Bombesin stimulates a high affinity GTPase activity in membranes of Swiss 3T3 fibroblasts

Peptides of the bombesin family are mitogenic for Swiss 3T3 fibroblasts and in these cells stimulate the turnover of polyphosphoinositides. Recent studies have suggested that G protein(s) may be involved in the signal transduction pathway triggered by bombesin. In this study we have found and characterized a high affinity GTPase activity stimulated by bombesin in membranes of Swiss 3T3 fibroblasts. Our results support the involvement of a G protein in the response of Swiss 3T3 cells to bombesin.     

Bombesin receptor from Swiss 3T3 cells. Affinity chromatography and reconstitution into phospholipid vesicles

Bombesin and its mammalian counterpart gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells in which distinct high affinity receptors have been identified. We developed here a probe for specific ligand affinity chromatography by coupling biotin to [lys3]bombesin. The resulting biotinylated [lys3]bombesin (BLB) retained biological activity as judged by inhibition of [125I]GRP binding to intact cells and membrane preparations and stimulation of rapid Ca2+ mobilization and DNA synthesis in intact cells. Using this ligand and magnetised beads coated with streptavidin, we extracted differentially a single protein from detergent-solubilized Swiss 3T3 membranes in a BLB-dependent manner. Visualization was achieved either after autoradiograph of metabolically labelled proteins with [35S]methionine or by silver staining of larger preparations. In other experiments, elution of BLB-receptor complexes bound to streptavidin beads was carried out at neutral pH and the eluted fraction was reconstituted into phospholipid vesicles. This procedure revealed [125I]GRP binding activity that exhibited saturability, specificity and a 1946-fold increase in specific activity.     

Solubilization of the bombesin receptor from Swiss 3T3 cell membranes. Functional association to a guanine nucleotide regulatory protein

Bombesin and structurally related peptides including gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells. Here we attempted to solubilize bombesin receptors under conditions in which the ligand (125I-labelled GRP) was prebound to the receptor prior to detergent extraction. We found that 125I-GRP-receptor complexes were solubilized from Swiss 3T3 cell membranes by using the detergents taurodeoxycholate or deoxycholate. These detergents promoted ligand-receptor solubilization in a dose-dependent manner. In contrast, a variety of other detergents including Triton X-100, octylglycoside, CHAPS, digitonin, cholic acid and n-dodecyl-beta-D-maltoside, were much less effective. Addition of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) to ligand-receptor complexes isolated by gel filtration enhanced the rate of ligand dissociation in a concentration-dependent and nucleotide-specific manner. Our results demonstrate for the first time the successful solubilization of 125I-GRP-receptor complexes from Swiss 3T3 cell membranes and provide evidence for the physical association between the ligand-receptor complex and a guanine nucleotide binding protein(s).     

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.     

Bombesin activates large-conductance chloride channels in Swiss 3T3 fibroblasts.

Using the patch-clamp technique (cell-attached patches), we found that bombesin, a Ca-mobilizing peptide mitogen, activates large-conductance Cl channels in Swiss 3T3 fibroblasts. The channel activation required a lag period of about 50 s and was equally observed whether bombesin was applied to the patch-pipette or to the bath. A23187 (10(-6)M) in the bath induced the similar currents with almost identical current-voltage relationship as bombesin: their slope conductances were 292 +/- 15 (bombesin) and 318 +/- 42 (A23187) pS. In inside-out patches, the induced channels were selective to Cl over gluconate (11:1). These observations strongly suggest that in Swiss 3T3 fibroblasts bombesin activates the Cl channels through a mechanism involving an increase in the intracellular free Ca concentration.     

