Coupling between the N- and C-terminal domains influences transducin-alpha intrinsic GDP/GTP exchange.
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.
Muradov
K G
KG
Department of Physiology, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Artemyev
N O
NO
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D015293:Transducin