| 1. |
- Bromée, Torun, 1973-
(författare)
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Evolution and Pharmacology of Receptors for Bradykinin and Neuropeptide Y in Vertebrates
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The bradykinin and neuropeptide Y (NPY) receptors belong to the superfamily of G-protein coupled receptors (GPCRs). The GPCRs form the largest class of therapeutic targets and it is therefore of great interest to investigate the pharmacological properties, functions and evolution of these receptors.Bradykinin (BK) is a nonapeptide that contributes to inflammatory responses, mediates pain signals and influences blood pressure. The two bradykinin receptor subtypes B1 and B2 are well characterized in mammals, but have received little attention in non-mammals. This thesis describes the cloning and characterization of the first piscine bradykinin receptor, from the Danio rerio (zebrafish). Ligand-receptor interactions were measured as production of intracellular inositol phosphate. Zebrafish BK activated the receptor with highest potency (pEC50=6.97±0.1) while mammalian BK was almost inactive. A complete alanine and D-amino acid scan of the BK peptide revealed important roles for receptor interaction for residues Gly4, Ser6, Pro7, Leu8 and Arg9. The receptor gene was mapped to chromosome 17 in the zebrafish genome in a region that shows conserved synteny to the human B1-B2 gene region on chromosome 14. The release of the zebrafish and pufferfish genomes enabled us to identify both B1 and B2 genes in Danio rerio and pufferfishes (Takifugu rubripes and Tetraodon nigroviridis) as well as the B1 gene in chicken. All of these species display conserved synteny of the gene region. Interestingly, the evolutionary rate is clearly greater for B1 than for B2. Kininogen, the precursor for bradykinin, is also located in a chromosome region with extensive conserved synteny.Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) comprise a family of related peptides and are involved in a variety of neuronal and endocrine functions. Receptor subtypes Y6 and Y7 were cloned and pharmacologically characterized in chicken. The genes are located one megabase apart on chromosome 13 in a region with conserved synteny to human chromosome 5. Porcine PYY bound to chicken Y6 with a Kd of 0.80±0.36 nM and chicken Y7 with a Kd of 0.14±0.01 nM. The Y6 mRNA is expressed in hypothalamus, gastrointestinal tract and adipose tissue and may be involved in appetite regulation like other NPY receptors. Chicken Y7 mRNA was only detected in adrenal gland. These results may help explain why these receptors have lost function in humans.
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| 2. |
- Bromée, Torun, et al.
(författare)
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Neuropeptide Y-family receptors Y6 and Y7 in chicken : Cloning, pharmacological characterization, tissue distribution and conserved synteny with human chromosome region
- 2006
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Ingår i: The FEBS Journal. - : Federation of European Biochemical Societies. - 1742-464X .- 1742-4658. ; 273:9, s. 2048-2063
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Tidskriftsartikel (refereegranskat)abstract
- The peptides of the neuropeptide Y (NPY) family exert their functions, including regulation of appetite and circadian rhythm, by binding to G-protein coupled receptors. Mammals have five subtypes, named Y1, Y2, Y4, Y5 and Y6, and recently Y7 has been discovered in fish and amphibians. In chicken we have previously characterized the first four subtypes and here we describe Y6 and Y7. The genes for Y6 and Y7 are located 1 megabase apart on chromosome 13, which displays conserved synteny with human chromosome 5 that harbours the Y6 gene. The porcine PYY radioligand bound the chicken Y6 receptor with a Kd of 0.80 ± 0.36 nm. No functional coupling was demonstrated. The Y6 mRNA is expressed in hypothalamus, gastrointestinal tract and adipose tissue. Porcine PYY bound chicken Y7 with a Kd of 0.14 ± 0.01 nm (mean ± SEM), whereas chicken PYY surprisingly had a much lower affinity, with a Ki of 41 nm, perhaps as a result of its additional amino acid at the N terminus. Truncated peptide fragments had greatly reduced affinity for Y7, in agreement with its closest relative, Y2, in chicken and fish, but in contrast to Y2 in mammals. This suggests that in mammals Y2 has only recently acquired the ability to bind truncated PYY. Chicken Y7 has a much more restricted tissue distribution than other subtypes and was only detected in adrenal gland. Y7 seems to have been lost in mammals. The physiological roles of Y6 and Y7 remain to be identified, but our phylogenetic and chromosomal analyses support the ancient origin of these Y receptor genes by chromosome duplications in an early (pregnathostome) vertebrate ancestor.
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| 3. |
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| 4. |
- Del Tredici, Andria L., et al.
(författare)
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Identification of novel selective V-2 receptor non-peptide agonists
- 2008
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Ingår i: Biochemical Pharmacology. - : Elsevier BV. - 0006-2952 .- 1356-1839. ; 76:9, s. 1134-1141
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Tidskriftsartikel (refereegranskat)abstract
- Peptides with agonist activity at the vasopressin V-2 receptor are used clinically to treat fluid homeostasis disorders such as polyuria and central diabetes insipidus. of these peptides, the most commonly used is desmopressin, which displays poor bioavailability as well as potent activity at the V-1b receptor, with possible stress-related adverse effects. Thus, there is a strong need for the development of small molecule chemistries with selective V-2 receptor agonist activity. Using the functional cell-based assay Receptor Selection and Amplification Technology (R-SAT (R)), a screening effort identified three small molecule chemotypes (AC-94544, AC-88324, and AC-110484) with selective agonist activity at the V-2 receptor. One of these compounds, AC-94544, displayed over 180-fold selectivity at the V-2 receptor compared to related vasopressin and oxytocin receptors and no activity at 28 other G protein-coupled receptors (GPCRs). All three compounds also showed partial agonist activity at the V-2 receptor in a cAMP accumulation assay. In addition, in a rat model of central diabetes insipidus, AC-94544 was able to significantly reduce urine output in a dose-dependent manner. Thus, AC-94544, AC-88324, and AC-110484 represent novel opportunities for the treatment of disorders associated with V-2 receptor agonist deficiency.
