The Construction Of HNPFF2 Receptor Cell Model And The Research Of HNPFF2 Receptor-induced Signal Transduction

Two endogenous peptides, an octapeptide (F8Famide or NPFF neuropeptide FF) and an octadecapeptide (A18Famide or NPVF), have been isolated from the bovine central nervous system by using an anti-serum directed against FMRFamide (Phe-Met-Arg-Phe-NH2), a peptide identified in a variety of mollusk species. Neuropeptide FF has many functions both in the CNS and periphery. The pharmacological effects of Neuropeptide FF result from its interactions with two G protein-coupled receptors, Neuropeptide FF1 and Neuropeptide FF2. Neuropeptide FF1 and Neuropeptide FF2 receptors, stably expressed in some cells, have been confirmed that these receptors are Gi/o-protein coupled, such as in CHO, HEK293 and SH-SY5Y cells. Analysis of the peptide and receptors, together with pharmacological and physiological data, imply that NPFF2 receptor would be the primary receptor for neuropeptide FF.The SK-N-SH and the SK-N-MC cell lines were developed from human central nervous system by J.L.Biedler in 1970 and 1971 respectively. Panula et al reported for the first time SK-N-MC cell line endogenously expressing NPFF and hNPFF2 receptor in 2006, and they also found that (1DMe) NPYF, a NPFF analogue, had activated the mitogen-activated protein kinase (MAPK) cascade. However, SH-SY5Y was derived from SH-N-SH cell line, it is implied that SH-SY5Y cell line might be endogenously express hNPFF2 receptor, and activated hNPFF2 receptor could activate the MAPK. Thus, we used RT-PCR and gene sequencing to confirm this presumption. Upon different concentrations of NPFF treatment, the MAPK cascade was activated. When SH-SY5Y cells were treated with 0.1μM NPFF, it significantly increased the amount of the phosphorylated form of ERK1/2, and the treatment of cells with 10μM NPFF resulted in the peak value. This result suggest that SH-SY5Y cell line endogenously express hNPFF2 receptor at the function level, and it confirm Panula et al s’report. Some results have been reported to activation of the Akt cascade through GPCRs, such asμ- andδ- opioid receptors, and the MAPK cascade has interacted with the Akt cascade. Upon NPFF treatment, it resulted in a dose-dependent activation of the Akt cascade, evidenced by the NPFF-induced phosphorylation of the Akt(T308). Only cells were treated with 100μM NPFF, it significantly increased the amount of the phosphorylated form of Akt. It implies that the Akt cascade was activatied by activated hNPFF2 receptor. Here, we also construct two kinds of cell model to better investigate to hNPFF2 receptor-induced cell signal events, one is stable express model (pCDNA3.1(+)-HA-hNPFFR2-HEK293), another is transient express model (pECFP-C1-HA-hNPFFR2-Hela).In conclusion, we show that SH-SY5Y cells endogenously express functional hNPFF2 receptor, and that the receptor stimulation leads to the activation of the MAPK and the Akt cascade.