Background and aim:
G protein-coupled receptors (GPCRs) are vital membrane proteins involved in cellular and physiological processes, including sensory perception, signaling, neurotransmission, and immune response. Consequently, understanding their function and regulation is particularly relevant for developing targeted therapies for human diseases. A family of three single-transmembrane proteins, called receptor activity-modifying proteins (RAMPs), has been shown to co-evolve and act as regulators of GPCR function. Still, system-wide GPCR-RAMP interactions have remained underexplored (Kotliar et al., 2023, PharmaRev).

Methods:
We developed a multiplexed system for a multi-modal analysis of the membranebound receptors. The versatile system is based on combinatorial epitope tags in a co-expression library of > 200 GPCRs and RAMPs. Following solubilization into micelles, we apply bead-based immuno-capture assays to determine the receptors' expression, epitope presentation, and RAMP interactions. The data is then implemented into interactive web interfaces to simplify the evaluation.

Results:
At first, we evaluated our concept on a set of 25 secretine-like GPCRs and their interactions with RAMPs (Lorenzen et al., 2019, SciAdv). We then expanded the versatility of our platform to > 200 GPCR and validated the specificity of > 400 anti-GPCR antibodies (Dahl et al., 2023, SciAdv). To demonstrate the system's utility, we studied the downstream effects of GPCR-RAMP binding for the Itch receptor (Kotliar et al., 2023, JBC). More recently, we used the full library to map the interactome of GPCRs and RAMPs system-wide (Kotliar et al., submitted).

Conclusion:
The developed multiplexed system has allowed us to study the analytically challenging membrane receptors and reveal interactions with system-wide implications for GPCR regulation.