Bombesin, diacylglycerols, and phorbol esters rapidly stimulate the phosphorylation of an Mr = 80,000 protein kinase C substrate in permeabilized 3T3 cells. Effect of guanine nucleotides

We have used digitonin permeabilization to study the mechanism of bombesin-induced activation of protein kinase C in Swiss 3T3 cells. Protein kinase C-mediated phosphorylations in permeabilized cells were identified using phorbol esters and diacylglycerols. Addition of phorbol 12,13-dibutyrate (PDBu) in the presence of [gamma-32P]ATP and digitonin caused a marked and rapid time- and dose-dependent increase in the phosphorylation of an Mr 80,000 cellular protein (maximum stimulation = 12.6 +/- 1.6-fold after 1 min, EC50 = 27 nM). 1-oleoyl-2-acetylglycerol substituted for PDBu in stimulating the phosphorylation of Mr 80,000 protein (EC50 = 13 microM). Bombesin also caused a striking increase in the phosphorylation of Mr 80,000 protein with a time course similar to that observed with PDBu. This phosphorylation was mimicked by mammalian bombesin-like peptides and blocked by the bombesin antagonists [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P and [Leu13 psi (CH2NH)Leu14]bombesin. Down-regulation of protein kinase C in intact cells by prolonged exposure to PDBu prevented Mr 80,000 protein phosphorylation upon subsequent bombesin addition in digitonin-permeabilized cells. Comigration on one- and two-dimensional gel electrophoresis and phosphopeptide mapping confirmed that the Mr 80,000 protein phosphorylated in permeabilized cells was indistinguishable from the Mr 80,000 protein which is the major protein kinase C substrate in intact cells. The GDP analogue guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) caused a 70% inhibition of the bombesin-induced phosphorylation of Mr 80,000 protein but had no effect on the phosphorylation induced by PDBu. Bombesin stimulated Mr 80,000 protein phosphorylation in permeabilized cells in a dose-dependent manner (EC50 = 4 nM), and GDP beta S shifted the bombesin dose response curve to higher bombesin concentrations (EC50 = 14 nM). These results demonstrate for the first time a growth factor receptor-mediated activation of protein kinase C in permeabilized cells and provide functional evidence for the involvement of a G protein in the transmembrane signaling pathway that mediates the stimulation of protein kinase C by bombesin in Swiss 3T3 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.     

Evidence for multiple, ras-like, guanine nucleotide-binding proteins in Swiss 3T3 plasma membranes. Stimulation of GTPase activity by cytosolic factors

A novel method for the analysis of putative G-proteins has been developed that reveals the existence of a large family of GTP/GDP-binding proteins with similar characteristics to those of p21ras in 3T3 cell plasma membranes. In the presence of Mg2+, exchange of [alpha-32P]GDP with prebound ligand was very slow, but, as with p21ras, exchange was dramatically accelerated by excess EDTA. In the presence of Mg2+, three classes of binding sites were distinguishable. However, no p21ras was detected in the membranes with the pan-reactive anti-ras antibody, Y13-259. Gel filtration analysis resolved two peaks of binding activity centering at 60 and 21 kilodaltons. High resolution anion exchange chromatography separated at least 11 unique GDP-binding proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-solubilized membranes, none of which cross-reacted with a pan-G alpha antiserum. Analogous to p21ras, the binding activities of 9 of the 11 species were sensitive to the thiol reagent N-ethylmaleimide, and six peaks possessed detectable GTPase activity in the absence of extrinsic factors. Addition of cytosol activated the GTPase activity of four of the peaks. We infer that the 11 peaks represent novel, small molecular weight guanine nucleotide-binding proteins, similar to those recently described in brain membranes.     

Bombesin induces c-fos and c-myc expression in quiescent Swiss 3T3 cells. Comparative study with other mitogens

We have studied the effect of the potent mitogen bombesin on the expression of c-fos and c-myc genes in quiescent mouse fibroblasts. We have demonstrated that bombesin rapidly induces a transient expression of c-fos mRNA followed by a more protracted elevation in c-myc mRNA levels. The intensity of the induction of expression of both proto-oncogenes depended on the dose of bombesin used. Prolonged treatment of the cells with TPA, which causes a selective decrease in protein kinase C activity, partially inhibited the induction of c-fos and c-myc gene expression by bombesin, similar to what has been observed with PDGF. However, a dramatic inhibition of the mitogenic response to bombesin--but not to PDGF--was found in TPA-treated cells. In contrast, TPA-treated cells showed an increased response to EGF with regard to proto-oncogene expression. The role of protein kinase C and Ca2+-dependent pathways in proto-oncogene induction by bombesin is discussed.     

