PT  - JOURNAL ARTICLE
AU  - Laura E Kilpatrick
AU  - Laura J Humphrys
AU  - Nicholas D Holliday
TI  - A G Protein Coupled Receptor Dimer Imaging Assay Reveals Selectively Modified Pharmacology of Neuropeptide Y Y1 / Y5 Receptor Heterodimers
AID  - 10.1124/mol.114.095356
DP  - 2015 Jan 01
TA  - Molecular Pharmacology
PG  - mol.114.095356
4099  - http://molpharm.aspetjournals.org/content/early/2015/01/30/mol.114.095356.short
4100  - http://molpharm.aspetjournals.org/content/early/2015/01/30/mol.114.095356.full
AB  - The ability of G protein coupled receptors (GPCRs) to form dimers, and particularly heterodimers, offers potential for targeted therapeutics with improved selectivity. However studying dimer pharmacology is challenging, because of signalling cross-talk, or because dimerization may often be transient in nature. Here we develop a system to isolate the pharmacology of precisely defined GPCR dimers, trapped by bimolecular fluorescence complementation (BiFC). Specific effects of agonist activation on such dimers are quantified using automated imaging and analysis of their internalisation, controlled for by simultaneous assessment of endocytosis of one co-expressed protomer population. We applied this BiFC system to study example neuropeptide Y (NPY) Y1 receptor dimers. Incorporation of binding site or phosphorylation site mutations into just one protomer of a Y1 / Y1 BiFC homodimer had no impact on efficient NPY stimulated endocytosis, demonstrating that single site agonist occupancy, and one phosphorylated monomer within the dimer, was sufficient. For two Y1 receptor heterodimer combinations (with the Y4 receptor, or β2-adrenoceptor), agonist and antagonist pharmacology was explained by independent actions on the respective orthosteric binding sites. However Y1 / Y5 receptor BiFC dimers, compared to the constituent subtypes, were characterised by reduced potency and efficacy of Y5 selective peptide agonists, the inactivity of Y1 selective antagonists, and a change from surmountable to non-surmountable antagonism for three unrelated Y5 antagonists. Thus allosteric interactions between Y1 and Y5 receptor binding sites modify the pharmacology of the heterodimer, with implications for potential anti-obesity agents that target centrally co-expressed Y1 and Y5 receptors to suppress appetite.