Arachidonic acid release by bombesin. A novel postreceptor target for heterologous mitogenic desensitization

Prolonged exposure of Swiss 3T3 cells to vasopressin causes heterologous mitogenic desensitization to bombesin and structurally related peptides including gastrin-releasing peptide (GRP) without down-regulation of the bombesin receptor. The number and affinity of bombesin/GRP receptor sites and modulation of 125I-GRP binding by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) are unaffected in membrane preparations from vasopressin-treated cultures. Stimulation of inositol phosphate accumulation, mobilization of intracellular calcium, production of diacylglycerol, and transmodulation of the epidermal growth factor receptor by bombesin are similarly unaffected. Thus, the heterologous mitogenic desensitization is not due to uncoupling of bombesin receptor from transducing G protein(s) or to an inability to activate phospholipase C. Bombesin, unlike vasopressin, causes a rapid dose-dependent release of [3H]arachidonic acid and prostaglandin E2 from Swiss 3T3 cells (EC50 approximately 4 nM), which is inhibited by the specific bombesin receptor antagonist [Leu13-psi(CH2NH)-Leu14]bombesin. Crucially, release of [3H]arachidonic acid and prostaglandin E2 by bombesin is completely suppressed by prolonged pretreatment with vasopressin (EC50 = 0.6 nM). The mitogenic action of bombesin is restored by adding arachidonic acid to vasopressin-treated cells. We conclude first that arachidonic acid release is an early signal in the mitogenic response to bombesin and second that pretreatment with vasopressin induces heterologous mitogenic desensitization to bombesin by a novel mechanism: inhibition of arachidonic acid release.     

Purification and characterization of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells

The bombesin/gastrin-releasing peptide (GRP) receptor was solubilized from Swiss mouse 3T3 cell membranes in an active form and was purified about 90,000-fold to near homogeneity by a combination of wheat germ agglutinin-agarose and ligand affinity chromatography. The purified receptor displayed a single diffuse band with a Mr of 75,000-100,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After treatment of the receptor with N-glycanase, removing N-linked oligosaccharide moieties, the protein yielded a Mr = 38,000 band. These results agree with the Mr value estimated for the GRP receptor that was labeled on Swiss 3T3 cells by cross-linking to 125I-GRP1-27. GRP1-27 bound to the purified receptor with a Kd of 0.038 +/- 0.019 nM. By comparison, the soluble receptor in unfractionated extracts and intact membranes displayed a Kd for GRP1-27 of 0.036 +/- 0.003 nM and 0.13 +/- 0.04 nM, respectively. The relative potencies of a series of GRP analogs for the soluble receptor and intact membranes indicated that the extraction procedure did not significantly alter the receptor's ligand binding specificity. However coupling of the receptor to its guanyl nucleotide regulatory protein was not maintained in the soluble extract, and a G-protein did not co-purify with the receptor. Physiological concentrations of NaCl greatly inhibited the binding of some GRP analogs to the receptor, while the binding of other analogs was not affected. A domain on the GRP molecule involving Lys-13 or Arg-17 was identified which promoted binding to the GRP receptor under conditions of low ionic strength. These findings aided the development of an effective ligand affinity resin for the purification of the GRP receptor.     

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.     

Characterization of the high-affinity receptors on Swiss 3T3 cells which mediate the binding, internalization and degradation of the mitogenic peptide bombesin.

Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides.     

Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking

We have previously identified by chemical cross-linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000-85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells. Because bombesin-like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000-85,000 cross-linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000-85,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with 125I-labelled gastrin-releasing peptide (125I-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,000 cross-linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I-GRP binding and the affinity labelling of the Mr 75,000-85,000 protein. We also show that the cross-linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000-85,000 complex applied to wheat germ lectin-sepharose columns was eluted by addition of 0.3 M N-acetyl-D-glucosamine. Second, treatment with endo-beta-N-acetylglucosaminidase F reduced the apparent molecular weight of the affinity-labelled band from 75,000-85,000 to 43,000, indicating the presence of N-linked oligosaccharide groups.     

