Communication between switch II and switch III of the transducin alpha subunit is essential for target activation.
Comparisons of the tertiary structures of the GDP-bound and guanosine 5'-O-(thiotriphosphate) (GTPgammaS)-bound forms of the alpha subunit of transducin (alphaT) indicate that there are three regions that undergo changes in conformation upon alphaT activation. Two of these regions, Switch I and Switch II, were originally identified in Ras, while Switch III appears to be unique to trimeric GTP-binding proteins (G proteins). We find that replacement of the Switch III region (aspartic acid 227 through asparagine 237) with a single alanine residue yields an alphaT subunit that fully binds and hydrolyzes GTP but no longer stimulates the activity of the cyclic GMP phosphodiesterase (PDE), the physiological target for transducin. We also show that changing glutamic acid 232 of alphaT to a leucine (E232L) had no effect on rhodopsin-stimulated GTP-GDP exchange nor on the GTP hydrolytic activity of alphaT. However, the GTPgammaS-bound form of the alphaTE232L mutant was unable to stimulate the activity of the cyclic GMP PDE. The lack of stimulation was not due to an inability of the alphaTE232L mutant to bind to the target. Taken together, these results indicate that glutamic acid 232 mediates a conformational coupling between Switch II and Switch III, which is essential for converting GTP-dependent G protein-target interactions into a stimulation of target/effector activity.
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Department of Pharmacology, Veterinary Medical Center, Cornell University, Ithaca, New York 14853-6401, USA.
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D000818:Animals; D001665:Binding Sites; D002417:Cattle; D020558:GTP Phosphohydrolases / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D018411:Spodoptera; D015293:Transducin / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020558:GTP Phosphohydrolases; D015293:Transducin