Work

Red and far-red cleavable fluorescent dyes for self-labelling enzyme protein tagging and interrogation of GPCR co-internalization

Year: 2024

Type: article

Abstract: Post-labelling cleavable substrates for self-labelling protein tags, such as SNAP- and Halo-tags, can be used to study cell surface receptor trafficking events by stripping dyes from non-internalized protein pools. Since the complexity of receptor biology requires the use of multiple and orthogonal approaches to simultaneously probe multiple receptor pools, we report the development of four membrane impermeable probes that covalently bind to either the SNAP- or the Halo-tag in the red to far-red range. These molecules bear a disulfide bond to release the non-internalized probe using the reducing agent sodium 2-mercaptoethane sulfonate (MESNA). As such, our approach allows the simultaneous visualization of multiple internalized cell surface proteins in two colors which we showcase using G protein-coupled receptors. We use this approach to detect internalized group II metabotropic glutamate receptor (mGluRs), homo- and heterodimers, and to reveal unidirectional crosstalk between co-expressed glucagon-like peptide 1 (GLP1R) and glucose-dependent insulinotropic polypeptide receptors (GIPR). In these applications, we translate our method to both high resolution imaging and quantitative, high throughput assays, demonstrating the value of our approach for a wide range of applications.

Source: RSC Chemical Biology

DOI: 10.1039/d4cb00209a

Authors: Kilian Roßmann, Ramona Birke, Joshua Levitz, Ben Jones, Johannes Broichhagen

Institutions: Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Weill Cornell Medicine, Imperial College London

Language: English

Topic: Receptor Mechanisms and Signaling

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,962