Detergent-free extraction of a functional low-expressing GPCR from a human cell line

• Published in: Biochimica et biophysica acta. Biomembranes 2020-03, Vol.1862 (3), p.183152-183152, Article 183152
• Main Authors: Bada Juarez, Juan Francisco, Muñoz-García, Juan C., Inácio dos Reis, Rosana, Henry, Alistair, McMillan, David, Kriek, Marco, Wood, Martyn, Vandenplas, Catherine, Sands, Zara, Castro, Luis, Taylor, Richard, Watts, Anthony
• Format: Article
• Language: English
• Subjects: Detergent-free; Dopamine receptor; GPCR; Lipodisq; Microscale thermophoresis; Native Nanodiscs; Neurotensin; SMA polymer
• ISSN: 0005-2736; 1879-2642
• DOI: 10.1016/j.bbamem.2019.183152

Dopamine receptors (DRs) are class A G-Protein Coupled Receptors (GPCRs) prevalent in the central nervous system (CNS). These receptors mediate physiological functions ranging from voluntary movement and reward recognition to hormonal regulation and hypertension. Drugs targeting dopaminergic neurotransmission have been employed to treat several neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, Huntington's disease, attention deficit hyperactivity disorder (ADHD), and Tourette's syndrome. In vivo, incorporation of GPCRs into lipid membranes is known to be key to their biological function and, by inference, maintenance of their tertiary structure. A further significant challenge in the structural and biochemical characterization of human DRs is their low levels of expression in mammalian cells. Thus, the purification and enrichment of DRs whilst retaining their structural integrity and function is highly desirable for biophysical studies. A promising new approach is the use of styrene–maleic acid (SMA) copolymer to solubilize GPCRs directly in their native environment, to produce polymer-assembled Lipodisqs (LQs). We have developed a novel methodology to yield detergent-free D1-containing Lipodisqs directly from HEK293f cells expressing wild-type human dopamine receptor 1 (D1). We demonstrate that D1 in the Lipodisq retains activity comparable to that in the native environment and report, for the first time, the affinity constant for the interaction of the peptide neurotransmitter neurotensin (NT) with D1, in the native state. •We report for the first time the Lipodisq detergent-free purification of a human wild-type GPCR from its native environment.•We demonstrate that microscale thermophoresis can be employed to characterize ligand binding for Lipodisq-embedded protein.•We show for the first time the binding of the neurotensin peptide to D1R, which might have important biological implications.