Cloning Of The Full-length ActRIP (Activin Receptor-interacting Protein) 2a Gene, And Study Of Comparison Between ActRIP2a And ActRIP1-3

Activin, a member of the TGF-β superfamily, plays important roles as a cell proliferation and differentiation factor during the early development of embryos, neurogenesis, hematopoiesis, apoptosis and hormone action from new born to adult. Two types of activin receptor have been identified, type Ⅰ and type Ⅱ, and both of them belong to the family of serine/threonine kinase receptors. Interaction between the type I and type Ⅱ receptors is necessary for activin-induced signal transduction. Activin binds directly to the type Ⅱ receptor, which then recruits the type I receptor to the complex and phosphorylates it, resulting in the activation of the type I receptor and the downstream propagation of the signal through a cascade response elicited by Smad proteins to the nucleus. Cellular function of activin in target cells is tissue specific, which is possibly related with the difference of intracellular signal transduction. A group of mouse PDZ proteins had been identified that interacts with the activin type ⅡA receptor (ActRIIA) named activin receptor-interacting proteins(ActRIPs). Because ActRIPs are different in tissue distribution pattern, expression style and biological effects, they are considered as the key molecules determining the activin tissue sepicifity. Identification of ActRIPs provided a better understanding of the regulation and mechanisms of serine / threonine kinase receptors -mediated signaling pathway.To approach the interactive characteristic of ActRIP2a with activin receptor, based on the identification and cloning a novel ActRIP (ActRIP2a) gene, we compared the tissue distribution pattern, expression profile, biological effects and activin-mediated signaling of the ARIP2a and ARIP1-3.Our data demonstrated that the ActRIPs were important signal proteins mediating activin effects, which strengthened the base of relative diseases therapies with theoretical and experimental evidence.Part I: Cloning and structure analysis of ActRIP2aIn the study of cytoplasmic proteins interacting with activin receptors, a novel cDNA clone encoding the full-length of ActRIP2a was obtained from a mouse brain cDNA library. ActRIP2a gene encodes a 153 amimo acid protein with an NH2-terminal PDZ domain (containing GLGF core peptide), which is considered essential for the protein-protein interactions through the NH2 - terminus. There is a PDZ domain in the NH2 - terminal region of ActRIP2a, ActRIP 2 and ActRIP 3, while there are five PDZ domain in the ActRIP 1, and only PDZ5 have ability to interact with ActRIIA. Amino acid sequence analysis revealed that the PDZ domain of ActRIP2a was 33% homologous with the PDZ of ActRIP 1 and 96% homologous with ActRlI binding domain of ActRIP3, but C-terminus of ActRIP2a showed no homology with that of ActRIP3, which suggested that ActRIP2a and ActRIP 1, 3 maybe exert different biologic effects. ActRIP2a is identical to ActRIP2 except that 15 and 99 amino acid position of encoding region. In addition, ActRIP2 lacks the nucleotides 826—989 in nonencoding region, and all of the differences indicate that ActRIP2a and ActRIP2 may be also differed in biological activity.Part II: Interaction of ActRIP2a with ActR IIATo determine whether ActRIP2a could bind ActR IIA in Mammalian cell or not, Mammalian two-hybrid assay was performed in CHO cell. Luciferase activity of the cell co-transfected with pACT-ActRIP2a and pBIND-ActRIIAwas higher than that of the cell only transfected with pACT-ActRIP2a or pBIND-ActRIIA, which indicated that ActRIP2a was capable of interacting with ActR II A. Moreover, pull-down assay showed that ActRIP2a could interact with COOH-terminal region of ActR II through the PDZ domain. Immunoprecipitation assay demonstrated that the COOH-terminal region of ActRIP2a is able to form ActRIP2a homodimer through a hydrophobic bridge or heterodimer with ActRIP2.Part III: The tissue distribution of ActRIP2a and ActRIPl-3 in miceBecause activin has significant tissue specificity, to determine whether the tissue distribution of ActRIPs is associated with functions of activin or not, the tissue distribution of ActRIP2a and ActRIPl-3 in mice was analysis by Northern blot, RT-PCR and immunohistochemical stain.Firstly, the result of Northern blot showed that ActRIP2 mRNA is widely expressed in various mouse tissues including heart, brain, liver, muscle, Kinney, testicle as well as in lung, but with a lower level. However, previous reports showed the ActRIPl mRNA was predominately expressed in neuronal tissue. Different gene expression of ActRIPl, ActRIP 2 and ActRIP 3 was confirmed.Secondly, results of immunohistochemical stain showed that ActRIP2 was detected in all of mouse tissue;ActRIPl was detected in all tissuesexcept lung, pamcreas and seminiferous tubule of testicle;ActRIP3 is mainly expressed in the hippocampi, hypothalamus and nerve fiber of cerebella