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Selective inactivation of guanine-nucleotide-binding regulatory protein (G-protein) alpha and betagamma subunits by urea.

ABSTRACT: G-protein-coupled receptors activate signal-transducing G-proteins, which consist of an alpha subunit and a betagamma dimer. Membrane extraction with 5-7 M urea has been used to uncouple receptors from endogenous G-proteins to permit reconstitution with purified G-proteins. We show that alpha(i) subunits are inactivated with 5 M urea whereas the betagamma dimer requires at least 7 M urea for its inactivation. There is no significant loss of receptors. Surprisingly, Western-blot analysis indicates that the urea-denatured alpha(i) subunit remains mostly membrane-bound and that beta is only partially removed. After 7 M urea treatment, both alpha(i1) and betagamma subunits are required to restore high-affinity agonist binding and receptor-catalysed guanosine 5'-[gamma-thio]triphosphate binding. We demonstrate the generality of this approach for four G(i)-coupled receptors (alpha(2A)-adrenergic, adenosine A1, 5-hydroxytryptamine(1A) and mu-opioid) expressed in insect cells and two mammalian cell lines. Thus a selectivity of urea for G-protein alpha versus betagamma subunits is established in both concentration and mechanism.

SUBMITTER: Lim WK

PROVIDER: S-EPMC1221661 | biostudies-other | 2001 Mar

REPOSITORIES: biostudies-other

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Project description:G proteins mediate the action of G protein coupled receptors (GPCRs), a major target of current pharmaceuticals and a major target of interest in future drug development. Most pharmaceutical interest has been in the development of selective GPCR agonists and antagonists that activate or inhibit specific GPCRs. Some recent thinking has focused on the idea that some pathologies are the result of the actions of an array of GPCRs suggesting that targeting single receptors may have limited efficacy. Thus, targeting pathways common to multiple GPCRs that control critical pathways involved in disease has potential therapeutic relevance. G protein betagamma subunits released from some GPCRs upon receptor activation regulate a variety of downstream pathways to control various aspects of mammalian physiology. There is evidence from cell- based and animal models that excess Gbetagamma signaling can be detrimental and blocking Gbetagamma signaling has salutary effects in a number of pathological models. Gbetagamma regulates downstream pathways through modulation of enzymes that produce cellular second messengers or through regulation of ion channels by direct protein-protein interactions. Thus, blocking Gbetagamma functions requires development of small molecule agents that disrupt Gbetagamma protein interactions with downstream partners. Here we discuss evidence that small molecule targeting Gbetagamma could be of therapeutic value. The concept of disruption of protein-protein interactions by targeting a "hot spot" on Gbetagamma is delineated and the biochemical and virtual screening strategies for identification of small molecules that selectively target Gbetagamma functions are outlined. Evaluation of the effectiveness of virtual screening indicates that computational screening enhanced identification of true Gbetagamma binding molecules. However, further refinement of the approach could significantly improve the yield of Gbetagamma binding molecules from this screen that could result in multiple candidate leads for future drug development.

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Selective inactivation of guanine-nucleotide-binding regulatory protein (G-protein) alpha and betagamma subunits by urea.

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