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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]