Probing the molecular mechanisms that regulate key steps in the GPCR-sensory response pathway responsible for vision in dim light

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 (GT). GTP-bound GT
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 GT. Binding of
GTP-bound GT 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 GT 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 GT-PDE6 interactions, (ii) studies with a bivalent GT antibody that enables us to form different
asymmetric configurations of GT-PDE6 complexes and (iii) site-directed spin probe labeling with electron spin
resonance spectroscopy, to test our model for how two GT 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 GT-PDE6 interactions to determine how membranes
facilitate PDE6 activation by GT, and (ii) FRET to examine the orientation of the PDE subunits on PDE6 in the
presence and absence of GTP-bound GT in a membrane environment. We will also: (iii) reconstitute GT-
stimulated PDE6 activity in nanodiscs, and (iv) undertake structure determinations of PDE6 alone and bound to
GT, 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 terminated when its stimulation has ceased,
as well as provide fundamentally important insights into key steps essential for other GPCR-sensory responses.

Grant Number: 5R01EY034867-03
NIH Institute/Center: NIH
Principal Investigator: RICHARD CERIONE