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

Abstract

The N-terminal regions of the heterotrimeric G-protein α-subunits represent one of the major Gβγ contact sites and have been implicated in an interaction with G-protein-coupled receptors. To probe the role of the N- terminal domain of transducin-α in G-protein function, a chimeric Gtiα subunit with the 31 N-terminal Gtα residues replaced by the corresponding 42 residues of Gsα (Ns-Gtiα) has been examined for the interaction with light- activated rhodopsin (R*). Gtiα displayed a somewhat higher R*-stimulated rate of GTPγS binding relative to Ns-Gtiα, suggesting modest involvement of the Gtα N-terminal sequence in recognition of the receptor. However, the intrinsic rate of nucleotide exchange in Ns-Gtiα was significantly faster (k(app) = 0.014 min-1) than that in Gtiα (k(app) = 0.0013 min-1) as judged by the GTPγS binding rates. Substitution of 42 N-terminal residues of Gsα by the Gtα residues in a reciprocal chimera, Nt-Gsα, had an opposite effect-notable reduction in the intrinsic GTPγS-binding rate (k(app) = 0.0075 min-1) in comparison with Gsα (k(app) = 0.028 min-1). Residue Val30 (His41 in Gsα) within the N-terminal region of Gtα interacts with the C-terminal residue, Ile339. To test the hypothesis that observed changes in the intrinsic nucleotide exchange rate in chimeric Gα subunits might be attributed to this interaction, GtiαVal30His, GtiαIle339Ala, and Ns- GtiαHis41Val mutants have been made and analyzed for basal GTPγS binding. GtiαVal30His and GtiαIle339Ala had increased GTPγS binding rates (k(app) = 0.010 and 0.009 min-1, respectively), whereas Ns-GtiαHis41Val had a decreased GTPγS binding rate (k(app) = 0.0011 min-1) relative to their parent proteins. These results suggest that the coupling between the N- terminal and C-terminal domains of Gtα is important for maintaining a low nucleotide exchange rate in unstimulated transducin.