SPECIFIC RECOGNITION OF THE HUMAN NEUROENDOCRINE RECEPTOR FOR VASOACTIVE-INTESTINAL-PEPTIDE BY ANTIPEPTIDE ANTIBODIES

Rabbit polyclonal IgG antibodies were generated to three distinct synt hetic peptide substituents of the human neuroendocrine-type 7 transmem brane-domain receptor for vasoactive intestinal peptide (VIP), includi ng a portion of the amino-terminus, first extracellular loop, and carb oxyl-terminus. Immunofluorescent staining of both human K293 cell tran sfectants, expressing recombinant VIP receptors, and HT-29 human intes tinal epithelial cells, bearing native VIP receptors, was observed wit h each of the antibodies and was eliminated specifically after absorpt ion of antibodies with the respective peptide immunogen. Each of the a ntibodies 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 cellu lar receptors at a concentration of 1 mu g/ml, that yielded optimal im munofluorescence, or at 5-300 mu g/ml. In contrast, 5-200 mu g/ml of a nti-peptide antibodies as IgG, but not Fab, significantly inhibited th e 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 o r alone altering the level of cyclic AMP. Antibodies to several peptid e 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 signal s, without altering the binding of VIP. (C) 1994 Academic Press, Inc.