Application of bioluminescence resonance energy transfer to quantitate cell-surface expression of membrane proteins.

We report a bioluminescence resonance energy transfer (BRET) assay to quantitate the fraction of an engineered membrane protein at the cell surface versus inside the cell. As test cases, we engineered two different G protein-coupled receptors (GPCRs) in which a NanoLuc luciferase (NLuc) and a HaloTag are fused to the extracellular amino-terminal tail of the receptors. We then employed a pulse-chase labeling approach relying on two different fluorescent dyes with distinctive cell permeability properties. The dyes are efficiently excited by luminescence from NLuc, but are spectrally distinct. Measuring BRET from the chemiluminescence of the NLuc to the fluorophores bound to the HaloTag minimizes the limitations of in-cell fluorescence resonance energy transfer (FRET)-based approaches such as photobleaching and autofluorescence. The BRET surface expression assay can quantitatively differentiate between the labeling of receptors at the cell surface and receptors inside of the cell. The assay is shown to be quantitative and robust compared with other approaches to measure cell surface expression of membrane proteins such as enzyme-linked immunosorbent assay or immunoblotting, and significantly increases the throughput because the assay is designed to be carried out in microtiter plate format.

Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins.

Receptor activity-modifying proteins (RAMPs) form complexes with G protein-coupled receptors (GPCRs) and may regulate their cellular trafficking and pharmacology. RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail. To elucidate a comprehensive GPCR-RAMP interactome, we created a library of 215 dual epitope-tagged (DuET) GPCRs representing all GPCR subfamilies and coexpressed each GPCR with each of the three RAMPs. Screening the GPCR-RAMP pairs with customized multiplexed suspension bead array (SBA) immunoassays, we identified 122 GPCRs that showed strong evidence for interaction with at least one RAMP. We screened for interactions in three cell lines and found 23 endogenously expressed GPCRs that formed complexes with RAMPs. Mapping the GPCR-RAMP interactome expands the current system-wide functional characterization of RAMP-interacting GPCRs to inform the design of selective therapeutics targeting GPCR-RAMP complexes.

The Coronavirus Calendar (CoronaCal): a simplified SARS-CoV-2 test system for sampling and retrospective analysis.

Objectives: To develop a biological diary (CoronaCal) that allows anyone in the community to collect and store serial saliva samples and chart symptoms on ordinary printer paper. Methods: Diaries were analyzed for the presence of SARS-CoV-2 RNA using established polymerase chain reaction (PCR) procedures. CoronaCal diaries were distributed to volunteer subjects in the community during the peak of the COVID-19 outbreak in New York. Volunteers collected their own daily saliva samples and self-reported symptoms. Results: SARS-CoV-2 RNA extracted from CoronaCals was measured using qPCR and RNA levels were correlated with reported symptoms. SARS-CoV-2 RNA was detected in CoronaCals from nine of nine people with COVID-19 symptoms or exposure to someone with COVID-19, and not in one asymptomatic person. CoronaCals were stored for up to 70 days at room temperature during collection and then frozen for up to four months before analysis, suggesting that SARS-CoV-2 RNA is stable once dried onto paper. Conclusions: Sampling saliva on simple paper provides a useful method to study the natural history and epidemiology of COVID-19. The CoronaCal collection and testing method is easy to implement, inexpensive, non-invasive and scalable. The approach can inform the historical and epidemiological understanding of infections in individuals and populations.