Human bombesin receptor subtype 3 (BRS-3) was cloned based on its homology to the human gastrin-releasing peptide (GRP) receptor and neuromedin B (NMB) receptor. Some bombesin-like peptides were shown to activate BRS-3 expressed in Xenopus laevis oocytes, but only at relatively high concentrations, which suggests that BRS-3 is an orphan receptor. To study the pharmacology of BRS-3 in the context of a mammalian cell, we used BR2 cells, which are Balb/3T3 fibroblasts transfected with BRS-3 cDNA. A number of bombesin-like peptides found in mammals and amphibians stimulated calcium mobilization in BR2 cells but exhibited no effect on nontransfected parental Balb/3T3 cells. Of these peptides, NMB (EC50 approximately 1-10 microM) was the most active for stimulation of calcium mobilization. Testing of a series of NMB analogs truncated at the amino terminus and carboxyl terminus indicated that the minimal size of NMB required for retention of full activity was Ac-NMB(3-10). Systematically replacing each residue with alanine, or changing its chirality, demonstrated that the carboxyl-terminal residues His8, Phe9, and Met10 of NMB are important for optimal activity. We also tested whether a number of bombesin (BN) analogs that are potent pure or partial antagonists of the GRP receptor can activate BRS-3 in BR2 cells. One such analog, D-Phe6-BN(6-13) propyl amide, activated BRS-3-mediated calcium mobilization with an EC50 level of 84 nM. Through additional synthesis, we generated a significantly more potent analog, D-Phe6-Phe13-BN(6-13) propyl amide, which displayed an EC50 level of 5 nM for activation of BRS-3. Taken together, our data show that the core portions of bombesin-like peptides required for activation of BRS-3 are similar to those necessary for activation of the GRP and NMB receptors and thus provide pharmacological evidence that BRS-3 is in the BN receptor family. Furthermore, we have identified an agonist of BRS-3, namely D-Phe6-Phe13-BN(6-13) propyl amide, which is roughly 1000-fold more potent than BRS-3 agonists described previously.
We demonstrate that gastrin-releasing peptide (GRP) can inhibit the proliferation of human immortal nontumorigenic (184-B5) mammary epithelial cells ectopically expressing the human GRP receptor. Growth of Balb 3T3 cells ectopically expressing relatively high levels of the GRP receptor was also inhibited by GRP; however, growth of transfectants expressing lower levels of the receptor was not inhibited. Compared with Balb 3T3 cells, mammary epithelial cells could be rendered sensitive to growth inhibition by GRP by the expression of fewer GRP receptors. GRP also stimulated DNA synthesis in quiescent, serum-starved Balb 3T3 transfectants. In clones that were sensitive to growth inhibition by GRP by virtue of their expression of relatively high levels of the GRP receptor, the dose-response curve of GRP-stimulated DNA synthesis was bell shaped. This is consistent with our conclusion that the growth-inhibiting activity of GRP required the activation of a relatively large pool of receptors in Balb 3T3 cells. Significantly, prostaglandin H synthase inhibitors, which block the production of prostaglandins from arachidonic acid, reduced GRP-inhibitory effects on DNA synthesis. We also compared a number of GRP-stimulated signaling pathways in Balb 3T3 clones that were sensitive or insensitive to growth inhibition by GRP, including cAMP formation, phospholipase C activation, calcium mobilization, and arachidonic acid formation. Taken together, these results demonstrate a novel GRP receptor-coupled signal pathway promoting growth inhibition in which prostaglandin H synthase plays a significant role.
Isolated chloroplast coupling factor 1 ATPase is known to retain about 1 mol of tightly bound ADP/mol of enzyme. Some experimental results have given evidence that the bound ADP is at catalytic sites, but this view has not been supported by observations of a slow replacement of the bound ADP when CaATP or MgATP is added. The experiments reported in this paper show why a slow replacement of ADP bound at a catalytic site can occur. When coupling factor 1, labeled with tightly bound [3H]ADP, is exposed to Mg2+ or Ca2+ prior to the addition of MgATP or CaATP, a pronounced lag in the onset of ATP hydrolysis is observed, and only slow replacement of the [3H]ADP occurs. Mg2+ or Ca2+ can induce inhibition very rapidly, as if an inhibited form of the enzyme results whenever the enzyme with tightly bound ADP encounters Mg2+ or Ca2+ prior to ATP. The inhibited form can be slowly reactivated by incubation with EDTA, although some irreversible loss in activity is encountered. In contrast, when MgATP or CaATP is added to enzyme depleted of Mg2+ and Ca2+ by incubation with EDTA, a rapid onset of ATP hydrolysis occurs and most of the tightly bound [3H]ADP is released within a few seconds, as expected for binding at a catalytic site. The Mg2+-induced inhibition of both the ATPase activity and the lack of replacement of tightly bound [3H] ADP can be largely prevented by incubation with Pi under conditions favoring Pi addition to the site containing the tightly bound ADP. Our and other results can be explained if enzyme catalysis is greatly hindered when MgADP or CaADP without accompanying Pi is tightly bound at one of the three catalytic sites on the enzyme in a high affinity conformation.
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.
