PTEN controls three-dimensional (3D) glandular morphogenesis by coupling juxtamembrane signaling to mitotic spindle machinery. While molecular mechanisms remain unclear, PTEN interacts through its C2 membrane-binding domain with the scaffold protein b-Arrestin1. Because b-Arrestin1 binds and suppresses the Cdc42 GTPase-activating protein ARHGAP21, we hypothesize that PTEN controls Cdc42-dependent morphogenic processes through a b-Arrestin1-ARHGAP21 complex. Here, we show that PTEN knockdown (KD) impairs b-Arrestin1 membrane localization, b-Arrestin1-ARHGAP21 interactions, Cdc42 activation, mitotic spindle orientation and 3D glandular morphogenesis. Effects of PTEN deficiency were phenocopied by b-Arrestin1 KD or inhibition of b-Arrestin1-ARHGAP21 interactions. Conversely, silencing of ARHGAP21 enhanced Cdc42 activation and rescued aberrant morphogenic processes of PTEN-deficient cultures. Expression of the PTEN C2 domain mimicked effects of full-length PTEN but a membrane-binding defective mutant of the C2 domain abrogated these properties. Our results show that PTEN controls multicellular assembly through a membrane-associated regulatory protein complex composed of b-Arrestin1, ARHGAP21 and Cdc42.

IntroductionThe annexins are a family of Ca 2+ /lipid-binding proteins that differ from most other Ca 2+ -binding proteins in their Ca 2+ -binding sites. These have a unique architecture that allows them to dock onto membranes in a peripheral and reversible manner. The conserved Ca 2+ -and membrane-binding module is the annexin core domain, which consists of four so-called annexin repeats, each of which is 70 residues in length. It is highly α-helical and forms a compact, slightly curved disc that has a convex surface harboring the Ca 2+ -and membranebinding sites and a concave side that points away from the membrane and is thereby available for other types of interaction/regulation (Fig. 1). The N-terminal region precedes the core domain and is diverse in sequence and length. It mediates regulatory interactions with protein ligands and regulates the annexin-membrane association (reviewed by Gerke and Moss, 2002;Raynal and Pollard, 1994). Although the N-terminal domain has long been considered a separately folded entity, recent crystal structures reveal that, at least in annexin A1, part of it can integrate into the folded core. Ca 2+ (and probably membrane) binding can then trigger exposure of the N-terminal region, making it available for additional interactions/activities ( Fig. 1) (Rosengarth and Luecke, 2003). The activity of the exposed N-terminal region could thus be tightly controlled through Ca 2+ /membrane binding.The annexin family comprises >500 different gene products expressed in most phyla and species (reviewed by Morgan and Fernandez, 1997). In vertebrates, 12 annexin subfamilies (A1-A11 and A13), which have different splice variants, have been identified. These have different N-terminal domains and differently positioned Ca 2+ /membrane-binding sites within the core domain. Analyses of the biochemical properties and subcellular localizations of annexins, and later studies of the effects of anti-annexin antibodies and annexin mutants, mainly in permeabilized cell systems, have allowed several potential physiological functions to be assigned to different annexins. Most of these take into account their regulated binding to membranes and a scaffold role at certain membrane domains is a common theme.Proposed to act as membrane-membrane or membranecytoskeleton linkers, annexins have been implicated in Ca 2+ -regulated exocytotic events, certain aspects of endocytosis and stabilization of specific domains of organelle membranes and the plasma membrane. However, other potential functions have been put forward -for example, those taking into account the RNA-binding capacity of some annexins (Filipenko et al., 2004;Vedeler and Hollas, 2000), their regulated nuclear localization (Eberhard et al., 2001;Mizutani et al., 1992;Tomas and Moss, 2003) or specific nucleotide-binding activities (Banderowicz-Pikula et al., 2001; Caohuy et al., 1996). Because some annexins occur extracellularly, they might also function outside the cell, although their (direct or indirect) secretion is not well understood. Detailed ...

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake.

TRPV5 and TRPV6 constitute the Ca 2+ in¯ux pathway in a variety of epithelial cells. Here, we identi®ed S100A10 as the ®rst auxiliary protein of these epithelial Ca 2+ channels using yeast two-hybrid and GST pull-down assays. This S100 protein forms a heterotetrameric complex with annexin 2 and associates speci®cally with the conserved sequence VATTV located in the C-terminal tail of TRPV5 and TRPV6. Of these ®ve amino acids, the ®rst threonine plays a crucial role since the corresponding mutants (TRPV5 T599A and TRPV6 T600A) exhibited a diminished capacity to bind S100A10, were redistributed to a subplasma membrane area and did not display channel activity. Using GST pull-down and co-immunoprecipitation assays we demonstrated that annexin 2 is part of the TRPV5±S100A10 complex. Furthermore, the S100A10±annexin 2 pair colocalizes with the Ca 2+ channels in TRPV5-expressing renal tubules and TRPV6-expressing duodenal cells. Importantly, downregulation of annexin 2 using annexin 2-speci®c small interfering RNA inhibited TRPV5 and TRPV6-mediated currents in transfected HEK293 cells. In conclusion, the S100A10±annexin 2 complex plays a crucial role in routing of TRPV5 and TRPV6 to plasma membrane.

The Coronavirus Disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Related Coronavirus 2 (SARS-CoV-2) is a global health emergency. As only very limited therapeutic options are clinically available, there is an urgent need for the rapid development of safe, effective, and globally available pharmaceuticals that inhibit SARS-CoV-2 entry and ameliorate COVID-19 severity. In this study, we explored the use of small compounds acting on the homeostasis of the endolysosomal host-pathogen interface, to fight SARS-CoV-2 infection. We find that fluoxetine, a widely used antidepressant and a functional inhibitor of acid sphingomyelinase (FIASMA), efficiently inhibited the entry and propagation of SARS-CoV-2 in the cell culture model without cytotoxic effects and also exerted potent antiviral activity against two currently circulating influenza A virus subtypes, an effect which was also observed upon treatment with the FIASMAs amiodarone and imipramine. Mechanistically, fluoxetine induced both impaired endolysosomal acidification and the accumulation of cholesterol within the endosomes. As the FIASMA group consists of a large number of small compounds that are well-tolerated and widely used for a broad range of clinical applications, exploring these licensed pharmaceuticals may offer a variety of promising antivirals for host-directed therapy to counteract enveloped viruses, including SARS-CoV-2.