Studies On The Molecular Mechanisms Underlying The Internalization And ERK1/2Activation By PKR2Receptor

PKs (Prokineticins), including PK1and PK2, share50%homology in the amino acid sequence. They are involved in a variety of biological functions including gastrointestinal smooth muscle contraction, angiogenesis, neurogenesis, nociception, feeding circadian, ischemia, inflammatory and emotional regulation by binding to two closely related G-protein-coupled receptors (GPCRs), PKR1and PKR2. Recent studies have shown that mutations in prokineticin2(PK2) and PKR2are associated with the Kallmann syndrome and idiopathic hypogonadotropic hypogonadism. Our recent studies have shown that stimulation of PKR2expressed HEK293cell with PK2resulted in a redistribution of receptors from the plasma membrane to an intracellular compartment, leading to a rapid decrease in surface PKR2. And PKR2recycles back to the plasma membrane after the removal of ligand. However, little is known about the molecular mechanisms underlying the PKR2endocytosis. It has been shown that stimulation with PK2is able to elicit a transient activation of ERK1/2(Extracellular signal regulated kinase1/2) on PKR2expressing HEK293cells. But the mechanization underlying the process still remains unknown.Objective:To understand the mechanisms underlie the internalization and ERK1/2activation by PKR2Receptor.Methods:(1) By using confocal fluorescence microscopy, we studied the PKR2endocytosis by analysis of the distribution of GFP-tagged PKR2on the cell membrane and cytoplasm. We interfered with the GRK2, P-arrestin, clathrin and protein kinase C by expressing dominant negative proteins (such as GRK2and P-arrestins), or small interference RNA, or compounds in order to study the involvement of these proteins in the PKR2internalization.(2) We studied the phosphorylation of ERK1/2by using Western blot. Various compounds were tested to understand the involvement of internalization, PKC, PLCβ, MEK, EGFR in the activation of ERK1/2by PRK2.Results:(1) PK2-induced PKR2endocytosis is GRK2-and clathrin-dependent, but β-arrestin-independent.(2) PKC activation also induced PKR2endocytosis; however, PKC activation is not necessary for the PK2-induced PKR2endocytosis.(3) The internalization, PKC activation and EGFR are not required for the PKR2-induced ERK1/2activation.(4) PKR2-induced ERK1/2activation depends on the released βγ subunit of G-protein, PLCβ and MEK.Conclusions:We demonstrated that the internalization of PKR2by PK2stimulating is in a GRK2and clathrin-dependent, but β-arrestin independent manner. We also revealed that, in HEK293cells, PKR2-induced ERK1/2activation depends on the released βγ subunit of G-protein, PLCβ and MEK. Our study will facilitate the understanding of these pathways in the physiopathological roles of PKR2receptor.