Biochimica et Biophysica Acta 1864 (2017) 217–230 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein Lisa Seipold a,MarkusDammea, Johannes Prox a, Björn Rabe a, Petr Kasparek b, Radislav Sedlacek b, Hermann Altmeppen c, Michael Willem d, Barry Boland e, Markus Glatzel c, Paul Saftig a,⁎ a Institut für Biochemie, Christian-Albrechts-Universität zu Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany b Czech Centre for Phenogenomics and Laboratory of Transgenic Models of Diseases, Division BIOCEV, Institute of Molecular Genetics of the CAS, v. v. i., Vestec, Czech Republic c Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany d Biomedical Center, Ludwig-Maximilians-University, 81337 Munich, Germany e Department of Pharmacology and Therapeutics, School of Medicine, University College Cork, Cork, Ireland article info abstract Article history: Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α- Received 12 July 2016 secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch Received in revised form 28 October 2016 signalling we have only limited information about its regulation. In this study, we have identified ADAM10 Accepted 2 November 2016 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed Available online 3 November 2016 in the murine brain and elevated in brains of Alzheimer´s disease (AD) patients, was identified and confirmed to γ Keywords: bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the - ADAM10 secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and Tetraspanin was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding APP tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Presenilin Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expres- sion of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway. © 2016 Elsevier B.V. All rights reserved. 1. Introduction to these protein interactions. Most likely, different tetraspanins act in concert in a tissue-specific context in the regulation of the stability Tetraspanins were discovered in the early 90s as proteins which and intracellular trafficking of membrane proteins. This affects the span the membrane four times. Today, the 33 known mammalian steady state level and localization of tetraspanin partner proteins in a members of the tetraspanin family are involved in diverse biological given cellular or membrane compartment [1]. processes including the regulation of immune responses, infection Members of the ADAM (A Disintegrin And Metalloproteinase) family with pathogens and cancer progression [1]. As glycosylated and which can mediate the proteolytic processing of membrane proteins palmitoylated proteins, tetraspanins interact with each other and with referred to as ectodomain shedding [6] were also found to interact many transmembrane proteins including integrins, cell adhesion with certain tetraspanins. In particular, the interaction of tetraspanins proteins, growth factor and cytokine receptors and membrane embed- with ADAM10 has raised attention due to its proteolytic function as an ded enzymes [2–5]. Such an interaction network of tetraspanins and amyloid precursor protein (APP) α-secretase, as the sheddase of the transmembrane proteins was previously referred to as the “tetraspanin cellular prion protein (PrPc) and as a Notch1 receptor site 2 proteinase web” [2]. The transmembrane domains and the large extracellular loop [7–10] was found to interact with members of the tetraspanin super- of tetraspanins as well as the surrounding lipid composition contribute family. A subfamily of tetraspanins, TspanC8, with eight cysteines in the large extracellular domain, appeared to have high affinity to ⁎ Corresponding author. ADAM10. Tspan5, 10, 14, 15, 17 and 33 interact with ADAM10 and E-mail address: [email protected] (P. Saftig). regulate its exit from the ER and transport to the plasma membrane http://dx.doi.org/10.1016/j.bbamcr.2016.11.003 0167-4889/© 2016 Elsevier B.V. All rights reserved. 