Journal
CHEMBIOCHEM
Volume 9, Issue 14, Pages 2276-2284Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.200800221
Keywords
G protein-coupled receptors; membrane proteins; structural biology; surface plasmon resonance; Y receptors
Funding
- Swiss National Science Foundation [3100A0-11173]
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Binding of peptide hormones to G protein-coupled receptors is believed to be mediated through formation of contacts of the ligands with residues of the extracellular loops of family 1 GPCRs. Here we have investigated whether additional binding sites exist within the N-terminal domain, as studied in the form of binding of peptides from the neuropeptide Y (NPY) family to the N terminus of the Y4 receptor (N-Y4). The N-terminal domain of the Y4 receptor has been expressed in isotopically enriched form and studied by solution NMR spectroscopy. The peptide is unstructured in solution, whereas a micelle-associated helical segment is formed in the presence of dodecylphosphocholine (DPC) or sodium dodecylsulfate (SDS). As measured by surface plasmon resonance (SPR) spectroscopy, N-Y4 binds with approximately 50 mu m affinity to the pancreatic polypeptide (PP), a high-affinity ligand to the Y4 receptor whereas binding to neuropeptide Y (NPY) and peptide YY (PYY) is much weaker. Residues critical for binding in PP and in N-Y4 have been identified by site-directed mutagenesis. The data indicate that electrostatic interactions dominate and that this interaction is mediated by acidic ligand and basic receptor residues. Residues of N-Y4 are likely to contribute to the binding of PP and in addition might possibly also help to transfer the hormone from the membrane-bound state into the receptor binding pocket.
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