Identification of effector residues on photoreceptor G protein, transducin.
Transducin is a photoreceptor-specific heterotrimeric GTP-binding protein that plays a key role in the vertebrate visual transduction cascade. Here, using scanning site-directed mutagenesis of the chimeric Galphat/Galphai1 alpha-subunit (Galphat/i), we identified Galphat residues critical for interaction with the effector enzyme, rod cGMP phosphodiesterase (PDE). Our evidence suggests that residue Ile208 in the switch II region directly interacts with the effector in the active GTP-bound conformation of Galphat. Residues Arg201, Arg204, and Trp207 are essential for the conformation-dependent Galphat/effector interaction either via direct contacts with the inhibitory PDE gamma-subunit or by forming an effector-competent conformation through the communication network between switch II and the switch III/alpha3-helix domain of Galphat. Residues His244 and Asn247 in the alpha3 helix of Galphat are responsible for the conformation-independent effector-specific interaction. Insertion of these residues rendered the Galphat/i chimera with the ability to bind PDE gamma-subunit and stimulate PDE activity approaching that of native Galphat. Comparative analysis of the interactions of Galphat/i mutants with PDE and RGS16 revealed two adjacent but distinct interfaces on transducin. This indicates a possibility for a functional trimeric complex, RGS/Galpha/effector, that may play a central role in turn-off mechanisms of G protein signaling systems, particularly in phototransduction.
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
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Natochin
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Granovsky
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Artemyev
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D017607:Aluminum Compounds / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005459:Fluorides / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D015293:Transducin / Q000737:chemistry* / Q000235:genetics
D017607:Aluminum Compounds; D020690:GTPase-Activating Proteins; D011506:Proteins; C050992:tetrafluoroaluminate; D006160:Guanosine Triphosphate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D020558:GTP Phosphohydrolases; D015293:Transducin; D005459:Fluorides