Mechanism of allosteric regulation of the rod cGMP phosphodiesterase activity by the helical domain of transducin alpha subunit.
The G protein alpha subunit (Galpha) is composed of two distinct folding domains: a GTP-binding Ras-like domain and an alpha helical domain (HD). We have recently reported that the helical domain (HDt) of the vertebrate visual transducin alpha subunit (Galphat) synergizes activation of retinal cyclic GMP phosphodiesterase (PDE) by activated Galphat (Liu, W., and Northup, J. K., (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12878-12883). Here, we examine the molecular basis for this HD-based signaling regulation, and we provide a new model for the activation of the target effector. The HD proteins derived from visual transducin or taste gustducin alpha subunits, but no other Galpha HD proteins, each attenuate the PDE catalytic core (Palphabeta) and synergize Galphat stimulation of the holoPDE (Palphabetagamma2) with similar apparent affinities. The data from studies of both HDt-mediated attenuation and stimulation indicate that the HDt and the PDE inhibitory subunit (Pgamma) interact with PDE at independent sites and that Palphabeta contains the binding sites for HD. The saturation of both processes by HDt displays positive cooperativity with Hill coefficients of 1.5 for the attenuation of Palphabeta activity and 2.1 for synergism of holoPDE activation. Our data suggest the that Galphat-HDt regulates PDE by allosterically decreasing the affinity of Palphabeta for Pgamma and thus simultaneously facilitating the interaction of the activated Galphat-Ras-like domain with Pgamma. Thus, we propose a new model for the high efficiency of PDE activation as well as deactivation, and, overall, a novel mechanism for controlling fidelity, sensitivity, and efficacy of G protein signaling.
Liu
W
W
Laboratory of Cellular Biology, NIDCD, National Institutes of Health, Rockville, Maryland 20850, USA.
Clark
W A
WA
Sharma
P
P
Northup
J K
JK
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D020134:Catalytic Domain; D002942:Circular Dichroism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D012243:Rhodopsin / Q000378:metabolism; D015398:Signal Transduction; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism*
D046911:Macromolecular Substances; D011994:Recombinant Proteins; C075192:gustducin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012243:Rhodopsin; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D019204:GTP-Binding Proteins; D015293:Transducin