Ligand-mimicking receptor variant discloses binding and activation mode of prolactin-releasing peptide.
The prolactin-releasing peptide receptor and its bioactive RF-amide peptide (PrRP20) have been investigated to explore the ligand binding mode of peptide G-protein-coupled receptors (GPCRs). By receptor mutagenesis, we identified the conserved aspartate in the upper transmembrane helix 6 (Asp(6.59)) of the receptor as the first position that directly interacts with arginine 19 of the ligand (Arg(19)). Replacement of Asp(6.59) with Arg(19) of PrRP20 led to D6.59R, which turned out to be a constitutively active receptor mutant (CAM). This suggests that the mutated residue at the top of transmembrane helix 6 mimics Arg(19) by interacting with additional binding partners in the receptor. Next, we generated an initial comparative model of this CAM because no ligand docking was required, and we selected the next set of receptor mutants to find the engaged partners of the binding pocket. In an iterative process, we identified two acidic residues and two hydrophobic residues that form the peptide ligand binding pocket. As all residues are localized on top or in the upper part of the transmembrane domains, we clearly can show that the extracellular surface of the receptor is sufficient for full signal transduction for prolactin-releasing peptide, rather than a deep, membrane-embedded binding pocket. This contributes to the knowledge of the binding of peptide ligands to GPCRs and might facilitate the development of GPCR ligands, but it also provides new targeting of CAMs involved in hereditary diseases.
Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology, Universität Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany.
Daniel
D
Rathmann
Diana
D
Lindner
Stephanie H
SH
DeLuca
Kristian W
KW
Kaufmann
Jens
J
Meiler
Annette G
AG
Beck-Sickinger
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D015195:Drug Design; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011388:Prolactin / Q000737:chemistry*; D056690:Prolactin-Releasing Hormone / Q000737:chemistry*; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D008024:Ligands; D010455:Peptides; D056690:Prolactin-Releasing Hormone; D043562:Receptors, G-Protein-Coupled; D011388:Prolactin