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GPCR kinase 2 (GRK2)/β-arrestins and protein kinase A (PKA)/protein kinase C (PKC) mediate homologous and heterologous regulations of GPCRs, respectively. Conventional protein kinase C enzymes (PKCs), as exemplified by PKCβII, selectively inhibit internalization of dopamine D2 receptor and β2 adrenoceptor in a β-arrestin- but not GRK2-dependent manner. PKCβII interacts with β-arrestin2 upon autophosphorylation at T250, and inhibits the receptor internalization by decreasing the ubiquitination of β-arrestin2. PKCβII interferes with the interaction between β-arrestin2 and MDM2 in the cytosol, resulting in the redistribution of MDM2 to the nucleus. Subsequently, deubiquitination of β-arrestin2 and inhibition of agonist-induced receptor internalization follow. Thus, our study suggests that the extent of β-arrestin ubiquitination and the autophosphorylation status of PKCs determine PKCβII-mediated inhibition of homologous regulatory processes of GPCRs., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Clathrin-mediated and caveolar endocytic pathways represent the major routes through which G protein-coupled receptors (GPCRs) could be internalized. GPCR kinase 2 (GRK2) and β-arrestins are representative proteins that mediate the GPCR endocytosis. However, the molecular mechanisms through which GRK2 and β-arrestin mediate clathrin-mediated and caveolar endocytosis remain unclear. In this study, we determined the cellular components and processes that mediate the selective interaction between clathrin/caveolin1 and GRK2/β-arrestins. For this we utilized the following: (i) mutant dopamine D 2 receptor and β 2 adrenoceptor in which the potential GRK2 phosphorylation sites were altered and (ii) cells in which clathrin, caveolin1, β-arrestins, or Mdm2 expression were knocked down. Our results showed that clathrin-mediated endocytosis occurs more rapidly than caveolar endocytosis. Clathrin-mediated endocytosis and the interaction between clathrin and GRK2/β-arrestin2 occurred in a GRK2-mediated receptor phosphorylation-dependent manner. In contrast, caveolar endocytosis and the interaction between caveolin1 and GRK2/β-arrestin2 were independent of receptor phosphorylation status. Mdm2-mediated ubiquitination of β-arrestin, which occurred in a receptor phosphorylation-dependent manner, was required for the interaction of arrestin with clathrin. Thus, this study shows that GRK2-mediated receptor phosphorylation accompanied by β-arrestin ubiquitination is a critical cellular event that links GRK2 and β-arrestins to clathrin-mediated endocytosis. A GPCR can undergo both phosphorylation-dependent and –independent endocytosis. Clathrin-mediated and caveolar endocytosis occur in phosphorylation and activation recognition modes, respectively. Ubiquitination of β-arrestin, which occurs in the nucleus [19], mediates its interaction with clathrin. WT, wildtype; Ago, agonist; β-Arr2, β-arrestin2; CHC, calthrin heavy chain; Cav1, caveolin1; Ub, ubiquitin. Image 1 • Receptor phosphorylation is determining factor for clathrin-mediated endocytosis. • Receptor phosphorylation mediates the clathrin vs. GRK2/β-arrestin2 interaction. • Caveolin1 interacts with GRK2/β-arrestin2 without need of receptor phosphorylation. • Ubiquitination is responsible for the interaction between β-arrestin2 and clathrin. [ABSTRACT FROM AUTHOR]