Isolation of the bombesin/gastrin-releasing peptide receptor from human small cell lung carcinoma NCI-H345 cells.

Purification of the gastrin-releasing peptide (GRP) or bombesin receptor has proved elusive in part due to technical difficulties. In the present studies, the problem of oxidized radioligand was avoided by the use of 125I-GRP, which was verified to be not oxidized by high performance liquid chromatography. Specific 125I-GRP binding (at 0 degrees C) to intact human small cell lung carcinoma NCI-H345 cells which had been subjected to a dilute acid wash was 6 fmol/10(6) cells. Inhibition of GRP degradation by human H345 cell membranes through the use of phenanthroline or phosphoramidon permitted the development of binding assays for the GRP receptor in detergent-solubilized crude membrane preparations. The solubilized GRP receptor exhibited saturable, high affinity (KD = 1.3 nM), temperature-dependent specific binding averaging 402 +/- 65 fmol/mg protein (mean +/- S.E. for eight separate membrane preparations with 125I-GRP concentration = 3 nM), with a Bmax = 434 fmol/mg protein using a gel filtration binding assay. That the GRP receptor had been solubilized was demonstrated by its failure to pellet when centrifuged at 100,000 x g for 60 min, its passage through a 0.22-micron filter without loss of binding activity, and its elution in the void volume of a Sephadex G-50 gel filtration column, but within the inclusion volume of a Sephacryl S-200 column (Ve/V0 = 1.1). Isolation of the GRP receptor from human H345 cell-solubilized membranes was achieved by ligand affinity chromatography. A unique 70-kDa band on silver-stained reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis was reproducibly eluted from GRP14-27 affinity columns by an acidic high salt buffer, but binding activity was denatured by these conditions. The protein nature of the GRP receptor was demonstrated by its sensitivity to proteases after isolation. In addition, two unique bands of 65 and 70 kDa were eluted from the GRP14-27 affinity column with GRP14-27 in neutral buffer, and this eluate possessed specific 125I-GRP binding with a stoichiometry of approximately 1:1. Thus, reported here is the isolation of a functional membrane-associated, saturable, high affinity GRP receptor with temperature-dependent binding from the solubilized membranes of human H345 cells.     

Identification of a receptor for peptides of the bombesin family in Swiss 3T3 cells by affinity cross-linking

The cross-linking agent ethylene glycol-bis(succinimidyl succinate) was used to covalently link 125I-labeled gastrin releasing peptide (125I-GRP) to an Mr 75,000-85,000 surface protein in Swiss 3T3 cells that displays many characteristics of a specific receptor for peptides of the bombesin family. This protein was not present in other cell lines which do not exhibit receptors for bombesin-like peptides. Unlabeled GRP competed for affinity labeling of the Mr 75,000-85,000 protein in a concentration-dependent manner, and other bombesin-related peptides also inhibited the cross-linking of 125I-GRP to this component. In contrast, high concentrations of a variety of other peptide hormones and mitogens had no effect. Affinity labeling of the Mr 75,000-85,000 protein was dependent on the concentration of 125I-GRP and exhibited saturability. 125I-GRP affinity labeling of this protein was also demonstrated by two-dimensional gel electrophoresis. These studies suggest that an Mr 75,000-85,000 surface protein with an isoelectric point of 6.0 to 6.5 is a major component of the receptor for peptides of the bombesin family in Swiss 3T3 cells.     

Characterization of the murine pancreatic receptor for gastrin releasing peptide and bombesin.

