University thesis:
Dissertation, Albert-Ludwigs-Universität Freiburg im Breisgau, 2018
Footnote:
Description:
Abstract: GABAB receptors are the most abundant G protein-coupled receptors in the mammalian brain that mediate important aspects of the action of GABA. They show a pronounced diversity in subcellular distribution, functional properties and cellular signaling. The heterodimeric receptor consists of GABAB1, which is responsible for agonist binding, and GABAB2, which triggers GDP/GTP exchange in a Gi/o-type G protein and therefore activates signaling. Alternative promoter usage creates two distinct GABAB1 subtypes, B1b and B1a. The latter contains two sushi-type protein domains at the extracellular N-terminus. This 160 amino acid extension drives trafficking of GABAB1a to axonal compartments, which is arguably mediated through protein-protein interactions at the sushi domains of GABAB1a.<br>In this thesis, GABAB1a-specific interactors were identified by comparing proteomic data from wild-type and GABAB1a knock-out mice with a mass spectrometric approach. For this purpose, GABAB receptors were effectively solubilized from membrane-enriched fractions of adult brain and subsequently purified with specific antibodies targeting the receptor. In addition, a method to express and purify the sushi domains as recombinant protein in mg-scale has been established. The domains were used as a bait in pull-down experiments and together with heterologous complex reconstitution verified these protein interaction strings. The approaches demonstrated unequivocally that adherence-junction associated protein-1 (AJAP1), amyloid precursor protein (APP) and PILRα-associated neural protein (PIANP) directly interact with GABAB1a-containing receptors. The dynamics of the receptor assembly was evaluated in a further step by a differential proteomic analysis. The comparison of the individual knock-outs of AJAP1 and APP with wild-type mice revealed that the GABAB1a-specific proteins split into distinct GABAB(1a/2) receptor assemblies. In contrast to AJAP1 and PIANP, APP recruits further proteins putatively responsible for the axonal transport machinery. In addition, structural analysis helped to decipher binding sites of the B1a-interactors and revealed a common WG sequence preceded by hydrophobic amino acids and followed by G/P and A/T that interacts with sushi domain 1, but not sushi domain 2, at the N-terminal end of GABAB1a.<br>Further characterization of these proteins point toward their functional relevance. They show different binding affinities to the sushi domains, a different ...