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. 2021 Apr 23;11:8870. doi: 10.1038/s41598-021-85408-x

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Codon optimisation increases ADGRL4/ELTD1 expression but FL and CTF forms of ADGRL4/ELTD1 do not activate canonical GPCR signalling. (A) Representative Western blot of co FL ADGRL4/ELTD1 compared to wt FL ADGRL4/ELTD1. (B) ADGRL4/ELTD1 signalling construct design showing FL and CTF forms with different tagging strategies. (C) Gαq HTRF FRET assays in ADGRL4/ELTD1 transiently transfected HEK293T cells. Positive controls used were US28 for Gαq (p < 0.0001) and the CTF form of ADGRLG6/GPR126 for Gαs (p < 0.0001) (D) Gαq, Gα12/13, Gαs luciferase assays in ADGRL4/ELTD1 transiently transfected HEK293T cells. Positive controls used were PMA/ionomycin for Gαq (p < 0.0001), US28 for Gα12/13 (p < 0.0001) and the CTF form of GPR126 for Gαs (p < 0.0001). (Abbreviations: Pos CT = positive control; Neg CT = negative control; FL = full length; wt = wild type; co = codon optimised; CTF = C-terminal fragment; HA = hemagglutinin; GFP = green fluorescent protein).