Functional reconstitution of purified metabotropic glutamate receptor expressed in the fly eye

Abstract

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.

In Vivo Reconstitution of γ-Secretase in Drosophila Results in Substrate Specificity

“…This tissue is especially suitable for the characterization of membrane proteins due to its high membrane content and suitability for biochemical studies (16,20,29). Furthermore, the Drosophila eye is a well-established system to model neurodegenerative diseases, including Alzheimer's disease (28), and requires Notch signaling and, thus, ␥-secretase activity for its development (5).…”

Agonist-dependent Signaling by Group I Metabotropic Glutamate Receptors Is Regulated by Association with Lipid Domains

“…Whereas cholesterol was shown to modify ligand binding affinity of some class A GPCRs, it may also act by stabilizing receptor dimers or the 7-TM region in active conformation favoring G protein coupling, two possibilities that are not mutually exclusive. DmGluRA, the Drosophila ortholog of vertebrate group II mGluRs (72), can be cross-linked to cholesterol (73) and associates with sterol-rich membranes that enhance its agonist affinity (73,74), indicating that cholesterol may contribute to stabilization of the ligand-bound active conformation of DmGluRA, although the structural elements supporting the interaction and its functional impact were not examined.…”

Glutamate-binding affinity ofDrosophilametabotropic glutamate receptor is modulated by association with lipid rafts

“…This model postulates the existence of modulators that act on the 7TMD, which in turn influences ligand binding. Interestingly, we have previously (8) shown for reconstituted DmGluRA, that ligand binding depends on the presence of ergosterol in the liposomes. This finding suggests that a sterol-7TMD interaction is involved in the regulation of ligand binding.…”

“…The first Drosophila mGluR cloned, (DmGluRA), shares the structural features characteristic for mammalian mGluRs (6). Recently, we have reported the expression of DmGluRA in insect cells by recombinant baculovirus infection (7), and in transgenic Drosophila melanogaster (8), where the receptor is directed to the photoreceptor cells in the fruit fly eye, and is colocalized with rhodopsin in specialized membrane stacks, called rhabdomeres.…”