Bombesin-like peptides (BLPs) can regulate the growth of normal and transformed cells. To compare the relative activities of the three known human BLP receptor subtypes [i.e., the gastrin-releasing peptide (GRP) receptor, neuromedin B (NMB) receptor, or BLP receptor subtype 3] in growth regulation, we expressed each receptor in a receptor-deficient host, Balb/3T3 cells. None of the receptor agonists used in our study promoted DNA synthesis by quiescent parental, nontransfected Balb/3T3 cells. Using clones stably transfected with the NMB receptor however, we found that NMB stimulated the incorporation of [3H]thymidine 2.5- to 8-fold over basal levels. The greatest net stimulation of [3H]thymidine incorporation occurred when the medium contained insulin. In quiescent Balb/3T3 cells transfected with the GRP receptor, GRP promoted a 15-fold increase in DNA synthesis in the absence of insulin or other growth factors. GRP also induced the labeling of a large percentage (53%) of the cells with bromodeoxyuridine. To determine the length of time that GRP receptor signaling was required to drive quiescent cells into the S phase of the cell cycle, we blocked GRP receptor signaling by addition of a competitive GRP receptor antagonist at different times after stimulating cells with GRP. Our data demonstrate that persistent GRP receptor signaling throughout a large part of the G1 phase of the cell cycle is important in the mitogenic effects of GRP in these cells. Hitherto uncharacterized GRP receptor signaling pathways may be important in this process. BLPs also stimulated a mitogenic response by transfectants expressing the BLP receptor subtype 3 if insulin was contained in the medium. Taken together, these studies indicate that all three BLP receptor subtypes may contribute to growth regulation in vivo.
To enable the detailed pharmacological characterization of five bombesin (BN) analogs with respect to the human gastrin-releasing peptide (GRP) receptor, we ectopically expressed the receptor in BALB/3T3 cells. In such cells (termed GR1 cells), GRP stimulated DNA synthesis and Ca2+ mobilization. Two of these analogs, D-Phe6-BN(6-13) methyl ester (Ki = 1.38 +/- 0.07 nM) and 4-pyridyl-CO-His7-D-Ala11-Lys12-COCH2CH2-phenyl- BN(7-13) methyl amide (Ki = 2.17 +/- 0.05 nM), were pure antagonists of GRP-stimulated DNA synthesis in GR1 cells (IC50 = 14 +/- 8.5 nM and 5.1 +/- 2.0 nM, respectively), whereas three analogs, Leu13-psi-Leu14-BN (Ki = 21.6 +/- 2.2 nM), D-Phe6-BN(6-13) ethyl amide (Ki = 5.17 +/- 0.64 nM), and D-Phe6-BN(6-13) propyl amide (Ki = 0.68 +/- 0.01 nM), displayed significant partial agonistic activity. Although three analogs promoted mitogenesis in GR1 cells, none of the analogs stimulated calcium mobilization in GR1 cells. This dichotomy was not limited to transfected cells, because the same result was obtained for D-Phe6-BN(6-13) propyl amide using human fetal lung cells, which naturally express the GRP receptor. We also assessed the effect of BN analogs on calcium mobilization in transfected GR9 cells expressing about 30 times higher levels of the GRP receptor, compared with GR1 cells. D-Phe6-BN(6-13) ethyl amide, D-Phe6-BN(6-13) propyl amide, and Leu13-psi-Leu14-BN were partial agonists of the intracellular Ca2+ mobilization response of GR9 cells. One conclusion consistent with our data is that GRP-stimulated DNA synthesis requires the activation of far fewer receptors than does GRP-stimulated calcium mobilization. Thus, analogs with a small amount of agonist activity can trigger a mitogenic response but not an intracellular Ca2+ mobilization response, unless cells express a high level of receptors. These studies also provide evidence that the promotion of DNA synthesis in quiescent GR1 or human fetal lung cells via the GRP receptor does not require mobilization of intracellular Ca2+.
We have shown previously that the AKT2 pathway is essential for cell survival and important in malignant transformation. In this study, we demonstrate elevated kinase levels of AKT2 and phosphatidylinositol-3-OH kinase (PI3K) in 32 of 80 primary breast carcinomas. The majority of the cases with the activation are estrogen receptor alpha (ERalpha) positive, which prompted us to examine whether AKT2 regulates ERalpha activity. We found that constitutively activated AKT2 or AKT2 activated by epidermal growth factor or insulin-like growth factor-1 promotes the transcriptional activity of ERalpha. This effect occurred in the absence or presence of estrogen. Activated AKT2 phosphorylates ERalpha in vitro and in vivo, but it does not phosphorylate a mutant ERalpha in which ser-167 was replaced by Ala. The PI3K inhibitor, wortmannin, abolishes both the phosphorylation and transcriptional activity of ERalpha induced by AKT2. However, AKT2-induced ERalpha activity was not inhibited by tamoxifen but was completely abolished by ICI 164,384, implicating that AKT2-activated ERalpha contributes to tamoxifen resistance. Moreover, we found that ERalpha binds to the p85alpha regulatory subunit of PI3K in the absence or presence of estradiol in epithelial cells and subsequently activates PI3K/AKT2, suggesting ERalpha regulation of PI3K/AKT2 through a nontranscriptional and ligand-independent mechanism. These data indicate that regulation between the ERalpha and PI3K/AKT2 pathway (ERalpha-PI3K/AKT2-ERalpha) may play an important role in pathogenesis of human breast cancer and could contribute to ligand-independent breast cancer cell growth.
