A GTPase-activating protein for the G protein Gα(z). Identification, purification, and mechanism of action

Jun Wang, Yaping Tu, Jimmy Woodson, Xiaoling Song, Elliott M. Ross

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Abstract

A GTPase-activating protein (GAP) specific for Gα(z) was identified in brain, spleen, retina, platelet, C6 glioma cells, and several other tissues and cells. G(z) GAP from bovine brain is a membrane protein that is refractory to solubilization with most detergents but was solubilized with warm Triton X-100 and purified up to 50,000-fold. Activity is associated with at least two separate proteins of M(r) ~22,000 and 28,000, both of which have similar specific activities. In an assay that measures the rate of hydrolysis of GTP pre-bound to detergent-soluble Gα(z), the GAP accelerates hydrolysis over 200-fold, from 0.014 to 3 min-1 at 15 °C, or to ≤20 min- 1 at 30 °C. It does not alter rates of nucleotide association or dissociation. When co-reconstituted into phospholipid vesicles with trimeric G(z) and m2 muscarinic receptor, G(z) GAP accelerates agonist-stimulated steady-state GTP hydrolysis as predicted by its effect on the hydrolytic reaction. In the single turnover assay, the K(m) of the GAP for Gα(z)-GTP is 2 nM. Its activity is inhibited by Gα(z)-guanosine 5'-O-thiotriphosphate (Gα(z)-GTPγS) or by Gα(z)-GDP/A1F4 with K(i) 1.5 nM for both species; Gα(z)-GDP does not inhibit. G protein βγ subunits inhibit G(z) GAP activity, apparently by forming a GTP-Gα(z)βγ complex that is a poor GAP substrate. G(z) GAP displays little GAP activity toward Gα(i1) or Gα(o), but its activity with Gα(z) is competitively inhibited by both Gα(i1) and Gα(o) at nanomolar concentrations when they are bound to GTPγS but not to GDP. Neither phospholipase C-β1 (a G(q) GAP) nor several adenylyl cyclase isoforms display G(z) GAP activity.

Original languageEnglish
Pages (from-to)5732-5740
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number9
DOIs
Publication statusPublished - Feb 28 1997
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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