Receptor for bombesin with associated tyrosine kinase activity.

The neuropeptide bombesin is known for its potent mitogenic activity on murine 3T3 fibroblasts and other cells. Recently it has been implicated in the pathogenesis of small cell lung carcinoma, in which it acts through an autocrine loop of growth stimulation. Phosphotyrosine (P-Tyr) antibodies have been successfully used to recognize the autophosphorylated receptors for known growth factors. In Swiss 3T3 fibroblasts, phosphotyrosine antibodies identified a 115,000-Mr cell surface protein (p115) that became phosphorylated on tyrosine as a specific response to bombesin stimulation of quiescent cells. The extent of phosphorylation was dose dependent and correlated with the mitogenic effect induced by bombesin, measured by [3H]thymidine incorporation. Tyrosine phosphorylation of p115 was detectable minutes after the addition of bombesin, and its time course paralleled that described for the binding of bombesin to its receptor. Immunocomplexes of phosphorylated p115 and phosphotyrosine antibodies bound 125I-labeled [Tyr4]bombesin in a specific and saturable manner and displayed an associated tyrosine kinase activity enhanced by bombesin. Furthermore, the 125I-labeled bombesin analog gastrin-releasing peptide, bound to intact live cells, was coprecipitated with p115. These data strongly suggest that p115 participates in the structure and function of the surface receptor for bombesin, a new member of the family of growth factor receptors with associated tyrosine kinase activity.     

Activation of extracellular signal-regulated kinase mediates bombesin-induced mitogenic responses in prostate cancer cells

Bombesin and its mammalian homologue gastrin-releasing peptide have been shown to be highly expressed and secreted by neuroendocrine cells in prostate cancer, and are thought to be related to the carcinogenesis and progression of this disease. We found, in this study, bombesin specifically induced mitogen-activated protein (MAP) kinase activation as shown by increased extracellular regulated kinase (ERK) phosphorylation and epidermal growth factor (EGF) receptor transactivation in prostate cancer cells, which express functional gastrin-releasing peptide receptor. The transactivation of EGF receptor was required for bombesin-induced ERK phosphorylation. Furthermore, non-receptor tyrosine kinase Src and cellular Ca2+ were shown to be involved in bombesin-induced EGF receptor transactivation and ERK phosphorylation. Inhibition of either EGF receptor transactivation or ERK activation blocked bombesin-induced DNA synthesis in these cells. Taken together, these data suggest bombesin may act as a mitogen in prostate cancer by activating MAP kinase pathway via EGFR transactivation.     

Identification of the bombesin receptor on murine and human cells by cross-linking experiments

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.     

Bombesin receptor in membranes from Swiss 3T3 cells. Binding characteristics, affinity labelling and modulation by guanine nucleotides.

Bombesin-like neuropeptides, including mammalian gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells. In this study, we have characterized the bombesin receptor in membrane preparations from these cells. Addition of Mg2+ during cell homogenization was essential to preserve 125I-GRP binding activity in the resulting membrane preparation. The effect of Mg2+ was concentration dependent, with a maximum at 5 mM. Specific binding of 125I-GRP was saturable; Scatchard analysis indicated a single class of high-affinity sites of Kd = (2.1 +/- 0.3) x 10(-10) M at 15 degrees C and Kd = (1.9 +/- 0.4) x 10(-10) M at 37 degrees C, and a maximum binding capacity of 580 +/- 50 fmol/mg of protein (15 degrees C) or 604 +/- 40 fmol/mg of protein (37 degrees C). The kinetically derived dissociation constant was 1.5 x 10(-10) M. 125I-GRP binding was inhibited in a concentration-dependent manner by various peptides containing the highly conserved C-terminal heptapeptide of the bombesin family, including bombesin, GRP, neuromedin B and the 8-14 fragment of bombesin. In contrast, a variety of structurally unrelated mitogens and neuropeptides had no effect. The cross-linking agent ethyleneglycolbis(succinimidylsuccinate) covalently linked 125I-GRP to a single Mr 75 000-85 000 protein in membrane preparations of 3T3 cells. Affinity labelling of this molecule was specific and dependent on the presence of Mg2+ during membrane preparation. Finally, the non-hydrolysable GTP analogue guanosine-5'-[gamma-thio]triphosphate (GTP[S]) caused a concentration-dependent inhibition of 125I-GRP binding and cross-linking to 3T3 cell membranes [concentration giving half-maximal inhibition (IC50) approximately 0.2 microM]. The inhibitory effect was specific (GMP, ATP or ATP[S] had no effect at 10 microM) and was due to an increase in Kd from (1.7 +/- 0.2) x 10(-10) M to (4.3 +/- 0.6) x 10(-10) M in the presence of 10 microM-GTP[S]. This modulation of ligand affinity and cross-linking implies that the bombesin receptors that mediate mitogenesis in Swiss 3T3 cells are coupled to a guanine-nucleotide-binding-protein signal-transduction pathway.     

