2',3'-Dialdehyde GTP as an irreversible G protein antagonist. Disruption and reconstitution of G protein-mediated signal transduction in cells and cell membranes

J Biol Chem. 1994 Dec 16;269(50):31999-2007.

Abstract

The 2',3'-dialdehyde analogue of GTP, oGTP, was devised as an irreversible antagonist of regulatory GTP-binding proteins (G proteins). Here, we show that oGTP uncouples transmembrane signaling mediated by a set of distinct G proteins both in isolated membranes and in whole cells. In human platelet membranes, pretreatment with oGTP suppressed receptor- and G protein-controlled regulation of adenylyl cyclase activity. In chick neuronal cells, inhibition of the voltage-sensitive Ca(2+)-current by various membrane receptors (alpha 2-adrenergic, somatostatin, GABAB) was eliminated when oGTP was applied intracellularly in the whole cell patch-clamp configuration. Disruption of endogenous signaling pathways by oGTP occurred through specific blockage of the GTP-binding site of G protein alpha-subunits by the following criteria: (i) pretreatment of membranes with oGTP blocked direct G protein activation by guanine nucleotides as well as labeling of Gs alpha and Gi alpha with the photoaffinity probe [alpha-32P]GTP azidoanilide. (ii) The effect of oGTP was antagonized by the simultaneous introduction of guanosine 5'-(3-O-thio)triphosphate into the patch-clamped cell. (iii) The time to onset of action was similar for oGTP and guanosine 5'-O-thio)diphosphate. (iv) Inactivation of G protein-dependent signaling was overcome by substituting G protein alpha-subunits. Addition of both the short and long form of recombinant Gs alpha (rGs alpha-s and rGs alpha-L) restored guanine nucleotide-dependent adenylyl cyclase activity to oGTP-treated platelet membranes with rGs alpha-L being approximately 3-10-fold more potent than rGs alpha-s. This apparent preference was due to the intrinsically different activation rates of rGs alpha-L and rGs alpha-s. When reconstituted with exogenous rGs alpha, the A2-adenosine receptor did not discriminate among the two forms of rGs alpha. Thus, Gs alpha-L is the primary determinant of basal cAMP formation in platelets. In contrast, neither the addition of various recombinant subtypes of Gi/o nor purified bovine brain beta gamma-dimers reconstituted adenylyl cyclase inhibition in oGTP-treated membranes. All subtypes of Gi alpha stimulated adenylyl cyclase. In the presence of rGs alpha, a conditional stimulation by beta gamma-dimers was observed. This pattern of stimulation shows that platelet adenylyl cyclase is a type II-like isoform. Either a differently modified G protein or an ancillary GTP-binding component is required for adenylyl cyclase inhibition in platelets. oGTP can be considered a useful tool for disruption and reconstitution of transmembrane signaling mediated by presumably all classes of heterotrimeric G proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclases / metabolism
  • Affinity Labels
  • Animals
  • Baclofen / pharmacology
  • Blood Platelets / drug effects
  • Blood Platelets / ultrastructure
  • Calcium Channels / metabolism
  • Cell Membrane / metabolism
  • Chick Embryo
  • GTP-Binding Proteins / antagonists & inhibitors*
  • GTP-Binding Proteins / chemistry
  • Guanosine Triphosphate / analogs & derivatives*
  • Guanosine Triphosphate / chemistry
  • Guanosine Triphosphate / pharmacology
  • Humans
  • In Vitro Techniques
  • Ion Channel Gating / drug effects
  • Neurons / physiology
  • Recombinant Proteins
  • Signal Transduction / drug effects*

Substances

  • Affinity Labels
  • Calcium Channels
  • Recombinant Proteins
  • 2-(guanylformylmethoxy)-3-(triphospho)propanal
  • Guanosine Triphosphate
  • GTP-Binding Proteins
  • Adenylyl Cyclases
  • Baclofen