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Cloning and functional characterization of a complementary DNA encoding the murine fibroblast
E R Spindel1, E Giladi, P Brehm
1Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton 97006.
Abstract:
The amphibian tetradecapeptide bombesin and its mammalian homolog gastrin-releasing peptide are neurotransmitters and paracrine hormones, and are mitogenic for fibroblast and small cell lung carcinoma cell lines. cDNAs encoding the bombesin/gastrin-releasing peptide receptor (BR) expressed by murine Swiss 3T3 fibroblasts were isolated using electrophysiological and luminometric Xenopus oocyte expression assays. Oocytes microinjected with BR transcripts responded to concentrations of bombesin from 1 x 10(-10) to 1 x 10(-6) M. These responses showed homologous desensitization and could be specifically blocked by bombesin antagonists. Sequence analysis showed that the BR has seven membrane-spanning domains and five potential N-linked glycosylation sites. Data base analysis showed that the BR is most homologous to the tachykinin receptors. Although tyrosine kinase activity has been associated with BR function, no tyrosine kinase homologies occur within the BR sequence.
Insights
Researchers isolated the bombesin/gastrin-releasing peptide receptor (BR) using Xenopus oocyte assays. This receptor, crucial for cell growth, shares homology with tachykinin receptors.
Area of Science:
- Molecular Biology
- Pharmacology
- Cell Biology
Background:
- Bombesin and gastrin-releasing peptide are key neurotransmitters and hormones.
- These peptides are mitogenic for fibroblast and small cell lung carcinoma cells.
- Understanding their receptor is vital for cancer research.
Purpose of the Study:
- To isolate and characterize the bombesin/gastrin-releasing peptide receptor (BR).
- To investigate the functional properties and sequence homology of the BR.
Main Methods:
- Xenopus oocyte expression assays (electrophysiological and luminometric).
- Microinjection of BR transcripts into oocytes.
- Bombesin stimulation, antagonist blocking, and sequence analysis.
Main Results:
- Successfully isolated cDNAs encoding the BR from murine Swiss 3T3 fibroblasts.
- Oocytes expressing BR responded to bombesin, exhibiting homologous desensitization.
- BR sequence revealed seven membrane-spanning domains and homology to tachykinin receptors.
Conclusions:
- The bombesin/gastrin-releasing peptide receptor (BR) was successfully isolated and functionally characterized.
- BR exhibits characteristic receptor properties including desensitization and specific antagonist blockade.
- Structural analysis places BR within the superfamily of G protein-coupled receptors, related to tachykinin receptors.