tissue, as well as oocyte and seminiferous tubule of testicle. Lower expression was also observed in other regions of brain. These results indicated that ActRIP3 may participate in regulation of hippocampi, hypothalamus and other neuron cells which play important roles in the control of the hypothalamus-pituitary-gonadal (HPG) axis.Because the difference between ActRIP2a and ActRIP2 was not seen through Northern blot and immunohistochemical stain, RT-PCR assay was carried out to compare the expression of ActRIPs, and results showed that high expression levels of ActRIP2a mRNA in brain, pituitary, and testicle and moderate expression level in pancreas and ovary. ActRIPl mRNA was widely expressed in brain stem, cerebrum, cerebellum, pituitary, hypothalamus, testical and adrenal gland. sActRIPl (short ActRIPl) mRNA was expressed in all of tissues except cardiac muscle and lung tissue. ActRIP2 mRNA and ActRIP3 mRNA was expressed in various tissues. Thus, these results suggested that the difference of tissue distribution of ActRIP2a, ActRIPl, ActRIP 2 and ActRIP 3 is relative to their diverse functions.Part IV: Expression pattern of ActRIP2a in cellTo further investigate the biological activity of ActRIP2a, we compared ActRIP2a, ActRIPl, ActRIP3 and ActRIP5 in cellular expression profile. CHO cell line was transfected with ActRIP2a, ActRIP3 and ActRIP5 expression plamids. Immunocytochemistry showed that ActRIP2a localized at the two poles of the cells, while ActRIP3 localized in cytoplasm in a spread manor, and ActRIP5 clustered near the perinuclear region. Thedifferences in the expression location of ActRIP2a, 3, 5 suggested their differences in function.Correlated data shown that activin exert diverse biological effects during the development of various tumors, with related to activin signaling pathway. Therefore, we selected RAW264.7, L929, Hepal-6 and NS-1 four cell lines derived from different tumors to detect and compare the expression of ActRIP2a at protein and mRNA levels. Immunocytochemistry showed that ActRIP2a expression location in the four tumor cell lines was similar to CHO cell line transfected with ActRIP2a, in which most protein was localized in one side of the cell. RT-PCR assay showed that ActRIP2a was expressed in all four tumor cell lines, but with different levels.Part V: Biological functions of ActRIP2a in cellsTo confirm the interaction of ActRIP2a with ActR IIA by PDZ like domain, mammalian two -hybrid assay was performed in CHO cells. To further investigate the effects of ActRIP2a on activin signaling transduction, CHO cells were transfected with pcDNA3-ActRIP2a or pcDNA3-ActRIP3 together with a reporter plasmid CAGA-lux, which specifically reponses to activin, and then transcript activity induced by activin was measured. ActRIP2a expression inhibited activin-induced specific gene transcript response in dose- dependant manner, while ActRIP3 enhanced the response in dose- dependant manner. On the other hand, overexpression of ActRIP2a in CHO cell co-transfected with pcDNA3-ActRIP3 and pcDNA3-ActRIP2a decreased the response. These data have shown that ActRIP2a and ActRIP3 are different in regulation of activin-inducded signaling transduction. In addition, co-expression of ActRIP2a and ActRIP3 in cells havs antagonistic effect on each other's expression, and therefore co-regulates the active-induced signal transduction.Because the secretion of NO from single activin-stimulated RAW264.7 cell was enhanced, we analyzed the effect of overexpression of ActRIP2a on the enhanced secretion. The result showed that ActRIP2a overexpression inhibited the enhanced secretion of NO from activin-stimulated RAW264.7 cell in dose-dependant manner. ActRIP2a overexpression inhibited the secretion of NO, while ActRIP3 overexpression enhanced the secretion. So we studied whether ActRIPs expression is related with activin in RAW264.7 cell. The results showed that the secrection of NO from activin-stimulated RAW264.7 cell and the expression of ActRIP2, 3 mRNA were significantly enhanced, at the same time ActRIP2a mRNA expression was inhibited. It is suggested the different functions of ActRIP2a and ActRIP2. The effects of activin on RAW264.7 are related with ActRIPs-mediated signaling transduction.Futhermore, the effects of ActRIP2a overexpression in RAW264.7 on the expression of ActRIIA in the cell membrane were evaluated by Flow Cytometry. The results suggested that ActRIP2a could inhibit the expression of ActR IIA in cell membrane.In general, these results suggest that ActRIP2a is capable of interacting with the COOH-terminal region of ActR IIA through PDZ-like domain, directly participates the expression regulation of ActRIIs in cells as a negative regulation protein. By comparison the difference of ARIP2a, ARIP1, 2 and 3 in tissue distribution pattern, expression profile and biological effects, as well as in the function in activin signaling pathway, ActRIPs participate the activin signal transduction as novel members of ActRIP family.