Screening Of Proteins Interacting With NPR-K By The Yeast Two-Hybrid System

Our understanding of the genetic basis of familial atrial fibrillation (AF) is mostlylimited to genes encoding ion channels. In2008, two non-ionchannel genes NPPA andNUP155were found to be disease causing gene of AF, which updated the understandingof the mechanism of AF. In a previous case control study, the candidate genes weresequenced and three new variants in patients were identified in NPPA.NPPA gene encodes for the precursor of atrial natriuretic peptide (ANP) whichregulates blood pressure, water and electrolyte balance and cardiovascular homeostasis.ANP stimulates natriuretic peptide receptor-A (NPR-A) and increases intracellular cGMP.However, the mechanism underling how the guanylyl cyclase activity was regulated indifferent tissue is unclear. We plan to figure out the role of NPR-A in atrial fibrillation bysearching the proteins interacting with it using Yeast Two-Hybrid System.Firstly, the intracellular domain (ICD) cDNA sequence of human NPR-A(NPR-A-ICD) was cloned into pGBKT7. Yeast strain AH109was transformed bypGBKT7-NPR-A-ICD. And no toxicity or self-activation was detected. Then,pGBKT7-NPR-A-ICD was expressed.13preliminary positive colonies were selected bySD nutrient medium screening. Finally, one positive colony was confirmed by a secondtime SD nutrient medium screening and β-galactosidase assay. The library plasmid ofpositive clone was extracted and sequenced. The sequence shared99%homology withHuman PLA2G2A. Further studies are needed to confirm the interaction betweenPLA2G2A and NPR-A, if these two proteins are really physically interacting with NPR-A,we will also investigate the physiological and the pathphysiological roles of these specificinteractions. NPR-A exists thyroid gland, adrenal gland, kidney, liver and other tissues.