Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane.
Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 microCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.
Lucca-Junior
W
W
Division of Neuroscience and Reproductive Sciences, Oregon National Primate Research Center, Oregon Health and Science University, ONPRC/OHSU, Beaverton, Oregon, USA. wlucca1@hotmail.com
Janovick
J A
JA
Conn
P M
PM
D000818:Animals; D002064:Buserelin / Q000378:metabolism / Q000494:pharmacology; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002522:Chlorocebus aethiops; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D009154:Mutation; D011490:Protein Disulfide Reductase (Glutathione) / Q000235:genetics / Q000378:metabolism*; D011966:Receptors, LHRH / Q000378:metabolism*
D007295:Inositol Phosphates; D011966:Receptors, LHRH; D011490:Protein Disulfide Reductase (Glutathione); C477009:TXNDC12 protein, human; D002064:Buserelin