Processing of the amyloid precursor protein (APP) by b-and c-secretases leads to the generation of amyloid-b (Ab) peptides with varying lengths. Particularly Ab42 contributes to cytotoxicity and amyloid accumulation in Alzheimer's disease (AD). However, the precise molecular mechanism of Ab42 generation has remained unclear. Here, we show that an amino-acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Ab species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of Ab42, leave the level of Ab40 unaffected, but increase Ab38 and shorter Ab species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that c-secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating Ab42 production.

The tubulin-binding protein gephyrin copurifies with the inhibitory glycine receptor (GlyR) and is essential for its postsynaptic localization. Here we have analyzed the interaction between the GlyR and recombinant gephyrin and identified a gephyrin binding site in the cytoplasmic loop between the third and fourth transmembrane segments of the beta subunit. GlyR alpha subunits and GABAA receptor proteins failed to bind recombinant gephyrin. However, insertion of an 18 residue segment of the GlyR beta subunit into the GABAA receptor beta 1 subunit conferred gephyrin binding both in an overlay assay and in transfected mammalian cells. These results indicate that beta subunit expression is essential for the formation of a postsynaptic GlyR matrix.

Purified preparations of the inhibitory glycine receptor (GlyR) contain alpha and beta subunits, which share homologous primary structures and a common transmembrane topology with other members of the ligand‐gated ion channel superfamily. Here, a beta subunit‐specific antiserum was shown to precipitate the [3H]strychnine binding sites localized on alpha subunits from membrane extracts of both rat spinal cord and mammalian cells co‐transfected with alpha and beta cDNAs. Further, inhibition of alpha homo‐oligomeric GlyRs by picrotoxinin, a non‐competitive blocker of ion flow, was reduced 50‐ to 200‐fold for alpha/beta hetero‐oligomeric receptors generated by cotransfection. Site‐directed mutagenesis identified residues within the second predicted transmembrane segment (M2) of the beta subunit as major determinants of picrotoxinin resistance. These data implicate the M2 segment in blocker binding to and lining of the GlyR chloride channel.

No abstract

A 93 kd polypeptide associated with the mammalian inhibitory glycine receptor (GlyR) is localized at central synapses and binds with high affinity to polymerized tubulin. This protein, named gephyrin (from the Greek gamma epsilon phi upsilon rho alpha, bridge), is thought to anchor the GlyR to subsynaptic microtubules. Here we report its primary structure deduced from cDNA and show that corresponding transcripts are found in all rat tissues examined. In brain, at least five different gephyrin mRNAs are generated by alternative splicing. Expression of gephyrin cDNAs in 293 kidney cells yields polypeptides reactive with a gephyrin-specific antibody, which coprecipitate with polymerized tubulin. Thus, gephyrin may define a novel type of microtubule-associated protein involved in membrane protein-cytoskeleton interactions.

