Project Abstract Our laboratory has used the phototransduction pathway in retinal rods, a beautifully designed sensory response system, to study how G protein coupled receptors (GPCRs) propagate highly amplified signals. This pathway starts with the absorption of a photon by the GPCR rhodopsin, resulting in its activation of the heterotrimeric G protein transducin by catalyzing GDP-GTP exchange on the transducin-alpha subunit (GT). GTP-bound GT subunits then interact with their effector protein, the cyclic GMP (cGMP) phosphodiesterase-6 (PDE6), a tetrameric enzyme with two catalytic subunits (PDE, PDE) and two subunits (PDE) that bind GT. Binding of GTP-bound GT subunits to PDE6 activates its ability to hydrolyze cGMP to GMP, thus closing cGMP-gated ion channels in retinal rod membranes and sending a signal to the optic nerve. We determined structures for the rhodopsin-transducin complex by cryo-electron microscopy (cryoEM), which together with efforts from other laboratories, led to a detailed picture of how GPCRs activate their G protein partners. However, there is still a great deal to learn about how activated G proteins execute a precise regulation of their effector proteins. Recently, we solved a cryoEM structure for a complex in solution that contains two GTP-bound GT subunits and PDE6, leading to a model describing how transducin activates its biological effector. We will now test important aspects of this model through two broad experimental aims, each comprised of a number of sub-aims: 1) Determine how activated G subunits of the retinal G protein transducin exert a highly tuned regulation of their biological effector PDE6. We will perform: (i) fluorescence read-outs we developed to monitor GT-PDE6 interactions, (ii) studies with a bivalent GT antibody that enables us to form different asymmetric configurations of GT-PDE6 complexes and (iii) site-directed spin probe labeling with electron spin resonance spectroscopy, to test our model for how two GT subunits activate PDE6, as well as (iv) determine if the model is consistent with how RGS9 deactivates signal propagation. 2) Establish a mechanistic basis for how a membrane environment influences the ability of the retinal G protein to activate its biological effector. We will use: (i) fluorescence read-outs to monitor GT-PDE6 interactions to determine how membranes facilitate PDE6 activation by GT, and (ii) FRET to examine the orientation of the PDE subunits on PDE6 in the presence and absence of GTP-bound GT in a membrane environment. We will also: (iii) reconstitute GT- stimulated PDE6 activity in nanodiscs, and (iv) undertake structure determinations of PDE6 alone and bound to GT, to test our model for PDE6 activation in a more physiological setting. The results of these studies will enable us to further develop a comprehensive mechanistic picture for how an activated G protein regulates its biological effector in phototransduction, and how this signal is rapidly ter...