Functional reconstitution of purified metabotropic glutamate receptor expressed in the fly eye.
G-protein-coupled receptors (GPCRs) form one of the largest superfamilies of membrane proteins. Obtaining high yields of GPCRs remains one of the major factors limiting a detailed understanding of their structure and function. Photoreceptor cells (PRCs) contain extensive stacks of specialized membranes where high levels of rhodopsins are naturally present, which makes them ideal for the overexpression of GPCRs. We have generated transgenic flies expressing a number of GPCRs in the PRCs. Drosophila melanogaster metabotropic glutamate receptor (DmGluRA) expressed by this novel strategy was purified to homogeneity, giving at least 3-fold higher yields than conventional baculovirus expression systems due to the higher membrane content of the PRCs. Pure DmGluRA was then reconstituted into liposomes of varying composition. Interestingly, glutamate binding was strictly dependent on the presence of ergosterol.
Eroglu
Cagla
C
European Molecular Biology Laboratory, Structural Biology and Computational Biology Programme, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Cronet
Philippe
P
Panneels
Valérie
V
Beaufils
Philippe
P
Sinning
Irmgard
I
D000818:Animals; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D004875:Ergosterol / Q000378:metabolism; D005123:Eye / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism*; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D018094:Receptors, Metabotropic Glutamate; D018698:Glutamic Acid; D004875:Ergosterol