A C-terminal peptide of bovine rhodopsin binds to the transducin α-subunit and facilitates its activation

Abstract

In order to investigate the possible roles of the intracellular domains of rhodopsin in the functional coupling of the photoreceptor to transducin, different peptides that correspond to parts of the known sequence of rhodopsin were synthesized. Since we have found that the binding of rhodopsin to the α subunit of transducin (α(T)) increases the susceptibility of α(T) to phosphorylation by protein kinase C-β1, we used this phosphorylation reaction as an initial screen for peptides that mimic the actions of rhodopsin. The results of this screen indicated that a peptide from the C-terminal tail of rhodopsin (amino acids 325-338; KNPLGDDEASTTVS-amide; designated as peptide 3) was capable of interacting with the α(T) subunit. Evidence that peptide 3 binds to α(T) at a site that overlaps the rhodopsin-binding domain was obtained from experiments showing that peptide 3 inhibited the rhodopsin-stimulated GTPase activity of α(T) and that this inhibition was overcome at high levels of rhodopsin. A potentially important outcome of the peptide 3/α(T) interaction is the facilitation of the activation of the α(T) subunit. This was first demonstrated in fluorescence experiments where the binding of peptide 3 was shown to strongly promote the enhancement of the tryptophan emission of α(T) that is elicited by the addition of NaF. Specifically, the EC50 for NaF was shifted from ~ 4 mM in the absence of peptide 3 to below 0.5 mM in the presence of peptide 3. Further verification that peptide 3 facilitated the ability of NaF to activate the α(T) subunit was obtained from experiments measuring the α(T)GDP/NaF-stimulated hydrolysis of cyclic GMP by the cyclic GMP phosphodiesterase.