Low affinity interactions of GDPβS and ribose-or phosphoryl-substituted GTP analogues with the heterotrimeric G protein, transducin

Abstract

We have examined the effects of three commonly used classes of guanine nucleotide analogues on the retinal G protein, transducin (Gt), 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) (GDPβS); rather than inhibiting activation of Gt, GDPβS addition activated Gt as a result of a trace contaminant. Even when the contaminant levels were reduced 5-fold by chromatography, its effects dominated those of GDPβS, which binds Gt at least 1500-fold more weakly than guanosine 5′-O-(3-thiotriphosphate) (GTPγS). The affinity of Gt for GDP was found to be at least 300-fold weaker than for GTPγS, while the affinities of GTP and GTPγS 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 a subunit of Gt. They were neither effective activators of Gt nor effective inhibitors of activation by GTP or GTPγS. A γ-phosphoryl-modified analogue, an adduct of GTPγS and (5-(2(iodoacetyl)aminoethyl)amino)-naphthalene-1-sulfonic acid (dnsGTP), also activated Gt 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 Gt, in contrast to those of numerous other GTP-binding proteins which are potently activated or inhibited by these analogues.