Novel soluble, high-affinity gastrin-releasing peptide binding proteins in Swiss 3T3 fibroblasts

Peptides,Vol. 15, No. 6, pp. 993-1001, 1994 Copyright© 1994 ElsevierScienceLtd Printed in the USA.All fightsl~,~,erved 0196-9781/94 $6.00 + .00

Pergamon O196-9781(94)EOO76-X

Novel Soluble, High-At nity Gastrin-Releasing Peptide Binding Proteins in Swiss 3T3 Fibroblasts M A D E L E I N E A. K A N E , l L Y N D A B. P O R T A N O V A , K I M B E R L Y K E L L E Y , M A R K H O L L E Y , S H E R M A N E. R O S S , D O R O T H Y B O O S E , A N T O N I O E S C O B E D O - M O R S E AND BALTAZAR ALVARADO

Medical Oncology Section, Denver Veterans Affairs Medical Center, University of Colorado Health Sciences Center and University of Colorado Cancer Center, Denver, CO 80220

R e c e i v e d 25 F e b r u a r y 1994

KANE, M. A., L. B. PORTANOVA, K. KELLEY, M. HOLLEY, S. E. ROSS, D. BOOSE, A. ESCOBEDO-MORSE AND B. ALVARADO. Novelsoluble, high-aj~nitygastrin-releasingpeptide bindingproteins in Swiss 3T3fibroblasts. PEPTIDES 15(6) 993-1001, 1994.--Swiss 3T3 cells contained substantial amounts of soluble and specific [~25I]GRPbinders. Like the membraneassociated GRP receptor, they were of high affinity, saturable, bound to GRP(14-27) affinity gels, and exhibited specificity for GRP(14-27) binding. They differed in that acid or freezing destroyed specific binding, specific binding exhibited different time and temperature effects, no detergent was required for their solubilization, ammonium sulfate fraetionation yielded different profiles, the M~s were lower, GRP(1-16) also blocked binding, and a polyclonal anti-GRP receptor antiserum did not bind on Western blots. The isolated, soluble GRP binding protein(s) rapidly degraded [~zSI]GRP.These soluble GRP binding proteins may play a role in the regulation of the mitogenic effects of GRP on these cells. Gastrin-releasing peptide

Binding protein

Receptor

Swiss 3T3 fibroblast

MURINE Swiss 3T3 cells have provided an excellent model for studies of the properties of gastrin-releasing peptide (GRP) interactions with its receptor. G R P binding stimulated proliferation, a process associated with protein kinase C activation, phosphoinositide turnover, intracellular calcium release, and oncogene activation (10,38,45,55). G R P receptors have been ligand cross-linked, solubilized, isolated, and cloned from Swiss 3T3 cells and have been confirmed to be members of the seven membrane-spanning superfamily of receptors (7,18,20,27, 30,40,49,54). Specific high-affinity, membrane-associated G R P

receptors have been identified and characterized in a number of other tissues and cells (6,11,12,14,21-23,25-29,36,41,4648,50,52,53,55). G R P receptor and neuromedin B receptor cDNAs have been cloned from rodent and human sources (4,49). The present studies identify soluble, high-affinity G R P binding proteins in Swiss 3T3 cells, and compare their properties with membrane-associated GRP receptors. Although the soluble G R P binding proteins are similar to GRP receptors in that they are saturable, of high affinity, and specific for GRP, their properties differ in a number ofother ways. This indicates that these

Requests for reprints should be addressed to Madeleine A. Kane, M.D., Ph.D., Denver VA Medical Center, 11 IF, 1055 Clermont St., Denver, CO 80220.

993

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KANE ET AL. tion, with the latter solubilized in detergent (M) as previously described (14). 1500

Ammonium Sulfate Fractionation

ooo1 o. CYTOSOL

MEMBRANES

I 0--25 • 25--5(} ~ 50-80 • B0 ~ SN AMMONIUM SULFATE FRACTIONATfON

FIG. 1. (A) The effect of passageof Swiss 3T3 cell subcellular fractions through a 0.22-~t filter on [12~I]GRPspecific binding. Solubilized membranes (M) and soluble cytosolic fractions (C) were prepared and analyzed for binding as described in the Method section. The white bars show values (mean _+SD from four-nine experiments) obtained before filtration, and the black bars after filtration. The percentage of the total binding that was nonspecific binding was 30% in the C fraction and 15% in the M fraction. Specific binding was defined as the difference between total binding and nonspecific binding as defined in the Method section. *Denotes difference from unfiltered, p < 0.01. (B) The effect of ammonium sulfate fractionation on [mI]GRP specific binding to cytosolic fraction (white bars) and solubilized (black bars). Samples were prepared and binding determined as described in the Method section. Each value is the mean + SEM from three different experiments.

are novel, previously undescribed G R P binding proteins. They possess enzymatic activity and may play a role in regulation of the effects of GRP.

