Fluorescence resonance energy transfer was used to determine the structural changes in the extracellular ligand-binding segment in a functional glutamate receptor that contains the ligand-binding, transmembrane, and C-terminal segments. These studies indicate that the structural changes previously reported for the isolated ligand-binding domain due to the binding of partial and full agonists are also observed in this functional receptor, thus validating the detailed structure-function

more »

... ips that have been previously developed based on the structure of the isolated ligand-binding domain. Additionally, these studies provide the first evidence that there are no significant changes in the extent of cleft closure between the activated and desensitized states of the glutamate bound form of the receptor consistent with the previous functional investigations, which suggest that desensitization is mediated primarily by changes in the interactions between subunits composing the receptor. Ionotropic glutamate receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system and hence play a key role in processes such as learning and memory (1-5). Glutamate binding to the extracellular soluble ligand-binding domain (S1S2) 1 segment of the protein triggers a series of conformational changes that leads to receptor activation (formation of a cation selective transmembrane channel) and subsequent desensitization. The determination of the structure of the S1S2 for the GluR2 subunit of the glutamate receptor (6 -9) complemented with spectroscopic investigations (10 -13), in combination with the vast existing electrophysiological data on the native receptor (9, 14, 15), have provided the first direct structural insight into how the changes at the ligand-binding site lead to the sequence of conformational changes that regulate ion flow in this class of important proteins. The validity of these inferences hinges on the assumption that the isolated ligand-binding domain is a good model for the domain in a functional receptor, i.e. the ligand-binding domain in the presence of the functional unit, namely the transmembrane segments forming the ion channel. The assumption that the S1S2 protein is a representative model of the domain in the full glutamate receptor is currently based on equilibrium ligand binding assays that suggest similar ligand binding properties for the two proteins (16, 17) and recent UV-visible spectroscopic investigations that indicate a similar electronic environment for the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline in the S1S2 protein as in the full-length receptors expressed in cells (18) . In this report we have used fluorescence resonance energy transfer (FRET) to provide the first direct confirmation that the ligand-binding domain of a functional glutamate receptor exhibits the same conformational changes (cleft closure) due to agonist binding as that observed in the structures of the S1S2 protein. For these FRET investigations we have modified the cysteine light ⌬N-GluR4 homomeric receptors. The ⌬N-GluR4 receptors have the extracellular Nterminal domain (residues 1-402) deleted from the GluR4 flip subunit of the ␣-amino-5-methyl-3-hydroxy-4-isoxazole propionate subtype of the glutamate receptor and represent the minimum model construct essential for the full function, namely agonist-induced activation and desensitization, of the receptor. The two extracellular non-disulfide-bonded cysteine residues (426 and 529) in this ⌬N-GluR4 construct were mutated to serine, providing a receptor with no accessible cysteines. Green fluorescent protein (GFP) was then introduced at the N terminus to this modified protein to serve as the acceptor, and cysteines were introduced at specific positions and tagged using a maleimide derivative of terbium chelate to serve as the donor molecule and distances between donor and acceptor measured based on the FRET efficiency. MATERIALS AND METHODS Mutations and eGFP-tagged Cysteine Light GluR4 Construct Preparation-Using the construct for the GluR4-flip receptor with the first 402 residues deleted (generously provided by Dr. Keinanen, University of Helsinki, Helsinki, Finland) the two accessible non-disulfide-bonded cysteine residues, Cys 426 present in the extracellular side as well as Cys 529 present in the first transmembrane segment of the receptor, were mutated to serine using the Stratagene QuikChange site-directed mutagenesis kit (Stratagene). For introducing the GFP at the N terminus of this double mutant, the ⌬N-GluR4-C426S-C529S construct was amplified with PCR using primers containing XhoI and BamHI restriction enzyme sites; the PCR product was digested by using the XhoI and BamHI restriction enzymes and subcloned into the corresponding sites of pEGFP-C1 plasmid (Invitrogen). This construct was digested with NheI and BamHI, and the 2.3-kb fragment, which contained the ⌬N-GluR4-C426S-C529S construct cDNA fused with coding sequence for eGFP at the N terminus, was cloned into pcDNA3.1. The construct thus obtained (⌬N*-GFP) served as the background construct on which residues 444, 446, 653, and 686 were then individually mutated to cysteine to yield the four constructs used for the FRET study. For the donor only experiments the same mutations were performed on the ⌬N-GluR4-C426S-C529S without the presence of GPF tag (⌬N*). Mutations in the plasmids were introduced using the Stratagene QuikChange site-directed mutagenesis kit (Stratagene, CA), and the integrity of the final