Screening And Functional Studies On The Proteins Interacting With The C-terminus Of HPROKR2

G protein-coupled receptors (GPCRs) are the largest family of receptors ever discovered, including more than1000members. The structure of GPCRs consists of seven transmembrane (TM) helices, three intracellular loops (IL1-3) and the cytoplasmic C-terminal tail. Almost all cells express various G-protein coupled receptors, which activated G protein after binding to ligands and induced a variety of biological effects through initiating different signal transduction pathways.G protein-coupled receptor not only interact with G proteins but also with some auxiliary proteins, the latter also were known as G protein-coupled receptor interacting proteins (GIPs). Category of GIPs are very rich, some are transmembrane proteins such as another GPCR, ionic channels, ionotropic receptors, single transmembrane protein and soluble proteins. These proteins participated in the trafficking, targeting, signaling and allosteric regulation of GPCRs. To date, proteins that interact with the GPCR C-terminus are the most abundant.PROKR2belongs to the family of GPCRs. After binding to its corresponding ligands, PROKR2involved in various physiological processes which included gastrointestinal mobility, angiogenesis, circadian rhythm regulation and migration of neural stem cell. Recently, genetic studies had shown that mutations in PROKR2were associated with the Kallmann syndrome (KS) and/or idiopathic hypogonadotropic hypogonadism (IHH), disorders characterized by delayed puberty and infertility. Therefore, study on the proteins interacting with PROKR2could not only help us understand the regulation of hPROKR2, but also promote the discovery of some new genes associated with the KS/IHH.In this research, we used the cytoplasmic C-terminal tail of hPROKR2(333aa-384aa) as a bait and screened the interactive proteins from the human cDNA library through the yeast two-hybrid system. We got8significant CDS regions and choose SNAPIN for further research. The interaction between SNAPIN and hPROKR2were later successfully identified in vitro and in vivo through GST pull down assay and co-immunoprecipitation techniques respectively. Given the highly homologous between hPROKR1and hPROKR2, the interaction between the C-terminal of hPROKRl and SNAPIN was identified later. In further research, the fragment of SNAPIN (37-136aa), which included H1and H2region, the two a helix, was necessary for their interaction;343YFK345and351HWR353on the cytoplasmic C-terminal domain of hPROKR2was also necessary for their interaction. It was identified that the mutant receptors,6A-PROKR2(343YFK/AAA345,351HWR/AAA353), could not interact with SNAPIN.Binding to ligands caused the endocytosis of hPROKR2. Receptors could quickly return back to the cell membrane after removing the ligands. As SNAPIN participated in the intracellular vesicle transport, we hypothesized that the interaction between Snapin with PROKR2may involve in the endocytosis or recycling. After making endogenous snapin gene silence, the endocytosis of hPROKR2was not affected, but the recycling was inhibited. Accordingly, the rate of receptors’degradation increase. These phenomena was also verified in the defective receptors which could not interact with SNAPIN. Thus, our results showed that SNAPIN promote the recycling of hPROKR2.We got conclusions from this reasearch:(1) We screened a PROKR2interactive protein, SNAPIN, through the yeast two-hybrid and identified the interaction between them by GST pulldown and co-immunoprecipitation;(2) The interaction region of SNAPIN is located at37-136amino acids, which included twoa helix region, H1and H2;(3)343YFK345and351HWR353on the cytoplasmic C-terminal domain of hPROKR2was also necessary for its interaction with SNAPIN. YFK and HWR were also two aromatic amino acids followed by a basic amino acid;(4) The functional studies indicated that SNAPIN help the recycling of PROKR2after endocytosis.