Studies of the human, rat, and guinea pig Y4 receptors using neuropeptide Y analogues and two distinct radioligands

The neuropeptide Y-family receptor Y4 differs extensively between human and rat in sequence, receptor binding, and anatomical distribution. We have investigated the differences in binding profile between the cloned human, rat, and guinea pig Y4 receptors using NPY analogues with single amino acid replacements or deletion of the central portion. The most striking result was the increase in affinity for the rat receptor, but not for human or guinea pig, when amino acid 34 was replaced with proline; [Ahx(8-20),Pro(34)]NPY bound to the rat Y4 receptor with 20-fold higher affinity than [Ahx(8-20)]NPY. Also, the rat Y4 tolerates alanine in position 34 since p[Ala(34)]NPY bound with similar affinity as pNPY while the affinity for hY4 and gpY4 decreased about 50-fold. Alanine substitutions in position 33, 35, and 36 as well as the large loop-deletion, [Ahx(5-24)]NPY, reduced the binding affinity to all three receptors more than 100-fold. NPY and PYY competed with (125)I-hPP at Y4 receptors expressed in CHO cells according to a two-site model. This was investigated for gpY4 by saturation with either radiolabeled hPP or pPYY. The number of high-affinity binding-sites for (125)I-pPYY was about 60% of the receptors recognized by (125)I-hPP. Porcine [Ala(34)]NPY and [Ahx(8-20)]NPY bound to rY4 (but not to hY4 or gpY4) according to a two-site model. These results suggest that different full agonists can distinguish between different active conformations of the gpY4 receptor and that Y4 may display functional differences in vivo between human, guinea pig, and rat.