METHOD

Cells Swiss 3T3 murine fibrobtasts (ATCC No. CCL 92) were obtained from American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's modified essential medium (low glucose) supplemented with 10% fetal calf serum but no antibiotics in a humidified 5% CO2 atmosphere at 37°C.

(NH4)2SO4 fractionation was performed on C and M fractions by the addition of the appropriate amount of the dry chemical, dissolution, and precipitation of the protein pellet at 20°C for 90 min, followed by centrifugation at 30,000 × g for 30 min. Pellets were dissolved in 1-3 ml PBS [phosphate-buffered saline (potassium phosphate, 0.01 M, pH 7.4, with NaCI, 0.15 M); containing 0.1% Triton X-100 and protease inhibitors as above and dialyzed against this buffer to remove (NH4)2SO4. To the 30,000 × g supernatant was added additional (NH4)2SO4 to give the desired final concentration, and so on. The fractions were designated with the starting and ending concentration of (NH4)2SO4 used to produce the precipitate.

[125I]GRP Binding to M and C Fractions ['25I]GRP specific binding was determined as previously described (14,16). Briefly, an aliquot of sample containing 10-20 #g protein was incubated with [125I]GRP with or without 1000fold excess GRP(14-27) for l0 rain at 37°C. Bound radioactivity was separated from free by gel filtration. Specific binding was calculated by subtracting nonspecific binding from total binding, and results were expressed as fmol/mg protein. To test the stability of ['2~I]GRP specific binding, binding was measured before and after samples were frozen at -20°C, before and after passage five times through a 0.22-# filter, and before and after exposure to ligand affinity gel elution conditions. The presence of o-phenanthroline (1 raM) and phenylmethylsulfonyl fluoride (PMSF) (0.1 raM) in the binding buffer inhibited degradation of [125I]GRP.

Isolation of GRP Receptors From Affinity Gels Ligand affinity gels (AG) and control AG were prepared using Affi-Gel 10 (Bio-Rad) and GRP(14-27), and samples prepared

A

B

400. Z

~3oo.

Chemicals Bombesin, GRP, GRP(14-27), acetyl-GRP(20--27), GRP( 116), and substance P were obtained from Peninsula Laboratories (Belmont, CA) or from Baehem (Torrance, CA). [t~5I]GRP labeled on Tyr '5 was purchased from Amersham (Arlington Heights, IL). The specific activity was 2000 Ci/mmol. RPMI 1640 medium and potassium phosphate were obtained from Fisher Scientific. Affi-Gel 10 was obtained from Bio-Rad (Richmond, CA). Centricon 30 units were obtained from Amicon (Danvers, MA). Other materials and chemicals were obtained from either Fisher Scientific or Sigma.

Preparation of Subcellular Fractions Swiss 3T3 cells were lysed by sonication or by Dounee homogenization, and soluble cytosolie supematant fraction (C) and particulate membrane pellet were obtained by ultracentrifuga-

CL~ [ ~200. ~--E

~...~~ 100. Q•

0 0

-A

2'o

io

INCUBATION TIME (rain)

~o

3'0

i0

~o

120

INCUBATION TIME (rain)

FIG. 2. The effect of time and temperature on [125I]GRPspecific binding to Swiss 3T3 solubilized membranes (A) and cyto,olic fraction (B). Sampies were incubated with [t2SI]GRP (3 riM) with or without 1000-fold excess GRP(14-27) for various times and temperatures as shown, and specific binding was calculated as described in the Method section. The temperatures were l °C (triangles), 20°C (squares), and 370C (circles). Values shown are the means of duplicate determinations that agreed within 10%.

