Coupling between the N- and C-terminal domains influences transducin-alphaintrinsic GDP/GTP exchange

The N-terminal regions of the heterotrimeric G-protein alpha-subunits repre sent one of the major G beta gamma contact sites and have been implicated i n an interaction with G-protein-coupled receptors, To probe the role of the N-terminal domain of transducin-alpha in G-protein function, a chimeric Gt i alpha subunit with the 31 N-terminal Gt alpha residues replaced by the co rresponding 42 residues of Gs alpha (Ns-Gti alpha) has been examined for th e interaction with light-activated rhodopsin (R*). Gtia displayed a somewha t higher R*-stimulated rate of GTP gamma S binding relative to Ns-Gti alpha , suggesting modest involvement of the Gt alpha N-terminal sequence in reco gnition of the receptor. However, the intrinsic rate of nucleotide exchange in Ns-Gti alpha was significantly faster (k(app) = 0.014 min(-1)) than tha t in Gti alpha (k(app) = 0.0013 min(-1)) as judged by the GTP gamma S bindi ng rates. Substitution of 42 N-terminal residues of Gs alpha by the Gt alph a residues in a reciprocal chimera, Nt-Gs alpha, had an opposite effect-not able reduction in the intrinsic GTP gamma S-binding rate (k(app) = 0.0075 m in(-1)) in comparison with Gs alpha (k(app) = 0.028 min(-1)). Residue Val30 (His41 in Gs alpha) within the N-terminal region of Gt alpha interacts wit h the C-terminal residue, Ile339, To test the hypothesis that observed chan ges in the intrinsic nucleotide exchange rate in chimeric G alpha subunits might be attributed to this interaction, Gti alpha Val30His, Gti alpha Ile3 39Ala, and Ns-Gti alpha His41Val mutants have been made and analyzed for ba sal GTP gamma S binding. Gti alpha Val30His and Gti alpha Ile339Ala had inc reased GTP gamma S binding rates (k(app) = 0.010 and 0.009 min(-1), respect ively), whereas Ns-Gti alpha His41Val had a decreased GTP gamma S binding r ate (k(app) = 0.0011 min(-1)) relative to their parent proteins, These resu lts suggest that the coupling between the N-terminal and C-terminal domains of Gt alpha is important for maintaining a low nucleotide exchange rate in unstimulated transducin.