Desensitization of G protein-coupled receptors (GPCRs) represents a gradual attenuation of receptor responsiveness by continuous or repeated exposure to agonists. The most widely accepted molecular mechanism responsible for desensitization is that of GRK2-mediated receptor phosphorylation followed by association with β-arrestins. However, in most cases, this mechanism cannot explain the desensitization of GPCRs. In this study, we investigated whether there exists a direct correlation between desensitization and certain cellular events that commonly observed with desensitizing receptors. Our study showed that constitutive ubiquitination of β-arrestin, accompanied by nuclear to cytoplasmic translocation of Mdm2, was observed in cells expressing desensitizing GPCRs (dopamine D 3 receptor, K149C-dopamine D 2 receptor, β 2 adrenoceptor, and lysophosphatidic acid receptor 1). In contrast, Mdm2 was observed in the nucleus in cells expressing non-desensitizing GPCRs (dopamine D 2 receptor, C147K-dopamine D 3 receptor, and dopamine D 4 receptor). Molecular manipulation to convert the characteristics of the dopamine D 4 receptor from non-desensitizing to desensitizing changed the status of subcellular localization of Mdm2 from nuclear to cytoplasmic. With repeated agonist treatments of desensitizing receptors, Mdm2 translocated from cytoplasm to nucleus, resulting in the deubiquitination of β-arrestins. This study suggests that the property of a receptor that causes a change in subcellular localization of Mdm2, from the nuclear to cytoplasmic, could be used as a biomarker to predict the desensitization of a receptor. Mdm2 is recruited near the GPCRs that can undergo desensitization in a Gβγ/β-arrestin-dependent manner. Under desensitization conditions, Mdm2 translocates to the nucleus, and β-arrestins are deubiquitinated and make a tight complex with Gβγ. This series of events interferes with G protein cycling [1]. Image 1 • Common cellular events involved in the desensitization of GPCRs were investigated. • Constitutive ubiquitination of β-arrestin selectively occurred with desensitizing GPCRs. • Desensitizing GPCRs mediated translocation of Mdm2 from nucleus to cytoplasm. • Cytosolic localization of Mdm2 occurred in a Gβγ/β-arrestin-dependent manner. • Cytoplasmic Mdm2 moved to the nucleus under desensitization conditions. [ABSTRACT FROM AUTHOR]

The GRK/β-arrestin and PKC/PKA mediate the homologous and heterologous regulation of G protein-coupled receptors (GPCRs), respectively. Interaction between the two pathways is one of the most important issues in understanding the regulation of GPCRs. The present study investigated the regulatory effect of GRK2 and β-arrestins on PKC activation. The roles of GRK2 and β-arrestins in the functional regulation of PKC were assessed by determining their influence on PKC autophosphorylation and intracellular translocation. Radioligand binding assay was utilized to characterize intracellular trafficking of dopamine D 2 R, D 3 R, and β 2 adrenergic receptor (β 2 AR). The subdomains involved in the mutual interactions among GRK2, β-arrestin2, and PKCβII were determined by in vitro binding assay. Various point mutants of key regulatory players were combined with knockdown cells of GRK2, β-arrestins, and Mdm2 to functionally correlate the biochemical changes with functional outcomes. GRK2 and β-arrestin2 mutually inhibited the PKCβII autophosphorylation, a hallmark of PKCβII activation. β-Arrestin2 ubiquitination was required for the inhibitory activities of GRK2 as well as β-arrestin2. Furthermore, GRK2 facilitated β-arrestin2 ubiquitination, thus to enhance the inhibitory actions of β-arrestin2 on PKCβII activity. Aforementioned processes were also involved in the GRK2/β-arrestin2-mediated inhibition of the D 2 R, D 3 R, and β 2 AR endocytosis. The present study provides new insights into the intricate interactions between the homologous and heterologous GPCR regulation pathways. In addition, a novel regulatory role of GRK2 was proposed for the ubiquitination of β-arrestin in the context of the PKC-mediated heterologous regulation of GPCRs. [ABSTRACT FROM AUTHOR]

Cellular stimuli that increase diacylglycerol levels activate several protein kinase C (PKC) isoforms; however, prolonged stimulation depletes cells of PKCs. Ubiquitination is a critical cellular event that mediates the degradation of numerous proteins, including PKCs, but little is known of the molecular mechanisms involved in PKC ubiquitination. PKCβII is the most widely expressed PKC isoform and regulates a variety of cellular functions. Here, we show that in response to stimulation of the Gq-coupled angiotensin II type 1 receptor or treatment with phorbol ester, Mdm2, E3 ubiquitin ligase, interacted with PKCβII isotype in the nucleus, resulting in ubiquitination of PKCβII at the C-terminal K668 and K672 residues and its subsequent downregulation. Ubiquitinated PKCβII mediated the clathrin-mediated endocytosis of G protein-coupled receptors like the D 2 and D 3 dopamine receptors; in contrast, non-ubiquitinated PKCβII mediated an as yet uncharacterized clathrin- and caveolar-independent endocytic pathway. In conclusion, we characterized the molecular mechanisms involved in the activity-dependent ubiquitination of PKCβII that determine its life span and endocytic roles. Considering that PKCβII plays an important role in the development of various diseases, including diabetic vasculitis, the results obtained in this study will contribute to better understanding the pathogenesis of PKCβII-related diseases. [ABSTRACT FROM AUTHOR]