The Participation of the Endoplasmic Reticulum Protein Chaperone Thio-oxidoreductase in Gonadotropin Releasing Hormone Receptor Expression at the Plasma Membrane

摘要

Protein disulfide isomerase (PDI) family chaperone members 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 members. ERp18 shows partial oxidative activity as a PDI. The aim of the present study was to evaluate the participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin releasing hormone receptor expression at the plasma membrane. COS-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) WT human GnRHR, 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 μCi myo-[2-3H(N)]-inositol in 0.25 ml DMEM and treated for 2 h with Buserelin. We observed a decrease in maximal IP production in response to Buserelin in the cells co-transfected with hGnRHR, and from 20 ng to 75 ng of hERp18 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 change the maximal IP production when they were co-transfected with hERp18. These results suggest that hERp18 has a reduction role on disulfide bond in wild type hGnRHR folding.