Structural and Functional Studies of Gaq Signaling and Regulation.

p63RhoGEF;G[alpha]q;x-ray crystallography;nuclear magnetic resonance;regulator of g protein signaling;small-angle x-ray scattering;Biological Chemistry;Science (General);Science;Biophysics

Taylor, Veronica GailSmith, Janet ;

University of Michigan

关键词: p63RhoGEF;    G[alpha]q;    x-ray crystallography;    nuclear magnetic resonance;    regulator of g protein signaling;    small-angle x-ray scattering;    Biological Chemistry;    Science (General);    Science;    Biophysics;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/133483/vgtaylor_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Hydrolysis of bound GTP to GDP inactivates a G protein. Regulator of G protein signaling (RGS) proteins interact with heterotrimeric G protein alpha subunits to increase this hydrolysis rate. The R4 subfamily of RGS proteins generally accepts both G[alpha]i/o and G[alpha]q/11 family subunits as substrates, while the R7 and R12 subfamilies select against G[alpha]q/11. One RGS protein, RGS2, is known to be selective for G[alpha]q/11. The molecular basis for this selectivity is not clear. Previously, the crystal structure of the RGS2-G[alpha]q complex revealed a non-canonical interaction due to interfacial differences imposed by RGS2, the G[alpha] subunit, or both. To resolve this ambiguity, the 2.6 angstrom crystal structure of an R4 subfamily RGS protein, RGS8, was determined in complex with G[alpha]q. This structure shows RGS8 and G[alpha]q in a canonical interaction, indicating that the non-canonical interaction of RGS2 with G[alpha]q is due to unique features of RGS2. Based on the RGS8-G[alpha]q structure, residues in RGS8 that contact a unique loop in the alpha-helical domain of G[alpha]q were converted to residues typically found in R12 subfamily members, and the reverse substitutions were introduced into an R12 subfamily RGS protein. These substitutions perturbed the ability of each RGS to stimulate GTP hydrolysis, but did not reverse selectivity of either. Instead, selectivity for G[alpha]q is likely determined by contacts between alpha-6 of the RGS domain and Switch III of G[alpha]q.RhoA, a key actin cytoskeleton regulator, is activated by GPCRs that couple to G[alpha]q. One pathway that links G[alpha]q to RhoA is via p63 Rho guanine nucleotide exchange factor (p63RhoGEF). Although the atomic structure of the G[alpha]q-p63RhoGEF-RhoA complex is known, the mechanism of activation is not clear, in part because the structure for the basal conformation of p63RhoGEF has not yet been determined. Work described in this thesis uses nuclear magnetic resonance spectroscopy and small-angle x-ray scattering to determine the solution structure of the p63RhoGEF catalytic core. Successful completion of this work would characterize a G[alpha]q effector in both its basal and activated states, and would reveal the molecular basis for regulation of an important link between heterotrimeric and small molecular weight G proteins.