NOVEL BOMBESIN-LIKE PEPTIDE BINDING-PROTEINS FROM LUNG

Gastrin-releasing peptide (GRP) and other bombesin-like peptides (BLP) play an important role in lung development, response to injury, and c arcinogenesis. However, the mRNAs from previously cloned BLP receptors are not detectable on Northern blots of normal lung. The purpose of t his study was to isolate and characterize BLP binding proteins from no rmal mouse lung. Soluble cytoplasmic and detergent-solubilized membran e fractions were prepared from mouse lung and evaluated for specific I -125-GRP binding. Unexpectedly, not only the solubilized membrane but also the soluble cytoplasmic fractions demonstrated saturable, high-af finity, specific GRP binding activity with K-d = 1.6 nM, B-max = 135 f mol/mg protein and K-d = 7.5 nM, B-max = 323 fmol/mg protein, respecti vely. BLP binding proteins were isolated using GRP(14-27) affinity chr omatography and analyzed by SDS-PAGE. In each fraction, a major unique band of approximate M(r) = 70 kD was obtained and flanked by two weak er bands of approximate M(r) = 65 and 75 kD. Preincubating samples of the cytoplasmic fraction with various neuropeptides demonstrated speci ficity in that only incubation with GRP(14-27), the bioactive portion of the molecule, blocked affinity purification of these BLP binding pr oteins. The BLP binding proteins isolated from the cytoplasmic fractio n were purified by HPLC, digested with trypsin, and sequenced via Edma n degradation. These BLP binding proteins yielded peptides with the se quences IXGIYTDGQNTPXG and RAIMVEXXSEAXXSLLTP, both of which are uniqu e compared with the GenBank/EMBL data base. These studies have (I) ide ntified BLP binding protein in the lung; (2) demonstrated presence of both soluble cytoplasmic and membrane-associated forms; (3) characteri zed saturable, high-affinity, specific I-125-GRP binding; and (4) defi ned partial protein sequences for a novel soluble BLP binding protein. These studies provide strong evidence for the presence of previously undescribed BLP binding proteins in the soluble cytoplasmic fraction o f normal lung. These BLP binding proteins could represent either an en zyme that degrades BLP, a binding protein, a transmembrane receptor, o r a cytoplasmic/nuclear receptor.