The murine pancreatic receptor for bombesin and gastrin releasing peptide (GRP) has been characterized. Analysis of the binding of 125I-GRP to membranes indicates a single class of sites (10(-13) mol/mg protein) with Kd of 43 pM. A 70 kDa membrane protein was cross-linked to 125I-GRP by bis(sulfosuccinimidyl) suberate; labeling was blocked by GRP, GRP (14-27), AcGRP(20-27), GRP(18-27), bombesin and ranatensin, was partially blocked by [Leu13 psi (CH2NH)Leu14]bombesin and was unaffected by GRP(21-27) and GRP(1-16). The IC50 values for the competitive displacement of 125I-GRP from intact membranes by these peptides were similar to those obtained by the cross-linking experiments showing that the 70 kDa protein is the GRP receptor. The GRP receptor is G-protein coupled; divalent cations are required for high-affinity binding and nonhydrolyzable GTP analogs decrease receptor affinity. In minced pancreas, GRP caused a dose-dependent increase in inositol phosphates implicating phospholipase C in signal transduction. We suggest that the murine pancreatic receptor for bombesin/GRP is a 70 kDa membrane protein, is associated with a G-protein and stimulates phosphatidylinositol turnover.     

Internalization and degradation of peptides of the bombesin family in Swiss 3T3 cells occurs without ligand-induced receptor down-regulation.

The binding of [125I]gastrin releasing peptide ([125I]GRP) to Swiss 3T3 cells at 37 degrees C increases rapidly, reaching a maximum after 30 min and decreasing afterwards. The decrease in cell-associated radioactivity at this temperature is accompanied by extensive degradation of the labelled peptide. At 4 degrees C equilibrium binding is achieved after 6 h and [125I]GRP degradation is markedly inhibited. Extraction of surface-bound ligand at low pH demonstrates that the iodinated peptide is internalized within minutes after addition to 3T3 cells at 37 degrees C. The rate of internalization is strikingly temperature-dependent and is virtually abolished at 4 degrees C. In addition, lysomotropic agents including chloroquine increase the cell-associated radioactivity in cells incubated with [125I]GRP. The binding of [125I]GRP to Swiss 3T3 cells was not affected by pretreatment for up to 24 h with either GRP or bombesin at mitogenic concentrations. Furthermore, pretreatment with GRP did not reduce the affinity labelling of a Mr 75,000-85,000 surface protein recently identified as a putative receptor for bombesin-like peptides. These results demonstrate that while peptides of the bombesin family are rapidly internalized and degraded by Swiss 3T3 cells, the cell surface receptors for these molecules are not down-regulated.     

Effects of bombesin on human small cell lung cancer cells: evidence for a subset of bombesin non-responsive cell lines

The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI-H69, NCI-H128, and NCI-H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro-gastrin releasing peptide (GRP), only the NCI-H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I-GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.     

Homologous and heterologous mitogenic desensitization of Swiss 3T3 cells to phorbol esters and vasopressin: role of receptor and postreceptor steps

Prolonged treatment of Swiss 3T3 cells with phorbol 12,13 dibutyrate (PDB) rendered the cells refractory to subsequent mitogenic stimulation by both PDB and vasopressin. In contrast, the cells retained full responsiveness to a wide variety of other mitogens. An early response to vasopressin and phorbol esters, inhibition of (125I)-labeled epidermal growth factor [(125I)-EGF] binding, was also substantially decreased in PDB pretreated cells. The cross desensitization was not produced by vasopressin; this ligand induced homologous but not heterologous desensitization. Exposure of Swiss 3T3 cells to PDB caused a down regulation of (3H)-PDB receptors but did not reduce the binding of vasopressin to refractory cells. The time-course (t1/2 = 7 h) and dependence on PDB concentration (half maximal at 20 nM) for this phorbol ester receptor loss paralleled the induction of the mitogenic desensitizations to both PDB and vasopressin. However, the time-course of recovery revealed an important dissociation between receptor presence and mitogenic response. When Swiss 3T3 cultures, which had been pretreated with PDB, were washed to remove this ligand and incubated in its absence for 24 h, both (3H)-PDB receptors and PDB or vasopressin inhibition of (125I)-EGF binding were almost completely restored to control levels. However the homologous and heterologous mitogenic desensitizations showed a very different reversal time. After a 24-h recovery period PDB-treated refractory cells were still unable to synthesize DNA in response to PDB or vasopressin. The mitogenic desensitizations were however completely reversible; after a 48-h incubation in the absence of PDB the cells responded fully to the mitogenic actions of PDB or vasopressin. This finding suggests that a further postreceptor step was also desensitized by prolonged PDB treatment. The presence of a low level of cycloheximide during the PDB pretreatment blocked induction of this postreceptor refractoriness. We propose that this refractory postreceptor step selectively blocks both PDB and vasopressin stimulation of DNA synthesis and may represent the point at which the mitogenic pathways of phorbol esters and vasopressin converge.     

