The use of site-directed mutagenesis, transient transfection, and radioligand binding. A method for the characterization of receptor-ligand interactions.
Receptor-ligand interactions have traditionally been evaluated using a number of biochemical techniques including radioligand binding, photoaffinity labeling, crosslinking, and chemical modification. In modern biochemistry, these approaches have largely been superseded by site-directed mutagenesis in the study of protein function, owing in part to a better understanding of the chemical properties of oligonucleotides and to the ease with which mutant clones can now be generated. The Altered Sites II in vitro Mutagenesis System from the Promega Corporation employs oligonucleotides containing two mismatches to introduce specific nucleotide substitutions in the nucleic acid sequence of a target DNA. One of these mismatches will alter the primary sequence of a given protein, whereas the second will give rise to a silent restriction site that is used to screen for mutants. Transient transfection of tsA201 cells with mutant cDNA constructs using calcium phosphate as a carrier for plasmid DNA permits expression of recombinant receptors that can be characterized using radioligand binding assays. In this article, we focus on site-directed mutagenesis, heterologous expression in eukaryotic cells, and radioligand binding as a methodology to enable the characterization of receptor-ligand interactions.
Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada.
J G
JG
Newell
M
M
Davies
A N
AN
Bateson
D002130:Calcium Phosphates / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D015247:Deoxyribonuclease HindIII / Q000737:chemistry; D008024:Ligands; D008967:Molecular Biology / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; D008024:Ligands; D009841:Oligonucleotides; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; D004247:DNA; C020243:calcium phosphate; D015247:Deoxyribonuclease HindIII; C485829:calcium phosphate, dibasic, anhydrous