Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1997 Nov 15;100(10):2530–2537. doi: 10.1172/JCI119795

Constitutive activation of the gastrin-releasing peptide receptor expressed by the nonmalignant human colon epithelial cell line NCM460.

H A Ferris 1, R E Carroll 1, M M Rasenick 1, R V Benya 1
PMCID: PMC508453  PMID: 9366567

Abstract

Gastrin-releasing peptide (GRP) causes multiple effects in humans by activating a specific heptaspanning receptor. Within the gastrointestinal tract, GRP receptors (GRP-R) are not normally expressed by mucosal epithelial cells except for those lining the gastric antrum. In contrast, recent studies have shown that up to 40% of resected colon cancers aberrantly express this receptor. This is important because the GRP-R can cause the proliferation of many, but not all, tissues in which it is expressed. Since GRP and other agonists are not known to exist in the colonic lumen, it has not been clear how or even if GRP-R expression in colon cancer contributes to cell proliferation. To evaluate the functional consequence of GRP-R expression on colonic epithelium, we transfected the recently isolated nonmalignant human colon epithelial cell line NCM460 with the cDNA for this receptor. All NCM460 cell lines expressing varying numbers of GRP-R bound selected agonists and antagonists indistinguishably from receptors expressed by other human tissues. Furthermore GRP-R-expressing transfected cell lines, but not wild-type NCM460 cells, proliferated independently of serum or other growth factors. Further evaluation revealed that GRP-R in these cells tonically stimulated G alpha q/11, resulting in increased phospholipase C activation. Since transfected cells do not secrete GRP, nor is their growth influenced by exposure to receptor-specific antagonists, these data indicate that GRP-R ectopically expressed by NCM460 cells are constitutively active. This report provides the first evidence of mutation-independent heptaspanning receptor constitutive activation resulting in cell proliferation, and identifies a potential mechanism whereby the GRP-R may act as an oncogene in human colon cancer.

Full Text

The Full Text of this article is available as a PDF (480.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Battey J. F., Way J. M., Corjay M. H., Shapira H., Kusano K., Harkins R., Wu J. M., Slattery T., Mann E., Feldman R. I. Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):395–399. doi: 10.1073/pnas.88.2.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benya R. V., Akeson M., Mrozinski J., Jensen R. T., Battey J. F. Internalization of the gastrin-releasing peptide receptor is mediated by both phospholipase C-dependent and -independent processes. Mol Pharmacol. 1994 Sep;46(3):495–501. [PubMed] [Google Scholar]
  3. Benya R. V., Fathi Z., Kusui T., Pradhan T., Battey J. F., Jensen R. T. Gastrin-releasing peptide receptor-induced internalization, down-regulation, desensitization, and growth: possible role for cyclic AMP. Mol Pharmacol. 1994 Aug;46(2):235–245. [PubMed] [Google Scholar]
  4. Benya R. V., Schmidt L. N., Sahi J., Layden T. J., Rao M. C. Isolation, characterization, and attachment of rabbit distal colon epithelial cells. Gastroenterology. 1991 Sep;101(3):692–702. doi: 10.1016/0016-5085(91)90527-r. [DOI] [PubMed] [Google Scholar]
