Mw. Geraci et al., NOVEL BOMBESIN-LIKE PEPTIDE BINDING-PROTEINS FROM LUNG, American journal of respiratory cell and molecular biology, 10(3), 1994, pp. 331-338
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.