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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]

GRK-mediated receptor phosphorylation followed by association with β-arrestins has been proposed to be the molecular mechanism involved in the desensitization of G protein-coupled receptors (GPCRs). However, this mechanism does not explain the desensitization of some GPCRs, such as dopamine D 3 receptor (D 3 R), which does not undergo GRK-mediated phosphorylation. Loss-of-function approaches and mutants of dopamine D 2 receptor and D 3 R, which exhibit different desensitization properties, were used to identify the cellular components and processes responsible for desensitization. D 3 R mediated the recruitment of Mdm2 to the cytosol, which resulted in the constitutive ubiquitination of β-arrestin2 in the resting state. Under desensitization conditions, cytosolic Mdm2 returned to the nucleus, resulting in the deubiquitination of cytosolic β-arrestins. Deubiquitinated β-arrestins formed a tight complex with Gβγ, thereby sequestering it, causing interference in D 3 R signaling. In conclusion, this study shows that β-arrestins, depending on their ubiquitination status, control the G protein cycling by regulating their interactions with Gβγ. This is a novel mechanism proposed to explain how certain GPCRs can undergo desensitization without receptor phosphorylation. • Dopamine D 3 receptor (D 3 R), a non-Gq-coupled GPCR, undergoes desensitization independent of receptor phosphorylation. • D 3 R and a dopamine D 2 receptor mutant, which have the tendency to undergo desensitization, recruit Mdm2 to the cytoplasm. • Mdm2 translocates to the nucleus under D 3 R desensitization conditions, resulting in the deubiquitination of β-arrestins. • Deubiquitinated β-arrestins form a stable complex with Gβγ and sequester it from Gα, resulting in D 3 R desensitization. [ABSTRACT FROM AUTHOR]

Conventional members of protein kinase C (PKC) family, including PKCβII, are constitutively phosphorylated on three major motifs and located in the cytosol in a primed state. In response to cellular stimuli, PKCβII is activated through inducible phosphorylation and Mdm2-mediated ubiquitination. In this study, we aimed to identify the activation mechanism of PKCβII, focusing on the signaling cascade that regulate the phosphorylation and ubiquitination. Loss-of-function approaches and mutants of PDK1/PKCβII that display different regulatory properties were used to identify the cellular components and processes responsible for endocytosis. Phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation and ubiquitination of PKCβII, which are needed for its translocation to the plasma membrane, required the presence of both Gβγ and 14-3-3ε. Gβγ and 14-3-3ε mediated the constitutive phosphorylation of PKCβII by scaffolding PI3K and PDK1 in the cytosol, which is an inactive but required state for the activation of PKCβII by subsequent signals. In response to PMA treatment, the signaling complex translocated to the nucleus with dissociation of PI3K from it. Thereafter, PDK1 stably interacted with 14-3-3ε and was dephosphorylated; PKCβII interacted with Mdm2 along with Gβγ, leading to its ubiquitination at two lysine residues on its C-tail. Finally, PDK1/14-3-3ε and ubiquitinated PKCβII translocated to the plasma membrane. As PKCβII mediates a wide range of cellular functions and plays important roles in the pathogenesis of various diseases, our results will provide clues to understand the pathogenesis of PKCβII-related disorders and facilitate their treatment. [Display omitted] [ABSTRACT FROM AUTHOR]

The fate and activity of β-arrestin2, a key player in the regulation of desensitization and endocytosis of G protein-coupled receptors (GPCRs), are regulated by mouse double minute 2 homolog (Mdm2)-mediated ubiquitination. However, details of the molecular mechanisms of β-arrestin2 ubiquitination remain unclear. Studies on β-arrestin2 and Mdm2 mutants with modified nucleocytoplasmic shuttling properties have revealed that β-arrestin2 ubiquitination occurs in the nucleus in a Gβγ- and clathrin-dependent manner. The nuclear entry of both β-arrestin2 and Mdm2 commonly relies on the presence of importin complex but can occur independently of each other. Gβγ and clathrin regulated the nuclear entry of β-arrestin2 by mediating the interaction between β-arrestin2 and importin β1. In contrast, Akt-mediated phosphorylation of two serine residues of Mdm2 partly regulated the nuclear entry of Mdm2. Ubiquitinated β-arrestin2 along with Mdm2 translocated to the cytoplasm where they play various functional roles including receptor endocytosis and ubiquitination of other cytoplasmic proteins. The nuclear export of Mdm2 required nuclear entry and interaction of β-arrestin2 with Mdm2. Ubiquitination was required for the translocation of β-arrestin2 toward activated receptors on the plasma membrane and for its endocytic activity. The current study revealed the cellular components and processes involved in the ubiquitination of β-arrestin2, and these findings could be quintessential for providing directions and detailed strategies for the manipulation of GPCR functions and development of GPCR-related therapeutics. [ABSTRACT FROM AUTHOR]

[Display omitted] AKT, also known as protein kinase B (PKB), serves as a crucial regulator of numerous biological functions, including cell growth, metabolism, and tumorigenesis. Increasing evidence suggests that the kinase activity of AKT is regulated via ubiquitination by various E3 ligase enzymes in response to different stimuli. However, the molecular mechanisms underlying insulin-induced AKT ubiquitination are not yet fully understood. Here, we show that activation of the insulin receptor (IR) leads to enhanced ubiquitination of AKT1 at K8 and K14 residues, facilitated by the cytosolic E3 ubiquitin ligase enzyme, TRAF6. Further investigation using AKT1 mutants with modified nucleocytoplasmic shuttling properties reveals that TRAF6-mediated AKT1 ubiquitination occurs within the nucleus in a β-Arr2-dependent manner. The nuclear entry of TRAF6 depends on importin β1, while β-Arr2 regulates this process by facilitating the interaction between TRAF6 and importin β1. Additionally, the ubiquitination of AKT1 is essential for its translocation to the activated IR on the plasma membrane, where it plays a functional role in recruiting Glut4 and facilitating glucose uptake. This study uncovers the cellular components and processes involved in insulin-induced ubiquitination and activation of AKT1, providing insights and detailed strategies for manipulating AKT1. [ABSTRACT FROM AUTHOR]