). GRK-mediated desensitization is evoked by agonist occupancy of the receptor, is independent of the generation of second messengers, and represents one mechanism of homologous desensitization of the ␣ 2 AR. The molecular basis of heterologous regulation of ␣ 2 AR has been largely unexplored. In the current study, we investigated the regulation of ␣ 2 AR signaling by PKC. This kinase was found to phosphorylate the ␣ 2A subtype, which resulted in a rapid desensitization of receptor function. These effect...

Agmatine Potentiates Morphine-Induced Conditioned Place Preference in Mice: Modulation by Alpha(2)-Adrenoceptors

“…In addition, we have recently reported that agmatine and morphine exert synergistic anticonvulsant effect, which is mediated via a 2 -adrenergic pathway (Riazi et al, 2005). Both opioid receptors and a 2 -adrenoceptors belong to a superfamily of G protein-coupled receptors and their interaction may involve reciprocal feedback regulation through shared signaling mechanisms (Saunders and Limbird, 1999;Liang et al, 1998).…”

Effects of the Noradrenergic System in Rat White Matter Exposed to Oxygen–Glucose DeprivationIn Vitro

“…One pathway by which agonist activation results in a desensitization of ␣2AR signaling involves receptor phosphorylation by protein kinase C (PKC) (Liang et al, 1998). This phosphorylation may represent a mechanism by which crosstalk between different subtypes of adrenergic receptors, or between adrenergic receptors and other G-protein-coupled receptors, can occur (Liang et al, 1998(Liang et al, , 2002. We investigated the possibility that in white matter injured by OGD, persistent stimulation of ␣2AR leads to an ultimate decrease of its function through PKC activation.…”

“…␣2 receptors can couple to several different G proteins, whose ␣ and ␤␥ subunits subsequently engage different effectors. The receptor's G-protein-coupling regions were demonstrated to be phosphorylation sites for PKC (Liang et al, 1998). This kinase phosphorylates Ser360 within the third intracellular loop of the ␣2a subunit (Liang et al, 2002), leading to rapid desensitization of receptor function.…”

“…One explanation may involve receptor desensitization in response to persistent activation by agonists, so that with either treatment, the net effect was a reduction of ␣2AR activity. One pathway by which agonist activation results in a desensitization of ␣2AR signaling involves receptor phosphorylation by protein kinase C (PKC) (Liang et al, 1998). This phosphorylation may represent a mechanism by which crosstalk between different subtypes of adrenergic receptors, or between adrenergic receptors and other G-protein-coupled receptors, can occur (Liang et al, 1998(Liang et al, , 2002.…”

Regulation of α2AR trafficking and signaling by interacting proteins

“…Phosphorylation of α 2A AR by PKC can cause heterologous desensitization of the α 2A AR [29], and may also affect constitutive coupling of G proteins [30]. Therefore, PKC phosphorylation may represent a mechanism for crosstalk between α 2A AR-mediated and other signaling pathways.…”