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

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 ␣ 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. ( J. Clin. Invest. 1997. 100: 2530-2537.)

Gα13 Stimulates Rho-dependent Activation of the Cyclooxygenase-2 Promoter

“…It is also known that constitutively activated G␣ 13 can act as a potent oncogene (31)(32)(33) and that Rho function is necessary for transformation (55). In addition, constitutive or autocrine stimulation of heptahelical receptors that couple to G␣ 13 and Rho are also implicated in tumorogenesis (56,57). Our results identifying a novel link between G␣ 13 , Rho, and COX-2 suggest that this pathway may contribute to tumor development in at least some cell types, a proposition that warrants further experimental work.…”

Gα13/PDZ-RhoGEF/RhoA Signaling Is Essential for Gastrin-Releasing Peptide Receptor–Mediated Colon Cancer Cell Migration

“…As a model we used Caco-2 and HT-29 colon cancer cell lines, which express functional GRP receptor and form moderately well-differentiated adenocarcinoma in nude mice (Carroll et al, 2000). To determine RhoA activation, we conducted a time-course experiment, stimulating Caco-2 and HT-29 cells in serum free conditions with a concentration of GRP (100 nM) that has been used for previous colon cancer studies (Ferris et al, 1997;Glover et al, 2005).The level of RhoA activation was assessed using RhoA pulldown assay (Ren and Schwartz, 2000) (Fig. 1, A and B).…”

Expression of V1A and GRP receptors leads to cellular transformation and increased sensitivity to substance-P analogue-induced growth inhibition

“…Apparent constitutive activity of a wild-type GPCR may vary substantially in various systems. Differences in GPCR expression level, GPCR desensitisation, G-protein complement and GPCR/G-protein stochiometry can all contribute to such differences (Seifert and Wenzel-Seifert, 2002) This could explain why GRP receptors expressed in NCM460 cells, a nonmalignant human colon epithelial cell line, are constitutively active (Ferris et al, 1997) but are not in other cell types (Benya et al, 1994). Our findings are unlikely to be due to an overexpression artefact as the receptors in this study were expressed at levels not greater than those measured from a variety of other cancers (100 -50 000 sites/ cell, Jensen et al, 2001) So at these moderate levels, wild-type receptor was sufficient to activate downstream signals that promote cell survival under anchorage-independent conditions.…”