The objectives are: (1) To solubilize functional receptors for vasoactive intestinal peptide (VIP) from the lung; (2) To raise monoclonal anti-receptor antibody; (3) To purify the receptor and analyze its structure; (4) To demonstrate signal transduction by the purified receptor; (5) To determine whether GTP-binding proteins (G-proteins) and phospholipids regulate receptor-affinity, receptor selectivity, and efficiency of receptor-adenylate cyclase coupling; (6) To compare the incidence, concentration and affinity of autoantibody to the VIP-receptor in healthy and asthmatic subjects. These objectives will be reached by: (1) Receptor-solubilization from guinea pig lung with the detergent CHAPS; (2) Receptor- fractionation by high performance liquid chromatography; (3) Characterization of binding properties using 125I-VIP; (4) Monoclonal anti-receptor antibody production by cell hybridization; (5) Receptor-purification by affinity chromatography on immobilized receptor-antibody or immobilized-ligand; (6) Structural analysis (molecular weight, subunit structure, presence of S-S bridges and sugar residues) by electrophoresis, isoelectric focussing, chromatography and ultracentrifugation; (7) Receptor-reconstitution with G-proteins and adenylate cyclase in phospholipid vesicles, soluble receptor:G- protein complexes, or receptor negative cell membranes, followed by assay of GTP-sensitive VIP-binding, VIP-sensitive GTP binding and GTPase activity, and VIP-sensitive cyclic AMP synthesis; (8) Comparison of receptor affinity, peptide selectivity and degree of receptor:G-protein:adenylate cyclase coupling, after: (i) receptor-reconstitution with and without G-proteins, (ii) delipidation of the receptor and reconstitution in vesicles made from different phospholipids, (iii) phospholipase-treatment of reconstituted phospholipid vesicles; (9) Radioimmunoassay of plasma autoantibody of VIP-receptor using purified 125I-receptor as tracer and anti-human Ig as precipitant. This work will: (i) yield new information on the structure and regulation of VIP-receptors, (ii) investigate VIP-receptor interactions with second messenger systems, (iii) assess whether VIP-receptor autoantibody may be a pathogenetic factor in asthma, (iv) provide experimental tools for further investigation of structure-function correlates of the receptor, receptor biosynthesis and genetics, and receptor-related lesions in disease.
