Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Abstract

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 (Cys 14 Ala and Cys 200 Ala) 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 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 (Cys 14 Ala and Cys 200 Ala) that could not form the Cys 14 -Cys 200 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.Key words: Protein chaperones; Gonadotropin-releasing hormone receptor; Calnexin; Calreticulin; ERp18Research supported by NIH grants HD-19899, RR-00163, HD-18185, and TW/HD-00668. Received February 18, 2008. Accepted December 11, 2008 After synthesis in the endoplasmic reticulum (ER), G protein-coupled receptors (GPCRs) are often folded and assembled to be packaged into the ER-derived COPIIcoated vesicles and transported through the Golgi apparatus and the trans-Golgi network in order to arrive at the plasma membrane. During transportation through the ER and Golgi structures, GPCRs are submitted to post-translational modifications to acquire the mature conformation (1). These events -folding and trafficking of newly synthesized proteins -are highly regulated processes that likely require a number of different chaperone molecules belonging to the cell's quality control system (QCS). These QCS chaperones recognize non-native conformations of newly synthesized proteins and prevent their aggregation and export of the incompletely folded chains from the ER (2). When the maturation of a newly synthesized protein is aborted or inefficiently performed, chaperones catalyze a covalent bond between ubiquitin and the unfolded protein.

Genome-Wide Linkage Analysis of Global Gene Expression in Loin Muscle Tissue Identifies Candidate Genes in Pigs

“…TXNDC12 has also been shown to be expressed in many human tissues including skeletal muscle [25] . While no studies have considered the function of TXNDC12 in skeletal muscle, its role as a thiol-disulfide oxidoreductase in other cell types has been demonstrated [26] , [27] , [28] . Our results indicate SSC6 pQTL for protein content, loin muscle area (LMA) and loin muscle chop weight coincident with an eQTL for TXNDC12 .…”

A structural overview of the PDI family of proteins

“…This conserved proline might be important for ERp18 function, although a specific requirement for ERp18 function has yet to be determined. ERp18 shows specificity for a component of the complement cascade, the pentraxin‐related protein PTX3 [45], and has been implicated in the reduction of gonadotropin‐releasing hormone [46].…”