The postsynaptic glycine receptor of rat spinal cord is a glycosylated membrane protein that, after affinity purification, contains membrane-spanning subunits of Mr 48,000 and 58,000 and an associated peripheral polypeptide of Mr 93,000. Here, the quaternary structure of the transmembrane core of the receptor was investigated by chemically crosslinking its subunits. Upon treatment with crosslinking reagents of different side-chain specificities and lengths, a consistent set of adducts up to M, 260,000 was detected after separation by NaDodSO4/PAGE. The observed pattern of adducts was similar irrespective of whether purified receptor protein or synaptosomal membranes were crosslinked. Compositional Recent results obtained by cDNA cloning of subunits of the nicotinic acetylcholine (1-3), the glycine (4), and the yaminobutyric acid (5) receptors revealed significant homology in primary structure and predicted transmembrane topology between these chemically gated ion channel proteins. In particular, four hydrophobic segments (M1-M4) presumed to span the lipid bilayer are conserved in the subunits of the different receptors. These data established the existence of a previously postulated superfamily ofphylogenetically related ion channel-forming receptor polypeptides (6, 7).Electron microscopy (8, 9) and crosslinking experiments (10) have demonstrated that the four subunits of Torpedo and muscle nicotinic acetylcholine receptor (nAcChoR) are arranged in a quasisymmetrical pentameric (a2f3y5) structure around a central pit presumed to represent the transmembrane channel. Little, however, is known about stoichiometry, spatial arrangement, and total number of subunits constituting neuronal ligand-gated ion channels. The postsynaptic y-aminobutyric acid receptor (GABAAR) has been proposed to be a tetrameric transmembrane protein (11). For neuronal nAcChoR, tetrameric and pentameric structures are discussed (12, 13).Another member of the chemically gated ion channel family, the glycine receptor (GlyR), mediates postsynaptic inhibition in spinal cord and other parts ofthe central nervous system (14). Binding of the agonist glycine induces an increase in chloride permeability of the postsynaptic membrane and thus antagonizes depolarization-i.e., reduces neuronal firing rate (15). The convulsive alkaloid strychnine antagonizesglycine-mediatedinhibition,andglycine-displaceable [3H]strychnine binding has been used to investigate and localize the GlyR in the nervous system of different species (16)(17)(18)(19). The GlyR has been purified from spinal cord of different mammals by affinity chromatography on aminostrychnine-agarose and shown to be a large glycoprotein containing polypeptides of Mr 48,000 (a), 58,000 ((3), and 93,000 (y) (20)(21)(22). The Mr 48,000 subunit can be covalently labeled with [3Hlstrychnine by UV illumination and thus contains the antagonist-binding site of the GlyR (23). The Mr 48,000 and 58,000 polypeptides are glycosylated (21, 24) and assumed to be homologous on the basis of immunological cr...

The etiology of Alzheimer’s disease depends on the relative abundance of different amyloid-β (Aβ) peptide species. These peptides are produced by sequential proteolytic cleavage within the transmembrane helix of the 99 residue C-terminal fragment of the amyloid precursor protein (C99) by the intramembrane protease γ-secretase. Intramembrane proteolysis is thought to require local unfolding of the substrate helix, which has been proposed to be cleaved as a homodimer. Here, we investigated the backbone dynamics of the substrate helix. Amide exchange experiments of monomeric recombinant C99 and of synthetic transmembrane domain peptides reveal that the N-terminal Gly-rich homodimerization domain exchanges much faster than the C-terminal cleavage region. MD simulations corroborate the differential backbone dynamics, indicate a bending motion at a di-glycine motif connecting dimerization and cleavage regions, and detect significantly different H-bond stabilities at the initial cleavage sites. Our results are consistent with the following hypotheses about cleavage of the substrate. First, the GlyGly hinge may precisely position the substrate within γ-secretase such that its catalytic center must start proteolysis at the known initial cleavage sites. Second, the ratio of cleavage products formed by subsequent sequential proteolysis could be influenced by differential extents of solvation and by the stabilities of H-bonds at alternate initial sites. Third, the flexibility of the Gly-rich domain may facilitate substrate movement within the enzyme during sequential proteolysis. Fourth, dimerization may affect substrate processing by decreasing the dynamics of the dimerization region and by increasing that of the C-terminal part of the cleavage region.

Abstract:The glycophorin A transmembrane segment homodimerizes to a right-handed pair of a-helices. Here, we identified the amino acid motif mediating this interaction within a natural membrane environment. Critical residues were grafted onto two different hydrophobic host sequences in a stepwise manner and self-assembly of the hybrid sequences was determined with the ToxR transcription activator system. Our results show that the motif LIxxGxxxGxxxT elicits a level of self-association equivalent to that of the original glycophorin A transmembrane segment. This motif is very similar to the one previously established in detergent solution. Interestingly, the central GxxxG motif by itself already induced strong self-assembly of host sequences and the three-residue spacing between both glycines proved to be optimal for the interaction. The GxxxG element thus appears to be the most crucial part of the interaction motif.