Synthesis, receptor binding, and crosslinking of photoactive analogues of neuropeptide Y.
Five photoactive analogues of porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreatic polypeptide family, have been synthesized by solid phase peptide synthesis method, Fmoc/tBu strategy and carefully characterized. The analogues contain the photoactivatable amino acid 4'-(3-trifluoromethyl)-3H-diazirine-3-yl-phenyl-alanine ((Tmd)Phe) individually at different positions (1, 20, 21, 27 or 36) instead of tyrosine in the wildtype sequence. Affinity to membranes prepared from SMS-KAN cells, which stably express the Y2 receptor has been investigated by measuring the displacement of 125I-Bolton Hunter-NPY. After incubation of the membranes with different concentrations of the crosslinker and subsequent photolysis, the specific binding of 125I-Bolton Hunter-NPY at those membranes was tested in order to quantify the crosslinking efficiency. Whereas [(Tmd)Phe20] NPY, [(Tmd)Phe21] NPY and [(Tmd)Phe27] NPY revealed highest affinity to the Y2 receptor, crosslinking was most efficient when Tyr36 was replaced by (Tmd)Phe. This is in good agreement with the previously suggested C-terminal binding site of neuropeptide Y.
Beck-Sickinger
A G
AG
Departement Pharmazie, Zurich, Switzerland.
Wieland
H A
HA
Brunner
J
J
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001389:Azirines; D001665:Binding Sites; D002460:Cell Line; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives; D010777:Photochemistry; D017476:Receptors, Neuropeptide Y / Q000378:metabolism*; D013552:Swine
D000345:Affinity Labels; D001389:Azirines; D003432:Cross-Linking Reagents; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D010649:Phenylalanine; C044966:3-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)alanine