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.
Jacobs
Christine
C
Institut de Recherche Interdisciplinaire en Biologie Humaine et Moleculaire, School of Medicine, University of Brussels, Route de Lennik 808, B-1070 Brussels, Belgium. chjacobs@ulb.ac.be
Pirson
Isabelle
I
D000818:Animals; D016477:Artifacts; D019556:COS Cells; D002352:Carrier Proteins; D002522:Chlorocebus aethiops; D004285:Dogs; D019204:GTP-Binding Proteins; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D015854:Up-Regulation; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011989:Receptors, Thyrotropin; D033921:Vesicular Transport Proteins; D019204:GTP-Binding Proteins; C108941:RABAC1 protein, human