Bungarotoxin Binding Site (BBS)-tagging of Proteins

A novel approach to tag proteins using a minimal bungarotoxin binding site (BBS-tag). In this method we have developed a new method for the visualization of protein trafficking. We used a 13 amino acid a-bungarotoxin binding site (BTX) derived from the nicotinic acetylcholine receptor to tag the extracellular domain of the GluR1 and GluR2 subunits of AMPA receptors. These BBS-tagged receptors bind BTX and using rhodamine-labeled a-BTX we were able to specifically label and detect surface receptors in transfected cells. In addition, using this tag we could image internalization and insertion of receptors in living cells. Moreover, we constructed doubly tagged BBS-GFP-tagged GluR1 and GluR2 subunits and used fluorescence resonance energy transfer (FRET) to BTX binding and visualize receptor trafficking in living neurons. As BTX is also commercially available in several fluorescently labeled forms as well as in biotinylated and radioactive (125I and 3H) forms, this tag can be used to image, isolate and quantify the expression of BBS-tagged proteins. This approach is a potentially powerful novel technique to tag proteins and study membrane protein trafficking. Description (Set) Proposed Use (Set) In recent studies, GFP tagged receptor subunits have been shown to be useful to observe the cellular trafficking and synaptic targeting of proteins. However, using this approach it has been hard to distinguish intracellular and extracellular membrane proteins in living neurons. With this invention, we have developed a new method for the visualization of membrane protein trafficking.

Inventor(s): Huganir, Richard

Type of Offer: Licensing



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