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Cloning and Characterization of Cdnas Encoding Human Gastrin-Releasing Peptide (Bombesin/Mrna/Neuropeptide) ELIOT R

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Cloning and Characterization of Cdnas Encoding Human Gastrin-Releasing Peptide (Bombesin/Mrna/Neuropeptide) ELIOT R Proc. Nati. Acad. Sci. USA Vol. 81, pp. 5699-5703, September 1984 Biochemistry Cloning and characterization of cDNAs encoding human gastrin-releasing peptide (bombesin/mRNA/neuropeptide) ELIOT R. SPINDEL*, WILLIAM W. CHIN*, JANET PRICE+, LESLEY H. REESt, GORDON M. BESSERt, AND JOEL F. HABENER* *Laboratory of Molecular Endocrinology, Massachusetts General Hospital and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02114, and Departments of tChemical Endocrinology and tEndocrinology, St. Bartholomew's Hospital, London, England Communicated by Herman M. Kalckar, June 6, 1984 ABSTRACT We have prepared and cloned cDNAs de- As is characteristic of many neuropeptides, mammalian rived from poly(A)+ RNA from a human pulmonary carcinoid bombesin-like peptides exist in multiple forms. Bombesin- tumpor rich in immunoreactivity to gastrin-releasing peptide, a related peptides of 10 and 27 amino acids have been isolated peptide closely related in structure to amphibian bombesin. from porcine (16) and canine (4) tissues. Analyses by high- Mixtures of synthetic oligodeoxyribonucleotides correspond- pressure liquid chromatography of fetal lung (17) and lung ing to amphibian bombesin were used as hybridization probes tumors (15, 18, 19) yield multiple peaks of immunoreactivity; to screen a cDNA library prepared from the tumor RNA. Se- one peak elutes near bombesin and one elutes near GRP. quencing of the recombinant plasmids shows that human gas- Multiple immunoreactive forms are also detected in gastroin- trin-releasing peptide (hGRP) mRNA encodes a precursor of testinal tissue (9, 20). In brain, immunohistochemistry re- 148 amino acids containing a typical signal sequence, hGRP veals some distinct regions that react to bombesin antisera consisting of 27 or 28 amino acids, and a carboxyl-terminal and other regions that react only with antisera to ranatensin extension peptide. hGRP is flanked at its carboxyl terminus by (21), another amphibian peptide structurally related to bom- two basic amino acids, following a glycine used for amidation besin. of the carboxyl-terminal methionine. RNA blot analyses of tu- To clarify these complexities of the bombesin-like family mor RNA show a major mRNA of 900 bases and a minor of peptides, we wish to analyze the gene(s) encoding mem- mRNA of 850 bases. Blot hybridization analyses using human bers of this peptide family. As an initial step in these studies, genomic DNA are consistent with a single hGRP-encoding we describe here the molecular cloning and characterization gene. The presence of two mRNAs encoding the hGRP precur- of cDNAs corresponding to mRNAs encoding the precursor sor protein in the face of a single hGRP gene raises the possi- of human GRP (hGRP; pre-proGRP). of of the RNA bility alternative processing single transcript. METHODS Gastrin-releasing peptide (GRP) is a mammalian equivalent Polyadenylylated RNA was obtained from a hepatic metas- of the amphibian tetradecapeptide bombesin (1). GRP was tasis of a pulmonary carcinoid tumor rich in bombesin-like originally isolated from porcine stomach by using the release immunoreactivity (§), by using the guanidine thiocyanate of gastrin as a bioassay (2). Peptides related in structure have procedure (23) and oligo(dT) chromatography (24). Double- been isolated from avian proventriculus (3) and canine small stranded cDNA was prepared enzymatically as described by intestine (4). Bombesin and the GRPs share similarities in Crabtree and Kant (25). A recombinant cDNA library was their sequences and contain a common carboxyl-terminal prepared using homopolymeric dC-dG extensions and Pst I heptapeptide. cut pBR322 (New England Nuclear). Escherichia coli MC1061 GRP and bombesin have similar biological effects (5). In- were transformed by the calcium-shock procedure (26). fusion of nanogram amounts of either peptide to dogs or hu- Oligodeoxynucleotide probes were synthesized by the mans increases plasma immunoreactive levels of gastrin, phosphate triester method (27). The probes (Fig. 1) corre- pancreatic polypeptide, glucagon, gastric inhibitory peptide, sponded to two different regions of amphibian bombesin and and insulin (5, 6). Bombesin is also a potent neuropeptide; ranatensin to allow double screening and thus decrease the intraventricular injections of 1 pM bombesin into rats ele- detection of false-positive recombinants. Duplicate high- These (28) and low-density (29) colony screening was carried out vates blood sugar (7) and produces hypothermia (8). (30) potent effects coupled with the wide distribution of bombe- with 5'-end-labeled probes using hybridization condi- sin-like peptides throughout the mammalian gastrointestinal tions as described by Hanahan and Meselson (28). Cell-free the role of bombe- translations using wheat germ extract and