Event Title

Determination of Dopamine D2 and D3 Receptor Heteroligomerization using FRET Optical Microspectrometry

Mentor 1

Valerică Raicu

Location

Union Wisconsin Room

Start Date

24-4-2015 2:30 PM

End Date

24-4-2015 3:45 PM

Description

Determination of Dopamine D2 and D3 Receptor Heteroligomerization using FRET Optical MicrospectrometryWilliam F Schmidt, Sugato Ray, Elisa Alvarez-Curto, Michael R Stoneman, Graeme Milligan, Valerică RaicuDepartment of Physics, University of Wisconsin-MilwaukeeFörster (or fluorescence) resonance energy transfer (FRET) is a nonradiative process of energy transfer from an optically excited donor molecule (D) to an unexcited acceptor molecule (A) within close proximity. FRET can be used to detect protein-protein interactions, wherein proteins of interest are fluorescently tagged with FRET constructs and imaged in vivo with a spectrally resolved two-photon microscope. Determination of relative dispositions within protein complexes is possible using pixel-level spectral data and the kinetic theory of FRET. In this study, the interaction between D2 and D3 Dopamine receptors to form an oligomer was assessed using FRET as a tool. Many G protein-coupled receptors (GPCRs) including the D2/D3 pairing have been shown to assemble into heterodimers which often demonstrate differing pharmacological profiles compared to their corresponding monomers. These two receptors were fluorescently tagged with spectral variants of the Green fluorescent protein (GFP) and imaged within human embryonic kidney (HEK) cells. Using specialized software, spectral unmixing was performed on the images in order to separate donor emission from acceptor emission. Averages of FRET efficiencies (Eapp) throughout the cell were calculated from these values for each pixel in the cellular images and compared to theoretical models through application of the kinetic theory of FRET. The results of this study showed a low amount of detected FRET activity (Eapp = 5-10%), more consistent with the existence of background noise than with functional FRET; as a result, we could not confirm the hetero-oligomerization of D2 and D3 receptors as past studies have. To confirm that background noise alone could lead to spurious contributions to FRET, we imaged yeast cells (S. cerevisiae) containing only donor molecules (D) and computed the FRET efficiency. An average Eapp of 4-5% was obtained, demonstrating systematic detection of a small amount of false FRET. Nevertheless, lack of detected FRET between D2 and D3 receptors does not necessarily indicate lack of hetero-oligomerization, as FRET may have been impaired due unfavorable positioning of and distance between D and A tags. Future studies will explore D2/D2 and D3/D3 homo-oligomerization, wherein receptors of the same type may form heterodimers. Studies of oligomerization in GCPRs help us to understand the complete role of these protein assemblies in biological systems and aids in the understanding of pharmacological action at receptor sites. D2 and D3 Dopamine receptors are implicated in various diseases, and understanding their related functions and structures aids in the study of their treatment.

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Apr 24th, 2:30 PM Apr 24th, 3:45 PM

Determination of Dopamine D2 and D3 Receptor Heteroligomerization using FRET Optical Microspectrometry

Union Wisconsin Room

Determination of Dopamine D2 and D3 Receptor Heteroligomerization using FRET Optical MicrospectrometryWilliam F Schmidt, Sugato Ray, Elisa Alvarez-Curto, Michael R Stoneman, Graeme Milligan, Valerică RaicuDepartment of Physics, University of Wisconsin-MilwaukeeFörster (or fluorescence) resonance energy transfer (FRET) is a nonradiative process of energy transfer from an optically excited donor molecule (D) to an unexcited acceptor molecule (A) within close proximity. FRET can be used to detect protein-protein interactions, wherein proteins of interest are fluorescently tagged with FRET constructs and imaged in vivo with a spectrally resolved two-photon microscope. Determination of relative dispositions within protein complexes is possible using pixel-level spectral data and the kinetic theory of FRET. In this study, the interaction between D2 and D3 Dopamine receptors to form an oligomer was assessed using FRET as a tool. Many G protein-coupled receptors (GPCRs) including the D2/D3 pairing have been shown to assemble into heterodimers which often demonstrate differing pharmacological profiles compared to their corresponding monomers. These two receptors were fluorescently tagged with spectral variants of the Green fluorescent protein (GFP) and imaged within human embryonic kidney (HEK) cells. Using specialized software, spectral unmixing was performed on the images in order to separate donor emission from acceptor emission. Averages of FRET efficiencies (Eapp) throughout the cell were calculated from these values for each pixel in the cellular images and compared to theoretical models through application of the kinetic theory of FRET. The results of this study showed a low amount of detected FRET activity (Eapp = 5-10%), more consistent with the existence of background noise than with functional FRET; as a result, we could not confirm the hetero-oligomerization of D2 and D3 receptors as past studies have. To confirm that background noise alone could lead to spurious contributions to FRET, we imaged yeast cells (S. cerevisiae) containing only donor molecules (D) and computed the FRET efficiency. An average Eapp of 4-5% was obtained, demonstrating systematic detection of a small amount of false FRET. Nevertheless, lack of detected FRET between D2 and D3 receptors does not necessarily indicate lack of hetero-oligomerization, as FRET may have been impaired due unfavorable positioning of and distance between D and A tags. Future studies will explore D2/D2 and D3/D3 homo-oligomerization, wherein receptors of the same type may form heterodimers. Studies of oligomerization in GCPRs help us to understand the complete role of these protein assemblies in biological systems and aids in the understanding of pharmacological action at receptor sites. D2 and D3 Dopamine receptors are implicated in various diseases, and understanding their related functions and structures aids in the study of their treatment.