A

S

M

B1

G1

B2 G2

B3

G3

1169766-

<

45-

im

D

4

C

S

B1

G1 B2 G2

B3 G3

116 97 66

45-

FIG. 3. Reduced SDS-PAGE analysis of affnity-purified GRP binding proteins. (A) Serial elution fractions from GRP (G ! G3) and blank (B1-B3) affinity gel columns incubated with Swiss 3T3 M fraction prepared and analyzed as described in the Method section. Lane M is the crude solubilized membranes before passage over the affinity columns. (B) Serial elution fractions from GRP AG (GI-G3) or blank AG (B1-B3) incubated with Swiss 3T3 C fraction prepared and analyzed as described in the Method section, Lane C is the cytosolie fraction before application to the columns. Molecular weight markers are in the lanes labeled S, with molecular weights in kDa indicated to the left. The arrows at fight indicate the positions of the unique bands isolated from the M and C fractions, 995

996

KANE ET AL.

1 O0 -

A

m

m

m

q

0 75-

100-

lulose were blocked, incubated with rabbit antiserum to GRP receptor isolated from human small cell lung carcinoma NCIH345 cells (14) or preimmune control serum, 1:1000 in blocking buffer, washed, and specific binding visualized using goat antirabbit antiserum conjugated to horse radish peroxidase and diaminobenzidine.

B

75

Z 0

~z 5o

50

i,i ZO O:2 ~_ 2 5

25 ¸

0

GRP

GRp

NM8 SUBP

VP

BK

NONCE

1 4 - 2 7 1--1B

PREINCUBATION

0 GRP

GRp

14-27 1-16

PEPTIDE

111

NMB SOap

PREINCUBATION

VP

BK

NONE

RESULTS AND DISCUSSION

Similarities in Specific [1251]GRPBinding to C and M

PEPTIDE

FIG. 4. The effects of preincubation with various peptides (100 ~tM) on the GRP AG eluates from M (A) or C (B) subcellular fractions from Swiss 3T3 cells. Eluates were analyzed by SDS-PAGE and the major bands quantified by densitomelry and expressed as a percent of no peptide control. For M (in A), the band was M, = 70 kDa; for C (in B), two bands, M, = 70 kDa (white bars) and Mr = 65 kDa (black bars) were analyzed. Non-GRP peptide abbreviations are: NMB, neuromedin B; SUBP, substance P; VP, vasopressin; BK, bradykinin.

as above were incubated with control AG and GRP(14-27) AG as previously described (14,16) except that buffers used for the C fraction did not contain detergent. Elution was accomplished with PBS containing 1 M NaC1 for experiments that screened for enzymatic activity. In some experiments, samples were incubated with 100 u M GRP(14-27), GRP( 1-16), neuromedin B, bradykinin, vasopressin, or substance P at 37°C for 10 min prior to their application to replicate G R P affinity columns.

SDS-PAGE SDS-PAGE was performed using 7.5% gels with reducing buffer according to the Laemmli method (19) using a Bio-Rad Miniprotean apparatus, and gels were silver stained (24). The relative amount of protein in the bands was estimated by densitometric analysis using a UVP image document system.

Evaluation of Proteolytic Activity of Soluble GRP Binding Proteins G R P binding proteins isolated from the C fraction of Swiss 3T3 cells by affinity chromatography by elution with potassium phosphate (0.01 M, pH 7.5), containing l M NaC1, were incubated with [~25I]GRP for 30 rain at 37°C before or after the addition of 17.8 M acetic acid. The reaction mixture was clarified by centrifugation at 10,000 × g, and an aliquot (20 t~l) was analyzed by HPLC (14).

Immunoblot Analysis of GRP AG Eluates of C and M Fractions GRP AG eluates from M and C fractions prepared as above were analyzed by reduced SDS-PAGE and electrotransferred to nitrocellulose using Bio-Rad Transblot (44). Strips of nitrocel-

