Functional Reconstitution of Dopamine D2 Receptor into a Supported Model Membrane in a Nanometric Confinement

Abstract

Dopamine D2 receptor (D2R), a G‐protein‐coupled receptor (GPCR), plays critical roles in neural functions and represents the target for a wide variety of drugs used to treat neurological diseases. However, its fundamental physicochemical properties, such as dimerization and affinity to different lipid environments, remain unknown. Here, reconstitution and characterization of D2R in a supported model membrane in nanometric confinement are reported. D2R is expressed in Chinese hamster ovary (CHO) cells and transferred into the supported model membrane as cell membrane blebs. D2R molecules are reconstituted with an elevated density in the cleft between the substrate and poly(dimethylsiloxane) (PDMS) elastomer. Reconstituted D2R retains the physiological functions, as evaluated from its binding to an antagonist and dimerization lifetime. The transient dimer formation of D2R, similar to the live cell, suggests that it is an innate property that does not depend on the cellular structures such as actin filaments. Although the mechanism of this unique reconstitution process is currently not fully understood, the finding points to a new possibility of using a nanometric space (<100 nm thick) as a platform for reconstituting and studying membrane proteins under the quasi‐physiological conditions, which are difficult to be created by other methods.

Extracellular vesicles adhere to cells predominantly through the interaction of CD151-associated integrin heterodimers and GM1 with laminin

“…The tagging of integrin β1 with Halo7 at this position is known to have no detrimental effects on integrin activity (Huet-Calderwood et al, 2017). Wild-type or integrin β1 KO PC3 cells were cotransfected with cDNA encoding Halo7 integrin β1 and transposase, yielding cell lines that exhibited stable expression of Halo7 integrin β1 (Yusa et al, 2009; Nagai et al, 2021). Cells exhibiting high expression of Halo7-integrin β1 were selected by flow cytometry.…”

Antiparallel dimer structure of CELSR cadherin in solution revealed by high-speed atomic force microscopy

“…Simultaneous observations of CELSR2-mGFP and CELSR2-Halo at the single-molecule level were performed by the objective-lens-type total internal reflection (TIR) system on an inverted microscope (IX-81; Olympus) equipped with a 100× objective lens (numerical aperture (NA) 1.49; UAPON100XOTIRF; Olympus) ( 59 , 60 ). To observe the cell–cell interface being formed at less than a few hundred nanometers above the glass surface, the incident angle of the laser was adjusted, which differs slightly from the TIR angle (the cell–cell interface is formed between the two plasma membranes where two facing cells are layered.…”

Integrated model membrane for biophysical studies and biomedical applications

“…In a different line of study, we directly transferred a mammalian GPCR, dopamine D2 receptor (D2R), from Chinese hamster ovary (CHO) cells into the patterned membrane using vesicles that bud off from the cell membrane (blebs). 28) D2R molecules were effectively reconstituted in a nanometer-sized thin cleft formed between a patterned SLB and PDMS. The density of D2R in the model membrane was high enough for the transient dimerization similar to the live plasma membrane.…”