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Endocrine Abstracts (2014) 34 OC1.4 | DOI: 10.1530/endoabs.34.OC1.4

SFEBES2014 Oral Communications Young Endocrinologists prize session (6 abstracts)

Single molecule analysis of GPCR transactivation reveals oligomeric complexes that regulate signal sensitivity

Kim Jonas 1 , Francesca Fanelli 2 , Ilpo Huhtaniemi 1,3 & Aylin Hanyaloglu 1


1Imperial College London, London, UK; 2University of Modena, Modena, Italy; 3University of Turku, Turku, Finland.


G protein-coupled receptors (GPCRs) are the largest family of mammalian receptors, modulating most aspects of endocrine homeostasis. GPCRs can associate to form dimers and higher order oligomers, acting to diversify receptor functionality. Most studies have explored the functional significance of GPCR dimerization in vitro and we have recently demonstrated the functional relevance of GPCR dimerization in vivo. Using the LH receptor (LHR), we have shown that targeted co-expression of ligand-binding deficient (LHRB−), and signalling-defective (LHRS−) LHR restored the gonadal function and fertility of male LHR knockout mice, showing that dimerisation is a physiologically relevant form of GPCR-mediated signalling. As transactivation is a complex form of signal regulation, we went on to study the molecular aspects of GPCR di/oligomerisation in vitro, utilising these LHRB− and LHRS− transactivating mutants. Using the single molecule imaging technique of photo-activated localisation microscopy (PALM), we have probed the molecular composition of both WT LHR, and transactivating LHRB−/S− complexes, with a localisation precision of ~20 nm. Both WT and transactivating LHR complexes formed dimers and higher order oligomeric complexes. Surprisingly, the transactivating mutant LHRB−/S− appeared to favour hetero-oligomeric (LHRB−/S−) complexes, therefore we determined if this difference in association impacted on ligand-dependent G-protein pathways using human chorionic gonadotrophin (hCG) and LH. Both LH and hCG produced comparable Gs-dependent responses in WT LHR and LHRB−/S, however, LH-dependent Gq pathways were attenuated in the transactivating model. No differences were observed in associated forms in either WT or LHRB−/S− complexes. Structural modelling and spatial arrangement analysis via PALM provided insight into how the transactivating mutant receptors associate into dimeric and higher order complexes. These studies show us how di/oligomerization may modulate the functionality of one receptor subtype, providing key insights into important mechanisms of how di/oligomerisation impacts on the physiology of GPCRs in endocrine systems.

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