Differential dependence on N-glycosylation of anthrax toxin receptors CMG2 and TEM8

ANTXR 1 and 2, also known as TEM8 and CMG2, are two type I membrane proteins, which have been extensively studied for their role as anthrax toxin receptors, but with a still elusive physiological function. Here we have analyzed the importance of N-glycosylation on folding, trafficking and ligand binding of these closely related proteins. We find that TEM8 has a stringent dependence on N-glycosylation. The presence of at least one glycan on each of its two extracellular domains, the vWA and Ig-like domains, is indeed necessary for efficient trafficking to the cell surface. In the absence of any N-linked glycans, TEM8 fails to fold correctly and is recognized by the ER quality control machinery. Expression of N-glycosylation mutants reveals that CMG2 is less vulnerable to sugar loss. The absence of N-linked glycans in one of the extracellular domains indeed has little impact on folding, trafficking or receptor function of the wild type protein expressed in tissue culture cells. N-glycans do, however, seem required in primary fibroblasts from human patients. Here, the presence of N-linked sugars increases the tolerance to mutations in cmg2 causing the rare genetic disease Hyaline Fibromatosis Syndrome. It thus appears that CMG2 glycosylation provides a buffer towards genetic variation by promoting folding of the protein in the ER lumen.

Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors

Julie Deuquet Ariosa, Laurence Gouzi Abrami, Nicolas Jean Philippe Guex, Suzanne Salvi, Françoise Gisou van der Goot Grunberg

Hyaline Fibromatosis Syndrome (HIS) is a human genetic disease caused by mutations in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein thought to be involved in the homeostasis of the extracellular matrix. Little is known about the structure and function of the protein or the genotype phenotype relationship of the disease. Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level. Altogether, we show that missense mutations that map to the extracellular von Willebrand domain or the here characterized lg-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the lg-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

Wiley Open Access2011

Advanced Fluorescence Microscopy to Study Plasma Membrane Protein Dynamics

Joachim Piguet

Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane.

EPFL2010

A Biochemical and Biophysical Investigation of 5-HT3 Receptor Stability

Menno Berend Tol

The 5-hydroxytryptamine 3 receptor (5-HT3R) is a member of the pentameric ligand-gated ion channel (pLGIC) family, that plays an important role in fast signal transduction and cell-cell communication in synapses: They convert a chemical signal from a neurotransmitter into the electrical signal that represents the action potential. Activation of 5-HT3R with serotonin triggers a range of molecular reorganizations that lead to channel opening and subsequent membrane depolarization. Because of their role, pLGICs are involved in a wide range of diseases and disorders and are therefore important drug targets. A typical pLGIC is composed of a β -sheet rich extracellular domain (ECD) where ligand binding takes place, a transmembrane domain that is composed of four α-helices (TM1-TM4). In vertebrates pLGICs often contain a third intracellular domain (ICD), located between TM3 and TM4. The 5-HT3R is active as a homopentameric receptor of five identical A subunits, or as a heteropentamer containing one or more of several subunits (B to E) together with an A subunit. The 5-HT3 receptor is naturally located in the cell membrane in low amounts. For in vitro experiments such as biophysical investigation, the receptor has to be overproduced and purified in a detergent-solubilized state. Therefore, a robust and scalable expression system for homo- and heteropentameric 5-HT3 receptors was developed, building upon a tetracycline inducible cell line and Strep-tag affinity purification technology. Production of solubilized and purified 5-HT3R is well suited for use in the structural and functional characterization of the receptor. The stability of the 5-HT3 receptor is an important parameter during structural and functional experimentation. Using thermal unfolding the stability of 5-HT3R was investigated in plasma membranes as well as during detergent-extraction, purification and reconstitution into artificial lipid bilayers. A large loss in thermostability was found that correlates with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of 5-HT3R occurred in consecutive steps at distinct protein locations: First a loss of ligand binding is detected, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally leads to the formation large receptor aggregates. It was also found that structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Thermal unfolding leads to the irreversible formation of aggregates which is known to prevent effective refolding. Therefore, an further investigation of 5-HT3R was made by unfolding the receptor in urea and sodium dodecyl sulfate (SDS). The results indicate iii iv ABSTRACT that, unlike thermal unfolding, unfolding in urea and SDS does not result in the formation of aggregates. Unfolding by urea shows a 50% loss of helical content while keeping the characteristic pentamer intact. Contrastingly, unfolding by SDS shows dissociation of the pentamer, but no noticeable change in secondary structure. Still, refolding from urea and SDS proved to be impossible. Two weak fluorophores, crystal violet (CrV) and ethidium bromide (EtBr), bind to the homologous nicotinic acetylcholine receptor (nAChR). Because the fluorescence of these compounds is also sensitive towards their environment they can be used as a molecular beacon for the functional state of a receptor. When applied to 5-HT3R, it was found that CrV and EtBr bind with micromolar affinity to the desensitized state of the receptor – similar to binding reported for the nAChR. It appeared that binding of CrV or EtBr to the 5-HT3R induces a certain functional state (resting, open or desensitized). Because no detectable change in fluorescence occurs, this prevents their use as molecular beacons. Still, non-competitive binding to the 5-HT3R was little reported on before and could be an important target in pharmacological research. The ICD of the 5-HT3R is predicted to contain a stretch of ∼60 disordered or flexible residues which might be involved in subunit assembly and membrane trafficking. These unstructured regions of a 5-HT3R were probed using limited proteolysis. Thereby two distinct fragments were formed that remain tightly associated with each other, while a small stretch of 35 residues between TM3 and TM4 was removed. Furthermore, it was shown that the thermal stability of the helical core is unchanged compared to untreated 5-HT3R, but that the extracellular ligand-binding domain is very sensitive to cleavage in the ICD. By probing the 5-HT3R with limited proteolysis a rigid core was identified, indicating the important role of the transmembrane domain for holding together the pLGIC scaffold. The results presented here contribute a significant body of knowledge to membrane proteins in general and to the 5-HT3 receptor in particular, upon which further research can be based leading to advances in the fields of biochemistry, biophysics and pharmacology.