Screening The PKR2-interactive Proteins By Yeast Two-Hybrid System

Kallmann syndrome (KS) is a clinically and genetically heterogeneous disorder that combines hypogonadotropic hypogonadism (IHH) with a deficiency of the sense of smell. The deficiency of the sense of smell related to the absence or hypoplasia of the olfactory bulbs and tracts. Hypogonadism is due to gonadotropin-releasing hormone (GnRH) deficiency, which presumably results from a failure of the embryonic migration of neuroendocrine GnRH cells from the olfactory epithelium to the forebrain. This failure could be a consequence of the early degeneration of olfactory nerve and terminal nerve fibres, because the latter normally act as guiding cues for the migration of GnRH cells. Franz Jozef Kallmann was the first who described this disease in1944. He suggested that this disease has hereditary background. The prevalence of KS is still unknown. KS has been roughly estimated at one out of8000in males. In females, the prevalence is thought to be five times lower. At present, ten genes are regarded as causal genes of KS. Mutations in the genes, however, account for barely30%of the KS patients.KS is caused by mutations in the PKR2gene. PKR2belongs to the seven-transmembrane, G protein-coupled receptors (GPCRs) family. The intracellular C-terminal domain of GPCRs is recognized as the significant domain interacting with cytosolic proteins, involved in the regulation of a wide range of functions including targeting, trafficking and signaling of GPCRs in specialized cells.To identify proteins regulating PKR2function, we screened a human cDNA library using the yeast two-hybrid system with the intracellular C-terminal domain of PKR2as bait. A protein called HSPD1was identified as a binding protein of PKR2preliminarily. We verified the direct interaction of PKR2and HSPD1by GST pull-down and co-immunoprecipitation experiments, and further characterized a region on the C-terminal of PKR2(1-13amino acid residues) is responsible for direct physical interaction with HSPD1by GST pull-down assays. HSPD1has been demonstrated as a typical molecular chaperone involved in protein folding. We further investigated the effect on PKR2function by over-expression of HSPD1. We did not observe direct regulation of PKR2function by HSPD1. It has been revealed that HSPD1usually funcions as a HSPD1-HSP10complex. And for this reason, we will explore the effect of HSPD1-HSP10complex on PKR2function in the future.Conclusion:We have identified HSPD1as a protein interacting with PKR2using the yeast two-hybrid system, laying a foundation for investigating HSPD1in the function PKR2and potential roles in the pathogenesis of Kallmann syndrome.