Proadrenomedullin N-terminal 20 peptide (PAMP) elevates blood glucose levels via bombesin receptor in mice

We found a potent hyperglycemic effect of proadrenomedullin N-terminal 20 peptide (PAMP) after intra-third cerebroventricular administration at a dose of 10 nmol in fasted mice. PAMP has four homologous residues with bombesin (BN), a hyperglycemic peptide. PAMP showed affinity for gastrin-releasing peptide preferring receptor (GRP-R) and neuromedin B preferring receptor. The PAMP-induced hyperglycemic effect was inhibited by [D-Phe(6), Leu-NHEt(13), des-Met(14)]-BN (6-14), GRP-R specific antagonist, indicating that the hyperglycemic effect is mediated at least in part via GRP-R. Furthermore, pretreatment of alpha-adrenergic blocker inhibited the PAMP-induced hyperglycemia and hyperglucagonemia, suggesting that the increase of glucagon secretion through alpha-adrenergic activation is involved in this hyperglycemic effect of PAMP.     

Gastrin-releasing peptide (GRP,mammalian bombesin) in the pathogenesis of lung cancer

Established human lung cancer exhibits a complex pattern of genetic changes as well as several distinct autocrine growth factor loops for regulatory peptides. The best studied example is that of gastrin-releasing peptide (GRP), the mammalian homolog of the amphibian bombesin. It is produced by up to 70% of small cell lung cancers and 10–20% of non-small cell lung cancers. GRP stimulates the growth of normal bronchial epithelium as well as that of small cell lung cancer, and its blockade with the use of antibodies or synthetic antagonists inhibits the growth of these tumors. Study of its molecular biology has revealed a complex pattern of mRNA processing which has lead to the recent isolation of a novel family of peptides termed gastrin-releasing peptide gene-associated peptides (GGAPs), present in normal and malignant human tissues. Additional efforts have been directed at characterizing the GRP receptor as well as its intracellular signaling pathways which have been reported both as G protein phospholipase C coupled events as well as activation of a membrane associated tyrosine kinase. In view of its expression in normal bronchial epithelium and its mitogenic effects on this tissue, GRP appears to play a central role in the early events of pulmonary carcinogenesis.     

Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding

Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an Mr 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent; enhancement in 80k phosphorylation could be detected as early as 10 s after the addition of peptide. Recently, a rapid increase in the phosphorylation of an 80k cellular protein after treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact fibroblasts (Rozengurt, E., A. Rodriguez-Pena, and K. A. Smith, 1983, Proc. Natl. Acad. Sci. USA., 80:7244-7248; Rozengurt, E., A. Rodriguez-Pena, M. Coombs, and J. Sinnett-Smith, 1984, Proc. Natl. Acad. Sci. USA., 81:5748-5752). The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of 125I-labeled epidermal growth factor (125I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. Peptides structurally related to bombesin, including gastrin-releasing peptide, also stimulated 80k phosphorylation and inhibited 125I-EGF binding; both effects were selectively blocked by a novel bombesin antagonist. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca2+ concentration does not mediate the bombesin inhibition of 125I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of 125I-EGF to its cellular receptor.     

Chronic desensitization to bombesin by progressive down-regulation of bombesin receptors in Swiss 3T3 cells. Distinction from acute desensitization

