首页|PKC-isoform specific regulation of receptor desensitization and KCNQ1/ KCNE1 K+ channel activity by mutant?1B-adrenergic receptors

PKC-isoform specific regulation of receptor desensitization and KCNQ1/ KCNE1 K+ channel activity by mutant?1B-adrenergic receptors

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  • NSTL
  • Elsevier
Activation of a specific protein kinase C (PKC) isoform during stimulation of Gq protein-coupled receptors (GqPCRs) is determined by homologous receptor desensitization that controls the spatiotemporal formation of downstream Gq signalling molecules. Furthermore, GqPCR-activated PKC isoforms specifically regulate receptor activity via a negative feedback mechanism. In the present study, we investigated the contribution of several phosphorylation sites in the alpha 1B-adrenergic receptor (alpha 1B-AR) for PKC and G protein coupled receptor kinase 2 (GRK2) to homologous receptor desensitization and effector modulation. We analyzed signalling events downstream to human wildtype alpha 1B-ARs and alpha 1BARs lacking PKC or GRK2 phosphorylation sites (Delta 391-401, alpha 1B-Delta PKC-AR and Delta 402-520, alpha 1B-Delta GRK-AR) by means of FRET-based biosensors in HEK293 that served as online-assays of receptor activity. K+ currents through KCNQ1/KCNE1 channels (IKs), which are regulated by both phosphatidylinositol 4,5-bisphosphate (PIP2)depletion and/or phosphorylation by PKC, were measured as a functional readout of wildtype and mutant alpha 1B-AR receptor activity. As a novel finding, we provide evidence that deletion of PKC and GRK2 phosphorylation sites in alpha 1B-ARs abrogates the contribution of PKC alpha to homologous receptor desensitization. Instead, the time course of mutant receptor activity was specifically modulated by PKC beta. Mutant alpha 1B-ARs displayed pronounced homologous receptor desensitization that was abolished by PKC beta-specific pharmacological inhibitors. IKs modulation during stimulation of wildtype and mutant alpha 1B-ARs displayed transient inhibition and current facilitation after agonist withdrawal with reduced capability of mutant alpha 1B-ARs to induce IKs inhibition. Pharmacological inhibition of the PKC beta isoform did not augment IKs reduction by mutant alpha 1B-ARs, but shifted IKs modulation towards current facilitation. Coexpression of an inactive (dominant-negative) PKC delta isoform (DNPKC delta) abolished IKs facilitation in alpha 1B-Delta GRK-AR-expressing cells, but not in alpha 1B-Delta PKC-AR-expressing cells. The data indicate that the differential modulation of IKs activity by alpha 1B-Delta GRK- and alpha 1B-Delta PKC-receptors is attributed to the activation of entirely distinct novel PKC isoforms. To summarize, specific phosphorylation sites within the wildtype and mutant alpha 1B-adrenergic receptors are targeted by different PKC isoforms, resulting in differential regulation of receptor desensitization and effector function.

alpha-Adrenergic receptorMutant? 1B-receptorsPKCReceptor desensitizationFRETIKsPROTEIN-KINASE-CRECTIFIER POTASSIUM CURRENTDEPENDENT PHOSPHORYLATIONMEDIATED DESENSITIZATIONQUANTITATIVE PROPERTIESSELECTIVE INHIBITORSCOUPLED RECEPTORANGIOTENSIN-IIINTERNALIZATIONACTIVATION

Renkhold, Lina、Kollmann, Rike、Inderwiedenstrasse, Leonie、Kienitz, Marie-Cecile

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Univ Klin Munster

Ruhr Univ Bochum

2022

10.1016/j.cellsig.2021.110228

Cellular Signalling

Cellular Signalling

SCI
ISSN：0898-6568
年,卷(期)：2022.91
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