Abstract
Y receptors (YRs) are G protein-coupled receptors whose Y(1)R, Y(2)R, and Y(5)R subtypes preferentially bind neuropeptide Y (NPY) and peptide YY, whereas mammalian Y(4)Rs show a higher affinity for pancreatic polypeptide (PP). Comparison of YR orthologs and paralogs revealed Asp(6.59) to be fully conserved throughout all of the YRs reported so far. By replacing this conserved aspartic acid residue with alanine, asparagine, glutamate, and arginine, we now show that this residue plays a crucial role in binding and signal transduction of NPY/PP at all YRs. Sensitivity to distinct replacements is, however, receptor subtype-specific. Next, we performed a complementary mutagenesis approach to identify the contact site of the ligand. Surprisingly, this conserved residue interacts with two different ligand arginine residues by ionic interactions; although in Y(2)R and Y(5)R, Arg(33) is the binding partner of Asp(6.59), in Y(1)R and Y(4)R, Arg(35) of human PP and NPY interacts with Asp(6.59). Furthermore, Arg(25) of PP and NPY is involved in ligand binding only at Y(2)R and Y(5)R. This suggests significant differences in the docking of YR ligands between Y(1/4)R and Y(2/5)R and provides new insights into the molecular binding mode of peptide agonists at GPCRs. Furthermore, the proposed model of a subtype-specific binding mode is in agreement with the evolution of YRs.
Highlights
This makes them interesting target molecules for therapeutic interventions
Identification of Conserved Residues in the Third Extracellular Loop—By comparing the amino acid sequences of ECL3 of human, rat, and mouse Y receptors (YRs) (Fig. 1) and further available sequences, the conservation of residue Asp6.59 became obvious for YRs, whereas Asp/Glu is not found in any other peptidebinding GPCRs except for the receptors of the neuropeptide FF (NPFF) and pyroglutamylated Arg-Phe-amide peptide (QRFP) family
IP formation revealed a more than 600-fold reduction in porcine NPY (pNPY) potency for Y2R-D6.59A, a 12-fold reduction for Y4R-D6.59A, and a 99-fold reduction for Y5RD6.59A compared with a 461-fold reduction for Y1 receptor (Y1R)-D6.59A (Table 1)
Summary
Peptide Synthesis—The following peptides were synthesized by automated solid phase peptide synthesis using the Fmoctert-butyl (9-fluorenylmethoxycarbonyl-tert-butyl) strategy [23]: porcine NPY (pNPY), [Ala4]pNPY, [Ala19]pNPY, [Ala25]pNPY, [Ala26]pNPY, [Ala33]pNPY, [Ala35] pNPY, human PP (hPP), [Ala25]hPP, [Ala26]hPP, [Ala33]hPP, and [Ala35]hPP. To confirm the correct position of C-terminal Ala residues, bromocyan digest followed by Edman sequencing ([Ala33]hPP and [Ala35]hPP) or tryptic digest combined with matrix-assisted laser desorption ionization mass spectrometry of the fragments ([Ala33]pNPY and [Ala35]pNPY) were performed. Radioligand binding studies were performed with [3H]propionylated pNPY ([3H]pNPY; 107 Ci/mmol; Amersham Biosciences) and [3H]propionylated hPP ([3H]hPP) on transiently transfected cells as described previously [34, 35] (for details see supplemental materials). Immunological and Immunofluorescence Studies—To ligand-independently estimate plasma membrane localization, a cell surface ELISA was performed with wild type and mutant receptors carrying an N-terminal hemagglutinin tag [40] (for details see supplemental materials). Fluorescence images were obtained with a confocal laser-scanning microscope (Leica TCS SP2; Heidelberg, Germany)
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