Prolonged exposure (40 h) of Swiss 3T3 cells to bombesin induced homologous desensitization to bombesin and structurally related peptides including mammalian gastrin releasing peptide (GRP). The ability of bombesin to mobilize intracellular Ca2+, inhibit epidermal growth factor binding, and stimulate DNA synthesis was profoundly and selectively inhibited. In contrast, Ca2+ mobilization by either vasopressin or bradykinin was unaffected, indicating that chronic desensitization is mechanistically distinct from acute desensitization of Ca2+ mobilization. Prolonged (24 or 40 h) pretreatment with bombesin also induced a 78 +/- 5% loss of bombesin receptor binding sites in both intact and plasma membrane preparations of Swiss 3T3 cells without an apparent change in receptor affinity (Kd = 1.9 +/- 0.1 x 10(-9) M and Kd = 1.8 +/- 0.2 x 10(-9) M for control and pretreated cells, respectively). Loss of 125I-GRP binding was slow and progressive with half-maximal loss of binding occurring after 7 h and maximal after approximately 14 h. Cross-linking of 125I-GRP to intact cultures and membrane preparations revealed an identical time-dependent loss of the Mr = 75,000-85,000 cross-linked band, previously identified as the bombesin receptor. Prolonged exposure of the cells to phorbol 12,13-dibutyrate, epidermal growth factor, cholera toxin, or mitogenic combinations of these agents did not alter 125I-GRP binding. Receptor down-regulation and loss of mitogenic responsiveness to bombesin were: (a) induced in a parallel dose-dependent manner by bombesin (ED50 = 1 nM), GRP (ED50 = 2 nM), and neuromedin B (ED50 = 20 nM), but not by the biologically inactive fragment GRP (1-16); (b) inhibited by the specific bombesin antagonist [Leu13-psi(CH2NH)-Leu14] bombesin, and (c) reversed upon removal of bombesin with a similar time course (full recovery after 15 h). On the basis of these observations, we propose that prolonged pretreatment of Swiss 3T3 cells with bombesin induces homologous desensitization to peptides of the bombesin family by down-regulation of cell surface bombesin receptors.     

Stimulation of tyrosine kinase activity in anti-phosphotyrosine immune complexes of Swiss 3T3 cell lysates occurs rapidly after addition of bombesin, vasopressin, and endothelin to intact cells.

Treatment of quiescent Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, endothelin/vasoactive intestinal contractor (VIC), and bradykinin strikingly increased the initial rate of tyrosine phosphorylation measured in anti-phosphotyrosine immunoprecipitates of a major band of Mr 115,000 (p115) and two minor components of Mr 90,000 and 75,000. Neuropeptides increased the labeling of p115 within seconds and with great potency; half-maximum concentrations were 0.1, 0.2 and 0.3 nM for bombesin, vasopressin, and VIC, respectively. Immunoblotting and peptide mapping showed that the p115 band phosphorylated in anti-phosphotyrosine immunoprecipitates is identical to a major Mr 115,000 substrate for neuropeptide-stimulated tyrosine phosphorylation in intact Swiss 3T3 cells. Furthermore, bombesin, vasopressin, and VIC markedly increased the rate of phosphorylation of Raytide, a broad specificity tyrosine kinase peptide substrate, by decreasing (8 +/- 1.3-fold) the apparent Km of the kinase for the substrate. Phorbol 12,13-dibutyrate and the Ca2+ ionophore A23187 had a weaker effect on tyrosine protein kinase activity in immune complexes compared with bombesin. Furthermore, down-regulation of protein kinase C blocked the small effect of phorbol esters but did not impair bombesin-stimulated tyrosine kinase activity. These results provide direct evidence for neuropeptide activation of a tyrosine kinase in cell-free preparations and identify a novel event in the action of this class of growth factors in Swiss 3T3 cells.     

Primary structures of the bombesin-like neuropeptides in frog brain show that bombesin is not the amphibian gastrin-releasing peptide

Peptides displaying gastrin-releasing peptide/bombesin-like immunoreactivity were isolated in pure form from an extract of the brain of the European green frog, Rana ridibunda. The primary structure of the more abundant peptide was established as: Gly-Ser-His-Trp-Ala-Val-Gly-His-Leu-Met. NH2. This sequence shows one substitution (Ser for Asn) compared with mammalian gastrin-releasing peptide (18-27) (neuromedin C). The extract also contained gastrin-releasing peptide but bombesin was absent. The data indicate that bombesin is not the amphibian counterpart of gastrin-releasing peptide.     

Bombesin and the C-terminal tetradecapeptide of gastrin-releasing peptide are growth factors for normal human bronchial epithelial cells

Bombesin and the C-terminal portion of gastrin-releasing peptide (GRP14-27) each increase clonal growth rate and colony-forming efficiency of normal human bronchial epithelial cells. These effects occur in the presence or absence of an optimal concentration (5 ng/ml) of epidermal growth factor (EGF). In contrast to EGF bombesin and GRP14-27 do not stimulate cell migration. Thus, bombesin and the C-terminal fragment of gastrin-releasing peptide represent a new class of peptides mitogenic for normal human epithelial cells.     

