Characterisation of neuropeptide Y receptor subtypes by synthetic NPY analogues and by anti-receptor antibodies

Neuropeptide Y (NPY), a 36-mer neuromodulator, binds to the receptors Y-1, Y-2, Y-4 and Y-5 with nanomolar affinity. They all belong to the rhodopsin-like G-protein coupled, seven transmembrane helix spanning receptors. In this study, Ala-substituted and centrally truncated NPY analogues were compared with respect to affinity to the Y-receptors. Furthermore, antibodies against the second (E2) and the third (E3) extracellular loop of NPY Y-1-, Y-2- and Y-5-receptor subtypes were raised and affinity to intact cells was tested by immunofluorescence assays. Both methods were applied in order to receive subtype selective tools and to characterise ligand binding. The analogues [A(13)]-pNPY and [A(27)]-pNPY showed subtype selectivity for the Yz-receptor. Sera against the E2 loop of the Y-1-receptor and against the E2 loop of the Y-2-receptor were subtype selective. Two antibodies against the Y-5 E2 and E3 loop recognised the Y-5- and Y-2-receptor subtypes. In combination, these sera are able to distinguish between the Y-1-, Y-2-, and Ys-receptor subtypes. The analogues and antibodies represent valuable tools to distinguish NPY receptors on membranes and intact cells.