Monitoring expression and clustering of the ionotropic 5HT3 receptor in plasma membranes of live biological cells

The ionotropic 5HT3 receptor was expressed in transiently transfected mammalian cells, yielding an unprecedented high concn. of up to 12 million receptors per cell. Receptor traffic in the plasma membrane of live cells was obsd. continuously over 24 h by fluorescence scanning confocal microscopy. This was possible by using 5HT3 receptor-specific fluorescent ligands with high binding affinity and low off-rate to pulse label receptors at any time after appearance on the cell surface, and label subsequently those receptors expressed later by another, spectrally distinguishable, high-affinity fluorescent ligand. Having reached a crit. cell surface concn. of .apprx.3000 receptors/mm2, the receptors started to aggregate in patches with a 4-fold increased surface concn. The clusters were constantly delivered from a pool of freshly expressed receptors isotropically distributed within the basolateral region of the cell membrane. From there, they migrated to and accumulated on the apical cell surface .apprx.9 h after transfection. Individual clusters grew until they reached a crit. size of 1-2 mm when they merged to form with 3-5 mm large macroclusters. Clustered receptors were immobile on the minute time scale but always coexisted with monomeric receptors in the regions surrounding the clusters as revealed by fluorescence correlation spectroscopy. Because the receptor d. of 12,000 receptors/mm2 in the patches is as high as that found in two-dimensional crystals of certain membrane proteins, such patches might be a proper source for direct crystn. of membrane proteins without prior purifn. [on SciFinder (R)]

Transmembrane Signaling Analysis in Model Membrane Systems

Luigino Grasso

Studying the complex mechanisms of transducing an extracellular signal across a plasma membrane to initiate an intracellular responce is of fundamental importance for a cell’s function. Transmembrane signaling is the key allowing cells to sense and communicate with its environment. Signal transduction is primarily mediated by numerous membrane receptors. In this thesis we investigated the function of G-protein-coupled receptors (GPCR). GPCRs represent the largest and most diverse group of membrane proteins, encoded by more than 800 genes. They allow cells to recognize as diverse extracellular stimuli as photons, organic odorants, nucleotides, nucleosides, peptides, lipids and proteins, conferring to GPCRs a fundamental role in signal transduction. They are broadly distributed across the entire body and thus involved in a wide range of physiological and pathological processes, which makes GPCRs one of the main targets of modern medicines. Activation of GPCRs initiate numerous intracellular signaling pathways, involving a large number of proteins and molecules. They act as a central molecular activators and integrators of a complex network of signaling pathways. While most of the studies have been performed on cells, the complexity of cellular systems has raised the need to develop simpler model systems to assess the role of individual signaling com- ponents. In this thesis we present different model systems to study the functionality of GPCR signal transduction from the ligand binding to the activation of downstream cellular reactions. The first part of the thesis concerns the isolation of single receptors from cultured cells by detergent solubilization followed by purification and the subsequent receptor reconstitution into giant unilamellar vesicles (GUV). Detergent micelle receptors were analyzed by fluorescence correlation spectroscopy (FCS) and surface plasmon resonance (SPR) measurements. We could determine the conditions of functional solubilization of GPCRs by characterizing the amount of obtained receptor-detergent micelles, their homogeneity and the capacity of the receptor to selectively bind ligands. The receptors were then reconstituted into GUVs and ligand binding was monitored by confocal microscopy. In the second part of the thesis, we investigated different aspects properties of native plasma membrane vesicles. These vesicles are derived from live mammalian cells using v cytochalasin B or optical tweezers; they comprise parts of a cell’s plasma membrane and cytosol. After the characterization of their overall composition, we investigated their ability to mediated transmembrane activation of intracellular signaling reactions upon agonist binding to a GPCR. We could successfully monitor the ligand binding to receptor by FCS, the subsequent G-protein activation by intermolecular FRET and the receptor desensitization by monitoring the arrestin recruitment to the plasma membrane. These vesicles thus represent the smallest autonomous container performing cellular signaling reactions thus functioning like a minimized cell. Finally, preliminary experiments demonstrated the potential of native vesicles to serve as novel drug delivery system since they meet most of the requirements for an efficient drug delivery platform.