Requirement of cortical actin organization for bombesin, endothelin, and EGF receptor internalization

The role of actinorganization in occupancy-induced receptor internalization remainspoorly defined. Here we report that treatment of mouse Swiss 3T3 cellswith latrunculin A, a potent inhibitor of actin polymerization(including cortical actin), inhibited the internalization of theendogenous bombesin/gastrin-releasing peptide (GRP) receptor, as judgedby uptake of ¹²⁵I-labeled GRP or fluorescent Cy3-labeledbombesin. In contrast, cells pretreated with cytochalasin D showedminimal inhibition of bombesin/GRP receptor internalization. Similarly,pretreatment of Swiss 3T3 cells with the potent Rho-kinaseinhibitor HA-1077, at concentrations (10-20 µM) thatabrogated bombesin-mediated stress fiber formation, did notsignificantly alter receptor-mediated internalization of¹²⁵I-GRP. These results indicate that bombesin/GRP receptorinternalization depends on latrunculin A-sensitive cortical actinrather than on rapidly turning over actin stress fibers that aredisrupted by either cytochalasin D or HA-1077. The rates andtotal levels of internalization of the endogenously expressedendothelin A receptor and epidermal growth factor receptor were alsomarkedly reduced by latrunculin A in Swiss 3T3 cells. The potency oflatrunculin A for inhibiting G protein-coupled receptor endocytosis wascomparable to that for reducing internalization of the epidermal growthfactor tyrosine kinase receptor. We conclude that cortical actinstructures, disrupted by latrunculin A, are necessary foroccupancy-induced receptor internalization in animal cells.

     

Signal transduction through the vasoactive intestinal peptide receptor stimulates phosphorylation of the tyrosine kinase pp60c-src.

The present study demonstrates that signal transduction through a receptor lacking intrinsic tyrosine protein kinase activity involves a rapid and potent phosphorylation of a non-receptor tyrosine protein kinase in the membranes. Vasoactive intestinal peptide (VIP) stimulates phosphorylation of a membrane protein with a M.W. of 56 KD (pp60) in the cultured chick embryonic retinal pigment epithelium. VIP stimulates phosphorylation of the pp60 with such efficiency and potency that the maximal phosphorylation has been observed at the earliest time (3 minutes at 1 x 10(-6)M VIP) and the lowest concentration (1 x 10(-11)M for 20 minutes) examined. Western blot analysis with a monoclonal antibody anti-pp60src (GD11, Parsons et al., J. Virol. 51, 272-282, 1984) indicates that the pp60 is the pp60c-src, a normal cell oncogene product with intrinsic tyrosine protein kinase activity.     

Bombesin: a possible role in wound repair

During tissue regeneration and wound healing of the skin, migration, proliferation and differentiation of keratinocytes are important processes. Here we assessed the effect of a neuropeptide, bombesin, on keratinocytes during regeneration from scratch wounding. Bombesin purified from amphibian skin, is homologous of mammalian gastrin-releasing peptide and is active in mammals. Its pharmacological effects mediate various physiological activities: hypertensive action, stimulating action on gastric secretion, hyperglycemic effect or increased insulin secretion. In vitro it shows a hyperproliferative effect on different experimental models and is involved in skin repair. The aim of this study was to elucidate the effect of Bombesin in an in vitro experimental model on a mechanically injured human keratinocyte monolayer. We evaluated different mediators involved in wound repair such as IL-8, TGFbeta, IL-1, COX-2, VEGF and Toll-like receptors 2 and 4 (TLR2 and TLR4). We also studied the effects of bombesin on cell proliferation and motility and its direct effect on wound repair by observing the wound closure after mechanical injury. The involvement of the bombesin receptors neuromedin receptor (NMBR) and gastrin-releasing peptide receptor (GRP-R) was also evaluated. Our data suggest that bombesin may have an important role in skin repair by regulating the expression of healing markers. It enhanced the expression of IL-8, TGFbeta, COX-2 and VEGF. It also enhanced the expression of TLR2, while TLR4 was not expressed. Bombesin also increased cell growth and migration. In addition, we showed that NMBR was more involved in our experimental model compared to GRP-R.     