  5. Berridge M. J., Dawson R. M., Downes C. P., Heslop J. P., Irvine R. F. Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Biochem J. 1983 May 15;212(2):473–482. doi: 10.1042/bj2120473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang E. B., Brown D. R., Wang N. S., Field M. Secretagogue-induced changes in membrane calcium permeability in chicken and chinchilla ileal mucosa. Selective inhibition by loperamide. J Clin Invest. 1986 Jul;78(1):281–287. doi: 10.1172/JCI112562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  8. Corjay M. H., Dobrzanski D. J., Way J. M., Viallet J., Shapira H., Worland P., Sausville E. A., Battey J. F. Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells. J Biol Chem. 1991 Oct 5;266(28):18771–18779. [PubMed] [Google Scholar]
  9. Cuttitta F., Carney D. N., Mulshine J., Moody T. W., Fedorko J., Fischler A., Minna J. D. Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer. 1985 Aug 29-Sep 4Nature. 316(6031):823–826. doi: 10.1038/316823a0. [DOI] [PubMed] [Google Scholar]
  10. Ferris H. A., Carroll R. E., Lorimer D. L., Benya R. V. Location and characterization of the human GRP receptor expressed by gastrointestinal epithelial cells. Peptides. 1997;18(5):663–672. doi: 10.1016/s0196-9781(97)00127-7. [DOI] [PubMed] [Google Scholar]
  11. Frucht H., Gazdar A. F., Park J. A., Oie H., Jensen R. T. Characterization of functional receptors for gastrointestinal hormones on human colon cancer cells. Cancer Res. 1992 Mar 1;52(5):1114–1122. [PubMed] [Google Scholar]
  12. Giacchetti S., Gauvillé C., de Crémoux P., Bertin L., Berthon P., Abita J. P., Cuttitta F., Calvo F. Characterization, in some human breast cancer cell lines, of gastrin-releasing peptide-like receptors which are absent in normal breast epithelial cells. Int J Cancer. 1990 Aug 15;46(2):293–298. doi: 10.1002/ijc.2910460226. [DOI] [PubMed] [Google Scholar]
  13. Halmos G., Pinski J., Szoke B., Schally A. V. Characterization of bombesin/gastrin-releasing peptide receptors in membranes of MKN45 human gastric cancer. Cancer Lett. 1994 Sep 30;85(1):111–118. doi: 10.1016/0304-3835(94)90246-1. [DOI] [PubMed] [Google Scholar]
  14. Huang N. N., Wang D. J., Heppel L. A. Role of adenosine 3':5'-monophosphate-dependent protein kinase and cAMP levels in ATP-dependent mitogenesis in Swiss 3T3 cells. J Biol Chem. 1994 Jan 7;269(1):548–555. [PubMed] [Google Scholar]
  15. Kachur J. F., Miller R. J., Field M., Rivier J. Neurohumoral control of ileal electrolyte transport. I. Bombesin and related peptides. J Pharmacol Exp Ther. 1982 Mar;220(3):449–455. [PubMed] [Google Scholar]
  16. Kjelsberg M. A., Cotecchia S., Ostrowski J., Caron M. G., Lefkowitz R. J. Constitutive activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J Biol Chem. 1992 Jan 25;267(3):1430–1433. [PubMed] [Google Scholar]
  17. Kosugi S., Van Dop C., Geffner M. E., Rabl W., Carel J. C., Chaussain J. L., Mori T., Merendino J. J., Jr, Shenker A. Characterization of heterogeneous mutations causing constitutive activation of the luteinizing hormone receptor in familial male precocious puberty. Hum Mol Genet. 1995 Feb;4(2):183–188. doi: 10.1093/hmg/4.2.183. [DOI] [PubMed] [Google Scholar]
  18. Lorimer D. D., Benya R. V. Cloning and quantification of galanin-1 receptor expression by mucosal cells lining the human gastrointestinal tract. Biochem Biophys Res Commun. 1996 May 15;222(2):379–385. doi: 10.1006/bbrc.1996.0752. [DOI] [PubMed] [Google Scholar]
  19. Lounsbury K. M., Casey P. J., Brass L. F., Manning D. R. Phosphorylation of Gz in human platelets. Selectivity and site of modification. J Biol Chem. 1991 Nov 15;266(32):22051–22056. [PubMed] [Google Scholar]