A chemoattractant receptor on macrophages exists in two affinity states regulated by guanine nucleotides

The binding characteristics of the oligopeptide chemoattractant receptor on guinea pig macrophages and macrophage membrane preparations were characterized using detailed binding studies and computer analysis. Viable macrophages bound the radiolabeled chemoattractant N-formyl-methionyl-leucyl-[3H]phenylalanine with single dissociation constant (KD) of 18.4 +/- 4.6 nM with 15,300 +/- 1,800 sites per cell. Binding data from membrane preparations indicated the presence of two classes of binding sites with KD of 1.5 +/- 0.4 nM and 25.5 +/- 11.0 nM. Approximately 23% of the receptors were in the high affinity state. In the presence of added guanine nucleotide di- or triphosphates, the high affinity receptors in the membrane preparations were converted to low affinity states with no change in the total receptor number. Nonhydrolyzable derivatives of GTP were most potent in converting the receptor from its high to low affinity state. These data suggest that the affinity state of the oligopeptide chemoattractant receptor in macrophages is regulated by guanine nucleotides and GTPase, implying that the transduction mechanisms of this receptor may be controlled by a guanine nucleotide regulatory unit.     

Bombesin stimulates the rapid activation of phospholipase A2-catalyzed phosphatidylcholine hydrolysis in Swiss 3T3 cells.

In Swiss 3T3 fibroblasts bombesin stimulated the release of arachidonic acid in a time- and dose-dependent manner. Arachidonate levels were significantly elevated after only a 2-s stimulation with the agonist. Furthermore, by measuring the arachidonate content of cellular phospholipids after cell activation, it was shown that there was selective depletion from phosphatidylcholine over the same time course. The corresponding production of lysophosphatidylcholine suggested the involvement of a phosphatidylcholine-specific phospholipase A2. Initial arachidonic acid release was not dependent on the presence of extracellular calcium, not activated by treatment of the cells with thapsigargin, and was unaffected by down-regulation of protein kinase C activity, or by treatment of the cells with the protein kinase C inhibitor staurosporine. These data strongly suggest that occupation of the bombesin receptor is closely coupled to activation of phospholipase A2 which results in the rapid release of arachidonic acid from phosphatidylcholine.     

Guanine nucleotides stimulate hydrolysis of phosphatidylinositol and polyphosphoinositides in permeabilized Swiss 3T3 cells

Hydrolysis-resistant analogues of GTP specifically stimulate the formation of [3H]inositol mono-, bis- and trisphosphates by saponin-permeabilized Swiss 3T3 cells prelabelled with [3H]inositol. Each inositol phosphate is formed largely by hydrolysis of its parent lipid and not by dephosphorylation of inositol 1,4,5-trisphosphate [(1,4,5)IP3]. Although hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is most sensitive to guanine nucleotides, hydrolysis of phosphatidyl-inositol (PI) and phosphatidylinositol 4-phosphate (PIP) is quantitatively more important. These results suggest that a guanine nucleotide-dependent regulatory protein(s) (G-protein) is involved in regulating the hydrolysis of PI and PIP, as well as PIP2, and so may allow formation of diacylglycerol (DG) without simultaneous production of (1,4,5)IP3 and mobilization of intracellular Ca2+.     

Bombesin induction of c-fos and c-myc proto-oncogenes in Swiss 3T3 cells: significance for the mitogenic response

