On the use of cells or membranes for receptor binding: growth hormone secretagogues.
Receptor binding techniques have been widely used in different biochemical applications, with isolated membranes being the most used receptor preparation in this type of assays. In this study, intact cells were compared with isolated membranes as receptor support for radioligand receptor binding assay. The growth hormone secretagogue receptor 1a (GHSR-1a) expressed in human embryonic kidney 293 (HEK293) cells was used as a model of G-protein-coupled receptors. Differences between using intact cells in suspension and using isolated membranes were evaluated for different aspects of the receptor binding assay: total binding variations while both receptor preparations remain on ice, modifications in incubation conditions, saturation, and competition using different agonists. Intact cells are more prone to variability. Although under optimized settings both preparations were equivalent, the K(d) value for intact cells was three times higher than that using isolated membranes. However, no significant differences were observed in competition assays obtaining practically identical K(i) values for all ligands tested. For the GHSR-1a, isolated membranes are the better choice if particular incubation conditions are required (less variability), whereas intact cells yield easy, fast, and physiological conditions for receptor binding assays.
Pinyot
A
A
Bioanalysis Group, Neuropsychopharmacology Program, Municipal Institute for Medical Research-Hospital del Mar, 08003 Barcelona, Spain.
Nikolovski
Z
Z
Bosch
J
J
Segura
J
J
Gutiérrez-Gallego
R
R
D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods*; D054440:Receptors, Ghrelin / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D054440:Receptors, Ghrelin