As shown in Fig. 1, specific ['25I]GRP binding to the C fraction was similar to that in the M fraction containing GRP receptor. The presence of significant amounts of specific [~25I]GRP binding to C was unexpected. That this was binding to soluble proteins, and not membrane miceUes, was demonstrated by: l) the specific binding activity was in the 100,000 × g supernatant; 2) no detergent was required for its solubilization; 3) specific binding was not reduced by five consecutive passages through a 0.22-~ filter, although the total protein was reduced by about 10% for both C and M; 4) the same specific binding activity was observed in the C fraction whether the cells were lysed by sonication or by Dounce homogenization. No putative G R P receptor was observed in the cytosolic fraction (100,000 × g supernatants) in the Swiss 3T3 affinity cross-linking studies (54). However, the cross-linking was carried out with intact cells for a relatively brief time ( 15 min) at a temperature (15 °C) at which receptor internalization would be slowed. [~25I]GRP specific binding to C, as well as M, was saturable and of high affinity (data not shown). The saturation curve data were analyzed by GraphPad InPlot Software (San Diego, CA). For the M fraction, the Ka was 0.86 nM, and the B~.,~ was 246 fmol/mg protein. For the C fraction, the Ka was 2.8 nM, with a B~x of 372 fmol/mg protein, both somewhat higher than for M. The B~x for M fraction was similar to that reported previously for the purified G R P receptor (7), but the differences in affinity constants may be due to differences in buffer, detergents, and assay procedures used.

Biochemical Differences in Specific [1251]GRPSpecific Binding to C and M Fractionation of M and C fractions by (NI-I4)2504 precipitation resulted in enrichment of the specific [12~I]GRP specific binding in the 50-80% fraction for M [Fig. l(B)] and in the 0 25% fraction for C fraction. Because the cloned G R P receptor appears to be of the seven membrane-spanning region type, the presence of a structurally related soluble form would necessitate a mechanism by which the very hydrophobic character of the membrane-associated form would be modified by, for example, phosphorylation, glycosylation, or sulfation to increase the aqueous solubility. The effects of ammonium sulfate support an unrelated G R P binding protein in C. In both Swiss 3T3 M and C fractions, [~25I]GRP specific binding was markedly temperature dependent. As shown in Fig. 2(A), at 37°C maximum specific binding to M occurred after l0 min and remained stable for at least 60 rain. Only a very small amount of specific binding was observed at 1°C for 60 rain, as seen with human small cell lung carcinoma NCI-H345 extracts and cells 04). Specific binding at 20oc was intermediate.

SOLUBLE GRP BINDING PROTEINS IN SWISS 3T3 CELLS

997

A GRP14_27 S

100

10

1

0.10

0

GRP1.16 100

10

100

10

S

1

0.10

0

S

0

0

S

NMB 1

0.10

B GRP14_27 100

10

1

0.10

GRP1_16 100

10

S

1

0.10

0

NMB 100

10

1

0.10

S

0

FIG. 5. The effect of pretreatment concentration of GRP(I 4-27), GRP(1-16), or neuromedin B (NMB) on the intensity of bands eluted from GRP AG after incubation with M (A) or C (B) fractions. The peptide concentrations in the preincubation are shown in uM. S denotes the 66 kDa molecular weight standard.

In the C fraction [Fig. 2(B)], specific binding of [~25I]GRP at 37°C reached a maximum at 30 min and then fell to no detectable specific binding by 60 rain. The amount of specific [~25I]GRP binding was unchanged after incubation from 10 to 120 min at 20°C, and little or no specific binding was observed at 1°C. Because the soluble lysosomal enzymes are present in C fraction, some degradation by this fraction is not unexpected,

as phenanthroline and PMSF would not inhibit all these enzymes. The soluble GRP binding proteins may be more susceptible to proteolytic degradation than the membrane-associated form at physiological temperatures. Both freezing and acid treatment completely destroyed [I~SI]GRP specific binding to C fraction. Freezing reduced binding by M fraction by 50%, and acid treatment altered binding

998

KANE ET AL.

Degradation of[1251]GRPby Is'olated Soluble GRP Binding Proteins

O 2000.0

j

z 0 o<

As shown in Fig. 6, GRP binding proteins isolated from Swiss 3T3 fibroblast C fraction completely degraded [~25I]GRP after incubation for 30 min at 37°C. Pretreatment of the sample with acetic acid prevented the degradation.