reticulocyte ly- tract (9) and nervous system (10) support sate, plasmid preparations, RNA hybridization, DNA hy- sin as an important neuroregulatory peptide. Bombesin-like immunoreactivity is also detected in the bridization, and DNA chemical sequence analyses were car- neuroendocrine cells of the lung (11). In humans, the highest ried out by standard techniques (31-37). levels are found in the lung just after birth (12) and levels RNA blots were hybridized for 18 hr at 42°C in 50% forma- then decrease in parallel with the observed decrease in the mide/5x NaCl/Cit (1x NaCI/Cit = 0.15 M NaCl/0.015 M number of neuroendocrine cells Bombesin Na citrate)/5 x Denhardt's solution (1x Denhardt's solution pulmonary (13). = 0.02% Ficoll-400/0.02% bovine serum albumin/0.02% immunoreactivity is also found in high levels in pulmonary sodium carcinoid tumors as well as in many small cell carcinomas of polyvinylpyrrolidone-40)/0.5% dodecyl sulfate/son- the lung (14, 15). Hence, bombesin-like immunoreactivity is icated denatured salmon sperm (100 /,g/ml). Southern blots a potential chemical marker for these tumors. Abbreviations: GRP, gastrin-releasing peptide; hGRP, human GRP; pre-proGRP, precursor of hGRP. The publication costs of this article were defrayed in part by page charge §Price, J., Nieuwenhuijsen-Kruseman, A. C., Clement-Jones, V. & payment. This article must therefore be hereby marked "advertisement" Rees, L. H. (1983) Endocrine Society, 65th Annual Meeting, San in accordance with 18 U.S.C. §1734 solely to indicate this fact. Antonio, TX, June 8-10, 188 (abstr.). 5699 Downloaded by guest on September 24, 2021 5700 Biochemistry: Spindel et al. Proc. NatL Acad Sci. USA 81 (1984) C 3' -CCN GTA AAG TAC CC - 5' G A Ranatensin (a) (4-11) gin trp ala val gly his phe met gly Bombesin (b) (7-14) gin trp ala val gly his leu met gly 3' - GTC ACC CGN CAN CC GTA GAN TAC CC - 5, T G AAT C A B FIG. 1. Design of oligodeoxyribonucleotide probes. Probe A was prepared as two 16-fold mixed sets (Al and A2); Al used the GTC and A2 used the GTT complement to the glutamine codon. Probe B was synthesized as a 32-fold mixed set. Probe C was made to ranatensin-(8-11), which is closely related to bombesin-(11-14), but requires only a 16-fold mixed synthesis. N indicates the inclusion of all 4 bases. Hybridization temperatures for probes A, B, and C were 41TC, 28TC, and 37TC, respectively. Sequences are ranatensin-(4-11) (a) and bombesin-(7-14) (b). were hybridized for 36 hr at 450C in the same hybridization sequence (38). At the carboxyl terminus, hGRP is flanked by solution. two lysines following a glycine, which likely provides the signal for amidation of the terminal methionine residue (39). RESULTS Pre-proGRP does not code for any additional GRP-like or From 3 g of tumor, 100 ,ug of poly(A)+ RNA was prepared. bombesin-like peptides, nor are there any other peptides set Addition of 1 Aug of RNA to cell-free translation systems re- off by two consecutive basic amino acids'characteristic of sulted in 10- to 30-fold increases in protein synthesis, thus sites that are cleaved during the post-translational process- demonstrating the integrity of the tumor mRNA. By using 10 ing of prohormones. The protein-coding region is followed jug of poly(A)+ RNA, 0.8 jug of double-stranded cDNA was by 289 untranslated bases before the poly(A) tail com- synthesized as described by Crabtree and Kant (25). Trans- mences; a typical A-A-A-T-A-A polyadenylylation signal formation of E. coli with 100 ng of cDNA produced 20,000 (40)' occurs 19 bases before the poly(A) tail. colonies. High- followed by low-density screening with Hybridization analysis (RNA blot) demonstrates the pres- probes A2 and C (Fig. 1) yielded 15 hybridizing colonies ence of two mRNAs: a predominant mRNA of 900 bases and (probe C hybridized 'to essentially the same colonies as a minor RNA of approximately 850 bases (Fig. 4). Hybrid- probe B but with less background). Sequence analyses of the ization analysis (Southern blot) of genomic DNA from hu- recombinant plasmids obtained from these colonies (Fig. 2) man lymphocytes shows only a single band with BamHI, yielded close to the entire sequences for the mRNA encod- HindIII, and EcoRI digests, consistent with a single gene ing pre-proGRP (Fig. 3). Both probes A2 and C hybridized coding for pre-proGRP. appropriately despite single base mismatches. The deduced amino acid sequence shows that hGRP is DISCUSSION part of a 148-amino acid polypeptide that is delimited by an Cloning of cDNA prepared from a pulmonary carcinoid tu- AUG start codon and three successive stop codons. The mor demonstrates that hGRP is encoded in a precursor pro- amino-terminal portion of this precursor contains a cluster of tein. There is a hydrophobic signal sequence'(38), although hydrophobic amino acids consistent with the "pre" or signal, the exact cleavage point of the "pre" sequence is not deter- Hinf I Avav Hint I Pvu IL Taq I Hp GRE| siLPI I 'Iv 5'' - 3' GRP 1:1'--".,'.. pB- I do-- Iq- 0 *b* b~MEM pp-7 0 40 0 p8-3 Ep P.R lim ps-12 *-cDNA 100 bp I - poly (dG: dC)Lnker FIG.
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