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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]

Filamin A (FLNA) is known to act as platform for the signaling and intracellular trafficking of various GPCRs including dopamine D 2 and D 3 receptors (D 2 R, D 3 R). To understand molecular mechanisms involved in the FLNA-mediated regulation of D 2 R and D 3 R, comparative studies were conducted on the signaling and intracellular trafficking of the D 2 R and D 3 R in FLNA-knockdown cells, with a specific focus on the roles of the proteins that interact with FLNA and the D 2 R and D 3 R. Lowering the level of cellular FLNA caused an elevation in RalA activity and resulted in selective interference with the normal intracellular trafficking and signaling of the D 2 R and D 3 R, through GRK2 and β-arrestins, respectively. Knockdown of FLNA or coexpression of active RalA interfered with the recycling of the internalized D 2 R and resulted in the development of receptor tolerance. Active RalA was found to interact with GRK2 to sequester it from D 2 R. Knockdown of FLNA or coexpression of active RalA prevented D 3 R from coupling with G protein. The selective involvement of GRK2- and β-arrestins in the RalA-mediated cellular processes of the D 2 R and D 3 R was achieved via their different modes of interactions with the receptor and their distinct functional roles in receptor regulation. Our results show that FLNA is a multi-functional protein that acts as a platform on which D 2 R and D 3 R can interact with various proteins, through which selective regulation of these receptors occurs in combination with GRK2 and β-arrestins. [ABSTRACT FROM AUTHOR]

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 D 2 receptor and β 2 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. [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]

Dopamine D2 receptor (D2R) has been shown to activate extracellular signal‐regulated kinases (ERKs) via distinct pathways dependent on either G‐protein or β‐arrestin. However, there has not been a systematic study of the regulatory process of D2R‐mediated ERKs activation by G protein‐ versus β‐arrestin‐dependent signaling since D2R stimulation of ERKs reflects the simultaneous action of both pathways. Here, we investigated that differential regulation of D2R‐mediated ERKs activation via these two pathways. Our results showed that G protein‐dependent ERKs activation was transient, rapid, reached maximum level at around 2 min, and importantly, the activated ERKs were entirely confined to the cytoplasm. In contrast, β‐arrestin‐dependent ERKs activation was more sustained, slower, reached maximum level at around 10 min, and phosphorylated ERKs translocated into the nucleus. Src was found to be commonly involved in both the G protein‐ and β‐arrestin‐dependent pathway‐mediated ERKs activation. Pertussis toxin Gi/o inhibitor, GRK2‐CT, AG1478 epidermal growth factor receptor inhibitor, and wortmannin phosphoinositide 3‐kinase inhibitor all blocked G protein‐dependent ERKs activation. In contrast, GRK2 and β‐Arr2 played a main role in β‐arrestin‐dependent ERKs activation. Receptor endocytosis showed minimal effect on the activation of ERKs mediated by both pathways. Furthermore, we found that the formation of a complex composed of phospho‐ERKs, β‐Arr2, and importinβ1 promoted the nuclear translocation of activated ERKs. The differential regulation of various cellular components, as well as temporal and spatial patterns of ERKs activation via these two pathways, suggest the existence of distinct physiological outcomes. [ABSTRACT FROM AUTHOR]

Background: Desensitization of G protein–coupled receptors (GPCRs) refers to a rapid attenuation of responsiveness that occurs with repeated or continuous exposure to agonists. GRK-mediated phosphorylation and subsequent binding with arrestins in the activated receptor cytoplasmic cavity in competition with G proteins has been suggested as the conventional mechanism of desensitization. Along with widely accepted conventional mechanism of desensitization, studies of various GPCRs including dopamine D2-like receptors (D2R, D3R, D4R) have suggested the existence of another desensitization mechanism. In this study, loss-of-function approaches and D2-like receptor mutants that display different desensitization properties were used to elucidate the molecular mechanisms responsible for desensitization. Results: Desensitization development entailed the signaling cascade composed of Src, PDK1, and Akt, the latter of which in turn interacted with USP33, an arrestin deubiquitinase, to promote arrestin deubiquitination. The deubiquitinated arrestin subsequently formed a complex with Gβγ and translocated to the nucleus via an importin complex, wherein it sequestered Gβγ from the receptor and Gα, thereby attenuating receptor signaling. As in D2-like receptors, both USP33 and importin β1 were involved in the desensitization of the β2 adrenoceptor. Conclusions: In addition to the conventional mechanism of desensitization, which occurs on the plasma membrane and in the cytosol, this study provides a new insight that another desensitization pathway in which nuclear trafficking plays a critical role is operating. It is plausible that multiple, complementary desensitization measures are in place to properly induce desensitization depending on receptor characteristics or the surrounding environment. -2RaxQqo6CD-C8c3iMTZGQ Video Abstract [ABSTRACT FROM AUTHOR]