Probing of the neuropeptide Y-Y1-receptors interaction with anti-receptor antibodies.
The Y1 receptor, which belongs to the family of rhodopsin-like GTP-binding protein-coupled, seven-transmembrane helix-spanning receptors, binds the 36-mer neuromodulator neuropeptide Y (NPY) with nanomolar affinity. Synthetic fragments of the N-terminus, extracellular loops and C-terminus of the Y1 receptor were used to generate 18 anti-receptor antibodies; ten of them recognize the receptor expressed on intact cells as well as on membranes that have been prepared (with the exception of one antibody raised against the intracellular C-terminus) as investigated by ELISA. SDS/PAGE of solubilized membranes, subsequent Western blotting and staining with the antibodies revealed two proteins of 73 kDa and 51 kDa for both, the rat and the human receptor. Competition with neuropeptide Y showed that the binding of seven antibodies is strongly inhibited in the presence of the native ligand. Using photoactivatible analogues, it could be demonstrated that the competition efficiency strongly depends on the position of the crosslinker within the ligand. Based on these studies, a model for the ligand-receptor interaction is suggested. These antibodies represent novel tools for the structural characterization of the Y1 receptor and its interaction with NPY and antagonists as well as for localization studies.
Wieland
H A
HA
Biological Research Department, Boehringer Ingelheim Pharma Deutschland, Biberach, Germany.
Eckard
C P
CP
Doods
H N
HN
Beck-Sickinger
A G
AG
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017476:Receptors, Neuropeptide Y / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000345:Affinity Labels; D007106:Immune Sera; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; C097212:neuropeptide Y-Y1 receptor