Bombesin is a potent mitogen for Swiss 3T3 cells and acts synergistically with insulin and other growth factors. We show here that addition of bombesin to quiescent Swiss 3T3 cells causes a striking increase in the levels of c-fos and c-myc mRNAs. Enhanced expression of c-fos (122 +/- 14-fold) occurred within minutes of peptide addition followed by increased expression of c-myc (82 +/- 16-fold). The concentrations of peptide required for half-maximal increase in the levels of c-fos and c-myc mRNAs were 1.0 and 0.9 nM, respectively. The peptide [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P which inhibits the binding of bombesin to its receptor and bombesin-stimulated DNA synthesis in Swiss 3T3 cells blocked the increase in c-fos and c-myc mRNA levels promoted by bombesin. Down-regulation of protein kinase C by long-term exposure to phorbol esters prevented c-fos and c-myc induction by bombesin. This and other results indicate that the induction of these proto-oncogenes by bombesin could be mediated by the coordinated effects of protein kinase C activation and Ca2+ mobilization. The marked synergistic effect between bombesin and insulin was used to assess whether the increase in the induction of c-fos and c-myc is an obligatory event in cell activation. In the presence of insulin, bombesin stimulated DNA synthesis at subnanomolar concentrations but had only a small effect on c-fos and c-myc mRNA levels. This apparent dissociation of mitogenesis from proto-oncogene induction was even more dramatic in 3T3 cells with down-regulated protein kinase C. In these cells bombesin stimulated DNA synthesis in the presence of insulin but failed to enhance c-fos and c-myc mRNA levels at comparable concentrations. Thus, the induction of c-fos and c-myc may be a necessary step in the mitogenic response initiated by ligands that act through activation of protein kinase C but the expression of these proto-oncogenes may not be an obligatory event in the stimulation of mitogenesis in 3T3 cells by mitogens that utilise other signalling pathways.     

Bombesin and platelet-derived growth factor stimulate formation of inositol phosphates and Ca2+ mobilization in Swiss 3T3 cells by different mechanisms.

Highly purified platelet-derived growth factor (PDGF) or recombinant PDGF stimulate DNA synthesis in quiescent Swiss 3T3 cells. The dose-response curves for the natural and recombinant factors were similar, with half-maximal responses at 2-3 ng/ml and maximal responses at approx. 10 ng/ml. Over this dose range, both natural and recombinant PDGF stimulated a pronounced accumulation of [3H]inositol phosphates in cells labelled for 72 h with [3H]inositol. In addition, mitogenic concentrations of PDGF stimulated the release of 45Ca2+ from cells prelabelled with the radioisotope. However, in comparison with the response to the peptide mitogens bombesin and vasopressin, a pronounced lag was evident in both the generation of inositol phosphates and the stimulation of 45Ca2+ efflux in response to PDGF. Furthermore, although the bombesin-stimulated efflux of 45Ca2+ was independent of extracellular Ca2+, the PDGF-stimulated efflux was markedly inhibited by chelation of external Ca2+ by using EGTA. Neither the stimulation of formation of inositol phosphates nor the stimulation of 45Ca2+ efflux in response to PDGF were affected by tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast, TPA inhibited phosphoinositide hydrolysis and 45Ca2+ efflux stimulated by either bombesin or vasopressin. Furthermore, whereas formation of inositol phosphates in response to both vasopressin and bombesin was increased in cells in which protein kinase C had been down-modulated by prolonged exposure to phorbol esters, the response to PDGF was decreased in these cells. These results suggest that, in Swiss 3T3 cells, PDGF receptors are coupled to phosphoinositidase activation by a mechanism that does not exhibit protein kinase C-mediated negative-feedback control and which appears to be fundamentally different from the coupling mechanism utilized by the receptors for bombesin and vasopressin.     

Lack of negative regulation of beta-adrenoreceptor affinity by guanine nucleotides in embryonic membranes and its induction by the GTP-binding protein from mature protein

The negative regulation of the beta-adrenoreceptor affinity by guanine nucleotides in the sarcolemmal fraction of chicken skeletal muscle at different stages of ontogenesis was studied. It was found that the negative regulation is absent in the embryonic period; the effect of GTP is manifested only before hatching, whereas that of Gpp(NH)p--at later periods, i.e., in 1-month-old chickens. Similar age-dependent dynamics was revealed with respect to the GTP effect on the dissociation rate of the [3H]DHA-beta-adrenoreceptor complex. An addition to the system containing embryonic muscle membranes of the GTP-binding protein isolated from skeletal muscle and liver of chickens whose age exceeds 20 days led to earlier manifestations of the above effects (on the 13th-15th embryonic days). The data obtained testify to the limiting role of GTP-binding proteins in the negative control of the hormone-receptor interaction and support the authors' hypothesis on the absence in the embryonic muscle of the 42 kD GTP-binding protein responsible for the functional coupling of the hormone-sensitive adenylate cyclase components.     

