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Corticotropin-releasing factor receptors in human small cell lung carcinoma cells: radioligand binding, second
K D Dieterich1, D E Grigoriadis, E B De Souza
1Neurocrine Biosciences, Inc., San Diego, California 92121.
Abstract:
Numerous peptides, growth factors, and receptors have been identified in small cell lung carcinoma (SCLC) cells. The present study was designed to examine the radioligand binding, second messenger, and messenger RNA (mRNA) characteristics of CRF receptors in a variety of SCLC lines and to compare their characteristics to CRF receptors in the mouse pituitary tumor AtT-20 cells. The human SCLC cell lines NCI-H69, H82, H146, H209, H345, H446, and H510A and control AtT-20 cells all demonstrated specific [125I]Tyr(o)-ovine CRF ([125I]oCRF) binding, which was linear with increasing protein concentrations, saturable, reversible, and of high affinity. NCI-H82 cells showed the highest level of specific [125I]oCRF binding (approximately 60% of the total binding). Scatchard analysis revealed a single homogeneous class of binding sites in NCI-H82 and AtT-20 cells, with Kd values of 263 +/- 48 and 285 +/- 75 pM, respectively, and binding capacities of 74 +/- 7 and 70 +/- 13 fmol/mg protein, respectively. [125I]oCRF-binding sites on NCI-H82 and AtT-20 cells had comparable pharmacological characteristics with the following rank order of inhibitory potencies: rat/human CRF approximately ovine CRF approximately bovine CRF > alpha-helical oCRF-(9-41) > bovine CRF-(1-41)OH >> vasoactive intestinal peptide, secretin, GH-releasing hormone. [125I]oCRF binding in the cell lines was inhibited by guanine nucleotides, suggesting a coupling of receptors to guanine nucleotide-binding proteins. The functional nature of the CRF receptor was demonstrated in second messenger studies in which rat/human CRF stimulated cAMP production in NCI-H82 and AtT-20 cells with comparable EC50 values of about 3 nM; the percent stimulation over basal activity was significantly higher in NCI-H82 cells (approximately 30-fold increase) than in AtT-20 cells (approximately 12-fold increase). Northern blot analysis of total RNA revealed the presence of a 2.6-kilobase mRNA band in NCI-H82 cells corresponding to the recently cloned human CRF receptor. In summary, the data demonstrate the presence of CRF receptors in SCLC cell lines with kinetic, pharmacological, second messenger, and mRNA characteristics comparable to those in pituitary and brain and suggest a possible role for CRF as a regulatory peptide in human SCLC.
Insights
This study found corticotropin-releasing factor (CRF) receptors in small cell lung carcinoma (SCLC) cells, similar to pituitary cells. These CRF receptors exhibit comparable binding, signaling, and mRNA characteristics, suggesting a potential regulatory role in SCLC.
Area of Science:
- Endocrinology
- Molecular Biology
- Oncology
Background:
- Small cell lung carcinoma (SCLC) is known to express various peptides, growth factors, and receptors.
- Corticotropin-releasing factor (CRF) receptors have been identified in various cell types, including tumors.
Purpose of the Study:
- To investigate the radioligand binding, second messenger, and mRNA characteristics of CRF receptors in human SCLC cell lines.
- To compare these characteristics with those of CRF receptors in mouse pituitary tumor AtT-20 cells.
Main Methods:
- Radioligand binding assays using [125I]Tyr(o)-ovine CRF ([125I]oCRF).
- Scatchard analysis to determine binding site characteristics.
- Pharmacological profiling of receptor binding.
- Second messenger studies measuring cAMP production.
- Northern blot analysis for mRNA expression.
Main Results:
- Specific [125I]oCRF binding was observed in all tested SCLC cell lines and AtT-20 cells, exhibiting high affinity and saturable kinetics.
- NCI-H82 SCLC cells showed the highest binding levels, with Scatchard analysis revealing a single class of binding sites comparable to AtT-20 cells.
- CRF stimulated cAMP production in NCI-H82 cells more significantly than in AtT-20 cells, and a 2.6-kilobase mRNA band for the CRF receptor was detected in NCI-H82 cells.
Conclusions:
- CRF receptors are present in human SCLC cell lines.
- These receptors share comparable kinetic, pharmacological, second messenger, and mRNA characteristics with those found in pituitary and brain tissues.
- CRF may play a regulatory role in human SCLC.