Regions of alpha-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 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 alpha-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.
Sheridan
D L
DL
Interdepartmental Neuroscience Program, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA; Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Robert
A
A
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Cho
C H
CH
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Howe
J R
JR
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Hughes
T E
TE
Department of Cell Biology and Neuroscience, Montana State University, 513 Leon Johnson Hall, Bozeman, MT 59717, USA. Electronic address: thughesgfp@mac.com.
D000595:Amino Acid Sequence; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression / Q000502:physiology; D018698:Glutamic Acid / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D016296:Mutagenesis / Q000502:physiology; D009842:Oligopeptides; D018408:Patch-Clamp Techniques / Q000379:methods; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D018091:Receptors, AMPA / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D009842:Oligopeptides; D010455:Peptides; D021122:Protein Subunits; D018091:Receptors, AMPA; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; C085706:FLAG peptide