Chemical modification of bovine transducin: effect of fluorescein 5'-isothiocyanate labeling on the activities of the transducin .alpha. subunit

Abstract

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residues of bovine transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells. The incorporation of FITC showed a stoichiometry of approximately 1 mol of FITC/mol of transducin. The labeling was specific for the T alpha subunit. There was no significant incorporation on the T beta gamma subunit. The modification had no effect on the transducin-rhodopsin interaction or on the binding of guanosine 5'-(beta, gamma-imidotriphosphate) [Gpp(NH)p] to transducin in the presence of photolyzed rhodopsin. The dissociation of the FITC-transducin-Gpp(NH)p complex from rhodopsin membrane remained unchanged. However, the intrinsic GTPase activity of T alpha and its ability to activate the cGMP phosphodiesterase were diminished by FITC modification. The rate of FITC labeling of the transducin-Gpp(NH)p complex was about 3-fold slower than that of transducin. Limited tryptic digestion and peptide mapping were used to localize the FITC labeling site. The majority of the FITC label was on the 23-kilodalton fragment, and a minor amount was on the 9-kilodalton fragment of the T alpha subunit. These results indicate that FITC labeling does not alter the activation of transducin by photolyzed rhodopsin but does affect the GTP hydrolytic activity as well as the GTP-induced conformational change of T alpha, which ultimately leads to the activation of cGMP phosphodiesterase.

Identification of a binding site on retinal transducin α for the phosphodiesterase inhibitory γ subunit

“…In order to identify the region of Ta that interacts with the basic region of PDEy (residues 24-45), whole Ta-GTP[S] was tested in a direct binding assay as well as the 33 kDa fragment of Ta. This Ta fragment has been shown to activate PDE but does not bind to rhodopsin or to T/Jy [29][30][31][32][33]. Binding to PDEy was observed using this direct binding assay with both Ta and the 33 kDa fragment (Table 1).…”

“…This model was developed using previously known sequence data and information obtained from partial proteolysis of Ta [30][31][32]. Ta can be functionally divided into a 1-2 kDa region on the N-terminus, which is needed for binding to T/?y [30][31][32][33], a 21 kDa region (approximately residues 19-204), which interacts with the PDE [33], a 12 kDa region, which binds guanine nucleotides, and a C-terminus, which binds to rhodopsin [32,46]. This model has, thus far, not been confirmed by tertiary structural analyses.…”

“…The 33 kDa piece still contained GTP[S] as assessed by the use of [35S]GTP[S]. The fragment still activates PDE [29], but does not bind to rhodopsin or to Tfly [30][31][32][33].…”

Chemical Modification of Transducin with Dansyl Chloride Hinders Its Binding to Light-activated Rhodopsin

“…However, the lysine residue(s) that is modified on T by each reagent evidently must be different, since DnsCl blocked the association of T with R* and PLP labeled the protein active site. In contrast, when fluorescein 5'-isothiocyanate was employed to modify the lysine residues of T, no effect on the T : R interaction or on the binding of GMPpNp to T was obtained (Hingorani and Ho, 1987). Similarly, phenyl isothiocyanate produced no effect on the GTP binding capability of T .…”

“…Similarly, phenyl isothiocyanate produced no effect on the GTP binding capability of T . On the other hand, fluorescein 5'-isothiocyanate labeling affected the GTP hydrolytic activity as well as the GTP-induced conformational change of T α , which ultimately led to the activation of cGMP phosphodiesterase (Hingorani and Ho, 1987). All of these observations suggest the existence of different functional lysines on T that are either located in the proximity of the interaction site with the photoreceptor protein, near the magnesium and guanine nucleotide binding sites in regions involved in the structural changes taking place upon protein activation, or near the contact region with cGMP phosphodiesterase.…”