Pitfalls in the use of transfected overexpression systems to study membrane proteins function: the case of TSH receptor and PRA1.

Thyrotropin (TSH) stimulates thyroid cell differentiation and proliferation by binding to its G-protein coupled receptor (GPCR) receptor, the thyrotropin receptor (TSHR). To understand some differences in transduction between human and dog cells a search for partners proteins was performed by a two-hybrid screen with the dog C-terminal tail of the receptor as bait. Prenylated Rab acceptor 1 (PRA1) was identified by this way. Pursuing with the characterization of the biochemical interaction, we performed co-transfection experiments in COS-7 cells and we observed, by measurement of the intracellular cAMP produced and by FACS experiments, that the co-expression of two membrane proteins (PRA1, NIS, LH/CGR with TSHR) alters their expression. This effect seems to be restricted to co-expression of two-membrane proteins because the expression does not decrease when the membrane protein is co-expressed with a cytosolic protein (Rab9, Cyclin D3). This pitfall should be considered in such expression experiments.