Kainate-binding human CNS receptors of the EAA4 family
a human cns receptor and kainate technology, applied in the field of kainate-binding human cns receptors of the eaa4, can solve the problems of limited availability of brain tissue of human origin, difficult development of therapeutics which modulate these processes, and complicated search for human therapeutics
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example 1
Isolation of DNA Coding for the Human EAA4a Receptor
[0050] cDNA coding for the human EAA4a receptor was identified by probing human fetal brain cDNA that was obtained as an EcoRI-based lambda phage library (lambda ZAP) from Stratagene Cloning Systems (La Jolla, Calif., U.S.A.). The cDNA library was screened using an oligonucleotide probe capable of annealing to the 3' region of the rat GluR5 receptor sequence reported by Bettler et al., supra. The specific sequence of the .sup.32P-labelled probe (SEQ ID NO: 9) is provided below:
[0051] 5'-ATCGGCGGCATCTTCATTGTTCTGGCTGCAGGACTCGTGC-3'
[0052] The fetal brain cDNA library was screened under the following hybridization conditions; 6.times. SSC, 25% formamide, 5% Denhardt's solution, 0.5% SOS, 100 ug / ml denatured salmon sperm DNA, 42C. Filters were washed with 2.times. SSC containing 0.5% SDS at 25C. for 5 minutes, followed by a 15 minute wash at 50C. with 2.times. SSC containing 0.5% SDS. The final wash was with 1.times. SSC containing 0.5%...
example 2
Construction of Genetically Engineered Cells Producing the Human EAA4a Receptor
[0054] For transient expression in mammalian cells, cDNA coding for the human EAA4a receptor was incorporated into the mammalian expression vector pcDNA1, which is available commercially from Invitrogen Corporation (San Diego, Calif., USA; catalogue number V490-20). This is a multifunctional 4.2 kb plasmid vector designed for cDNA expression in eukaryotic systems, and cDNA analysis in prokaryotes. Incorporated on the vector are the CMV promoter and enhancer, splice segment and polyadenylation signal, an SV40 and Polyoma virus origin of replication, and M13 origin to rescue single strand DNA for sequencing and mutagenesis, Sp6 and T7 RNA promoters for the production of sense and anti-sense RNA transcripts and a Col E1-like high copy plasmid origin. A polylinker is located appropriately downstream of the CMV promoter (and 3' of the T7 promoter).
[0055] The strategy depicted in FIG. 2 was employed to facilita...
example 3
Ligand Binding Assays
[0059] Transfected cells in the frozen state were resuspended in ice-cold distilled water using a hand homogenizer and centrifuged for 20 minutes at 50,000 g. The supernatant was discarded and the membrane pellet stored frozen at -70.degree. C.
[0060] COS cell membrane pellets were suspended in ice cold 50 mM Tris-HCl (pH 7.55, 5.degree. C.) and centrifuged again at 50,000 g for 10 minutes in order to remove endogenous glutamate that would compete for binding. Pellets were resuspended in ice cold 50 mM Tris-HCl (pH 7.55) buffer and the resultant membrane preparation was used as tissue source for binding experiments described below. Proteins were determined using the Pierce Reagent with BSA as standard.
[0061] Binding assays were then performed, using an amount of COS-derived membrane equivalent to 25-100 ug as judged by protein determination and selected radiolabelled ligand. In particular, for kainate binding assays, incubation mixtures consisted of 25-100 pg tis...
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