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| 5. |
- Fällmar, Helena, 1980-, et al.
(författare)
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Identification of positions in the human neuropeptide Y/peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes
- 2011
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Ingår i: Neuropeptides. - : Elsevier BV. - 0143-4179 .- 1532-2785. ; 45:4, s. 293-300
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Tidskriftsartikel (refereegranskat)abstract
- The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In humans this family consists of NPY, peptide YY (PYY) and pancreatic polypeptide (PP) which interact with distinct preference for the four receptors showing very low sequence identity, i.e. Y1, Y2, Y4 and Y5. The binding of similar peptides to these divergent receptors makes them highly interesting for mutagenesis studies. We present here a site-directed mutagenesis study of four amino acid positions in the human Y2 receptor. T(3.40) was selected based on sequence alignments both between subtypes and between species and G(2.68), L(4.60) and Q(6.55) also on previous binding studies of the corresponding positions in the Y1 receptor. The mutated receptors were characterized pharmacologically with the peptide agonists NPY, PYY, PYY(3-36), NPY(13-36) and the non-peptide antagonist BIIE0246. Interestingly, the affinity of NPY and PYY(3-36) increased for the mutants T(3.40)I and Q(6.55)A. Increased affinity was also observed for PYY to Q(6.55)A. PYY(3-36) displayed decreased affinity for G(2.68)N and L(4.60)A whereas binding of NPY(13-36) was unaffected by all mutations. The antagonist BIIE0246 showed decreased affinity for T(3.40)I, L(4.60)A and Q(6.55)A. Although all positions investigated were found important for interaction with at least one of the tested ligands the corresponding positions in hY1 seem to be of greater importance for ligand binding. Furthermore these data indicate that binding of the agonists and the antagonist differs in their points of interaction. The increase in the binding affinity observed may reflect an indirect effect caused by a conformational change of the receptor. These findings will help to improve the structural models of the human NPY receptors.
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| 6. |
- Fällmar, Helena, 1980-, et al.
(författare)
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Neuropeptide Y/peptide YY receptor Y2 duplicate in zebrafish with unique introns displays distinct peptide binding properties
- 2011
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Ingår i: Comparative Biochemistry and Physiology - Part B. - : Elsevier BV. - 1096-4959 .- 1879-1107. ; 160:4, s. 166-173
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The neuropeptide Y-family peptides and receptors are involved in a broad range of functions including appetite regulation. Both the peptide genes and the receptor genes are known to have duplicated in early vertebrate evolution. The ancestral jawed vertebrate had 7 NPY receptors but the number varies between 4 and 7 in extant vertebrates. Herein we describe the identification of an additional NPY receptor in two fish species, zebrafish and medaka. They cluster together with the Y2 receptors in phylogenetic analyses and seem to be orthologous to each other that is why we have named them Y2-2. Their genes differ from Y2 in having introns in the coding region. Binding studies with zebrafish Y2-2 receptors show that the three endogenous peptides NPY, PYYa and PYYb have similar affinities, 0.15-0.66nM. This is in contrast to the Y2 receptor where they differed considerably from one another. N-terminally truncated NPY binds poorly and the Y2 antagonist BIIE0246 binds well to Y2-2, results that are reversed in comparison to Y2. Zebrafish Y2-2 mRNA was detected by PCR in the intestine and the eye, but not in the brain. In conclusion, we have found a novel Y2-like NPY/PYY receptor that probably arose in early teleost fish evolution.
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| 7. |
- Fällmar, Helena, 1980-
(författare)
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Studies of the Neuropeptide Y Receptor Y2 in Human and Zebrafish
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The G-protein coupled receptors (GPCRs) comprise the largest family of receptors in humans and other vertebrates. They are embedded in the cell membrane and are activated by many different signaling molecules. Activation modulates cellular signal transduction pathways and influences many physiological processes. Therefore the GPCRs are important as targets for numerous drugs. The receptors for NPY (neuropeptide Y) belong to GPCRs of Class A (rhodopsin-like). NPY and its related peptides PYY and PP are involved in the regulation of appetite, blood pressure and many other processes. They share a common structure and interact with the receptors Y1, Y2, Y4 and Y5 in mammals, and, in addition, Y7 and Y8 in amphibians and bony fishes. This thesis is focused on the human Y2 receptor, known to reduce appetite, by investigating the importance of thirteen amino acid residues for ligand binding. Mutagenesis followed by functional expression and receptor binding was conducted. During the course of this work several new GPCR crystal structures have been resolved, thereby improving the receptor modeling in papers I-III. The major finding is that even though the Y1 and Y2 receptors have evolved from a common ancestor, their points of ligand interaction differ and have thus changed during evolution. In general, the positions investigated resulted in milder changes in the ligands’ affinities for Y2 compared to Y1. These findings were incorporated in the design of new Y1 and Y2 receptor models, leading to improved understanding of how such divergent receptors, sharing only 30 percent sequence identity, can still interact with the same ligands. Notably, several of the mutations introduced in Y2 resulted in increased affinity. A novel NPY receptor gene named Y2-2 was identified in the genomes of zebrafish and medaka. This brings the number of zebrafish NPY receptors to seven. The binding characteristics of zebrafish Y2-2 differed from zebrafish Y2 mainly in the interaction with NPY13-36 and the antagonist BIIE0246. In conclusion, these results increase our understanding of ligand interactions with GPCRs and will be useful for refinement of ligand-receptor models for future development of receptor subtype-selective drugs.
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