Bombesin and platelet-derived growth factor induce association of endogenous focal adhesion kinase with Src in intact Swiss 3T3 cells.

Stimulation of quiescent Swiss 3T3 cells with bombesin induces a rapid increase in the formation of complexes between focal adhesion kinase (FAK) and Src family members, which can be extracted with a buffer containing Triton, deoxycholate, and SDS but not with a buffer containing Triton alone. An increase in complex formation between FAK and Src in response to bombesin could be detected within 1 min, reached a maximum after 10 min, and declined toward base-line levels after 60 min of bombesin treatment. Bradykinin, endothelin, and lysophosphatidic acid also stimulated FAK-Src complex formation. Bombesin stimulated FAK/Src association through a Ca(2+) and phosphatidylinositol 3'-kinase-independent pathway that requires the integrity of the actin filament network and is partly dependent on functional protein kinase C. Treatment with the selective Src kinase inhibitor PP-2 inhibited both FAK activation and phosphorylation of FAK at Tyr(577) induced by bombesin in intact cells. Platelet-derived growth factor at low concentrations (1-10 ng/ml) also induced FAK-Src complex formation via a pathway that depended on the integrity of the actin cytoskeleton and phosphatidylinositol 3'-kinase. Thus, G protein-coupled receptor agonists and platelet-derived growth factor promote complex formation between endogenous FAK and Src in attached cells through different signal transduction pathways.     

Binding of radiolabeled antagonist to platelet-activating-factor receptor in human lung membranes: shifting of agonist binding affinity states by cations and guanine nucleotide

Binding of the radiolabeled platelet-activating-factor (PAF) receptor antagonist RP52770, [( 3H]-N-(3-chlorophenyl)-3-(3-pyridinyl)-1H, 3H-pyrrolo- [1,2-c]thiazole-7-carboxamide) to receptors in human lung membranes was time- dependent, protein-dependent, reversible and saturable. The dissociation constant and maximal binding density were 14 +/- 2 nM and 2.1 +/- 0.6 pmol/mg protein, respectively. [3H]-RP52770 binding to the PAF receptor was competitively displaced by PAF and receptor antagonists. The rank order of the binding affinities were PAF greater than RP52770 (+) greater than RP52770 (-) greater than CV3988, equivalent to the PAF receptor specificities determined from functional studies. Binding of PAF to [3H]-RP52770 labeled receptors was regulated by sodium, guanylylimido- diphosphate (GppNHp) and divalent cations. In the presence of EDTA, Na+ and GppNHp, in combination, binding of PAF to the receptor was maximally shifted to the right. These results clearly demonstrate that cations and guanine nucleotide can regulate the affinity states of the PAF receptor in human lung membranes.     

Bombesin, vasopressin, and endothelin stimulation of tyrosine phosphorylation in Swiss 3T3 cells. Identification of a novel tyrosine kinase as a major substrate.

Neuropeptide-stimulated tyrosine phosphorylation of specific components in Swiss 3T3 cells was investigated using monoclonal antibodies directed against the src transformation-associated substrates p125 focal adhesion kinase (FAK), a novel type of cytosolic tyrosine kinase, and p130. Treatment of Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, and endothelin caused a striking increase in the tyrosine phosphorylation of p125FAK, as judged either by anti-phosphotyrosine (anti-Tyr(P)) Western blots of anti-p125FAK immunoprecipitates, or by anti-p125FAK immunoblots of anti-Tyr(P) immunoprecipitates. Bombesin-stimulated tyrosine phosphorylation of p125FAK was detectable within seconds and concentration-dependent (half-maximum effect of 0.3 nM). Neuropeptides also stimulated the tyrosine phosphorylation of a second component of M(r) 130,000, previously identified as the major p130 phosphotyrosyl protein in src-transformed cells. Bombesin stimulated p130 tyrosine phosphorylation with kinetics and concentration dependence similar to those observed for p125FAK. This is the first report to identify substrates for neuropeptide-stimulated tyrosine phosphorylation; the finding that one of these substrates is a tyrosine kinase suggests the existence of a novel signal transduction pathway in the action of mitogenic neuropeptides.