| Abstract
| - The N-terminal regions of the heterotrimeric G-protein α-subunits represent one of the majorGβγ contact sites and have been implicated in an interaction with G-protein-coupled receptors. To probethe role of the N-terminal domain of transducin-α in G-protein function, a chimeric Gtiα subunit with the31 N-terminal Gtα residues replaced by the corresponding 42 residues of Gsα (Ns-Gtiα) has been examinedfor the interaction with light-activated rhodopsin (R*). Gtiα displayed a somewhat higher R*-stimulatedrate of GTPγS binding relative to Ns-Gtiα, suggesting modest involvement of the Gtα N-terminal sequencein recognition of the receptor. However, the intrinsic rate of nucleotide exchange in Ns-Gtiα wassignificantly faster (kapp = 0.014 min-1) than that in Gtiα (kapp = 0.0013 min-1) as judged by the GTPγSbinding rates. Substitution of 42 N-terminal residues of Gsα by the Gtα residues in a reciprocal chimera,Nt-Gsα, had an opposite effectnotable reduction in the intrinsic GTPγS-binding rate (kapp = 0.0075min-1) in comparison with Gsα (kapp = 0.028 min-1). Residue Val30 (His41 in Gsα) within the N-terminalregion of Gtα interacts with the C-terminal residue, Ile339. To test the hypothesis that observed changesin 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 basalGTPγS binding. GtiαVal30His and GtiαIle339Ala had increased GTPγS binding rates (kapp = 0.010 and0.009 min-1, respectively), whereas Ns-GtiαHis41Val had a decreased GTPγS binding rate (kapp = 0.0011min-1) relative to their parent proteins. These results suggest that the coupling between the N-terminaland C-terminal domains of Gtα is important for maintaining a low nucleotide exchange rate in unstimulatedtransducin.
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