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 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)
Geraci
M W
MW
Division of Pulmonary Sciences, University of Colorado Health Sciences Center, Denver.
Miller
Y E
YE
Escobedo-Morse
A
A
Kane
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D019886:Gastrin-Releasing Peptide; D008168:Lung / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D018000:Receptors, Peptide / Q000737:chemistry / Q000302:isolation & purification*
D009479:Neuropeptides; D010455:Peptides; D018000:Receptors, Peptide; D019886:Gastrin-Releasing Peptide