Leu13-psi(CH2NH)Leu14]bombesin is a specific bombesin receptor antagonist in Swiss 3T3 cells

The pseudopeptide [Leu13-psi(CH2NH)Leu14]bombesin blocks bombesin-stimulated mitogenesis in Swiss 3T3 cells in a competitive and reversible manner, but not that of other mitogens. It inhibits the mobilization of intracellular Ca2+ and activation of protein kinase C by bombesin-like peptides. It acts at receptor level, as shown by inhibition of [125I]GRP binding and reduction in cross-linking of the Mr 75-85,000 receptor-associated protein. Thus [Leu13-psi(CH2NH)Leu14]bombesin is a specific bombesin receptor antagonist in Swiss 3T3 cells which blocks long-term growth promoting effects of bombesin-like peptides.     

Conveyance of partial agonism/antagonism to bombesin/gastrin-releasing peptide analogues on Swiss 3T3 cells by a carboxyl-terminal leucine insertion.

Gastrin-releasing peptide (GRP) is a neuroendocrine hormone that may be involved in the pathophysiology of small cell lung carcinoma. We describe carboxylterminal peptide analogues of GRP and bombesin, a 14-residue amphibian homologue, that were modeled after the antagonist [Leu13-psi(CH2NH)-Leu14]bombesin and retained the psi bond. Three novel peptides contained a Leu insertion amino to the psi bond, i.e. ... Leu13Leu14 psi X (residues numbered after bombesin) where X = LeuNH2 or norleucine-NH2). The Leu-insertion analogues behaved as pure partial agonists/antagonists when examined for the ability to stimulate [3H]thymidine incorporation into quiescent Swiss 3T3 cells (agonist activity) and to diminish the agonist response of GRP (antagonist activity). A time course of [3H]thymidine incorporation into quiescent cells indicated maximal incorporation at 20-h post-peptide addition for bombesin and GRP and a Leu-insertion peptide, but the extent of the incorporation for the Leu-insertion peptide was half that of GRP and bombesin. The agonist dose responses of the Leu-insertion peptides (EC50 values of 1-10 nM) paralleled GRP and bombesin, but the maximal response of the Leu-insertion peptides, even at concentrations as high as 10(-4) M, was half the maximal value of GRP or bombesin. High concentrations of the Leu-insertion peptides antagonized 10 nM GRP (a concentration that produced a near-maximal GRP response) yielding a response that was half the maximal value of GRP and equivalent to the maximal response of the Leu-insertion peptides alone. Analogues of the form ... Leu13 psi X behaved as complete antagonists. The KD values of the Leu-insertion peptides for competitive binding versus 125I-GRP (2-50 nM) were as potent as parent ... Leu14 agonists. Stability studies indicated that peptide potencies for both agonist and antagonist activities diminished upon peptide incubation in medium or on cells. The results suggested that, for the Leu-insertion peptides, degradation into distinct products with different activities was not responsible for their partial agonist/antagonist behavior. Computer-generated molecular modeling studies indicated that the novel structures could adopt energy minimized conformations for either an agonist or an antagonist as proposed earlier (Coy, D.H., Heinz-Erian, P., Jiang, N.-Y., Sasaki, Y., Taylor, J., Moreau, J.-P., Wolfrey, W.T., Gardner, J.D., and Jensen, R. T. (1988) J. Biol. Chem. 263, 5056-5060).     

Interconversion of GRP78/BiP. A novel event in the action of Pasteurella multocida toxin, bombesin, and platelet-derived growth factor.

