A mutation that prevents GTP-dependent activation of the α chain of Gs

Abstract

Membrane-bound G proteins carry information from receptors on the outside of cells to effector proteins inside cells. The alpha subunits of these heterotrimeric proteins bind and hydrolyse GTP and control the specificity of interactions with receptor and effector elements. Signalling by G proteins involves a cycle in which the inactive alpha beta gamma-GDP complex dissociates to produce alpha*-GTP, which is capable of activating the effector enzyme or ion channel; the alpha*-GTP complex hydrolyses bound GTP and reassociates with beta gamma to form the inactive complex. We have characterized a mutation that interrupts this GTP-driven cycle in alpha s, the alpha-chain of Gs, the G protein that stimulates adenylyl cyclase. The mutation converts a glycine to an alanine residue in the presumed GDP-binding domain of alpha s. The location and biochemical consequences of this mutation suggest a common mechanism by which binding of GTP or ATP may induce changes in the conformation of a number of nucleoside triphosphate binding proteins.

Coordinate expression of the α and β subunits of heterotrimeric G proteins involves regulation of protein degradation in CHO cells

“…Constitutively active mutants, GKs R201E (GKs RE) and GKs Q227L (GKs QL), were constructed by replacing arginine at 201 with glutamic acid [9] and glutamine at 227 with leucine [10], respectively. Dominant negative mutants, GKs G226A (GKs GA) and GKs A366S/G226A/E268A (GKs TM), were formed by replacing glycine at 226 with alanine [11] and by combining the three mutations, respectively [12]. The plasmids containing the cDNAs of GL1 and GQ2 were kindly provided by Dr. William F. Simonds (NIDDK, NIH, Bethesda, MA, USA).…”

Positive regulation of human T cell activation by Gi2 proteins and interleukin-8

“…A first approach consisted in studying the responses to CD3 cross-linking of PB T lymphocytes in which G i protein signaling had been inactivated through the use of low doses of PT (Յ10 ng/mL), so as to eliminate any toxoid effect. Next, the ␣ subunits of the G i family (G ␣i1 , G ␣i2 , G ␣i3 ) were stably inactivated in Jurkat cells through mutations in one of their GTP-binding domains: for example, the G204A mutation in G ␣i2 prevents the protein from undergoing the conformation change necessary to achieve activated state after GTP-binding and the protein is blocked in inactive state [30,31]. Dominantnegative mutant forms of the three G ␣i subunits [32,33] were expressed in Jurkat cells and effects on cell responses to TCR activation studied.…”

Gem: an Induced, Immediate Early Protein Belonging to the Ras Family

“…Gem contains an unusual motif in the G3 (DXXG) region, which putatively participates in binding and hydrolysis of the GTP y phosphate (5 glycine residue is thought to play a central role in inducing conformational changes in guanosine triphosphatases (GTPases) as they cycle between GTP-and guanosine diphos- phate-bound states (7,8). Although the sequence EQDG occurs in Rad in a position corresponding to ENKG in Gem, the sequence DIWE has been proposed to correspond to the G3 motif in this protein (3).…”