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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]

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]