Mutation of the highly conserved Arg165 and Glu168 residues of human Gsα disrupts the αD-αE loop and enhances basal GDP/GTP exchange rate

• Published in: Journal of cellular biochemistry Vol. 93; no. 2; pp. 409 - 417
• Main Authors: Hinrichs, María Victoria, Montecino, Martin, Bunster, Marta, Olate, Juan
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-10-2004
• Subjects: adenylyl cyclase; G-protein; Gsα
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.20193

G protein signalling regulates a wide range of cellular processes such as motility, differentiation, secretion, neurotransmission, and cell division. G proteins consist of three subunits organized as a Gα monomer associated with a Gβγ heterodimer. Structural studies have shown that Gα subunits are constituted by two domains: a Ras‐like domain, also called the GTPase domain (GTPaseD), and an helical domain (HD), which is unique to heterotrimeric G‐proteins. The HD display significantly higher primary structure diversity than the GTPaseD. Regardless of this diversity, there are small regions of the HD which show high degree of identity with residues that are 100% conserved. One of such regions is the α helixD–α helixE loop (αD–αE) in the HD, which contains the consensus aminoacid sequence R*‐[RSA]‐[RSAN]‐E*‐[YF]‐[QH]‐L in all mammalian Gα subunits. Interestingly, the highly conserved arginine (R*) and glutamic acid (E*) residues form a salt bridge that stabilizes the αD–αE loop, that is localized in the top of the cleft formed between the GTPaseD and HD. Because the guanine nucleotide binding site is deeply buried in this cleft and those interdomain interactions are playing an important role in regulating the basal GDP/GTP nucleotide exchange rate of Gα subunits, we studied the role of these highly conserved R and E residues in Gα function. In the present study, we mutated the human Gsα R165 and E168 residues to alanine (A), thus generating the R165 → A, E168 → A, and R165/E168 → A mutants. We expressed these human Gsα (hGsα) mutants in bacteria as histidine tagged proteins, purified them by niquel‐agarose chromatography and studied their nucleotide exchange properties. We show that the double R165/E168 → A mutant exhibited a fivefold increased GTP binding kinetics, a higher GDP dissociation rate, and an augmented capacity to activate adenylyl cyclase. Structure analysis showed that disruption of the salt bridge between R165 and E168 by the introduced mutations, caused important structural changes in the HD at the αD–αE loop (residues 160–175) and in the GTPaseD at a region required for Gsα activation by the receptor (residues 308–315). In addition, other two GTPaseD regions that surround the GTP binding site were also affected. © 2004 Wiley‐Liss, Inc.