Incubation of Swiss 3T3 cells with [2-3H]adenine, as in other cell types, reveals the ADP-ribosylation of GRP78 (the 78-kDa glucose-regulated protein, also known as BiP, the immunoglobulin heavy chain-binding protein), a resident endoplasmic reticulum protein that assists in the processing of proteins destined for secretion or cell surface expression. Here we show that Pasteurella multocida toxin, a potent growth factor for cultured fibroblasts, decreased the ADP-ribosylation of GRP78/BiP to 16 +/- 6% of the control value (n = 23). The action of the toxin occurred after a lag period, was blocked by lysosomotrophic agents, and potentiated by increased incubation time (ED50 4 ng/ml and 1 ng/ml in 4 and 8 h, respectively), thus indicating that the toxin enters the cells to act. Bombesin and platelet-derived growth factor (PDGF) similarly decreased the ADP-ribosylation of GRP78/BiP (ED50 0.5 nM and 2.5 ng/ml, respectively) but acted more rapidly than the toxin. Signaling pathways activated by the toxin, bombesin, and PDGF had effects on the ADP-ribosylation of GRP78/BiP. Thus, activation of protein kinase C alone by phorbol 12,13-dibutyrate was partially effective, and down-regulation of protein kinase C attenuated but did not block the action of the toxin, bombesin, and PDGF. Agents that mobilize Ca2+ from the endoplasmic reticulum (A23187, ionomycin, and thapsigargin) caused a decrease in the ADP-ribosylation of GRP78/BiP that was similar in magnitude to that achieved by the toxin, bombesin, and PDGF, implicating a role for inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization in the action of the mitogenic agents. The growth factor-induced decrease in the ADP-ribosylation of GRP78/BiP may represent its conversion from an inactive to an active state.     

Solubilization of the receptor for the neuropeptide gastrin-releasing peptide (bombesin) with functional ligand binding properties

The receptor for the neuropeptide gastrin-releasing peptide, the mammalian homologue of bombesin, was solubilized from rat brain and Swiss 3T3 cells by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) and the cholesteryl hemisuccinate ester (CHS). Only the combination of the detergent CHAPS and the cholesteryl ester CHS in a glycerol-containing buffer satisfactorily preserved the binding activity upon solubilization. Specific binding activity was only solubilized from cell lines and tissue preparations known to express the GRP receptor. The dissociation constant (Kd) for the receptor solubilized from rat brain and Swiss 3T3 cells was 0.6 nM, similar to the value of 0.8 nM calculated for the membrane-bound receptor. Binding was saturable and reached equilibrium after approximately 2 h at 4 degrees C. The identity of the solubilized receptor with the membrane-bound one was further confirmed by the concordance of the relative binding affinities of various established bombesin analogues.     

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.

     

A structure function study of C-terminal extensions of bombesin.

Synthetic C-terminal extensions of BN were synthesized and the biological potency evaluated using Swiss 3T3 and small cell lung cancer cells. BN, which has an amidated C-terminal, inhibited specific [125I-Tyr4]BN binding activity to Swiss 3T3 cells with an IC50 value of 1 nM, whereas the IC50 of BN-OH, which has a free C-terminal, was 1800 nM. The IC50 values of BNG, BNGK and BNGKK were 1400, 4700 and 500 nM, respectively. Similar binding data were obtained using SCLC cell line NCI-H345 and the bombesin analogues functioned as agonists based on the ability to elevate cytosolic Ca2+ in Fura-2 AM loaded SCLC cells. Also, the bombesin analogues stimulated 3H-thymidine uptake in Swiss 3T3 cells and the ED50 values for BN, BNG, BNGK and BNGKK were 1, 1300, 3900 and 400 nM. These data suggest that an amidated C-terminal is essential for high affinity binding and potency of BN.     