  20. Marceau F. Kinin B1 receptors: a review. Immunopharmacology. 1995 Jun;30(1):1–26. doi: 10.1016/0162-3109(95)00011-h. [DOI] [PubMed] [Google Scholar]
  21. Miura Y., Kikuchi A., Musha T., Kuroda S., Yaku H., Sasaki T., Takai Y. Regulation of morphology by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in Swiss 3T3 cells. J Biol Chem. 1993 Jan 5;268(1):510–515. [PubMed] [Google Scholar]
  22. Moody T. W., Carney D. N., Cuttitta F., Quattrocchi K., Minna J. D. High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer. Life Sci. 1985 Jul 15;37(2):105–113. doi: 10.1016/0024-3205(85)90413-8. [DOI] [PubMed] [Google Scholar]
  23. Moyer M. P., Manzano L. A., Merriman R. L., Stauffer J. S., Tanzer L. R. NCM460, a normal human colon mucosal epithelial cell line. In Vitro Cell Dev Biol Anim. 1996 Jun;32(6):315–317. doi: 10.1007/BF02722955. [DOI] [PubMed] [Google Scholar]
  24. Narayan S., Draviam E., Rajaraman S., Singh P. High-affinity binding sites for bombesin on mouse colonic mucosal membranes. Mol Cell Biochem. 1991 Jul 24;106(1):31–39. doi: 10.1007/BF00231186. [DOI] [PubMed] [Google Scholar]
  25. Paschke R., Tonacchera M., Van Sande J., Parma J., Vassart G. Identification and functional characterization of two new somatic mutations causing constitutive activation of the thyrotropin receptor in hyperfunctioning autonomous adenomas of the thyroid. J Clin Endocrinol Metab. 1994 Dec;79(6):1785–1789. doi: 10.1210/jcem.79.6.7989485. [DOI] [PubMed] [Google Scholar]
  26. Pinski J., Schally A. V., Halmos G., Szepeshazi K. Effect of somatostatin analog RC-160 and bombesin/gastrin releasing peptide antagonist RC-3095 on growth of PC-3 human prostate-cancer xenografts in nude mice. Int J Cancer. 1993 Dec 2;55(6):963–967. doi: 10.1002/ijc.2910550615. [DOI] [PubMed] [Google Scholar]
  27. Popova J. S., Garrison J. C., Rhee S. G., Rasenick M. M. Tubulin, Gq, and phosphatidylinositol 4,5-bisphosphate interact to regulate phospholipase Cbeta1 signaling. J Biol Chem. 1997 Mar 7;272(10):6760–6765. doi: 10.1074/jbc.272.10.6760. [DOI] [PubMed] [Google Scholar]
  28. Preston S. R., Woodhouse L. F., Jones-Blackett S., Miller G. V., Primrose J. N. High-affinity binding sites for gastrin-releasing peptide on human colorectal cancer tissue but not uninvolved mucosa. Br J Cancer. 1995 May;71(5):1087–1089. doi: 10.1038/bjc.1995.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Qin Y., Halmos G., Cai R. Z., Szoke B., Ertl T., Schally A. V. Bombesin antagonists inhibit in vitro and in vivo growth of human gastric cancer and binding of bombesin to its receptors. J Cancer Res Clin Oncol. 1994;120(9):519–528. doi: 10.1007/BF01221028. [DOI] [PubMed] [Google Scholar]
  30. Radulovic S. S., Milovanovic S. R., Cai R. Z., Schally A. V. The binding of bombesin and somatostatin and their analogs to human colon cancers. Proc Soc Exp Biol Med. 1992 Jul;200(3):394–401. doi: 10.3181/00379727-200-43447. [DOI] [PubMed] [Google Scholar]
  31. Radulovic S., Miller G., Schally A. V. Inhibition of growth of HT-29 human colon cancer xenografts in nude mice by treatment with bombesin/gastrin releasing peptide antagonist (RC-3095). Cancer Res. 1991 Nov 1;51(21):6006–6009. [PubMed] [Google Scholar]
  32. Radulovic S., Schally A. V., Reile H., Halmos G., Szepeshazi K., Groot K., Milovanovic S., Miller G., Yano T. Inhibitory effects of antagonists of bombesin/gastrin releasing peptide (GRP) and somatostatin analog (RC-160) on growth of HT-29 human colon cancers in nude mice. Acta Oncol. 1994;33(6):693–701. doi: 10.3109/02841869409121784. [DOI] [PubMed] [Google Scholar]