Comparison of the actions of bombesin, gastrin-releasing peptide-27, neuromedin B, and gastrin-releasing peptide-10 in causing release of gastrin and gastric inhibitory peptide in rats

The objective of this study was to compare the gastrin- and gastric inhibitory peptide (GIP)-releasing actions of bombesin, gastrin-releasing peptide (GRP)-27, neuromedin B, and GRP-10 in rats. Both bombesin and GRP-27 are potent stimulants of gastrin and GIP release, whereas neuromedin B and GRP-10 are less effective, on a molar basis.     

Early phosphorylation events following the treatment of Swiss 3T3 cells with bombesin and the mammalian bombesin-related peptide, gastrin-releasing peptide.

Bombesin and the related mammalian peptides, such as gastrin-releasing peptide (GRP), are potent mitogens for some fibroblast cell lines. Here we have examined the bombesin- and GRP-mediated changes in the phosphorylation of proteins in Swiss 3T3 cells and compared these to the events observed after platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and tumor promoter treatment. In agreement with previous reports, bombesin, GRP and PDGF, but not EGF, increased the activity of protein kinase C. This was assayed by an inhibition of [125I]EGF binding, stimulation in phosphorylation of pp60c-src on serine 12 and stimulation in phosphorylation of a group of 80 kd proteins. The different phosphorylated forms of the 80 kd proteins were examined by tryptic peptide mapping and shown to contain multiple phosphorylation sites. An investigation of the tyrosine phosphorylation events following mitogen treatment revealed a significant difference between PDGF and the bombesin peptides. PDGF treatment caused a marked increase in total cellular phosphotyrosine levels, and tyrosine phosphorylation both of known substrates and its own receptor. In contrast, bombesin and GRP treatments resulted in only a weak or undetectable increase in tyrosine phosphorylation of total cellular protein or known substrates. In this respect bombesin and GRP were more similar to EGF. The fact that the bombesin peptides do not induce a phosphorylation response identical with either PDGF or EGF suggests that there is not a single common signal pathway which is activated by all these mitogens.     

Leupaxin is similar to paxillin in focal adhesion targeting and tyrosine phosphorylation but has distinct roles in cell adhesion and spreading

Focal adhesion (FA) formation is induced by extracellular matrix-stimulated integrin clustering and activation of receptors for diffusible factors. Leupaxin (LPXN) is a member of the paxillin family of FA proteins expressed in many cancer cell lines. We found activation of gastrin-releasing peptide receptor (GRPr) by bombesin (BN) stimulated LPXN translocation from cytoplasm to FAs. Using mutagenesis, we identified LIM3 as the primary FA targeting domain for LPXN and showed BN-induced LPXN tyrosine phosphorylation on residues 22, 62 and 72. A LIM3 point mutant of LPXN failed to target to FAs and had no BN-stimulated tyrosine phosphorylation. Conversely, a non-phosphorylatable mutant (Y22/62/72F) translocated to FAs after BN addition. Stimulation of FA formation using vinblastine also induced LPXN translocation and tyrosine phosphorylation. Therefore, dynamic LPXN tyrosine phosphorylation requires translocation to FAs. LPXN and paxillin had opposite roles in adhesion to collagen I (CNI) in MDA-MB-231 breast cancer cells. LPXN siRNA stimulated whereas paxillin siRNA inhibited cell adhesion. Knockdown of both LPXN and paxillin behaved similarly to paxillin knockdown alone, suggesting LPXN''s function in adhesion might depend on paxillin. Additionally, LPXN regulated cell spreading on CNI but not on fibronectin whereas paxillin knockdown suppressed spreading on both substrates. These results demonstrate that although LPXN and paxillin''s FA targeting and tyrosine phosphorylation are similar, each protein has distinct functions.Key words: focal adhesion, tyrosine phosphorylation, bombesin, adhesion, spreading     

MicroPET imaging of a gastrin-releasing peptide receptor-positive tumor in a mouse model of human prostate cancer using a 64Cu-labeled bombesin analogue

