Abstract
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.
Highlights
Brane [1,2,3]
This paper is available on line at http://www.jbc.org switch II domain and the ␣3-5 region as major regions for G␣/effector interactions that emerged from biochemical studies has just been confirmed by the crystal structure of G␣s complexed with catalytic domains of adenylyl cyclase [27, 28]
Expression and Characterization of Mutant G␣t/G␣i1 Proteins with Substitutions in the G␣t Switch II Region—The following amino acid residues of G␣t switch II that are surfaceexposed based on the crystal structure of G␣tGTP␥S, were selected for the scanning mutagenesis [12]: Arg201, Ser202, Arg204, Lys205, Lys206, Trp207, Ile208, His209, Glu212, and Gly213
Summary
G␣t, rod G protein (transducin) ␣-subunit; PDE, rod outer segment cGMP phosphodiesterase; P␣ and P␥, ␣-, -, and ␥-subunits of PDE; ROS, rod outer segment(s); uROS, ureastripped ROS membranes; P␥BC, P␥ labeled with 3-(bromoacetyl)-7diethyl aminocoumarin (BC); GTP␥S, guanosine 5Ј-O-(3-thiotriphosphate); RGS proteins, regulators of G protein signaling; GAP, GTPase activating protein; PCR, polymerase chain reaction; GST, glutathione S-transferase. A member of the RGS family (4 – 6), RGS9, and perhaps other retina-specific RGS proteins serve as GTPase-activating proteins (GAPs) for transducin [7,8,9] They target a transitional intermediate conformation of G␣t during GTP hydrolysis to accelerate GTP hydrolysis and expedite the signal termination [8, 10, 11]. The involvement of the switch II region of G␣ subunits in effector recognition has been supported recently by identification of conserved switch II residues in both G␣i and G␣s required for interaction with adenylyl cyclase [26]. To define roles of the switch II and ␣3-5 regions in the G␣t/PDE interaction and identify specific residues required for the effector binding we carried out scanning mutational analysis of these regions in the G␣t/G␣i1 chimera (G␣t/i) containing G␣t switch II and G␣i1 ␣3-5. G␣i1 residues that are different between G␣i1 and G␣t within the ␣3-5 region of G␣t/i were replaced by corresponding G␣t residues to identify effector-specifying residues of G␣t
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