  33. Rasenick M. M., Talluri M., Dunn W. J., 3rd Photoaffinity guanosine 5'-triphosphate analogs as a tool for the study of GTP-binding proteins. Methods Enzymol. 1994;237:100–110. doi: 10.1016/s0076-6879(94)37055-9. [DOI] [PubMed] [Google Scholar]
  34. Regoli D., Calo G., Rizzi A., Bogoni G., Gobeil F., Campobasso C., Mollica G., Beani L. Bradykinin receptors and receptor ligands (with special emphasis on vascular receptors). Regul Pept. 1996 Aug 27;65(1):83–89. doi: 10.1016/0167-0115(96)00076-6. [DOI] [PubMed] [Google Scholar]
  35. Reile H., Armatis P. E., Schally A. V. Characterization of high-affinity receptors for bombesin/gastrin releasing peptide on the human prostate cancer cell lines PC-3 and DU-145: internalization of receptor bound 125I-(Tyr4) bombesin by tumor cells. Prostate. 1994 Jul;25(1):29–38. doi: 10.1002/pros.2990250105. [DOI] [PubMed] [Google Scholar]
  36. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  37. Rozengurt E., Sinnett-Smith J. Bombesin stimulation of DNA synthesis and cell division in cultures of Swiss 3T3 cells. Proc Natl Acad Sci U S A. 1983 May;80(10):2936–2940. doi: 10.1073/pnas.80.10.2936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Severi C., Jensen R. T., Erspamer V., D'Arpino L., Coy D. H., Torsoli A., Delle Fave G. Different receptors mediate the action of bombesin-related peptides on gastric smooth muscle cells. Am J Physiol. 1991 May;260(5 Pt 1):G683–G690. doi: 10.1152/ajpgi.1991.260.5.G683. [DOI] [PubMed] [Google Scholar]
  39. Seybold V. S., Parsons A. M., Aanonsen L. M., Brown D. R. Characterization and autoradiographic localization of gastrin releasing peptide receptors in the porcine gut. Peptides. 1990 Jul-Aug;11(4):779–787. doi: 10.1016/0196-9781(90)90195-b. [DOI] [PubMed] [Google Scholar]
  40. Shenker A., Laue L., Kosugi S., Merendino J. J., Jr, Minegishi T., Cutler G. B., Jr A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature. 1993 Oct 14;365(6447):652–654. doi: 10.1038/365652a0. [DOI] [PubMed] [Google Scholar]
  41. Spindel E. R., Giladi E., Brehm P., Goodman R. H., Segerson T. P. Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrin-releasing peptide receptor. Mol Endocrinol. 1990 Dec;4(12):1956–1963. doi: 10.1210/mend-4-12-1956. [DOI] [PubMed] [Google Scholar]
  42. Tiberi M., Caron M. G. High agonist-independent activity is a distinguishing feature of the dopamine D1B receptor subtype. J Biol Chem. 1994 Nov 11;269(45):27925–27931. [PubMed] [Google Scholar]
  43. Traynor T. R., O'Grady S. M. Regulation of colonic ion transport by GRP. I. GRP stimulates transepithelial K and Na secretion. Am J Physiol. 1996 Mar;270(3 Pt 1):C848–C858. doi: 10.1152/ajpcell.1996.270.3.C848. [DOI] [PubMed] [Google Scholar]
  44. Wang Z., Bilsky E. J., Porreca F., Sadée W. Constitutive mu opioid receptor activation as a regulatory mechanism underlying narcotic tolerance and dependence. Life Sci. 1994;54(20):PL339–PL350. doi: 10.1016/0024-3205(94)90022-1. [DOI] [PubMed] [Google Scholar]
  45. Zhang M. L., Sugawa H., Kosugi S., Mori T. Constitutive activation of the thyrotropin receptor by deletion of a portion of the extracellular domain. Biochem Biophys Res Commun. 1995 Jun 6;211(1):205–210. doi: 10.1006/bbrc.1995.1797. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES