Charakterisierung der proteolytischen Spaltung des Glycin-Rezeptors im Endozytoseweg von Xenopus-Oozyten

The Glycin-Receptor (GlyR) is a ligand-gated chloride channel which mediates postsynaptic inhibition in spinal cord, retina and other regions of mammalian central nervous system. It is activated by the addition of glycine. Presently, four ligand-binding Alpha subunits (Alpha1- Alpha4) and a single Beta subunit are known, which can assemble to form homopentameric and hetereopentameric channels in different regions of the mammalian central nervous system. Each subunit shares with the other members of the nAChR superfamiliy a large glycosylated N-terminal ectodomain, which is responsible for ligand recognition, and four C-terminal transmembrane domains (M1-M4). M3 and M4 are separated by a cytosolic loop of approximately 85 amino acids, harbouring one tyrosine residue and ten lysine residues. Recently, our group demonstrated that the homopentameric Alpha1-GlyR is ubiquitinized at the plasma membrane, and subsequently internalized and proteolytically cleaved, yielding an N-terminal 35kDa- and a C-terminal 13kDa-fragment. The aim of this work was to localize the proteolytic cleavage site of the GlyR and to determine the role of the only tyrosine residue within the intracellular loop between M3 and M4. To identify the cleavage site, a single radioactively labelled [35S]-methionine was introduced through point mutation at different positions within the M3-M4 loop of the GlyR Alpha2-subunits. The point mutants were then expressed through cRNA-injection in Xenopus laevis oocytes. Their cleavage products were purified from oocyte extracts by Ni2+-NTA-Agarose chromatography and analysed by Tricine-SDS-Page and autoradiography. The incorporation of [35S]-methionine into the 13kDa fragment was essayed, thereby indirectly indicating the position of the cleavage site of the GlyR Alpha2-subunit. Using this technique, the cleavage site was narrowed to the three amino acid sequence 334EED336. Addition of PMSF, NH4Cl and concanamycin to the chase medium almost entirely suppressed proteolytic cleavage. Thus, a lysosomal serine protease seems most likely to be responsible for proteolytic cleavage of the GlyR. To explore the role of ubiquitination, and of the tyrosine residue within the M3-M4 loop, for internalisation of the GlyR, two mutants of the Alpha1-subunit were expressed: one ubiquitin-deficient mutant, in which all ten cytosolic lysine residues were replaced through arginines, and a second mutant, in which the tyrosine residue Y339 was replaced by arginine. As a measure of internalisation, proteolytic cleavage of the Alpha1-subunit mutants was then quantified. While both mutants individually were internalised to a similar degree as the wildtype-GlyR, the Y339A replacement within the ubiquitin-deficient Alpha1-GlyR lead to an extreme reduction of internalisation. Thus, it can be hypothesized that the GlyR is internalised through two different pathways: an Y339-dependant and an ubiquitin-dependant way, both of which seem to be able to compensate the other when blocked.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2005

Author dc:creator

• Leyendecker, Anne Larissa

Contributors dc:contributor

• Schmalzing, Günther

• info:eu-repo/classification/ddc/610
• Medizin
• Glycin Rezeptor
• Ubiquitinierung
• Endozytose

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123714%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:62121