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Endocrine Abstracts (2015) 37 GP09.04 | DOI: 10.1530/endoabs.37.GP.09.04

ECE2015 Guided Posters Nuclear receptors and signalling (8 abstracts)

Characterisation of insulin and β2-adrenergic receptor heteromers: experimental and bioinformatics approach

Maja Mandić 1 , Sanja Glisic 2 , Christina Pedersen 3 , Nevena Veljkovic 2 , Jane Nøhr 3 & Milka Vrecl 1


1Veterinary Faculty, Institute of Anatomy, Histology and Embryology, University of Ljubljana, Ljubljana, Slovenia; 2Institute of Nuclear Sciences VINCA, Center for Multidisciplinary Research, University of Belgrade, Belgrade, Serbia; 3Department of Incretin Biology, Novo Nordisk A/S, Måløv, Denmark.


Evidences for a functional link between insulin receptor (IR) and β2-adrenergic receptor (β2AR) exist in the mouse heart and in retina and are correlated with the cardiovascular dysfunction under insulin-resistant states1 and a diabetic retinopathy phenotype2. In our recent study we showed that i) IR coexpression reduced β2AR surface expression and accelerated its internalization, ii) provided evidence for direct interaction and higher-order β2AR:IR oligomer formation, likely comprising heteromers of homodimers, and iii) identified prospective intracellular interaction domains engaged in heteromerisation3. In this study we examined the involvement of the cytoplasmic part of the IR β chain in heteromerisation and addressed the role of β2AR:IR heteromer in signalling. Evidence suggesting that the cytoplasmic part of the IR β chain is prerequisite for the interaction with the β2AR was provided by BRET2 saturation and Heteromer Identification Technology (HIT) assays using a IR 1–1271 mutant lacking the IR C-terminal tail region (amino acids from 1272 to 1360). In BRET2 saturation assays, IR 1–1271–Rluc8 mutant displayed lower BRETmax value and slightly reduced affinity compared to the WT IR. For the complex consisting of IR 1–1271–RLuc8:β2AR-GFP2 the saturation was not reached; most likely reflecting only random collisions. Furthermore, in the IR 1–1271–Rluc8:β2AR HIT assay with β-arrestin 2 no substantial agonist-induced increase in BRET2 signal was detected that would be indicative of β-arrestin 2 recruitment to the IR 1–1271–Rluc8:β2AR heteromer, arguing against ability of IR 1–1271 to heteromerise with the β2AR. Additional in silico mutation analysis of IR predicted substitutions mostly affecting the binding to the β2AR. β2AR:IR heteromerization also influenced the pharmacological phenotype of the β2AR i.e. its ability to recruit β-arrestin 2 and cAMP signalling.

References: 1. Fu Q, Xu B, Liu Y, Parikh D, Li J, et al. Diabetes 2013 63 2676–2689.

2. Jiang Y, Zhang Q, Liu L, Tang J, Kern TS & Steinle JJ. PLoS One 2013 8 (7) e70555.

3. Mandić M, Drinovec L, Glisic S, Veljkovic N, Nøhr J & Vrecl M. PLoS One 2014 9 (11) e112664.

Disclosure: We acknowledge funding from the Basileus program to M Mandic, Slovenian Research Agency program P4-0053 to M Vrecl and the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant number 173001) to S Glisic.

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