Establishment Of Cell Model Stably Expressing PrRP Receptor And Primary Research On The MAPK(ERK) Signal Pathway

Prolactin-releasing peptide (PrRP) can bind to the orphan receptor PrRP receptor (also known as GPR10), which belongs to the G protein-coupled receptor family. As PrRP promotes the release of prolactin, it was named Prolactin-releasing peptide (PrRP). Since it was discovered in 1998, more than 150 papers had been reported about various fouctions of PrRP so far. In addition to specifically promoting anterior pituitary cells to release prolactin, PrRP also play an important role in multiple fields, which including cardiovscular system, stress respond, food intake, sleep and awakening, pain control, neuroendocrine and others. GPR10 are widely distributed, mainly in the pituitary, thalamic reticular nucleus, solitary tract nucleu, anterior pituitary and so on. In order to study the signal transduction mechanism of PrRP receptor on celluar level and to facilitate the research of PrRP’s structure-activity relationship in future, following work was done. First of all, the human PrRP31 peptide and human PrRP20 peptide were synthesized by solid phase synthesis method and confirmed by HPLC and MS (mass spectrum). Secondly, the Flag-tagged PrRPR expression vector (Flag-PrRPR-pcDNA3.1) were constructed and transfected into HEK293 cells, following several weeks of selection with G418, a positive cell line which stably expressing the PrRP receptor was acquired and confirmed by multiple levels, such as genome level, transcriptional level and protein expression level. Finally, using this cell model, primary studies on the signal transduction of PrRP receptor was conducted. Upon exposure of hPrRP, obvious activation of ERK1/2 was observed. The altitude of the activation of the ERK1/2 varied with the concentration and duration of hPrRPs. Pretreatment of PKC inhibitor lead to the attenuation of ERK1/2 activation, suggesting a PKC-dependent mechanism in ERK1/2 activation.