Cloning and structure-activity relationship studies of neuropeptide Y family receptors belonging to the Y1 subfamily

Abstract: Neuropeptide Y (NPY), peptide YY (PYY), and the pancreatic polypeptide (PP) belong to afamily of structurally related 36-amino-acid peptides. NPY acts as a neurotransmitter both inthe central and the peripheral nervous system and influences many functions includingfeeding, reproduction, and blood pressure while PYY and PP mainly act as hormones in thegut.NPY, PYY, and PP bind to a family of G-protein coupled receptors. This thesis describesthe cloning of five NPY-family receptors, three of which are new subtypes, using degenerateprimers and homology screenings. The rapidly evolving PP-preferring receptor Y4 fromhuman (h) and guinea pig (gp) were found to be more closely related to each other than eitherwas to the rat receptor. The gpY1 receptor is highly conserved with 91-92% identity to Y1from other orders of mammals. Two highly homologous zebrafish (z) receptors, zYb and zYc(75% identical), were cloned. These receptors form together with mammalian Y1, Y4, and y6and a third zebrafish receptor, zYa, a subfamily of receptors with 50% identity betweensubtypes.Structure-activity relationships of the gpY1, zYa, zYb, and zYc as well as rY4, rY4, andgpY4 receptors were investigated using peptide analogues with deletions or replacements ofvarious parts of the NPY molecule. The gpY1 receptor has previously been reported to displaydeviant pharmacological properties. However, the binding properties of the cloned gpY1expressed in CHO cells conforms to other cloned mammalian Y1 receptors. Receptors zYband zYc display properties similar to Y1 with a gradual loss of affinity with progressive N-terminal truncation of the NPY molecule. In contrast, NPY and truncated analogues likeNPY13-36 as well as loop-deleted peptides like [Ahx5-24]NPY bind to the zYa receptor withsimilar affinity as NPY. Despite the Y1-like binding profile and amino acid sequences of zYband zYc, neither of these bound the Y1-selective antagonists BIBP3226 and SR120819A.The different ligand-binding profiles of Y1-subfamily receptors together with the highdegree of sequence homology between the subtypes will be very useful in future modeling ofthese receptors. This will promote the development of new drugs, agonists and antagonists,directed against primarily the Y1 receptor but may also have relevance for drugs directedagainst the Y2 and Y5 receptors.