Magnus M. Berglund

Origins of the many NPY-family receptors in mammals

The NPY system has a multitude of effects and is particularly well known for its role in appetite regulation. We have found that the five presently known receptors in mammals arose very early in vertebrate evolution before the appearance of jawed vertebrates 400 million years ago. The genes Y(1), Y(2) and Y(5) arose by local duplications and are still present on the same chromosome in human and pig. Duplications of this chromosome led to the Y(1)-like genes Y(4) and y(6). We find evidence for two occasions where receptor subtypes probably arose before peptide genes were duplicated. These observations pertain to the discussion whether ligands or receptors tend to appear first in evolution. The roles of Y(1) and Y(5) in feeding may differ between species demonstrating the importance of performing functional studies in additional mammals to mouse and rat.

Neuropeptide Y receptor subtype with unique properties cloned in the zebrafish: the zYa receptor

Neuropeptide Y (NPY) belongs to a family of structurally related neuroendocrine peptides for which five different G-protein-coupled receptor subtypes have been cloned in mammals. To identify additional subtypes we have performed PCR with degenerate primers in different species. We describe here the cloning and pharmacological profile of a unique NPY receptor subtype in the zebrafish that has tentatively been called the zYa receptor. It has 46-50% amino acid identity to the mammalian Y1, Y4 and y6 receptors and the previously cloned zebrafish receptors zYb and zYc, and only about 27% to Y2 and Y5. The zYa receptor binds NPY and PYY from mammals as well as zebrafish with high affinities and has a K(d) of 28 pM for porcine (125)I-PYY. It has a unique binding profile displaying some features in common with each of the mammalian Y1, Y2 and Y5 receptors. In a microphysiometer assay the receptor responds with extracellular acidification. Chromosomal mapping in the zebrafish genome of zYa, zYb and zYc receptor genes indicates a possible orthologous relationship between zYc and mammalian y6, but identifies no obvious mammalian ortholog for zYa (zYb is a recent copy of zYc in the fish lineage). These results imply that previous studies of NPY in fishes, which have striven to interpret the effects within the framework of mammalian Y1, Y2, and Y5 receptors, need to be reevaluated. Thus, the sequence comparisons, pharmacological properties, and chromosomal localization suggest that the zYa receptor is a novel NPY receptor subtype which is likely to be present also in mammals.

Chicken neuropeptide Y receptor Y2: structural and pharmacological differences to mammalian Y21

Here we report the molecular cloning of the chicken (Gallus gallus) neuropeptide Y (NPY) receptor Y2, the first non‐mammalian Y2 receptor. It displays 75–80% identity to mammalian Y2 and has a surprisingly divergent cytoplasmic tail. Expression of the receptor protein in a cell line showed that the receptor did not bind the mammalian Y2 selective antagonist BIIE0246. Furthermore, porcine [Leu31, Pro34]NPY, which binds poorly to mammalian Y2, exhibited an unexpectedly high affinity for chicken Y2. In situ hybridisation revealed expression in the hippocampus. Thus, the chicken Y2 receptor exhibits substantial differences with regard to sequence and pharmacological profile in comparison to mammalian Y2 receptors, while the expression pattern in the central nervous system resembles that observed in mammals.

The cloned guinea pig pancreatic polypeptide receptor Y4 resembles more the human Y4 than does the rat Y4

Pancreatic polypeptide (PP) is involved in gastrointestinal functions and forms, together with neuropeptide Y (NPY) and peptide YY (PYY), the PP-fold family of peptides. The PP-binding receptor subtype Y4 has so far been cloned in human, rat, and mouse, and displays extensive species differences regarding sequence, pharmacology, and distribution. To explore this variability further, we have cloned the Y4 receptor in the guinea pig, which is evolutionarily equally distantly related to both humans and rodents. The guinea pig Y4 receptor is 84% identical to the human Y4 receptor, but only 74-75% identical to the rat and mouse receptors. The two latter are 75-76% identical to human Y4. The guinea pig Y4 receptor bound 125I-hPP with a dissociation constant (Kd) of 29+/-3 pM. The pharmacological profile of guinea pig Y4 has the following rank order of potencies: PP > NPY approximately = PYY approximately = LP-NPY approximately = LP-PYY > NPY2-36 >> [D-Trp32]NPY. Thus, the guinea pig receptor is more similar to the human Y4 than to the rat Y4 both in sequence and pharmacology. This agrees with the greater identity between guinea pig and human PP compared to rat PP. These comparisons suggest that the rodent PPs and Y4 receptors have an accelerated replacement rate.

