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Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

ABSTRACT: The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor is a member of the guanine nucleotide binding protein-coupled receptor superfamily with seven predicted hydrophobic transmembrane domains. In vitro transcripts from cloned cDNA templates encompassing the predicted protein coding domain, when injected into Xenopus oocytes, resulted in expression of functional GRP receptors. The predicted amino acid sequence of the open reading frame in cDNA clones matches the amino-terminal sequence as well as the sequence of four tryptic fragments isolated from the purified protein. Expression of the GRP receptor cDNA in model systems potentially provides a powerful assay for the development of subtype-specific receptor antagonists that may prove to be of therapeutic importance in human small cell lung carcinoma.

SUBMITTER: Battey JF

PROVIDER: S-EPMC50817 | biostudies-other | 1991 Jan

REPOSITORIES: biostudies-other

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Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

Battey J F JF Way J M JM Corjay M H MH Shapira H H Kusano K K Harkins R R Wu J M JM Slattery T T Mann E E Feldman R I RI

Proceedings of the National Academy of Sciences of the United States of America 19910101 2

The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor is a ...[more]

PMID: 1671171

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Project description:The gastrin-releasing peptide receptor (GRPR), a G protein-coupled receptor, is overexpressed in solid malignancies and particularly in prostate cancer. We synthesized a novel bombesin derivative, [68Ga]Ga-ProBOMB1, evaluated its pharmacokinetics and potential to image GRPR expression with positron emission tomography (PET), and compared it with [68Ga]Ga-NeoBOMB1. ProBOMB1 (DOTA-pABzA-DIG-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ(CH2N)-Pro-NH2) was synthesized by solid-phase peptide synthesis. The polyaminocarboxylate chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to the N-terminal and separated from the GRPR-targeting sequence by a p-aminomethylaniline-diglycolic acid (pABzA-DIG) linker. The binding affinity to GRPR was determined using a cell-based competition assay, whereas the agonist/antagonist property was determined with a calcium efflux assay. ProBOMB1 was radiolabeled with 68GaCl3. PET imaging and biodistribution studies were performed in male immunocompromised mice bearing PC-3 prostate cancer xenografts. Blocking experiments were performed with coinjection of [d-Phe6,Leu-NHEt13,des-Met14]bombesin(6-14). Dosimetry calculations were performed with OLINDA software. ProBOMB1 and the nonradioactive Ga-ProBOMB were obtained in 1.1 and 67% yield, respectively. The K i value of Ga-ProBOMB1 for GRPR was 3.97 ± 0.76 nM. Ga-ProBOMB1 behaved as an antagonist for GRPR. [68Ga]Ga-ProBOMB1 was obtained in 48.2 ± 10.9% decay-corrected radiochemical yield with 121 ± 46.9 GBq/μmol molar activity and >95% radiochemical purity. Imaging/biodistribution studies showed that the excretion of [68Ga]Ga-ProBOMB1 was primarily through the renal pathway. At 1 h postinjection (p.i.), PC-3 tumor xenografts were clearly delineated in PET images with excellent contrast. The tumor uptake for [68Ga]Ga-ProBOMB1 was 8.17 ± 2.57 percent injected dose per gram (% ID/g) and 9.83 ± 1.48% ID/g for [68Ga]Ga-NeoBOMB1, based on biodistribution studies at 1 h p.i. This corresponded to tumor-to-blood and tumor-to-muscle uptake ratios of 20.6 ± 6.79 and 106 ± 57.7 for [68Ga]Ga-ProBOMB1 and 8.38 ± 0.78 and 39.0 ± 12.6 for [68Ga]Ga-NeoBOMB1, respectively. Blockade with [d-Phe6,Leu-NHEt13,des-Met14]bombesin(6-14) significantly reduced the average uptake of [68Ga]Ga-ProBOMB1 in tumors by 62%. The total absorbed dose was lower for [68Ga]Ga-ProBOMB1 in all organs except for bladder compared with [68Ga]Ga-NeoBOMB1. Our data suggest that [68Ga]Ga-ProBOMB1 is an excellent radiotracer for imaging GRPR expression with PET. [68Ga]Ga-ProBOMB1 achieved a similar uptake as [68Ga]Ga-NeoBOMB1 in tumors, with enhanced contrast and lower whole-body absorbed dose.

Project description:Bombesin-like neuropeptides, including mammalian gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells. In this study, we have characterized the bombesin receptor in membrane preparations from these cells. Addition of Mg2+ during cell homogenization was essential to preserve 125I-GRP binding activity in the resulting membrane preparation. The effect of Mg2+ was concentration dependent, with a maximum at 5 mM. Specific binding of 125I-GRP was saturable; Scatchard analysis indicated a single class of high-affinity sites of Kd = (2.1 +/- 0.3) x 10(-10) M at 15 degrees C and Kd = (1.9 +/- 0.4) x 10(-10) M at 37 degrees C, and a maximum binding capacity of 580 +/- 50 fmol/mg of protein (15 degrees C) or 604 +/- 40 fmol/mg of protein (37 degrees C). The kinetically derived dissociation constant was 1.5 x 10(-10) M. 125I-GRP binding was inhibited in a concentration-dependent manner by various peptides containing the highly conserved C-terminal heptapeptide of the bombesin family, including bombesin, GRP, neuromedin B and the 8-14 fragment of bombesin. In contrast, a variety of structurally unrelated mitogens and neuropeptides had no effect. The cross-linking agent ethyleneglycolbis(succinimidylsuccinate) covalently linked 125I-GRP to a single Mr 75 000-85 000 protein in membrane preparations of 3T3 cells. Affinity labelling of this molecule was specific and dependent on the presence of Mg2+ during membrane preparation. Finally, the non-hydrolysable GTP analogue guanosine-5'-[gamma-thio]triphosphate (GTP[S]) caused a concentration-dependent inhibition of 125I-GRP binding and cross-linking to 3T3 cell membranes [concentration giving half-maximal inhibition (IC50) approximately 0.2 microM]. The inhibitory effect was specific (GMP, ATP or ATP[S] had no effect at 10 microM) and was due to an increase in Kd from (1.7 +/- 0.2) x 10(-10) M to (4.3 +/- 0.6) x 10(-10) M in the presence of 10 microM-GTP[S]. This modulation of ligand affinity and cross-linking implies that the bombesin receptors that mediate mitogenesis in Swiss 3T3 cells are coupled to a guanine-nucleotide-binding-protein signal-transduction pathway.

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Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

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Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

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