Poster Presentation ESA-SRB-ANZBMS 2024 in conjunction with ENSA

Investigating pharmacological changes in the AT1-LOX-1 receptor heteromer using bioluminescence resonance energy transfer (#508)

Julyan Tan 1 , Kevin DG Pfleger 1 2 3 , Elizabeth KM Johnstone 1 2 4
  1. Harry Perkins Institute of Medical Research and Centre for Medical Research, Nedlands, WA, Australia
  2. Australian Research Council Centre for Personalised Therapeutics Technologies, Australia
  3. Dimerix Limited, Nedlands, WA, Australia
  4. School of Biomedical Sciences, The University of Western Australia, Nedlands, WA, Australia

A significant determinant of atherosclerosis has been identified in the inflammatory signalling produced from the activation of Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) by its cognate ligand, oxidised LDL (OxLDL) (1).  Recent studies support the idea that G protein-coupled receptors (GPCRs) form heteromers with, and transactivate, non-GPCR partner receptors to cause varying changes to their pharmacology, signalling and intracellular trafficking (2).  Of interest is the idea that LOX-1 can form heteromeric complexes with GPCRs, such as angiotensin II type 1 (AT1) receptor (3). These may lead to a plethora of changes to receptor pharmacology and an upregulation in inflammatory signalling, contributing to atherosclerotic outcomes.

The present study aimed to investigate interactions between LOX-1 and the AT1 receptor, using the Receptor-Heteromer Investigation Technology (Receptor-HIT) (4) assay in HEK293FT cells.  Receptor-HIT detects receptor heteromers through the ligand-induced recruitment of interacting proteins to the heteromer, using a proximity-based biophysical technique such as bioluminescence resonance energy transfer (BRET) (5).  By co-expressing one luciferase-labelled receptor and one unlabelled receptor, as well as a fluorophore-labelled interacting protein, Receptor-HIT detects a BRET signal upon treatment with a ligand specific for the unlabelled receptor. This indicates recruitment of the interacting protein to the receptor heteromer, which may provide insights into pharmacological changes such as with G protein-signalling.

It was found that LOX-1 produced Receptor-HIT signals indicative of heteromerisation when co-transfected with the AT1 receptor and various signalling proteins.  Additionally, AT1 and LOX-1 co-transfection selectively altered some of the downstream signalling properties of the receptors.  These findings support the existence of the AT1-LOX-1 heteromer and provide evidence of novel pharmacological changes which may be related to atherosclerosis pathogenesis.

  1. 1. Kattoor AJ, Goel A, Mehta JL. LOX-1: Regulation, Signaling and Its Role in Atherosclerosis. Antioxidants (Basel). 2019 Jul 11;8(7). doi:10.3390/antiox8070218.
  2. 2. Pickering RJ, Tikellis C, Rosado CJ, Tsorotes D, Dimitropoulos A, Smith M, Huet O, Seeber RM, Abhayawardana R, Johnstone EK, Golledge J, Wang Y, Jandeleit-Dahm KA, Cooper ME, Pfleger KD, Thomas MC. Transactivation of RAGE mediates angiotensin-induced inflammation and atherogenesis. J Clin Invest. 2019 Jan 2;129(1):406-421. doi:10.1172/JCI99987.
  3. 3. Yamamoto K, Kakino A, Takeshita H, Hayashi N, Li L, Nakano A, Hanasaki-Yamamoto H, Fujita Y, et al. Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin-like oxLDL receptor. FASEB J. 2015 Aug;29(8):3342-56. doi:10.1096/fj.15-271627.
  4. 4. Johnstone EK, Pfleger KD. Receptor-Heteromer Investigation Technology and its application using BRET. Front Endocrinol (Lausanne). 2012 Aug 22;3:101. doi:10.3389/fendo.2012.00101.
  5. 5. Pfleger KD, Eidne KA. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods. 2006 Mar;3(3):165-74. doi: 10.1038/nmeth841.