EPFL2012

Investigation of the neurokinin-1 receptor by fluorescence techniques

Bruno Meyer

G protein-coupled receptors (GPCR) constitute by far the largest family of transmembrane cell-surface proteins involved in signal transduction and are the most important targets for the development of novel therapeutic compounds. Many processes mediated by GPCR signal transduction are still not well understood at the molecular level and novel methods for their investigation are of general interest. The thesis presented here describes investigations on the neurokinin-1 receptor (NK1R), a member of the GPCR family, using fluorescence techniques. Fluorescence resonance energy transfer (FRET), the technique of preference in this work, is used to measure ligand-protein and protein-protein interactions in living cells. In order to reach this goal, the NK1R has been labelled with either fluorescent proteins or by using the novel post-translational ACP labelling technique. A FRET-based method for monitoring specific ligand binding to NK1R is described. In this context, a fluorescent agonist for NK1R was synthesized and characterized. The ACP labelling technique was shown to be superior for this kind of experiments compared to labelling with fluorescent proteins. The same ACP labelling approach was shown to be ideally suited to study oligomerization of membrane proteins by FRET. It was shown, that the NK1R does not form homo-dimers, as many other GPCRs do. From these experiments we furthermore conclude, that the NK1R is present in small microdomains in the membrane. Interactions between the NK1R and the different subunits of the heterotrimeric Gαqβ1γ2-complex were investigated by FRET using different instrumentation. Proximity of the NK1R and the G protein could be detected, but no change in FRET was observed after activation of the receptor. Total internal reflection fluorescence (TIRF), was used to develop an assay for studying ligand binding in vitro. Here, the NK1R was immobilized directly on a surface using a cell membrane preparation. This method was shown to be highly sensitive.

EPFL2005

Investigating molecular interactions of G protein coupled receptors by fluorescence techniques

Jean-Baptiste Perez

G Protein coupled receptors (GPCRs) constitute an abundant family of membrane proteins which play a central role in cellular signaling and are important targets for modern medicine. Despite intensive research, central questions of the molecular mechanism of GPCR mediated signal transduction remain unresolved. This thesis concerns α1b-adrenergic receptor (α1b-AR) as a prototypic GPCR. The question whether ARs form homo- or hetero-oligomers was investigated using fluorescence resonance energy transfer (FRET). An important finding was that both α1a and α1b-AR subtypes form homo-oligomers. Also hetero-oligomers have been observed between the α1b- and the α1a-AR subtypes, but not with less related receptors. Another interesting finding concerns α1-AR co-internalization which correlates with the ability of the receptor to hetero-oligomerize. Investigating particular processes in living cells is often complicated by the complex cellular environment. Here we have developed a rather simple approach to study transmembrane signaling by using supported cell membrane sheets. They were prepared by pressing a glass coverslip on the apical part of cultured cells, ripping off large regions of the native plasma membrane. For the α1b-AR we still could observe ligand binding to such sheets, indicating that GPCR functionality was at least partly conserved. Due to the absence of cytosolic autofluorescence of the cells, supported membrane sheets were also found to be suited for single-molecule microscopy. Because both leaflets of the supported membranes remained fluid, it was possible to follow the diffusion of certain membrane proteins. For example using fluorescence recovery after photobleaching (FRAP) and single-molecule microscopy, we investigated how G proteins diffused along the membrane and compartmentalized. The predominant role of lipid anchors in G protein localization was highlighted by measuring the diffusion of two Gαq proteins fused with citrine at two different positions and citrine based constructs designed to mimic the monomeric and the trimeric form of a G protein. Finally, first results were obtained showing the feasibility to form supported cell membrane sheets on microstructured devices consisting of a planar silicon support perforated with arrays of holes. This geometry provides a full accessibility to both intra- and extracellular sides of the bilayer. This concept could potentially be applied for the development of chip-based screening assays.

EPFL2005

Investigation of the neurokinin-1 receptor by fluorescence techniques

Bruno Meyer

EPFL2005

Transmembrane Signaling Analysis in Model Membrane Systems

Luigino Grasso

EPFL2012

Investigating molecular interactions of G protein coupled receptors by fluorescence techniques

Jean-Baptiste Perez

EPFL2005