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

> **NIH NIH R01** · CORNELL UNIVERSITY · 2024 · $377,050

## Abstract

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 ter...

## Key facts

- **NIH application ID:** 10851975
- **Project number:** 5R01EY034867-02
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** RICHARD A. CERIONE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $377,050
- **Award type:** 5
- **Project period:** 2023-06-01 → 2027-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10851975

## Citation

> US National Institutes of Health, RePORTER application 10851975, Probing the molecular mechanisms that regulate key steps in the GPCR-sensory response pathway responsible for vision in dim light (5R01EY034867-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10851975. Licensed CC0.

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