• IDENTIFICATION OF THE CAP1-BINDING DOMAIN OF HUMAN ADENYLYL CYCLASE 3

      Gunby, Kimberly; Sabbatini, Maria E.; College of Science and Mathematics; Biological Sciences; Sabbatini, Maria; Augusta University (1/30/2020)
      My research is aimed at finding the cyclase-associated protein 1 (CAP1) binding domain on human adenylyl cyclase 3 (AC3). Previous studies in our lab show that the interaction between CAP1 and AC3 inhibits migration and invasion of pancreatic cancer cells. The inhibitory mechanism is thought to involve the binding of AC3 and CAP1, causing the inhibition of globular-actin polymerization needed for filopodia formation and cell motility. A better understanding of this interaction will help facilitate the discovery for drugs that inhibit the migration and invasion of pancreatic cancer cells. To locate the binding region, we constructed mutants of WT AC3 plasmid using a Site-Directed Mutagenesis kit. We substituted a highly conserved proline residue at position 307 for an arginine residue (P307R) and a glutamate residue at position 308 for an alanine residue (E308A). The mutations were confirmed by sequencing. We then transfected pancreatic cancer cell line PANC-1 with WT and mutant AC3 plasmids and confirmed the expression using Western-blotting. To test whether the mutated AC3 could still interact with CAP1, we performed co-immunoprecipitation. We found that the residues proline and arginine in AC3 are not required for the interaction with CAP1. Further substitutions of other conserved residues are underway.