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@PHDTHESIS{Friedrich:668346,
      author       = {Friedrich, Katharina},
      othercontributors = {Gründer, Stefan and Müller-Newen, Gerhard},
      title        = {{I}ntrazelluläre {L}okalisation und {T}argeting von
                      {ASIC}4},
      school       = {Rheinisch-Westfälische Technische Hochschule Aachen},
      type         = {Dissertation},
      address      = {Aachen},
      reportid     = {RWTH-2016-06711},
      pages        = {1 Online-Ressource (VII, 74 Seiten) : Illustrationen,
                      Diagramme},
      year         = {2016},
      note         = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
                      University; Dissertation, Rheinisch-Westfälische Technische
                      Hochschule Aachen, 2016},
      abstract     = {Acid-sensing ion channels (ASICs) are proton-sensitive,
                      voltage-independent and amiloride-sensitive sodium channels
                      of the DEG/ENaC gene family. ASIC4 is a member of the ASIC
                      family, and which mRNA is expressed in the central nervous
                      system. The strongest localisation of ASIC4-mRNA is found in
                      the pituitary gland. In contrast to other ASICs, ASIC4 can
                      not be activated by protons. Furthermore, ASIC4 comprises
                      only a rather low sequence identity with other ASICs of
                      about $45\%.$ In general, the sequence homology of ASIC1 –
                      ASIC3 is in the range of 50 – $65\%.$ The function of the
                      channel is unknown until now.In this study the subcellular
                      localisation and the targeting of ASIC4 were examined. After
                      transient transfection of ASIC4 in the heterologous cell
                      lines HEK293 and COS-7, an accumulation in vacuolar-like
                      structures has been observed. This was strongly different to
                      the reticular distribution pattern that is usually observed
                      for other members oft he ASIC family.To identify this
                      vacuolar localisation, cells were stained with antibodies or
                      fluorescent organelle markers and were examined with
                      confocal microscopy. Thereby, a co-localisation of ASIC4 and
                      mRFP-Rab5, a marker for early endosomes, has been found.In
                      the next step, the protein sequence was analysed to identify
                      motives that could be responsible for the special
                      distribution of ASIC4 in the cell. ASIC4 consists of two
                      transmembrane domains, an extracellular loop and short
                      intracellular amino- and carboxy-termini. Chimeras between
                      ASIC4 and either the amino- or the carboxy-terminus of
                      ASIC2a were constructed. ASIC2a, in contrast to ASIC4, is an
                      ion channel with a reticular distribution pattern,
                      Furthermore, point mutations of di-arginine- and
                      di-leucine-motives, which are known as trafficking signals,
                      were generated. In addition, the amino- and carboxy-termini
                      of native ASIC4 were truncated step by step. The
                      amino-terminus, as well as the carboxy-terminus could be
                      identified as important for the targeting of ASIC4. The
                      amino- and carboxy-terminal chimeras showed a reticular
                      distribution pattern, similar to the distribution pattern of
                      ASIC2a. This pattern suggested a localisation in the
                      endoplasmatic reticulum (ER). The first 18 amino acids of
                      the amino-terminus could be identified as important for the
                      anterograde transport from ER/Golgi to early endosomes.
                      Furthermore, a di-arginine-motif on position 478/479 of the
                      carboxy-terminus has an important function for the retention
                      of ASIC4 in early endosomes. By mutation of this
                      di-arginine-motif, ASIC4 shows a co-localisation with
                      RFP-Rab7, a marker for late endosomes. By truncation of the
                      carboxy-terminus including the mentioned di-arginine-motif,
                      ASIC4 was directed into another compartment of the cell,
                      which could be identified as lysosomes.Taken together, this
                      study could identify trafficking signals at the amino- as
                      well as at the carboxy-terminus that are responsible for the
                      localisation of ASIC4 in early endosomes. Thereby it can be
                      suggested that ASIC4 could play a role in this intracellular
                      compartment and not at the plasma membrane, as it is usual
                      for other members of the ASIC family.},
      cin          = {512000-2},
      ddc          = {610},
      cid          = {$I:(DE-82)512000-2_20140620$},
      typ          = {PUB:(DE-HGF)11},
      urn          = {urn:nbn:de:hbz:82-rwth-2016-067117},
      url          = {https://publications.rwth-aachen.de/record/668346},
}