The cloned guinea pig neuropeptide Y receptor Y1 conforms to other mammalian Y1 receptors

We have cloned the guinea pig neuropeptide Y (NPY) Y1 receptor and found it to be 92-93% identical to other cloned mammalian Y1 receptors. Porcine NPY and peptide YY (PYY) displayed affinities of 43 pM and 48 pM, respectively. NPY2-36 and NPY3-36 had 6- and 46-fold lower affinity, respectively, than intact NPY. Functional coupling was measured by using a microphysiometer. Human NPY and PYY were equipotent in causing extracellular acidification with EC50 values of 0.59 nM and 0.69 nM, respectively, whereas NPY2-36 and NPY3-36 were about 15-fold and 500-fold less potent, respectively, than NPY. The present study shows that the cloned guinea pig Y1 receptor is very similar to its orthologues in other mammals, both with respect to sequence and pharmacology. Thus, results from previous studies on guinea pig NPY receptors might imply the existence of an additional Y1-like receptor sensitive to B1BP3226.

Agonist internalization by cloned Y1 neuropeptide Y (NPY) receptor in Chinese hamster ovary cells shows strong preference for NPY, endosome-linked entry and fast receptor recycling.

In Chinese hamster ovary (CHO) cells expressing the cloned guinea-pig Y1 receptor, the saturable, receptor-linked internalization of NPY (NPY)-related peptides showed the rank order of human/rat neuropeptide Y (hNPY)>pig/rat peptide YY (pPYY)>=(Pro(34))human PYY>(Leu(31),Pro(34))hNPY>(Leu(31),Pro(34))hPYY>>BVD-11 (a selective Y1 antagonist). All agonists accessed similar numbers of Y1 sites in particulates from disrupted cells, with relatively small affinity variation. The rate of internalization could significantly depend on the overall interactivity of the agonist peptide (reflected in sensitivity to chaotropic agents, as well as in the level of non-saturable binding and internalization). Concentration-dependent inhibition of the agonist-driven CHO-Y1 internalization was found with filipin III (a cholesterol-complexing macrolide), and confirmed with inhibitors of clathrin lattice formation, phenylarsine oxide (PAO) and sucrose. In the concentration range affecting Y1 internalization, none of the above treatments or agents significantly alter agonist affinity for Y1 cell surface or particulate receptors. Largely similar responses to the above inhibitors were observed in CHO-Y1 cells for internalization of human transferrin. Internalization of CHO-Y1 receptor apparently is driven by NPY in strong preference to other naturally encountered agonists. At 37 degrees C, most of the internalized receptor is rapidly recycled through endosome-like membrane elements, detectable in Percoll gradients.

Reciprocal mutations of neuropeptide Y receptor Y2 in human and chicken identify amino acids important for antagonist binding

The neuropeptide Y (NPY) receptor Y2 antagonist BIIE0246 has sub‐nanomolar affinity for the human Y2 (hY2) receptor but binds very poorly to chicken Y2 (chY2) with micromolar affinity. Sequence comparisons identified several amino acids for investigation by mutagenesis. Reciprocal mutagenesis between hY2 and chY2 revealed that three of these, individually and in combination, are important for BIIE0246 binding, namely positions Gln135 in transmembrane (TM) 3, Leu227 in TM5, and Leu284 in TM6. Mutagenesis of hY2 to the corresponding amino in chY2 (generating hY2[Q135H,L227Q,L284F]) made the affinity of BIIE0246 as low as for chY2. Introduction into chY2 of the three human residues resulted in antagonist affinity almost as high as for hY2. To distinguish between direct and indirect effects, each of the three residues in hY2 was replaced with alanine. BIIE0246 bound with 28‐fold lower affinity to hY2[L227A], suggesting the Leu227 interacts directly with the antagonist. The other two alanine mutants bound with unaltered affinity, suggesting that the corresponding chY2 residues abolish binding through steric hindrance or charge repulsion. Thus, three amino acid residues can in an additive manner completely account for the difference in antagonist binding between the hY2 and chY2 receptors. These results will be useful for construction of three‐dimensional models of the widely divergent NPY receptor subtypes.

