Examination of the role of the amphipathic alpha-helix in the interaction of neuropeptide Y and active cyclic analogues with cell membrane receptors and dimyristoylphosphatidylcholine.
To test the potential importance of the putative C-terminal amphipathic alpha-helical region of neuropeptide Y (NPY) in receptor binding, the interactions of porcine NPY and several peptide analogues with lipid and cell membrane receptors were compared. Cyclic analogues were designed to constrain the N- and C-terminal regions of the peptide and to retain the folded conformation of NPY predicted from its sequence analogy with pancreatic polypeptide and its similar spectral behavior. The three cyclic peptides were [Cys2, 8-aminooctanoic acid5-24, D-Cys27]-NPY (C2-NPY), [Cys5, 8-aminooctanoic acid7-20, D-Cys24]-NPY (C5-NPY), and [D-Cys7, 8-aminooctanoic acid8-17, Cys20]-NPY (C7-NPY). All of the peptides bind with high affinity to pig spleen membranes, but only NPY and [Glu16, Ser18, Ala22, Leu28,31]-NPY (ESALL-NPY) bind quantitatively to dimyristoylphosphatidylcholine (DMPC) liposomes. C7-NPY and NPY20-36 bind with moderate affinity to liposomes, but only NPY and C7-NPY bind with high affinity to mouse brain receptors. Thus, lipid binding and receptor binding are not correlated in this series of peptides, and binding to the pig spleen receptor appears to require only the C-terminal region of the peptide. Simple lipid binding, as in NPY20-36, is insufficient for binding to the mouse brain receptor, suggesting that the N-terminal region of the peptide is required for high-affinity binding to this receptor. Data from fluorescence, differential scanning calorimetry, and liposome clearing experiments suggest that, although the interaction of NPY with lipid is consistent with formation of an amphipathic alpha-helix, a simple amphipathic alpha-helical model for the interaction with the high-affinity NPY receptor is insufficient to explain the data.(ABSTRACT TRUNCATED AT 250 WORDS)
McLean
L R
LR
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Buck
S H
SH
Krstenansky
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002462:Cell Membrane; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000031:analogs & derivatives / Q000378:metabolism*; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013154:Spleen / Q000378:metabolism; D013552:Swine
D008081:Liposomes; D009478:Neuropeptide Y; D011956:Receptors, Cell Surface; D004134:Dimyristoylphosphatidylcholine