Coupling between the N- and C-Terminal Domains Influences Transducin-α Intrinsic GDP/GTP Exchange

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Abstract
The N-terminal regions of the heterotrimeric G-protein alpha-subunits represent one of the major Gbetagamma 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-alpha in G-protein function, a chimeric Gtialpha subunit with the 31 N-terminal Gtalpha residues replaced by the corresponding 42 residues of Gsalpha (Ns-Gtialpha) has been examined for the interaction with light-activated rhodopsin (R). Gtialpha displayed a somewhat higher R-stimulated rate of GTPgammaS binding relative to Ns-Gtialpha, suggesting modest involvement of the Gtalpha N-terminal sequence in recognition of the receptor. However, the intrinsic rate of nucleotide exchange in Ns-Gtialpha was significantly faster (k(app) = 0.014 min(-)(1)) than that in Gtialpha (k(app) = 0.0013 min(-1)) as judged by the GTPgammaS binding rates. Substitution of 42 N-terminal residues of Gsalpha by the Gtalpha residues in a reciprocal chimera, Nt-Gsalpha, had an opposite effect-notable reduction in the intrinsic GTPgammaS-binding rate (k(app) = 0.0075 min(-)(1)) in comparison with Gsalpha (k(app) = 0.028 min(-)(1)). Residue Val30 (His41 in Gsalpha) within the N-terminal region of Gtalpha interacts with the C-terminal residue, Ile339. To test the hypothesis that observed changes in the intrinsic nucleotide exchange rate in chimeric Galpha subunits might be attributed to this interaction, GtialphaVal30His, GtialphaIle339Ala, and Ns-GtialphaHis41Val mutants have been made and analyzed for basal GTPgammaS binding. GtialphaVal30His and GtialphaIle339Ala had increased GTPgammaS binding rates (k(app) = 0. 010 and 0.009 min(-)(1), respectively), whereas Ns-GtialphaHis41Val had a decreased GTPgammaS 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 Gtalpha is important for maintaining a low nucleotide exchange rate in unstimulated transducin.