Michael D. Edgerton
Necker-Enfants Malades Hospital
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Featured researches published by Michael D. Edgerton.
Journal of Biological Chemistry | 1996
Gerardo Turcatti; Karin Nemeth; Michael D. Edgerton; Ulrich Meseth; François Talabot; Manuel C. Peitsch; Jonathan Knowles; Horst Vogel; André Chollet
A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligand-receptor interactions in the prototypic seven-transmembrane neurokinin-2 receptor (NK2) using this fluorescence-based approach is presented. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants containing an unique 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dap) residue at either site 103, in the first extracellular loop, or 248, in the third cytoplasmic loop, were functionally active. The fluorescent NK2 mutants were investigated by microspectrofluorimetry in a native membrane environment. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calculated by the theory of Förster, permit to fix the ligand in space and define the structure of the receptor in a molecular model for NK2 ligand-receptor interactions. Our data are the first report of the incorporation of a fluorescent unnatural amino acid into a membrane protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of experimental distances between a G protein-coupled receptor and its ligand by FRET. The method presented here can be generally applied to the analysis of spatial relationships in integral membrane proteins such as receptors or channels.
FEBS Letters | 1994
Michael D. Edgerton; Christian Chabert; André Chollet; Steve Arkinstall
Gqα and G11α differ from other G protein α subunits in that they have unique, conserved 6 residue amino‐terminal extensions. Wild‐type and amino‐terminal mutants of Gqα expressed in COS cells were analyzed for their ability to functionally couple with co‐expressed neurokinin NK2 receptor. Wild‐type, T2A and Δ2–7 Gqα were able to stimulate agonist driven phospholipase C (PLC) activity in identical manners. Other activities of these two amino‐terminal mutants including aluminum fluoride stimulated PLC activity, palmitoylation, interaction with Gβγ subunits and GTPγS‐induced trypsin resistance are also similar to the wild‐type α subunit. This demonstrates that the NK2 receptor is able to functionally interact with the α subunit of Gq and that the first seven amino‐acids of Gqα are not required for any of the α subunit functions tested. In contrast to the T2A and Δ2–7 mutants, a C9,10A Gqα mutant was not able to couple to either the NK2 receptor or PLC, as assessed by high‐affinity agonist binding and activation of PLC either in intact cells or in vitro. The C9,10A protein was able to assume a GTPγS‐induced trypsin‐resistant conformation and partitioned primarily to the pelletable fraction in a manner similar to the wild‐type protein. However, it was not labeled with [3H]palmitic acid. This suggests that blocking palmitoylation at the amino‐terminus of Gqα results in a loss of functional activity which reflects an inability to interact with both the receptor and downstream signaling targets.
Nature Biotechnology | 1998
Fabrizio Arigoni; François Talabot; Manuel C. Peitsch; Michael D. Edgerton; Eric Meldrum; Elisabeth Allet; Richard Fish; Therese Jamotte; Marie-Laure Curchod; Hannes Loferer
Journal of Biological Chemistry | 1995
Gonzalo Mazzei; Michael D. Edgerton; Christophe Losberger; Sybille Lecoanet-Henchoz; Pierre Graber; Anne Durandy; Jean-Francois Gauchat; Alain Bernard; Bernard Allet; Jean-Yves Bonnefoy
Journal of Biological Chemistry | 1996
Fabienne Pietravalle; Sybille Lecoanet-Henchoz; Horst Blasey; Jean-Pierre Aubry; Greg Elson; Michael D. Edgerton; Jean-Yves Bonnefoy; Jean-François Gauchat
Receptors & Channels | 1997
Gerardo Turcatti; Karin Nemeth; Michael D. Edgerton; Jonathan Knowles; Horst Vogel; André Chollet
Methods of Molecular Biology | 2000
Michael D. Edgerton; Lars-Ole Gerlach; Thomas Boesen; Bernard Allet
Archive | 1999
Fabrizio Arigoni; Michael D. Edgerton; Hannes Loferer; Manuel C. Peitsch
Archive | 1999
Fabrizio Arigoni; Michael D. Edgerton; Hannes Loferer; Manuel C. Peitsch
Archive | 1999
Fabrizio Arigoni; Michael D. Edgerton; Hannes Loferer; Manuel C. Peitsch