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

探索调节负责弱光视觉的 GPCR 感觉反应通路关键步骤的分子机制

基本信息

批准号：
10635707
负责人：
RICHARD A. CERIONE
金额：
$ 37.92万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10635707
关键词：
3-Dimensional Address Antibodies Binding Biochemical Biological Biological Assay C-terminal Catalysis Catalytic Domain Complex Coupled Coupling Cryoelectron Microscopy Cyclic GMP Disease Electron Spin Resonance Spectroscopy Environment Enzymes Eye diseases Fluorescence Fluorescence Resonance Energy Transfer G-Protein Signaling Pathway G-Protein-Coupled Receptors GTP Binding GTP-Binding Proteins Goals Guanosine Triphosphate Heterotrimeric GTP-Binding Proteins Ion Channel Gating Label Laboratories Learning Light Link Liposomes Membrane Membrane Lipids Modeling Molecular Probes Monitor Noise Optic Nerve Output Pathway interactions Photons Photoreceptors Phototransduction Physiologic pulse Physiological Play Proteins Regulation Retina Retinal Degeneration Rhodopsin Rod Outer Segments Role Sensory Sensory Receptors Signal Pathway Signal Transduction Site Structure System Testing Time Transducin Vision Vision Disorders Work absorption alpha Subunit Transducin clinically relevant design insight nanodisk novel novel therapeutic intervention phosphodiesterase 6 phosphoric diester hydrolase reconstitution response retinal rods three dimensional structure

项目摘要

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.

项目摘要