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a variety of carcinomas and has been the target for detection and treatment of these neoplasms in animals. In particular, analogues of the tetradecapeptide bombesin (BN) have been radiolabeled with (99m)Tc and (111)In for detection of GRPR-positive tumors by gamma ray scintigraphy. The goal of this study was to evaluate the potential of the bombesin analogue, DOTA-Aoc-BN(7-14), for positron-emission tomographic (PET) imaging after radiolabeling with the positron-emitter (64)Cu. A saturation binding assay on PC-3 human prostate cancer cells showed that (64)Cu-DOTA-Aoc-BN(7-14) had an equilibrium binding constant (K(d)) of 6.1 +/- 2.5 nM and a receptor concentration (B(max)) of 2.7 +/- 0.6 x 10(5) receptors/cell. The radiolabeled analogue also showed rapid internalization with 18.2% internalized into 10(5) PC-3 cells by 2 h. The tumor localization of (64)Cu-DOTA-Aoc-BN(7-14) was 5.5% injected dose per gram in athymic nude mice bearing PC-3 xenografts at 2 h postinjection. The tumor retention with respect to the 2 h value was 76% and 45% at 4 and 24 h, respectively, and was GRPR-mediated as shown by inhibition with a coinjection of excess peptide. MicroPET imaging of (64)Cu-DOTA-Aoc-BN(7-14) in athymic nude mice bearing subcutaneous PC-3 tumors showed good tumor localization. Further studies with (64)Cu-pyruvaldehyde-bis(N(4)-methylthiosemicarbazone) ((64)Cu-PTSM) suggested that low blood flow to the PC-3 tumors may have limited the localization of (64)Cu-DOTA-Aoc-BN(7-14). This study demonstrates that (64)Cu-DOTA-Aoc-BN(7-14) can be used to detect GRPR-positive tumors by PET imaging.     

Bombesin and substance P analogues differentially regulate G-protein coupling to the bombesin receptor. Direct evidence for biased agonism

Substance P analogues including [d-Arg1,d-Phe5,d-Trp7,9,Leu11]substance P (SpD) act as "broad spectrum neuropeptide antagonists" and are potential anticancer agents that inhibit the growth of small cell lung cancer cells in vitro and in vivo. However, their mechanism of action is controversial and not fully understood. Although these compounds block bombesin-induced mitogenesis and signal transduction, they also have agonist activity. The mechanism underlying this agonist activity was examined. SpD binds to the ligand-binding site of the bombesin/gastrin-releasing peptide receptor and blocks the bombesin-stimulated increase in [Ca2+]i within the same concentration range that causes sustained activation of c-Jun N-terminal kinase and extracellular signal-regulated protein kinase (ERK). The activation of c-Jun N-terminal kinase by SpD and bombesin is blocked by dominant negative inhibition of G(alpha12). The ERK activation by SpD is pertussis toxin-sensitive in contrast to ERK activation by bombesin, which is pertussis toxin-insensitive but dependent on epidermal growth factor receptor phosphorylation. SpD does not simply act as a partial agonist but differentially modulates the activation of the G-proteins G(alpha12), G(i), and G(q) compared with bombesin. This unique ability allows the bombesin receptor to couple to G(i) and at the same time block receptor activation of G(q). Our results provide direct evidence that SpD is acting as a "biased agonist" and that this has physiological relevance in small cell lung cancer cells. This validation of the concept of biased agonism has important implications in the development of novel pharmacological agents to dissect receptor-mediated signal transduction and of highly selective drugs to treat human disease.     

Inhibition of bombesin-stimulated gastrin release from isolated canine G cells by bombesin antagonists

This study investigated the effects of two putative bombesin antagonists, [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P and [Leu13-psi-CH2NH-Leu14]bombesin, on bombesin-stimulated gastrin release from isolated canine G cells following short-term culture. Canine antral tissue was dispersed by sequential collagenase and EDTA treatment, and counterflow elutriation was used to enrich for G cells. Plates were seeded with 2 x 10(6) cells/mL in each well and cultured for 2 days prior to testing. Gastrin-containing and somatostatin-containing cells were identified by immunocytochemistry using the biotin-avidin-peroxidase method and accounted for 8.5 and 1%, respectively, of adhered cells. Basal gastrin secretion was 1.91 +/- 0.48% of total cell content. After a 2-h incubation period, bombesin (0.01-100 pM) stimulated gastrin release in a concentration-dependent fashion. The substance P analog, at a concentration of 1 microM, modestly inhibited bombesin-stimulated gastrin release from canine G cells. This analog also produced weak stimulation of basal gastrin release. In contrast, the bombesin analog, at a concentration of 1 microM, did not affect basal gastrin secretion. The bombesin analog completely blocked bombesin-stimulated gastrin release from 0.01 to 1 pM and produced greater than 50% inhibition at higher doses. The ability of the bombesin analog to directly inhibit bombesin-stimulated gastrin release from cultured canine G cells underscores its usefulness in studies involving the role of bombesin and its mammalian counterpart, gastrin-releasing peptide, in the control of gastrin cell function.