The regulatory mechanisms and functional roles of agonist-induced internalization of G protein-coupled receptors (GPCRs) were analyzed using mutant dopamine D(2) receptors (D(2)Rs) in which all possible GPCR kinase (GRK) phosphorylation sites were mutated or the affinity for beta-arrestins was altered. Agonist-induced internalization of D(2)Rs involved a phosphorylation-dependent component, which was mediated by serine/threonine (S/T) residues in the second loop and T225 in the third loop, and a phosphorylation-independent component. GRK2-mediated enhancement of the internalization and inhibition of D(2)R signaling did not involve receptor phosphorylation, and only the former required the enzymatic activity of GRK2. The phosphorylation-deficient mutant (D(2)R-intracellular loop 2/3) recycled more slowly and showed more agonist-induced desensitization than did the wild-type D(2)R, suggesting that receptor phosphorylation mediates the recycling of the internalized receptors and enhances receptor resensitization. Blockade of the agonist-induced internalization of D(2)R-intracellular loop 2/3 provoked desensitization as in wild-type D(2)R, suggesting that certain cellular processes other than receptor dephosphorylation occurring within the endocytic vesicle are responsible for the resensitization of D(2)R. When dissociation between D(2)R and beta-arrestin was inhibited or when the expression of cellular beta-arrestins was decreased, agonist-induced desensitization of D(2)R did not occur, suggesting that dissociation from beta-arrestin is the main cellular process required for resensitization of D(2)R and is achieved through agonist-induced internalization. These results indicate that, in the regulation of some GPCRs, phosphorylation-independent association with beta-arrestin plays a major role in agonist-induced desensitization.

Medical staff members should pay more attention to stigma in stoma patients. Coping self-efficacy, family members' acceptance of the stoma, and participation in activities with other stoma patients are influencing factors that protect these patients against stigma, whereas body image loss and stool leakage place them at higher risk for stigma. Interventions aimed at improving protective factors and decreasing risk factors should be considered to reduce the level of stigma in patients with stoma.

Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the β2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of β2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

This study provides important information to guide practice and education. The themes extracted from the qualitative content analysis provide a useful framework to guide the nurse in discussing care with the stoma patients and ensuring patients' adjustment to the permanent stoma. Further work can be done using this framework to develop protocols as standards of care and education materials when educating nurse specialists in stoma care.

Among the characterized dopamine receptor subtypes, D₂ receptor (D₂R) and D₃ receptor (D₃R) are the main targets of neuroleptics that are currently in use. In particular, D₃R is closely related to the etiology of schizophrenia and drug addiction. The spatial expression patterns of D₂R and D₃R are distinct in certain areas of the brain. D₂R are heavily expressed in the regions responsible for motor functions, whereas D₃R are more selectively expressed in the limbic regions, which are associated with cognitive and emotional functions. Therefore, disturbances in the motor and endocrine functions, which are the most serious problems caused by the current neuroleptics, are likely to result from the non-selective blockade of D₂R. Selective regulation of D₃R is needed to separate the desired therapeutic activities from unwanted side effects that result from promiscuous blockade of other receptors. D₂R and D₃R possess high sequence homology and employ similar signaling pathways, and it is difficult to selectively regulate them. In this review, we discuss the signaling mechanisms, intracellular trafficking, and desensitization properties of D₂R and D₃R. In addition, the proteins interacting with D₂R or D₃R are discussed in relation to their roles in the regulation of receptor functions, followed by the current status of the development of selective D₃R ligands.

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Adipose tissue is a specialized organ that synthesizes and stores fat. During adipogenesis, Rho and Rho-associated kinase (ROCK) 2 are inactivated, which enhances the expression of pro-adipogenic genes and induces the loss of actin stress fibers. Furthermore, pan ROCK inhibitors enhance adipogenesis in 3T3-L1 cells. Here, we show that KD025 (formerly known as SLx-2119), a ROCK2-specific inhibitor, suppresses adipogenesis in 3T3-L1 cells partially through a ROCK2-independent mechanism. KD025 downregulated the expression of key adipogenic transcription factors PPARγ and C/EBPα during adipogenesis in addition to lipogenic factors FABP4 and Glut4. Interestingly, adipogenesis was blocked by KD025 during days 1~3 of differentiation; after differentiation terminated, lipid accumulation was unaffected. Clonal expansion occurred normally in KD025-treated cells. These results suggest that KD025 could function during the intermediate stage after clonal expansion. Data from depletion of ROCKs showed that KD025 suppressed cell differentiation partially independent of ROCK’s activity. Furthermore, no further loss of actin stress fibers emerged in KD025-treated cells during and after differentiation compared to control cells. These results indicate that in contrast to the pro-adipogenic effect of pan-inhibitors, KD025 suppresses adipogenesis in 3T3-L1 cells by regulating key pro-adipogenic factors. This outcome further implies that KD025 could be a potential anti-adipogenic/obesity agent.

Summary Background Dermal papilla cells (DPCs) play a key role in hair regeneration and morphogenesis. Therefore, tremendous efforts have been made to promote DPC hair inductivity. Objectives The aim of this study was to investigate the mitogenic and hair inductive effects of hypoxia on DPCs and examine the underlying mechanism of hypoxia‐induced stimulation of DPCs. Methods DPCs' hair inductivity was examined under normoxia (20% O2) and hypoxia (2% O2). Results Hypoxia significantly increased the proliferation and delayed senescence of DPCs via Akt phosphorylation and downstream pathways. Hypoxia upregulated growth factor secretion of DPCs through the mitogen‐activated protein kinase pathway. Hypoxia‐preconditioned DPCs induced the telogen‐to‐anagen transition in C3H mice, and also enhanced hair neogenesis in a hair reconstitution assay. Injected green fluorescent protein‐labelled DPCs migrated to the outer root sheath of the hair follicle, and hypoxia‐preconditioning increased survival and migration of DPCs in vivo. Conditioned medium obtained from hypoxia increased the hair length of mouse vibrissa follicles via upregulation of alkaline phosphatase, vascular endothelial growth factor, and glial cell line‐derived neurotrophic factor. We examined the mechanism of this hypoxia‐induced stimulation, and found that reactive oxygen species (ROS) play a key role. For example, inhibition of ROS generation by N‐acetylcysteine or diphenyleneiodonium treatment attenuated DPCs' hypoxia‐induced stimulation, but treatment with ROS donors induced mitogenic effects and anagen transition. NADPH oxidase 4 is highly expressed in the DPC nuclear region, and NOX4 knockout by CRISPR‐Cas9 attenuated the hypoxia‐induced stimulation of DPCs. Conclusions Our results suggest that DPC culture under hypoxia has great advantages over normoxia, and is a novel solution for producing DPCs for cell therapy. Whatʼs already known about this topic? Dermal papilla cells (DPCs) play a key role in hair regeneration and morphogenesis, but they are difficult to isolate and expand for use in cell therapy. Tremendous efforts have been made to increase proliferation of DPCs and promote their hair formation ability. What does this study add? Hypoxia (2% O2) culture of DPCs increases proliferation, delays senescence and enhances hair inductivity of DPCs. Reactive oxygen species play a key role in hypoxia‐induced stimulation of DPC. What is the translational message? Preconditioning DPCs under hypoxia improves their hair regenerative potential, and is a novel solution for producing DPCs for cell therapy to treat hair loss.