Re-evaluation of receptor-ligand interactions of the human neuropeptide Y receptor Y1: a site-directed mutagenesis study

Interactions of the human NPY (neuropeptide Y) receptor Y1 with the two endogenous agonists NPY and peptide YY and two non-peptide antagonists were investigated using site-directed mutagenesis at 17 positions. The present study was triggered by contradictions among previously published reports and conclusions that seemed inconsistent with sequence comparisons across species and receptor subtypes. Our results show that Asp287, at the border between TM (transmembrane) region 6 and EL3 (extracellular loop 3) influences peptide binding, while two aspartic residues in EL2 do not, in agreement with some previous studies but in disagreement with others. A hydrophobic pocket of the Y1 receptor consisting of Tyr100 (TM2), Phe286 (TM6) and His298 (EL3) has been proposed to interact with the amidated C-terminus of NPY, a theory that is unsupported by sequence comparisons between Y1, Y2 and Y5. Nevertheless, our results confirm that these amino acid residues are critical for peptide binding, but probably interact with NPY differently than proposed previously. Studies with the Y1-selective antagonist SR120819A identified a new site of interaction at Asn116 in TM3. Position Phe173 in TM4 is also important for binding of this antagonist. In contrast with previous reports, we found that Phe173 is not crucial for the binding of BIBP3226, another selective Y1 receptor antagonist. Also, we found that position Thr212 (TM5) is important for binding of both antagonists. Our mutagenesis results and our three-dimensional model of the receptor based on the high-resolution structure of bovine rhodopsin suggest new interactions for agonist as well as antagonist binding to the Y1 receptor.

Blockade of pancreatic polypeptide-sensitive neuropeptide Y (NPY) receptors by agonist peptides is prevented by modulators of sodium transport. Implications for receptor signaling and regulation

Ligand binding to rodent pancreatic polypeptide-responding neuropeptide Y (NPY) receptors (here termed PP/NPY receptors), or to cloned Y4 or Y5 receptors, is selectively inhibited by amiloride, peptide or alkylating modulators of sodium transport. The PP/NPY and Y4 receptors are also selectively blocked by human or rat pancreatic polypeptide (PP) and the blocking peptides are not dissociated by high concentrations of alkali chlorides (which restore most of the binding of subtype-selective agonists to Y1 and Y2 sites). The PP/NPY receptors could also be blocked by NPY and related full-length peptides, including Y1-selective agonists (IC50 300-400 pM). The cloned Y(4) receptors from three species are much less sensitive to NPY or PYY. The sensitivity of both the PP/NPY sites and the Y(4) sites to Y2-selective peptides is quite low. The ligand attachment to PP/NPY sites is also very sensitive to peptidic Y1 antagonist ((Cys31,NVal34NPY27-36))2, which however blocks these sites at much higher molarities. Blockade of PP/NPY and Y4 sites by agonist peptides can be largely prevented by N5-substituted amiloride modulators of Na+ transport, and by RFamide NRNFLRF.NH2, but not by Ca2+ channel blockers, or by inhibitors of K+ transport. Protection of both PP/NPY and Y4 sites against blockade by human or rat pancreatic polypeptide is also afforded by short N-terminally truncated NPY-related peptides. The above results are consistent with a stringent and selective activity regulation for rabbit PP/NPY receptor(s) that may serve to differentiate agonists and constrain signaling, and could involve transporter-like interactants.

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.