Resistance to inhibitors of cholinesterase-8a (Ric-8a) is a guanine nucleotide exchange factor for several heterotrimeric G protein α subunits, including the subfamilies Gαi, Gαq, and Gα12/13. G proteins have been shown to drive pathways in cancer cells leading to unregulated cell growth, migration and adhesion. Also, G proteins are frequently a pharmaceutical target for treating conditions such as cancer, diabetes, depression, allergies, and heart disease. Recently Ric-8 was found to be a G protein chaperone and needed for the G protein to be transported to the cell membrane; without Ric-8, G proteins malfunctioned and were destroyed. For Gα12 (part of the G12 subfamily of G proteins) literature has shown that the final nine amino acids at the C-terminus are necessary for binding to Ric-8. To assess the Gα12-Ric-8a interaction and binding affinity, protein binding assays and Western Blot analyses were conducting using Gα12 constructs in which the C-terminus was altered. Two Gα12 mutants, engineered with the substituted amino acid sequence Asn-Ala-Ala-Ile-Arg-Ser (NAAIRS), and a Gα12 with Gαs C-terminus construct all showed inhibited binding with Ric-8a while maintaining normal binding with the positive control leukemia-associated Rho guanine nucleotide exchange factor (LARG). Such a construct could potentially be beneficial to future research on the cell biological role of Gα12-Ric-8 interaction.
