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.     

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.     

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.     

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 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.     

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).     

Bombesin binding and biological effects on pancreatic acinar AR42J cells

The effects of bombesin on amylase release and the receptor binding of 125I-[Tyr4]bombesin in the rat pancreatic acinar carcinoma cell line AR42J were examined. Bombesin-like peptides stimulated amylase release from AR42J cells in a dose-dependent manner; a maximal 2-fold stimulation occurred at a bombesin concentration of 300 pM. Binding of 125I-[Tyr4]-bombesin to AR42J cells was specific, saturable and temperature dependent. The relative potencies with which various structurally related peptides stimulated amylase release correlated well with their relative abilities to compete for the bombesin receptor.     

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.     

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.     

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).     

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.     

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.     

Characterization of bombesin receptors on canine antral gastrin cells

Dispersed canine antral mucosal cells were prepared by sequential steps of collagenase digestion and EDTA treatment. Cell preparations enriched in gastrin cells were made by centrifugal elutriation followed by step density gradient centrifugation. Specific, saturable, and reversible binding of 125I-[Tyr4]-bombesin was found in all preparations. This saturable binding was time, temperature, and cell number dependent. In both velocity (elutriator) and density cell separation experiments, saturable binding of bombesin correlated with the distribution of cells containing gastrin- but not somatostatin-like immunoreactivity. Maximal specific binding to gastrin (G) cell-enriched fractions was reached in 45 min at 37 degrees C and constituted 90% of total binding. Addition of 100 nM nonradioactive bombesin to cells incubated with 50 pM 125I-[Tyr4]-bombesin for 45 min resulted in time-dependent dissociation of specifically bound tracer to about 40% of the maximal equilibrium binding. Analysis of saturable equilibrium binding yielded a best fit to a one-site model of high affinity binding sites with an apparent Kd of 85 +/- 14 pM and a Bmax of 231,000 +/- 71,000 receptors/gastrin cell. Nonradioactive [Tyr4]-bombesin and related analogs inhibited the specific binding of the tracer in a dose-related manner. The rank order of potency, determined at the IC50, of [Tyr4]-bombesin and related analogs for inhibition of specific binding was bombesin greater than [Tyr4]-bombesin = hGRP-27 greater than GRP-10 greater than ranatensin much greater than neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 microM did not inhibit bombesin binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

An analogue of substance P with broad receptor antagonist activity

[DPro4,DTrp7,9,10]Substance P-4-11 functions as a substance P receptor antagonist in several different systems. Because some analogues of substance P can function as receptor antagonists for bombesin as well as substance P, we tested [DPro4,DTrp7,9,10]substance P-4-11 for its ability to modify the interaction of various pancreatic secretagogues with their receptors in dispersed acini from guinea pig pancreas. [DPro4,DTrp7,9,19]Substance P-4-11 did not stimulate amylase secretion and did not alter the stimulation of amylase secretion caused by secretin, vasoactive intestinal peptide, calcitonin gene-related peptide or carbachol, but did inhibit the stimulation of amylase secretion caused by substance P, bombesin or cholecystokinin. With substance P, bombesin and cholecystokinin, [DPro4,DTrp7,9,10]substance P-4-11 caused a parallel rightward shift in the dose-response curve for stimulation of amylase secretion with no change in the maximal response. Schild plots of these results gave straight lines with slopes that were not significantly different from unity. [DPro4,DTrp7,9,10]Substance P-4-11 inhibited binding of 125I-labeled substance P, 125I-[Tyr4]bombesin and 125I-cholecystokinin octapeptide over the same range of concentrations as that in which it inhibited biologic activity of each of these peptides. Half-maximal inhibition of binding of 125I-substance P occurred with 4 microM, of 125I-[Tyr4]bombesin with 17 microM and of 125I-cholecystokinin octapeptide with 5 microM. With each radiolabeled peptide the value of Ki for inhibition of binding by [DPro4,DTrp7,9,10]substance P-4-11 was not significantly different from the corresponding value of Ki calculated from the appropriate Schild plot. The present results indicate that [DPro4,DTrp7,9,10]substance P-4-11 is a competitive antagonist at receptors for substance P, for bombesin and for cholecystokinin. Thus, these receptors must share a common peptide recognition mechanism even though they interact with agonists that have no obvious structural similarity.     

High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes.

The binding of the radiolabelled bombesin analogue [125I-Tyr4]bombesin to guinea-pig lung membranes was investigated. Binding of [125I-Tyr4]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (Bmax = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (KD = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [Tyr4]bombesin, neuromedin B and neuromedin C inhibited the binding of [125I-Tyr4]bombesin in an order of potencies as follows: [Tyr4]bombesin greater than bombesin greater than or equal to neuromedin C much greater than neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.     

Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.