Specific recognition of the human neuroendocrine receptor for vasoactive intestinal peptide by anti-peptide antibodies.
Rabbit polyclonal IgG antibodies were generated to three distinct synthetic peptide substituents of the human neuroendocrine-type 7 transmembrane-domain receptor for vasoactive intestinal peptide (VIP), including a portion of the amino-terminus, first extracellular loop, and carboxyl-terminus. Immunofluorescent staining of both human K293 cell transfectants, expressing recombinant VIP receptors, and HT-29 human intestinal epithelial cells, bearing native VIP receptors, was observed with each of the antibodies and was eliminated specifically after absorption of antibodies with the respective peptide immunogen. Each of the antibodies recognized the same approximately 70-kDa membrane proteins, extracted from both K293 cell transfectants and HT-29 cells, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis blots. Neither IgG nor Fab preparations of the antibodies inhibited VIP binding to cellular receptors at a concentration of 1 microgram/ml, that yielded optimal immunofluorescence, or at 5-300 micrograms/ml. In contrast, 5-200 micrograms/ml of anti-peptide antibodies as IgG, but not Fab, significantly inhibited the increase in concentration of cyclic AMP in HT-29 cells elicited by 1 nM VIP, without affecting the greater increase evoked by 100 nM VIP or alone altering the level of cyclic AMP. Antibodies to several peptide substituents thus bind specifically to VIP receptors in immunoblots and permeabilized cells, and may affect the cellular functions of VIP receptors with sufficient selectivity to reduce transduction of signals, without altering the binding of VIP.
Goetzl
E J
EJ
Department of Medicine, University of California, San Francisco 94143-0711.
Patel
D R
DR
Kishiyama
J L
JL
Smoll
A C
AC
Turck
C W
CW
Law
N M
NM
Rosenzweig
S A
SA
Sreedharan
S P
SP
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D009490:Neurosecretory Systems / Q000378:metabolism*; D011817:Rabbits; D018005:Receptors, Vasoactive Intestinal Peptide / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013194:Staining and Labeling
D000906:Antibodies; D018005:Receptors, Vasoactive Intestinal Peptide