| Title: | Second site suppressor mutations of a GTPase-deficient G-protein alpha-subunit. Selective inhibition of Gbeta gamma-mediated signaling |
| Author(s): | Apanovitch DM; Iiri T; Karasawa T; Bourne HR; Dohlman HG; |
| Address: | "Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06536, USA" |
| ISSN/ISBN: | 0021-9258 (Print) 0021-9258 (Linking) |
| Abstract: | "G proteins transmit signals from cell surface receptors to intracellular effectors. The intensity of the signal is governed by the rates of GTP binding (leading to subunit dissociation) and hydrolysis. Mutants that cannot hydrolyze GTP (e.g. GsalphaQ227L, Gi2alphaQ205L) are constitutively activated and can lead to cell transformation and cancer. Here we have used a genetic screen to identify intragenic suppressors of a GTPase-deficient form of the Galpha in yeast, Gpa1(Q323L). Sequencing revealed second-site mutations in three conserved residues, K54E, R327S, and L353Delta (codon deletion). Each mutation alone results in a complete loss of the beta gamma-mediated mating response in yeast, indicating a dominant-negative mode of inhibition. Likewise, the corresponding mutations in a mammalian Gi2alpha (K46E, R209S, L235Delta) lead to inhibition of Gbeta gamma-mediated mitogen-activated protein (MAP) kinase phosphorylation in cultured cells. The most potent of these beta gamma inhibitors (R209S) has no effect on Gi2alpha-mediated regulation of adenylyl cyclase. Despite its impaired ability to release beta gamma, purified recombinant Gpa1(R327S) is fully competent to bind and hydrolyze GTP. These mutants will be useful for uncoupling Gbeta gamma- and Galpha-mediated signaling events in whole cells and animals. In addition, they serve as a model for drugs that could directly inhibit G protein activity and cell transformation" |
| Keywords: | Animals Base Sequence COS Cells Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors/metabolism DNA Primers Escherichia coli/genetics GTP Phosphohydrolases/*metabolism GTP-Binding Proteins/chemistry/genetics/*metabolism Mating Factor Muta; |
| Notes: | "MedlineApanovitch, D M Iiri, T Karasawa, T Bourne, H R Dohlman, H G eng R01 GM055316/GM/NIGMS NIH HHS/ CA54427/CA/NCI NIH HHS/ GM 55316/GM/NIGMS NIH HHS/ GM27800/GM/NIGMS NIH HHS/ Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 1998/10/24 J Biol Chem. 1998 Oct 30; 273(44):28597-602. doi: 10.1074/jbc.273.44.28597" |
