An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin-1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin-1 is controlled by phosphoinositide-dependent membrane recruitment. An interacting protein was identi®ed, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re-localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin-1 strictly requires co-expression of CYTIP. Consequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule-1 is repressed by CYTIP. These ®ndings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by speci®c protein±protein interactions.

Key Points• mTORC1 activity in DCs by conditional deletion of Raptor leads to a progressive loss of LCs in the skin of mice.• mTORC1 but not mTORC2 is required for epidermal LC homeostasis.The PI3K/Akt/mTOR pathway has emerged as a critical regulator of dendritic cell (DC) development and function. The kinase mTOR is found in 2 distinct complexes, mTORC1 and mTORC2. In this study, we show that mTORC1 but not mTORC2 is required for epidermal Langerhans cell (LC) homeostasis. Although the initial seeding of the epidermis with LCs is not affected, the lack of mTORC1 activity in DCs by conditional deletion of Raptor leads to a progressive loss of LCs in the skin of mice. Ablation of mTORC2 function by deletion of Rictor results in a modest reduction of LCs in skin draining lymph nodes. In young mice Raptor-deficient LCs show an increased tendency to leave the skin, leading to a higher frequency of migratory DCs in skin draining lymph nodes, indicating that the loss of LCs results from enhanced migration. LCs lacking Raptor are smaller and display reduced expression of Langerin, E-cadherin, ␤-catenin, and CCR7 but unchanged levels of MHC-II, ruling out enhanced spontaneous maturation. Ki-67 and annexin V stainings revealed a faster turnover rate and increased apoptosis of Raptor-deficient LCs, which might additionally affect the preservation of the LC network. Taken IntroductionDendritic cells (DCs) are specialized antigen-presenting cells essential for the initiation of adaptive immune responses and the maintenance of tolerance to self-antigens. 1 The outcome of antigen recognition by a T cell is dependent on the expression of distinct costimulatory and coinhibitory molecules as well as pro and anti-inflammatory cytokines expressed by the DC. 2 DCs develop from hematopoietic stem cells through specialized progenitor cells. Under steady-state conditions 3 major types of DCs differing in their location, phenotype, and function exist, the plasmacytoid DCs (pDCs), lymphoid organ resident DCs (rDCs), and peripheral tissue migratory DCs (mDCs). 3 In skin draining lymph nodes (sLNs) the mDC subset includes epidermal Langerhans cells (LCs) and Langerin ϩ and Langerin Ϫ dermal DCs (dDCs). LCs and Langerin ϩ dDCs can be distinguished by the absence and presence of the integrin ␣ E subunit CD103, respectively. 4 In recent years it has become increasingly evident that the PI3K/Akt/mTOR pathway plays an important role in DC development and function. 5 The mammalian target of rapamycin mTOR is a conserved serine/threonine kinase found in 2 distinct multiprotein complexes termed mTORC1 and mTORC2. 6 Raptor and Rictor are components essential for the assembly and the specific functions of mTORC1 and mTORC2, respectively. The macrolide drug rapamycin specifically blocks the function of mTORC1. Although originally described as rapamycin insensitive, 7,8 increasing evidence indicates that prolonged rapamycin treatment also inhibits mTORC2. 9,10 MTORC1 is regulated by extracellular signals, which trigger the phosphatidylinositol 3Ј-ki...

Cytohesins are a family of highly homologous guanine nucleotide exchange factors (GEFs) that act on ADP-ribosylation factors (ARFs). The small ARF-GEFs are involved in integrin signaling, actin cytoskeleton remodeling, and vesicle transport. Here, we selected and applied a specific inhibitor for ARF nucleotide-binding site opener (ARNO)͞cytohesin-2, an RNA aptamer that clearly discriminates between cytohesin-1 and cytohesin-2. This reagent bound to an N-terminal segment of cytohesin-2 and did not inhibit ARF-GEF function in vitro. When transfected into HeLa cells, it persisted for at least 6 h without requiring stabilization. Its effect in vivo was to down-regulate gene expression mediated through the serumresponse element and knockdown mitogen-activated protein kinase activation, indicating that cytohesin-2 acts by means of mitogen-activated protein kinase signaling. We conclude that the N-terminal coiled-coil and parts of the Sec7 domain of cytohesin-2 are required for serum-mediated transcriptional activation in nonimmune cells, whereas cytohesin-1 is not. Our results indicate that intramer technology can be used not only for assigning novel biological functions to proteins or protein domains but also to prove nonredundancy of highly homologous proteins.

The ␤ 2 integrin LFA-1 is an important cell-cell adhesion receptor of the immune system. Evidence suggests that the molecule also participates in signaling and costimulatory function. We show here that clustering of the intracellular domain of the ␤ 2 chain but not of the ␣ L -or ␤ 1 -cytoplasmic domains, respectively, triggers intracellular Ca 2؉ mobilization in Jurkat cells. A ␤ 2 -specific NPXF motif, located in the C-terminal portion of the ␤ 2 tail, is required for Ca 2؉ signaling, and we show that this motif is important for the induction of allospecific target cell lysis by cytotoxic T cells in vitro. Significantly, the Ca 2؉ -signaling capacity of the ␤ 2 integrin is abrogated in T cells that do not express the T cell receptor but may be reconstituted by co-expression of the T cell receptor-chain. Our data suggest a specific function of the cytoplasmic domain of the ␤ 2 integrin chain in T cell signaling.