Dopamine D2 receptor (D2R), a G-protein coupled receptor (GPCR), plays critical roles in the neural functions and represents the target for a wide variety of drugs used to treat neurological diseases, including schizophrenia and Parkinson’s disease. However, fundamental physicochemical properties of D2R such as dimerization and affinity to different lipid environments remain unknown. Here we report reconstitution and characterization of D2R in a supported model membrane confined in a nanometric cleft between substrate and poly(dimethylsiloxane) (PDMS) elastomer. We expressed D2R in CHO cells and transferred it into the supported model membrane as cell membrane blebs. D2R molecules were reconstituted with an elevated density and lateral mobility in the cleft between the substrate and PDMS. Reconstituted D2R retained the physiological functions, as evaluated from its binding to an antagonist and dimerization lifetime. The transient dimer formation of D2R similar to that observed in the live cell suggests that it is an innate property that does not depend on the cellular structures such as actin filaments or clathrin coated pits. Although the mechanism of this unique reconstitution process is currently not fully understood, the finding points to a new possibility to use a nanometric space (smaller than 100 nm thickness and 10 fL volume) as a platform for reconstituting and studying membrane proteins under the quasi-physiological conditions, which is difficult to be created by other methods.

Functional reconstitution of dopamine D2 receptor into a supported model membrane in a nanometric confinement

ACS Spring Meeting 2021

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