Regions of α-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits that are permissive for the insertion of green fluorescent protein

The green fluorescent protein can be fused to the ends of a mature glutamate receptor subunit to produce functional, fluorescent receptors. However, there are good reasons to search for internal regions of receptor subunits that can tolerate green fluorescent protein insertion. First, internal inser...

Full description

Saved in:

Bibliographic Details
Published in:	Neuroscience Vol. 141; no. 2; pp. 837 - 849
Main Authors:	Sheridan, D.L., Robert, A., Cho, C.H., Howe, J.R., Hughes, T.E.
Format:	Journal Article
Language:	English
Published:	Oxford Elsevier Ltd 01-01-2006 Elsevier
Subjects:	Amino Acid Sequence AMPA receptor Biological and medical sciences Blotting, Western - methods Cell Line Flow Cytometry - methods Fundamental and applied biological sciences. Psychology fusion protein Gene Expression - physiology glutamate receptor subunit Glutamic Acid - pharmacology green fluorescent protein Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism Humans Immunohistochemistry - methods insertional tagging Membrane Potentials - drug effects Membrane Potentials - genetics Membrane Potentials - physiology Mutagenesis - physiology Oligopeptides Patch-Clamp Techniques - methods Peptides - genetics Peptides - metabolism Protein Subunits - genetics Protein Subunits - metabolism Receptors, AMPA - chemistry Receptors, AMPA - metabolism Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Tn 5 transposon Transfection - methods Vertebrates: nervous system and sense organs YFP AMPA GluR glutamate receptor subunit CFP green fluorescent protein mGluR GFP ATD AMPA receptor fusion protein CTD insertional tagging ME Tn 5 transposon FRET Green fluorescent protein Glutamate receptor Subunit Tn5 transposon Biological receptor
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	The green fluorescent protein can be fused to the ends of a mature glutamate receptor subunit to produce functional, fluorescent receptors. However, there are good reasons to search for internal regions of receptor subunits that can tolerate green fluorescent protein insertion. First, internal insertions of green fluorescent protein may produce functional, fluorescent subunits that traffic more correctly. Second, fluorescent proteins inserted near interacting surfaces of subunits could potentially create reagents suitable for fluorescence resonance energy transfer measurements. Finally, internal green fluorescent protein insertions could potentially produce subunits capable of signaling conformational changes through intrinsic changes in fluorescence intensity. To identify regions of receptor subunits that are permissive for green fluorescent protein insertion, we used a series of recombinant transposons to create fluorescent protein insertions in three α-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits. A combined analysis of the relative fluorescence intensity and glutamate-gated ion channel function of 69 different green fluorescent protein fusion proteins identified permissive zones for the creation of bright and fully functional receptor subunits in the C-terminal portion of the amino terminal domain, the intracellular tail of the carboxy terminal domain, and within the pore-forming regions of the channel.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0306-4522 1873-7544
DOI:	10.1016/j.neuroscience.2006.04.052