Characterization of bombesin receptors in a rat pituitary cell line

Bombesin is a tetradecapeptide which stimulates prolactin secretion in rats and man and in cultures of GH4C1 cells, a clonal strain of rat pituitary tumor cells. We have utilized [125I-Tyr4]bombesin to identify and characterize specific high affinity receptors in GH4C1 cells. Scatchard analysis of equilibrium binding data at 4 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (RT = 3600 +/- 500 sites/cell). The value for the equilibrium dissociation constant (Kd = 1.2 +/- 0.4 nM) agreed closely with the ED50 (0.5 nM) for bombesin stimulation of prolactin release. [125I-Tyr4]Bombesin binding at steady state at 37 degrees C was inhibited by increasing concentrations of unlabeled bombesin in a dose-dependent manner, with an ID50 = 1.4 +/- 0.2 nM. However, binding of [125I-Tyr4] bombesin was not inhibited by 100 nM thyrotropin-releasing hormone, vasoactive intestinal peptide, epidermal growth factor, or somatostatin. Therefore, [125I-Tyr4]bombesin binds to a receptor distinct from the receptors for other peptides which regulate hormone secretion by GH4C1 cells. The analog specificity for high affinity binding showed that the receptors for bombesin recognize the COOH-terminal octapeptide sequence in the molecule. Among five pituitary cell strains tested, two which contained saturable binding sites for [125I-Tyr4]bombesin (GH4C1 and GH3) had previously been shown to respond to bombesin with increased hormone secretion, whereas three which lacked receptors (GC, F4C1, and AtT20/D16v) were unresponsive. Therefore, the [125I-Tyr4]bombesin binding sites appear to be necessary for the biological actions of bombesin. Examination of the processing and metabolism of receptor-bound peptide demonstrated that at 4 degrees C [125I-Tyr4]bombesin binds to receptors on the surface of GH4C1 cells. At 37 degrees C, receptor-bound peptide is rapidly internalized and subsequently degraded in lysosomes. In summary, we have characterized for the first time specific, high affinity pituitary bombesin receptors which are necessary for the biological action of bombesin.     

The bombesin receptor is coupled to a guanine nucleotide-binding protein which is insensitive to pertussis and cholera toxins

The neuropeptide bombesin acts on a variety of target cells to stimulate the processes of secretion and cell proliferation. In this study we determined whether bombesin receptors interact with known guanine nucleotide-binding proteins in four different cell types: GH4C1 pituitary cells, HIT pancreatic islet cells, Swiss 3T3 fibroblasts, and rat brain tissue. Maximal concentrations of nonhydrolyzable GTP analogs decreased agonist binding to bombesin receptors in membranes from all four sources. In GH4C1 and HIT cell membranes GTP analogs inhibited bombesin receptor binding with IC50 values of about 0.1 microM, whereas GDP analogs were approximately 10-fold less potent. In contrast, GMP and the nonhydrolyzable ATP analog adenylyl-imidodiphosphate had no effect at 100 microM. Equilibrium binding experiments in GH4C1 and HIT cell membranes indicated a single class of binding sites with a dissociation constant (Kd) for [125I-Tyr4]bombesin of 24.4 +/- 7.0 pM and a binding capacity of 176 +/- 15 fmol/mg protein. Guanine nucleotides decreased the apparent affinity of the receptors without significantly changing receptor number. Consistent with this observation, guanine nucleotides also increased the rate of ligand dissociation. Pretreatment of GH4C1 or HIT cells with either pertussis toxin (100 ng/ml) or cholera toxin (500 ng/ml) for 18 h did not affect agonist binding to membrane bombesin receptors, its regulation by guanine nucleotides, or bombesin stimulation of hormone release. Although pertussis toxin pretreatment has been reported to block bombesin stimulation of DNA synthesis in Swiss 3T3 cells, it did not alter the binding properties of bombesin receptors in Swiss 3T3 membranes or inhibit the rapid increase in intracellular [Ca2+] produced by bombesin in these cells. In summary, our results indicate that the bombesin receptor interacts with a guanine nucleotide-binding protein which exhibits a different toxin sensitivity from those which regulate adenylate cyclase as well as those which couple some receptors to phospholipases.