# Protocol to visualize and quantify the COPII concentration and anterograde transport of nascent G protein-coupled receptors

## TL;DR

This paper provides a detailed protocol to visualize and measure how new GPCRs are transported from the endoplasmic reticulum to the cell surface using COPII vesicles.

## Contribution

A novel protocol using the RUSH system to study the anterograde transport of GPCRs and other cargoes.

## Key findings

- The protocol enables visualization of GPCR recruitment to COPII vesicles.
- It allows quantification of GPCR transport from the ER through the Golgi to the cell surface.
- The method is applicable to non-GPCR cargoes as well.

## Abstract

Here, we present a protocol for visualization and quantification of the recruitment of newly synthesized G protein-coupled receptors (GPCRs) to coat protein complex II vesicles and GPCR transport from the endoplasmic reticulum through the Golgi to the cell surface in the retention using the selective hooks assay. We describe steps for plasmid construction, cell transfection, transport synchronization, confocal microscope imaging, and quantification. This protocol is also applicable for studying the transport of non-GPCR cargoes.

For complete details on the use and execution of this protocol, please refer to Xu et al.1,2

•Application of the RUSH system in studying the forward delivery of nascent GPCRs•Steps to visualize the concentration of GPCRs in COPII vesicles•Introductions for characterizing the ER-to-Golgi transport of GPCRs•Guidance on measuring the post-Golgi traffic of GPCRs

Application of the RUSH system in studying the forward delivery of nascent GPCRs

Steps to visualize the concentration of GPCRs in COPII vesicles

Introductions for characterizing the ER-to-Golgi transport of GPCRs

Guidance on measuring the post-Golgi traffic of GPCRs

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for visualization and quantification of the recruitment of newly synthesized G protein-coupled receptors (GPCRs) to coat protein complex II vesicles and GPCR transport from the endoplasmic reticulum through the Golgi to the cell surface in the retention using the selective hooks assay. We describe steps for plasmid construction, cell transfection, transport synchronization, confocal microscope imaging, and quantification. This protocol is also applicable for studying the transport of non-GPCR cargoes.