[125I]-GR231118:: a high affinity radioligand to investigate neuropeptide YY1 and Y4 receptors

1 GR231118 (also known as 1229U91 and GW1229), a purported Y-1 antagonist and Y-4 agonist was radiolabelled using the chloramine T method. 2 [I-125]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. 3 Saturation binding experiments showed that [I-125]-GR231118 binds with very high affinity (K-d of 0.09-0.24 nM) in transfected HEK293 cells with the rat Y-1 and Y-4 receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y-2 or Y-5 receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. 4 Competition binding experiments revealed that specific [I-125]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y-1, but not rat Y-4, receptor cDNA. 5 Autoradiographic studies demonstrated that [I-125]-GR231118 binding sites were fully inhibited by the Y-1 antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [I-125]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [I-125]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y-4 receptor subtype. 6 In summary, [I-125]-GR231118 is a new radiolabelled probe to investigate the Y-1 and Y-4 receptors; its major advantage being its high affinity. Using highly selective Y-1 antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [I-125]-GR231118 to the Y-1 receptor allowing for the characterization and visualization of the purported Y-4 subtype.