Titrating the expression of a Gi protein-coupled receptor using an ecdysone-inducible system in CHO-K1 cells.
Changes in receptor density are often associated with pathological conditions. For example, high levels of the G protein-coupled somatostatin receptor, sst2, have been detected in a number of malignant cell types, a characteristic feature that is routinely utilised as a diagnostic tool. However, how the increased receptor expression affects cellular function through alterations in G protein-coupling or changes in the intensity or duration of activated signalling pathways is poorly understood. The current report details the use of an ecdysone-inducible expression system in CHO-K1 cells, whereby the consequence of modulating the level of human sst2 receptor expression on specific transduction events can be examined. A time- and concentration-dependent induction of sst2 receptor expression was attained by exposure of cells to the ecdysteroid-inducing agent, muristerone A (MuA). Increases in sst2 receptor expression were determined by immunoassay, immunoblotting and immunocytochemical analysis. Maximal sst2 receptor expression was obtained after treatment of cells with 7 microM MuA for 24 h. Functionality of the sst2 receptor was assessed by immunoblot analysis of phosphorylated forms of MAP kinase. Following receptor activation, time-dependent increases in the level of MAP kinase phosphorylation were shown to correlate with the degree of sst2 receptor induction. Confirmation of receptor activation was determined by visualisation of ligand-induced redistribution of sst2 receptors from the plasma membrane to discrete intracellular compartments. However, in a series of further studies, both immunocytochemical and fluorescence-activated cell sorting (FACS) analyses demonstrated that over a prolonged period, stable receptor expression could not be maintained in CHO-K1 cells using this expression system. Thus, routine analysis of the sst2 receptor expressing cell population is required to derive comparable results between assays, especially when some assays provide information from the whole cell population whilst others are based at the single cell level. On the basis of these observations we conclude that, providing such quality control measurements are taken, the ecdysone inducible expression system is a useful tool to modulate functional sst2 receptor expression in an in vitro environment over short time periods.
Cole
S L
SL
Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QJ, United Kingdom. slc77453@glaxowellcome.co.uk
Schindler
M
M
Sellers
L A
LA
Humphrey
P P
PP
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004441:Ecdysterone / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D017481:Receptors, Somatostatin / Q000235:genetics* / Q000378:metabolism; D017186:Titrimetry
D017481:Receptors, Somatostatin; C106140:somatostatin receptor sst2A; C055348:muristerone A; D004441:Ecdysterone; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go