# G protein selectivity profile of GPR56/ADGRG1 and its effect on downstream effectors

## TL;DR

This study explores how different ligands activate the GPR56 receptor, leading to distinct G protein selectivity but converging on the Rho pathway activation in cancer cells.

## Contribution

The study reveals distinct activation modes of GPR56 by different ligands, leading to differential G protein selectivity and downstream signaling.

## Key findings

- GPR56 constitutively activates G12 and G13, but antibody 10C7 preferentially activates G13.
- The autoproteolytically deficient mutant GPR56-T383A is activated by 10C7 but not by 3αDOG.
- 10C7 activates the Rho pathway in breast cancer cells despite favoring G13 over G12.

## Abstract

GPR56, an adhesion G-protein coupled receptor (aGPCRs) with constitutive and ligand-promoted activity, is involved in many physiological and pathological processes. Whether the receptor’s constitutive or ligand-promoted activation occur through the same molecular mechanism, and whether different activation modes lead to functional selectivity between G proteins is unknown. Here we show that GPR56 constitutively activates both G12 and G13. Unlike constitutive activation and activation with 3-a-acetoxydihydrodeoxygedunin (3αDOG), stimulation with an antibody, 10C7, directed against GPR56’s extracellular domain (ECD) led to an activation that favors G13 over G12. An autoproteolytically deficient mutant, GPR56-T383A, was also activated by 10C7 indicating that the tethered agonist (TA) exposed through autocatalytic cleavage, is not required for this activation modality. In contrast, this proteolysis-resistant mutant could not be activated by 3αDOG indicating different modes of activation by the two ligands. We show that an N-terminal truncated GPR56 construct (GPR56-Δ1–385) is devoid of constitutive activity but was activated by 3αDOG. Similarly to 3αDOG, 10C7 promoted the recruitment of b-arrestin-2 but GPR56 internalization was β-arrestin independent. Despite the slow activation mode of 10C7 that favors G13 over G12, it efficiently activated the downstream Rho pathway in BT-20 breast cancer cells. These data show that different GPR56 ligands have different modes of activation yielding differential G protein selectivity but converging on the activation of the Rho pathway both in heterologous expressions system and in cancer cells endogenously expressing the receptor. 10C7 is therefore an interesting tool to study both the processes underlying GPR56 activity and its role in cancer cells.