THE EFFECT OF CYTOHESIN-2/ARNO COILED-COIL DOMAIN MUTATIONS ON ITS INTERACTION WITH SCAFFOLD PROTEINS GRASP AND IPCEF

Open Access
Author:
Marikunte, Sanjana S
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Lorraine C Santy, Thesis Supervisor
  • David Scott Gilmour, Honors Advisor
Keywords:
  • Cell Migration
  • HGF Signaling
  • ARF6
  • Cytohesin-2/ARNO
  • GRASP
  • IPCEF
Abstract:
Epithelial cells line the surface of the skin, lungs, and the digestive tract. While normally stationary, epithelial cells can become migratory in instances of wound healing, cancer metastasis, and early development. This migration is regulated by small GTPases, including Arf6 and Rac1. GTPases act as molecular switches that are active when bound to GTP, and inactive when bound to GDP. GTPases are activated by guanine nucleotide exchange factors, including cytohesin-2/ARNO for Arf6 and Dock180 for Rac1. Epithelial cell migration is stimulated by hepatocyte growth factor, which causes the formation of a multi-protein complex involving cytohesin-2/ARNO, Dock180, and the scaffold proteins GRASP and IPCEF. The coiled-coil domain of cytohesin-2/ARNO binds to both GRASP and IPCEF. Previous research has shown that deletion of the cytohesin-2/ARNO coiled-coil domain prevents the formation of the multi-protein complex and thus prevents epithelial cell migration. This work investigates the role of four different mutations in the coiled-coil domain of cytohesin-2/ARNO and its effects on GRASP binding, IPCEF binding, and hepatocyte growth factor-stimulated cell migration. It was hypothesized that expression of the cytohesin-2/ARNO mutants would inhibit its binding with GRASP and IPCEF. The results suggest that mutations of cytohesin-2/ARNO from residues 28 through 32 and residues 54 through 58 might significantly decrease its binding with GRASP. The results also suggest that mutations of cytohesin-2/ARNO from residues 14 through 18, 28 through 32, and 54 through 58 significantly decrease its binding with IPCEF.