~ 1000.0-

Western Blots of GRP Binding Proteins 0.0 I 0

~

~ 2'0

4'0

6'0

---~'-~T 80

RETENTION TIME (rnin)

FIG. 6. Degradation of [~25I]GRPby GRP binding protein(s) isolated from C fraction. Protease inhibitors (o-phenanthroline and PMSF) were omitted from the elution buffer (l M NaC1 in 0.01 M potassium phosphate, pH 7.5). An aliquot of eluate was incubated with [~2SI]GRPfor 30 min at 37°C before (closed circles) or after (open circles) the addition of glacial acetic acid to a final concentration of 2 M. Samples were analyzed by HPLC, and serial 2-rain fractions were collected and counted. Unexposed [125I]GRPis shown by the open triangles.

very little. Both freezing and acid treatment had a greater effect on NCI-H345 cell GRP receptors (14).

GRP Affinity Gel (GRP AG) Chromatography of C and M Fractions As shown in Fig. 3(A), when they were analyzed by reduced SDS-PAGE, serial elution fractions (G I--G3) from the GRP AG incubated with M were enriched for a unique 70 kDa band compared with serial elution fractions (BI-B3) from control AG. This M, was similar to that reported for GRP receptor isolated from Swiss 3T3 cells (7) and NCI-H345 cells (14). In addition, the same unique band plus two or three bands of slightly smaller molecular weight on reduced SDS-PAGE (60-65 kDa) were observed in the eiuates of GRP AG incubated with the cytosolic fraction [Fig. 3(B)]. To demonstrate the specificity of the GRP binding of the proteins eluted from the GRP AGs, aliquots of C and M fractions were incubated with 100 ~ GRP(14-27), GRP(I-16), neuromedin B, bradykinin, vasopressin, or substance P prior to incubation with and elution from the GRP AG. Pretreatment with GRP(14-27) prevented binding of proteins from both M and C fractions (Fig. 4). GRP(1-16) also reduced binding by the C fraction [Fig. 4(B)]. Inhibition of binding by GRP(14-27), GRP(I-16), and neuromedin B was concentration dependent, as shown in Fig. 5. For the M, = 70 kDa protein from the M fraction, 50% inhibition occurred with approximately 0.5 GRP(14-27), 5 ~MGRP(I-16), and 10 t~M neuromedin B; results were similar for the C fraction. However, for the M, = 65 kDa protein from the C fraction, 50% inhibition required higher concentrations: approximately 10 w~/ GRP(14-27), 50 tzM GRP(1-16), and > 100 w~/neuromedin B.

As shown in Fig. 7, rabbit polyclonal antibodies to GRP receptor isolated from human small cell lung carcinoma NCIH345 cells (14) bound to protein isolated from M, but not C, fraction from Swiss 3T3 cells. This observation again supports the presence of novel GRP binding protein(s) in C. The true soluble nature of the novel GRP binding protein(s) from C fraction, its properties that differ from the membraneassociated receptor, and its enzymatic activity suggest a separate role for intracellular modification of GRP. The identity and function ofa GRP-specific protease in Swiss 3T3 cells, fibroblasts that also express GRP receptor, are not clear. Cytosolic receptors for steroids, retinoids, and thyroid hormone have been well characterized. Soluble forms of many peptide receptors have been found in the circulation ( 1-3,17,31,3335,37,43), but reports of intracellular soluble peptide receptor forms or peptide binding proteins are more limited. Intracellular forms of the prolactin receptor in ovary (5) and the thyrotropin (TSH) receptor in thyroid (51) have been observed. Proteolytic activity has not been described for these soluble receptor forms. Protein disulfide isomerase (PDI) resides in the lumen of the endoplasmic reticulum (32). Although it binds a variety of peptide motifs, the specificity of the soluble GRP binding protein described here makes it unlikely that it is PDI. Members of the heat shock protein (hsp) or stress protein family bind to various peptides and proteins and act as molecular chaperones, facilitating proper folding of proteins, oligomerization, and removal of aggregated or incorrectly folded proteins (42). hsc 73, a cytosolic member of the hsp family, enhances lysosomal degradation of polypeptides and may have proteolytic activity itself. However, the specificity for binding of GRP, but not unrelated peptides, of the Swiss 3T3 soluble GRP binding protein is inconsistent with the specificities of known hsp family members. An insulin-binding, insulin-degrading cytosolic protein of M, = 110 kDa has been reported in several tissues and in human hepatoma HepG2 cells (9,39). Insulin binding to its receptor and internalization was required before processing by this protease. The GRP binding protein described here could be responsible for further processing of internalized GRP. Soluble GRP binding proteins have also been reported in human small cell lung carcinoma NCI-H345 cells (13) and human bronchial epithelial BEAS B2B cells (15). Also, we have recently found soluble GRP binding proteins in normal mouse lung (8). The function (s) of these GRP binding proteins has not been elucidated. Like the soluble GRP binding protein (s) from Swiss 3T3 cells, however, these proteins could have proteolytic activity and could play a role in degradation of internalized iigand. It is not known whether this proteolysis would contribute

