Low Affinity Interactions of GDPβS and Ribose- or Phosphoryl-substituted GTP Analogues with the Heterotrimeric G Protein, Transducin

• Published in: The Journal of biological chemistry Vol. 271; no. 22; pp. 12925 - 12931
• Main Authors: Zera, Evelyn M., Molloy, David P., Angleson, Joseph K., Lamture, Jagannath B., Wensel, Theodore G., Malinski, Justine A.
• Format: Journal Article
• Language: English
• Published: 1996
• ISSN: 0021-9258
• DOI: 10.1074/jbc.271.22.12925

We have examined the effects of three commonly used classes of guanine nucleotide analogues on the retinal G protein, transducin (G sub(t)), and found them to be quite different from those that might be expected from results with other GTP-binding proteins. The most surprising results were with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S); rather than inhibiting activation of G sub(t), GDP beta S addition activated G sub(t) as a result of a trace contaminant. Even when the contaminant levels were reduced 5-fold by chromatography, its effects dominated those of GDP beta S, which binds G sub(t) at least 1500-fold more weakly than guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). The affinity of G sub(t) for GDP was found to be at least 300-fold weaker than for GTP gamma S, while the affinities of GTP and GTP gamma S were similar. Ribose-modified GTP analogues, including 2'(3')-O-(N-methylanthraniloyl) GTP (mant-GTP), 2'(3')-O-[(2-aminoethyl)carbamyl] GTP (edGTP), and adducts of fluorescein 5-isothiocyanate and rhodamine B-isothiocyanate with edGTP, interacted extremely weakly, if at all, with the GTP binding site of the alpha subunit of G sub(t). They were neither effective activators of G sub(t) nor effective inhibitors of activation by GTP or GTP gamma S. A gamma -phosphoryl-modified analogue, an adduct of GTP gamma S and (5-(2(iodoacetyl)aminoethyl)amino)-naphthalene-1-sulfonic acid (dnsGTP), also activated G sub(t) weakly, if at all, and did not inhibit its activation. The exclusion of these analogues points to the highly restrictive and specific nature of the GTP binding site of G sub(t), in contrast to those of numerous other GTP-binding proteins which are potently activated or inhibited by these analogues.