Abstract
Most drugs target a receptor for a hormone or neurotransmitter. A newer strategy for drug development is to target a downstream signaling element, such as the G protein associated with a receptor. Suramin is considered a lead compound targeting this moiety. It inhibits binding of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) to G proteins and reduces agonist binding to G protein-coupled receptors. Suramin is thought to uncouple the G protein from its associated receptor, although there is no direct evidence for this mechanism. We have now examined the effect of suramin on G protein signaling for the vasoactive intestinal peptide (VIP) receptor in lung. The primary experimental strategy was a two-step cross-linking reaction that covalently captures the VIP-receptor-G protein ternary complex. Such cross-linking provided the first direct evidence that suramin physically disrupts receptor-G protein coupling. We investigated how this uncoupling relates to the inhibition of GTPγS binding. Suramin indiscriminately hindered the dissociation of various guanine nucleotides from the G protein, implying that its action is not allosteric. Further cross-linking studies suggested that suramin does not obstruct the receptor docking site directly but appears to block the interface between G protein α and βγ subunits. Observations with a purified system of recombinant G protein subunits without a receptor yielded direct evidence that suramin suppresses the association between these subunits. This action can explain how it both disrupts receptor-G protein coupling and inhibits guanine nucleotide release. The improved understanding of suramin's action advances the development of selective inhibitors of G protein signaling.
Footnotes
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The initial part of this work was supported by a research grant (to J.C.K.) from the American Lung Association (National Program). The work of W.-C.C. constitutes part of his Doctor of Philosophy dissertation at the University of Mississippi Medical Center.
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doi:10.1124/jpet.104.078311.
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ABBREVIATIONS: GTPγS, guanosine 5′-O-(3-thiotriphosphate); VIP, vasoactive intestinal peptide; NF007, 8-(3-nitrobenzamido)-1,3,5-naphthalenetrisulfonic acid; DSS, disuccinimidyl suberate; BS3, bis(sulfosuccinimidyl) suberate; PAGE, polyacrylamide gel electrophoresis; EGS, ethylene glycol bis(succinimidylsuccinate); ANOVA, analysis of variance; GDPβS, guanosine 5′-O-(2-thiodiphosphate).
- Received September 23, 2004.
- Accepted December 20, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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