SOLUBLE G R P B I N D I N G P R O T E I N S IN SWISS 3T3 CELLS

A

C

999

A

C

-205

-116 -97

-66

-45 k

FIG. 7. Western blot analysis of eluates ofGRP AG incubated with M fraction (left) or C fraction (right). Strips labeled A were incubated with rabbit antiserum to NCI-H345 GRP receptor ( 1:1000); C, preimmune control serum. Specific antibody binding was detected using goat antirabbit antiserum conjugated to horseradish peroxidase. The positions of molecular weight markers are shown at right in kDa.

1000

K A N E ET AL.

to turning off signal transduction activated by G R P - G R P receptor binding or whether fragments of G R P could affect intracellular controls of proliferation, for example, by binding to specific response elements in DNA.

ACKNOWLEDGEMENTS This work was supported by Department of Veterans Affairs Research Career Development and Merit Review Awards, NCI First Award R29CA44763, and NCI R55-CA54502.

REFERENCES 1. Banner, D. W.; D'Arcy, A.; Janes, W.; et al. Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex: Implications for TNF receptor activation. Cell 73:431-445; 1993. 2. Caccia, P.; Cletini, O.; Isacchi, A.; Bergonzoni, L.; Orsini, G. Biochemical characterization of the molecular interaction between recombinant basic fibroblast growth factor and a recombinant soluble fibroblast growth factor receptor. Biochem. J. 294(Pt. 3):639-644; 1993. 3. Chilosi, M.; Semenzato, G.; Vinante, F.; et at. Increased levels of soluble interleukin-2 receptor in non-Hodgkin's lymphomas. Relationship with clinical, histologic, and phenotypic features. Am. J. Clin. Pathol. 92:186-191; 1989. 4. Corjay, M. H.; Dobrzanski, D. J.; Way, J. M.; et al. Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells. J. Biol. Chem. 266:18771-18779; 1991. 5. Dunaif, A. E.; Zimmerman, E. A.; Friesen, H. G.; Frantz, A. G. IntraceUular localization of prolactin receptor and prolactin in the rat ovary by immunocytocbemistry. Endocrinology 110:1465-1471; 1982. 6. E1-Domeiri, A. A. H.; Das Gupta, T. K. Reversal by melatonin of the effect of pinealectomy on tumor growth. Cancer Res. 33:28302833; 1973. 7. Feldman, R. I.; Wu, J. M.; Jensen, J. C.; Mann, E. Isolation of the bombesin/gastrin releasing peptide receptor from Swiss 3T3 cells. J. Biol. Chem. 265:17364-17372; 1990. 8. Geraci, M. W.; Miller, Y. E.; Escobedo-Morse, A.; Kane, M. A. Novel bombesin-like peptide binding proteins from lung. Am. J. Respir. Cell. Molec. Biol. 10:331-338; 1994. 9. Haft, J.; Shii, K.; Roth, R. A. In vivo association of [125Ii-insulin with a cytosolic insulin-degrading enzyme: Detection by covalent cross-linking and immunoprecipitation with a monoclonal antibody. Endocrinology 120:829-831; 1987. 10. Heikkila, R.; Trepel, J. B.; Cuttitta, F.; Neckers, L. M.; Sausviile, E. A. Bombesin related peptides induce calcium mobilization in a subset of human small cell lung cancer cell lines: Structure activity analysis and relationship to myc family. J. Biol. Chem. 262:1645616460; 1987. 11. Heimbrook, D. C.; Saari, W. S.; Balishin, N. L.; et al. Carboxylterminal modification of a gastrin releasing peptide derivative generates potent antagonists. J. Biol. Chem. 264:11258-11262; 1989. 12. Jensen, R. T.; Moody, T.; Pert, C.; Rivier, J. E.; Gardner, J. D. Interaction of bombesin and litorin with specific membrane receptors on pancreatic acinar cells. Proc. Natl. Acad. Sci. USA 75:61396143; 1978. 13. Kane, M. A.; Aguayo, S. M.; Portanova, L. B.; et al. Isolation of cytosolic as well as membrane-associated forms of human small cell lung carcinoma gastrin releasing peptide receptor. Proc. AACR 31: 54; 1990. 14. Kane, M. A.; Aguayo, S. M.; Portanova, L. B.; et al. Isolation of the bombesin/gastrin releasing peptide receptor from human small cell lung carcinoma NCI-H345 cells. J. Biol. Chem. 266:9486-9493; 1991. 15. Kane, M. A.; Kelley, K. Comparison of bombesin-like peptide (BLP) receptors from nonmalignant human bronchial epithelial BEAS B2B cells and human small cell lung carcinoma NCI-H345 cells. FASEB J. 6:A1636; 1992. 16. Kane, M. A.; Kelley, K.; Ross, S. E.; Portanova, L. B. Isolation of a gastrin releasing peptide receptor from normal rat pancreas. Peptides 12:207-213; 1991. 17. Kendall, R. L.; Thomas, K. A. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc. Natl. Acad. Sci. USA 90:10705-10709; 1993.