218 L. Seipold et al. / Biochimica et Biophysica Acta 1864 (2017) 217–230 where ectodomain shedding occurs [11–14]. Overexpression or RNAi- was cloned into a yeast expression vector (pTMBV4) and C-terminally mediated downregulation of the TspanC8s caused changes in the matu- fused to a Cub-LexA-VP16 cassette. Expression of the ADAM10 bait ration process and stabilized the surface expression of ADAM10 [12]. construct was monitored using positive (NubI) and negative (NubG) The interaction between different TspanC8 proteins and ADAM10 starts controls as supplied by the manufacturer. ADAM10 bait construct was in the ER [12] and mainly involves the large extracellular loop at the side co-transfected with N-terminally NubG-tagged murine cDNA brain of the TspanC8s and the membrane-proximal domain as well as the library (MoBiTec) into NMY51 yeast and seeded on selective media disintegrin domain in the ADAM10 protein [15]. The interaction of plates (−Leu, −Trp, −His). After incubation for 3–5daysat30°C ADAM10 with different TspanC8s may differentially affect the shedding clones were selected and expanded. Prey plasmids were extracted and of ADAM10 substrates including Notch1 [16]. It is of note that the analyzed by sequencing (GATC). TspanC8 family was also recently implicated in a possible functional clustering of the γ-secretase complex and the APP α-secretase, ADAM10 [17]. 2.3. Cell culture, transfection and plasmids Tetraspanin-3 (Tspan3) is another member of the tetraspanin super- fi family but only contains six cysteines in the large luminal domain. It was All cells were cultured in DMEM (Thermo Fisher Scienti c, Waltham, shown to associate with claudin-11 and beta1 integrin thereby regulat- USA) with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at ing the proliferation and migration of oligodendrocytes [18,19]. Tspan3 37 °C, 5% CO2 atmosphere and 95% relative humidity. HEK293 and N2A is expressed in all stages in oligodendrocyte development but is most cells stably overexpressing APP695 or APP695swe (Swedish mutant prominently expressed in neurons and astrocytes [19]. In brain, provided by Michael Willem, DZNE, Munich) were additionally supple- μ fi Tspan3 associates with the Nogo-A receptor sphingosine-1-phosphate mented with 200 g/ml of the antibiotic G418. HEK293 cells de cient − − − − receptor 2 (S1PR2). Tspan3 knockdown reduced the Nogo-A-induced for ADAM10 (A10 / ) or ADAM17 (A17 / )were generated by S1PR2 clustering, cell spreading and the inhibition of neurite outgrowth using the CRISPR/Cas9 system. The cells were transiently transfected [20].InXenopus leavis embryos Tspan3 is expressed in migratory neural with Polyethylenimin Max (PEI, Polysciences, Hirschberg, Germany). crest cells [21]. Tspan3 was also found to be a causative factor in the For inhibition of ADAM10 cell culture medium was supplemented μ development of acute myelogenous leukemia (AML) and deficiency of with 10 M of the hydroxamate-based inhibitor GI254023X (Iris Tspan3 led to an impaired stem cell-renewal and disease propagation Biotech, Marktredwitz, Germany) for 12 h. Mammalian expression [22]. vectors were pCI-neo (human APP695), pcDNA3.1Hygro (ADAM10) Using a yeast split-ubiquitin based screening system we identified and pFrog3 (murine Tspan3-myc and Tspan15-myc) derived from fi Tspan3 as a binding partner of ADAM10. This interaction did not affect pcDNA3 (Thermo Fisher Scienti c, Waltham, USA) [25]. The myc- the trafficking or maturation of the protease or its ability to cleave the epitope was inserted after the last amino acid of Tspan3 or Tspan15, neuronal cadherin (N-cadherin). However, cell-based experiments respectively. The myc-tagged murine Tspan7 expression plasmid was revealed that Tspan3 affected the stabilization of ADAM10 and APP at purchased from OriGene Technologies Rockville, USA (MR221859). the cell surface. Our experiments suggest that Tspan3 is part of an interaction scaffold that associates with APP and α- and γ-secretases 2.4. Generation of the Tspan3Y243L mutant at the plasma membrane and within the endocytic pathway. Tspan3Y243L was generated by mutating the cDNA sequence of myc- 2. Material and methods tagged wild type Tspan3. cDNA sequence coding for the amino acid Tyrosine (Y) at position 243 was exchanged by the sequence coding 2.1. Antibodies for Leucine (L). Myc-tagged Tspan3Y243L was again subcloned into the pFrog3