Am. J. Respir. Cell Mol. Biol., Vol 10, No. 3, Mar 1994, 331-338.
Novel bombesin-like peptide binding proteins from lung
MW Geraci, YE Miller, A Escobedo-Morse and MA Kane
Division of Pulmonary Sciences, University of Colorado Health Sciences Center, Denver.
Gastrin-releasing peptide (GRP) and other bombesin-like peptides (BLP) play
an important role in lung development, response to injury, and
carcinogenesis. However, the mRNAs from previously cloned BLP receptors are
not detectable on Northern blots of normal lung. The purpose of this study
was to isolate and characterize BLP binding proteins from normal mouse
lung. Soluble cytoplasmic and detergent-solubilized membrane fractions were
prepared from mouse lung and evaluated for specific 125I-GRP binding.
Unexpectedly, not only the solubilized membrane but also the soluble
cytoplasmic fractions demonstrated saturable, high-affinity, specific GRP
binding activity with Kd = 1.6 nM, Bmax = 135 fmol/mg protein and Kd = 7.5
nM, Bmax = 323 fmol/mg protein, respectively. BLP binding proteins were
isolated using GRP14- 27 affinity chromatography and analyzed by SDS-PAGE.
In each fraction, a major unique band of approximate M(r) = 70 kD was
obtained and flanked by two weaker bands of approximate M(r) = 65 and 75
kD. Preincubating samples of the cytoplasmic fraction with various
neuropeptides demonstrated specificity in that only incubation with
GRP14-27, the bioactive portion of the molecule, blocked affinity
purification of these BLP binding proteins. The BLP binding proteins
isolated from the cytoplasmic fraction were purified by HPLC, digested with
trypsin, and sequenced via Edman degradation. These BLP binding proteins
yielded peptides with the sequences IXGIYTDGQNTPXG and RAIMVEXXSEAXXSLLTP,
both of which are unique compared with the GenBank/EMBL data base.(ABSTRACT
TRUNCATED AT 250 WORDS)
