Isolation And Characterization Of Proteins That Interact With Activin TypeII Receptors And The Induction Of Dopaminergic Neurons In Striatal Stem Cells By ActivinA And BFGF

Activin is a multifunctional growth and differentiation factor that belongs to the transforming growth factor β (TGF-β) superfamily. Activin has a broad range of physiological activities: regulation of secretion of follicle-stimulating hormone from pituitary cells, stimulation of differentiation of erythroblasts, and mesoderm induction in Xenopus embryos. Activin has also been reported to act on neural cells. Activin is a survival factor for neural cells derived from rat brain, regulating neuronal development in the avian iris and ciliary body and expression of neuropeptides by cultured sympathetic neurons. These varied actions on cell proliferation, differentiation and apotosis depend upon target cell. Activin transduces its signal via heteromeric complexes composed of two different serine/threonine kinase receptors, termed type Ⅰ and type Ⅱ. Upon ligand binding, the type Ⅱ receptor transphosphorylates and activates the type Ⅰ receptor kinase at the membrane region. Then, the type Ⅰ receptor cytophasmic domain interacts with Smads , which are intracellular signaling molecules and regulate transcription of selected genes in a cell-specific manner. In the current report, the function of the activin type Ⅱ receptor(ActR Ⅱ)are limited to ligand binding, type Ⅰ receptor recruitment and transphosphorylation, ActR Ⅱs exist as two subtype, type ⅡA and ⅡB, each of which is encoded by individual genes .Several alternative splicing variants have also been found in each subtype. For example, activin type ⅡA-N receptor(ActR ⅡA-N) that is a splicing product of the ActR ⅡA, is specifically expressed in neural cells and thought to mediate neuronal-specific activin action. Finally , we previously reported the indentification of a PDZ protein, called ARIP1 (activin receptor interacting protein 1), that interacts specifically with the ActR ⅡA among the receptors for the TGF-β family. ARIP1 is a scaffolding protein that unites activin receptors with an intracellular signaling molecule, Smad. These findings clearly indicated that delicate regulation of the functions of the ActRⅡs is important for maintenance of the proper activin signaling. Ⅰ Isolation and characterization of proteins that interact with activin type Ⅱ receptors To determine the specific functions of activin type IIA receptors in activin signal transduction, we searched for cytoplasmic proteins that interact with these receptors by using yeast two-hybrid screening. We identified a novel mouse PDZ protein that interacts with activin tyep Ⅱ receptor, which we named activin receptor-interacting protein 2(ARIP2). To obtain a full-length cDNA for ARIP2, a mouse brain cDNA library in the ZAP Ⅱvector was screened using the fragment of a ARIP2 as the probe. One clone covering the full length cDNA of the ARIP2 and another novel cDNA were identified, which we named ARIP2-short, since it lack the C terminal of ARIP2.The ARIP2 cDNA encodes a protein of 153 amino acids and the ARIP2-short encodes a protein of 118 amino acids,both of ARIP2 and ARIP2-short contain a single PDZ domain at NH2-terminal region. Our results of ELISA, mammalian two hybrid and immunoprecipitation indicated that ARIP2 and ARIP2-short can interact specifically with C-terminal region of ActR Ⅱs through a PDZ domain in the NH2-terminal region and the result of immunoprecipitation shown ARIP2 forms Homo-oligomers via motifs such as the leucine zipper-like motifes found outside PDZ domain.To determine whether ARIP2 interacts with ActRIIA in the native environment,We performed the immunoprecipitation of ARIP2 from the detergent soluble extract of the mouse liver lysate, and found it can coimmunoprecipitated with anti-ActRIIA antibody , which indicates that ARIP2 interact with ActRIIA in vivo. We used reporter assay to study effects of ARIP2 on Activin-induced Transcriptional Response, and the results showed that overexpression of ARIP2 in CHO-K1 cells decreased the activin-induced transcriptional activities in a dose dependent manner, however, ARIP2-short increased the activin induced transcriptional activities in a dose dependent manner. Overexpression of ARIP2 in LΒT2 cells suppresses the activity of FSH promoter and reduces the expression of FSHΒ mRNA. In contranst ARIP-2 short activates the activity of FSH promoter and increases the expression of FSHΒ mRNA in LΒT2 cells.These data suggest that the C-terminal region of ARIP2 is involved in the regulation of activin signaling. To examine whether ARIP2 and ARIP2-short are involved in endocytosis ofactivin type IIA receptor, we performed internalization assays,the results indicate the overexpression of ARIP2 enhances endocytosis of ActR Ⅱ A. In contrast, ARIP2-short reduces endocytosis of ActR ⅡA. Ⅱ Induction of dopaminergic neurons in E14 mice striatal stem cells by activinA and bFGF. To study the effects of activin A in the induction of dopaminergic neurons, we cultrured the striatal stem cells derived from E14 mice and stimulated them with activin A and bFGF. The factors that associtated with dopaminergic neurons were identified by RT-PCR and Immunofluorescence staining. These results showed the synergy of activinA and bFGF in the induction of TH positive neurons in striatal stem cells. Ⅲ Diffrentiation of Embryonic stem cells to insulin-secreting cells induced by activinA. RT-PCR was used to identify the expression of insulin mRNA in stem cells that derived from E14 mice cerebrum and striatum stimulated by activinA and bFGF. These results indicated activinA induces the differentiation of Embryonic stem cells to insulin-secreting cells.