The mechanism underlying G protein-coupled receptor (GPCR)-mediated signaling systems is still unresolved despite the intense focus these systems have received over the past century. This is due in part to the difficulties in studying membrane proteins in their native context by methods that provide molecular details. The focus of the current proposal is to apply novel biophysical methodologies to unravel the molecular and temporal mysteries of GPCR-mediated signaling pathways. Rhodopsin and the visual system will be the initial focus of the research program. This prototypical GPCR signaling system offers several advantages that will allow for the application of novel biophysical approaches. Atomic force microscopy will result in high-resolution images of individual molecules that will provide structural and organizational information of the system. Single-molecule force spectroscopy will provide detailed information on the molecular interactions in rhodopsin that stabilize the protein and promote its function. Cryo-electron tomography will gain access to an unperturbed rod outer segment to provide structural information on this compartment and on the macromolecules that carry out their function at this venue. Fluorescence resonance energy transfer will be utilized to detect protein-protein interactions of signaling proteins to monitor the dynamic interactions that define the signaling process and the timeframe in which this takes place. Together the information obtained by this unique combination of methodologies will provide key pieces of molecular information that is currently unavailable for these systems. This will help define the molecular mechanism underlying the signaling events that govern important physiological processes regulated by GPCRs. G protein-coupled receptors (GPCRs) represent the largest class of cell surface proteins and drug targets currently on the market. This family of proteins is involved in virtually every physiological process, and dysfunctions in these systems can lead to diseases such as blindness, addiction, diabetes, and heart disease. Despite the importance of GPCRs an accurate molecular description of their action is still lacking. Understanding the molecular mysteries of these systems will lead to the development of more effective therapeutic solutions.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Transition Award (R00)
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Special Emphasis Panel (NSS)
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Mariani, Andrew P
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Case Western Reserve University
Schools of Medicine
United States
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Kawamura, Shiho; Gerstung, Moritz; Colozo, Alejandro T et al. (2013) Kinetic, energetic, and mechanical differences between dark-state rhodopsin and opsin. Structure 21:426-37
Park, P S-H (2012) Ensemble of G protein-coupled receptor active states. Curr Med Chem 19:1146-54
Kawamura, Shiho; Colozo, Alejandro T; Ge, Lin et al. (2012) Structural, energetic, and mechanical perturbations in rhodopsin mutant that causes congenital stationary night blindness. J Biol Chem 287:21826-35
Maeda, Akiko; Okano, Kiichiro; Park, Paul S-H et al. (2010) Palmitoylation stabilizes unliganded rod opsin. Proc Natl Acad Sci U S A 107:8428-33
Hovan, Stephanie C; Howell, Scott; Park, Paul S-H (2010) Fo?rster resonance energy transfer as a tool to study photoreceptor biology. J Biomed Opt 15:067001
Kawamura, Shiho; Colozo, Alejandro T; Muller, Daniel J et al. (2010) Conservation of molecular interactions stabilizing bovine and mouse rhodopsin. Biochemistry 49:10412-20
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Park, Paul S-H; Sapra, K Tanuj; Jastrzebska, Beata et al. (2009) Modulation of molecular interactions and function by rhodopsin palmitylation. Biochemistry 48:4294-304
Park, Paul S-H; Lodowski, David T; Palczewski, Krzysztof (2008) Activation of G protein-coupled receptors: beyond two-state models and tertiary conformational changes. Annu Rev Pharmacol Toxicol 48:107-41
Sapra, K Tanuj; Park, Paul S-H; Palczewski, Krzysztof et al. (2008) Mechanical properties of bovine rhodopsin and bacteriorhodopsin: possible roles in folding and function. Langmuir 24:1330-7

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