G-protein-coupled receptors (GPCRs
The tumour suppressor PTEN (phosphatase and tensin deleted on chromosome 10) regulates major cellular functions via lipid phosphatase-dependent and -independent mechanisms. Despite its fundamental pathophysiological importance, how PTEN's cellular activity is regulated has only been partially elucidated. We report that the scaffolding proteins β-arrestins (β-arrs) are important regulators of PTEN. Downstream of receptor-activated RhoA/ROCK signalling, β-arrs activate the lipid phosphatase activity of PTEN to negatively regulate Akt and cell proliferation. In contrast, following wound-induced RhoA activation, β-arrs inhibit the lipid phosphatase-independent anti-migratory effects of PTEN. β-arrs can thus differentially control distinct functional outputs of PTEN important for cell proliferation and migration.
The association of CD4, a glycoprotein involved in T-cell development and antigen recognition, and CC chemokine receptor 5 (CCR5), a chemotactic G proteincoupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for HIV. We observed that the majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relatively low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentrationdependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell-surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cellular distribution of CXCR4, the other HIV coreceptor. These results reveal a previously unappreciated role of CD4, which contributes to regulating CCR5 export to the plasma membrane. (Blood. 2009;113:1938-1947 IntroductionThe CC chemokine receptor 5 (CCR5) is a 7 membrane-spanning domain, G protein-coupled receptor (GPCR), which regulates chemotaxis and effector functions of T lymphocytes, macrophages, and dendritic cells (reviewed in Oppermann 1 ). Upon activation by its cognate chemokines, CCL5 (formerly regulated upon activation, normal T cell expressed and secreted [RANTES]), CCL3, and CCL4 (formerly macrophage inflammatory proteins 1␣ and 1, respectively), 2 CCR5 leads to cellular signaling through G proteins 3 as well as G protein-independent pathways. 4 In addition to its physiologic functions, CCR5 is also the main coreceptor of HIV, in association with CD4, 5 a cell-surface glycoprotein that participates in molecular complexes involved in both T-cell development and antigen recognition by T cells. CD4 is a single-membrane spanning domain receptor interacting with class II major histocompatibility complex (MHC) molecules 6 and with its cognate ligand interleukin-16 (IL-16). So far, no specific physiologic function has been attributed to the CCR5-CD4 complex.In the context of immune cell chemotaxis in vivo, recent studies have shown that the sustained (hours) lymphocyte motility within lymph nodes is driven by the lymphoid chemokine-rich environment and mediated by the activation of chemokine receptors. 7 Paradoxically, studies in reconstituted models have shown that stimulated chemokine receptors, including CCR5, are rapidly desensitized (seconds to minutes) via phosphorylation and -arrestin translocation before being internalized and eventually degraded. 1,[8][9][10] In fact, an adaptive mechanism to escape inactivation and maintain prolonged reactivity in vivo upon sustained stimulation has been reported for other GPCRs, based on the retention of important stocks of functional receptors within intracellular compartments (reviewed in Achour et al 11 ). Initially described for protease-activated receptors (such as the thrombin receptor 12 ), this phenomenon was subsequently reported for dopamine D1 recep...
Endoplasmic reticulum (ER) release and cell-surface export of many G protein-coupled receptors (GPCRs) are tightly regulated. For gamma-aminobutyric acid (GABA)B receptors of GABA, the major mammalian inhibitory neurotransmitter, the ligand-binding GB1 subunit is maintained in the ER by unknown mechanisms in the absence of hetero-dimerization with the GB2 subunit. We report that GB1 retention is regulated by a specific gatekeeper, PRAF2. This ER resident transmembrane protein binds to GB1, preventing its progression in the biosynthetic pathway. GB1 release occurs upon competitive displacement from PRAF2 by GB2. PRAF2 concentration, relative to that of GB1 and GB2, tightly controls cell-surface receptor density and controls GABAB function in neurons. Experimental perturbation of PRAF2 levels in vivo caused marked hyperactivity disorders in mice. These data reveal an unanticipated major impact of specific ER gatekeepers on GPCR function and identify PRAF2 as a new molecular target with therapeutic potential for psychiatric and neurological diseases involving GABAB function.
Over a period of 15 years the concept of G protein-coupled receptor (GPCR) dimerization moved from a challenging hypothesis to a scientific fact, which is now accepted by the vast majority of the scientists working in the field. However, several important issues remain debated such as the biological function of dimerization, or the actual complexity of the oligomeric organization. Because of its major potential implications in physiology and pharmacology, the question of GPCR heterodimerization (or hetero-oligomerization) is currently one of the most central. Several complementary experimental approaches are used to investigate these novel important aspects of GPCR biology. In this context, Bioluminescence Resonance Energy Transfer-based techniques are extremely powerful, provided that they are conducted with the appropriate (numerous) controls and correctly interpreted.
A recent study by Pilch-Cooper et al 1 refuting the existence of intracellular pools of CCR5 in human T cells challenged some of our observations reported in Blood. 2 Although these authors observed the same intracellular accumulation of CCR5 using our cell permeabilization and fixation conditions before FACS analysis, they concluded that this intracellular labeling was likely nonspecific binding. This was based on the observations that our experimental conditions in their hands led to nonspecific signals in osteosarcoma cells, which do not express CCR5, and that confocal immunofluorescence (IF) microscopy studies in untransfected Chinese hamster ovary (CHO) cells showed an off-target nuclear signal. 1 Correct permeabilization conditions are indeed critical to observe intraluminal epitopes while avoiding background. However, we do not believe that these results challenge our previous conclusions. First, antibody combinations showing background, such as fluochrome-conjugated anti-CCR5 2D7 antibodies used in FACS studies or nonconjugated 1/85a antibody in IF experiments 1 were not used in Achour et al. 2 In addition, our multiple controls were not considered. In our Figure 1A 2 the strong signal obtained in permeabilized T lymphocytes with 2 different anti-CCR5 antibodies was not found for the anti-CD4 antibody, excluding a general nonspecific signal. 2 Our confocal IF studies in T cells and THP-1 monocytes, revealed abundant intracellular CCR5, whereas no signal was visible in control CCR5-negative Jurkat T cells ( Figure 1B Nevertheless, we conducted new FACS and IF experiments in THP-1 and CHO cells (Figure 1), using the "mild" permeabilization procedure recommended by the authors. 1 Confirming the specificity of CCR5 labeling, YFP-tagged CCR5 expressed in CHO cells was labeled by antibodies, and no off-target signal was visible by confocal IF, including in CHO cells transfected with control plasmid. Under these conditions, intracellular endogenous CCR5 was visible in THP-1 cells and again no staining was observed in CCR5-negative Jurkat cells. After mild permeabilization, FACS studies performed with the antibody combinations used in our previous study did not show any background in CHO cells and confirmed the presence of endogenous intracellular CCR5 in THP-1 cells ( Figure 1C).The existence of abundant intracellular stores of CCR5 is consistent with similar observations for many G protein-coupled receptors, the family of receptors CCR5 belongs to. 3 In particular, intracellular stores of the chemokine receptor CXCR4 were reported in human T lymphocytes. 4 Interestingly, L-selectin stimulation of T cells induced cell surface mobilization of CXCR4 within 10 minutes. Because such a fast effect, incompatible with receptor neosynthesis, represents major evidence for the existence of internal stores, we performed a similar experiment using THP-1 monocytes ( Figure 1D-F). Adhesion of THP-1 cells on fibronectincoated slides for 10 minutes was sufficient to increase 5-fold the fluorescence signal of surface CCR5, wher...
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