Identification Of Interaction Between Angiogenin And Progranulin

Angiogenin, a member of ribonuclease superfamily, has known to be an important angiogenic factor. It's the only angiogenic factor that is a ribonuclease and is also the only member of the ribonuclease superfamily that is angiogenic. Angiogenin is a very basic, single-chain protein with molecular weight about 14 kDa. It can stimulate neovascularization in malignant tumors, which is an indispensable condition for cancer development. Extensive investigation on biological activities of angiogenin will help to elucidate the molecular network regulating tumor angiogenesis and faciliate discovery of novel interference target for antiangiogenesis cancer therapy.Progranulin (Granulin precursor, PGRN) was formly identified to be an angiogenin-binding protein by yeast two-hybrid screening, which was employed to screen its potential interacting molecules. It is a secreted growth factor with molecular weight about 68.5 kDa, and plays an important role in regulation of cell growth and development, wound repaire, and tumorigenesis.In order to confirm the binding between ANG and PGRN by Flourescence Resonance Energy Transfer (FRET) method, we first constructed plasmids of YFP-ANG and CFP-PGRN. Then, CFP-PGRN and YFP-ANG were co-transfected into COS-7 cells. The results showed that FRET did happen between ANG and PGRN in the cells. Meanwhile, we utilized colocalization analysis to further investigate the interaction in the cells. Data revealed that ANG was localized in thenucleus when the cells were transfected with YFP-ANG, and PGRN was localized in the cytoplasm in CFP-PGRN transfected cells. However, co-transfection with YFP-ANG and CFP-PGRN showed that both proteins were co-localized in the cytoplasm, demonstrating that the interaction between ANG and PGRN did happen, and PGRN can retard the nuclear localization of ANG To map the precise binding site of PGRN with ANG, we generated four deletion mutants of PGRN using pEXP-AD502 as a vector. The plasmids were co-transfected with pDBLeu-ANG into MaV203 yeast cells, and the cells were plated on selective media. Robust growth was observed in cells co-transfected with ANG and either GrnCDE, GmPGFBA, or GrnDE, but not with GrnC, suggesting that the later did not interact with ANG.Tumorigenesis includes many pathways, such as tumor angiogenesis and cell proliferation. PGRN is a secreted growth factor and it plays an important role in cell proliferation. Thus, the interaction between ANG and PGRN provides new clue for understanding the molecular mechanism of tumorigenesis, and may offer a new interference target for antiangiogenesis cancer therapy.