Comparison Between the Structure‐Function Relationships in Oncogenic and Wild‐Type G_α Subunits

Abstract

Guanine nucleotide binding proteins (G‐proteins), in conjunction with their receptors (GPCR), are one of the most prevalent signaling systems and are involved in the regulation of an array of cellular processes. Upon binding of an extracellular stimulus to the GPCR, the activated α‐subunit (Gα) exchanges GDP for GTP, releases both the βγ‐complex and the GPCR, and associates with an effector that further relays the signal. The signal terminates with the hydrolysis of GTP to GDP, causing the α‐subunit to reunite with the βγ‐subunits and the GPCR. G‐protein signaling must be tightly regulated to ensure the appropriate responses to extracellular stimuli. Any misregulation caused by mutations can be detrimental. Gsα and Giα1 are G‐protein subunits that, respectively, stimulate or inhibit adenylyl cyclase and regulate the production of the secondary messenger, cAMP. We studied two mutations observed in cancers of the intestine (R231H mutation in Gsα) and the colon (R208Q mutation in Giα1). Spectroscopic and crystallographic approaches were used to further understand these disease states.Fluorescence spectroscopy, circular dichroism, and UV/Vis spectroscopy showed lower melting temperature in the active conformation of the oncogenic mutants, indicating disruptions in the noncovalent interactions. Using malachite green and fluorescence GTP assays, we found that the kcat values for oncogenic Giα1 were lower than for the WT protein; the trend was the opposite for the WT and oncogenic Gsα proteins. When comparing the x‐ray structure of the R208Q mutant with that of the WT Giα1, the oncogenic point mutations do not show significant differences in the structure of the protein as a whole. However, molecular dynamics simulations modeling the interactions of T181 in Giα1 and T204 in Gsα (key residues in the hydrolysis of GTP) show drastically different interaction energies between mutants and WT proteins. Taken together, these findings suggest that the altered rates of GTP hydrolysis lead to an imbalance of cAMP in tumor cells.Support or Funding InformationNIH R15GM112025This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.