18. Kris, R. M.; Hazan, R.; Villines, J.; Moody, T. W.; Schlessinger, J. Identification of the bombesin receptor on murine and human cells by cross-linking experiments. J. Biol. Chem. 262:11215-11220; 1987. 19. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685; 1970. 20. Larsen, J. K.; Munch-Petersen, B.; Chftstiansen, J.; Jorgensen, K. Flow cytometric discrimination of mitotic cells. Cytometry 7:5463; 1986. 21. Mahmoud, S.; Palaszynski, E.; Fiskum, G.; Coy, D. H.; Moody, T. W. Small cell lung cancer bombesin receptors are antagonized by reduced peptide bond analogues. Life Sci. 44:367-373; 1989. 22. Markwell, M. A. K.; Haas, S. M.; Bieber, L. L.; Tolbert, N. E. A modification of the Lowry procedure to simplify protein determination in membranes and lipoprotein samples. Anal. Biochem. 87: 206-210; 1978. 23. Mendoza, S. A.; Schneider, J. A.; Lopez-Rivas, A.; Sinnett-Smith, J. W.; Rozengurt, E. Early events elicited by bombesin and structurally related peptides. II. Changes in Na + and Ca 2+ fluxes, Na+/ K + pump activity and intracellular pH. J. Cell Biol. 102:2223-2233; 1986. 24. Merril, C.; Goldman, D.; Keuren, M. Gel protein stains: Silver stain. Methods Enzymol. 104:441-447; 1984. 25. Micheletti, R.; Gilder, J. R.; Makhlouf, G. M. Identification of bombesin receptors on isolated muscle cells from human intestine. Regul. Pept. 21:219-226; 1988. 26. Moody, T. M.; Carney, D. N.; Cuttitta, F.; Quattrocchi, K.; Minna, J. High at~nity receptors for bombesin/GRP-like peptides on human small cell lung carcinoma. Life Sci. 37:105-113; 1985. 27. Moody, T. W.; Kris, R. M.; Fiskum, G.; Linden, C. D.; Berg, M.; Schlessinger, J. Characterization of receptors for bombesin/gastrinreleasing peptide in human and murine cells. Methods Enzymol. 168:481-493; 1989. 28. Moody, T. W.; Pert, C. B.; Rivier, J.; Brown, M. R. Bombesin: Specific binding to rat brain membranes. Proc. Natl. Acad. Sci. USA 75:5372-5376; 1978. 29. Moran, T. H.; Moody, T. W.; Hostetter, A. M.; Robinson, P. H.; Goldrick, M.; McHugh, P. R. Distribution of bombesin binding sites in the rat gastrointestinal tract. Peptides 21:219-226; 1988. 30. Naldini, L.; CiriUo, D.; Moody, T. W.; Comoglio, P. M.; Schlessinger, J.; Kfts, R. Solubilization of the receptor for the neuropeptide gastrinreleasing peptide (bombesin) with functional ligand binding properties. Biochemistry 29:5153-5160; 1990. 31. Narazaki, M.; Yasukawa, K.; Saito, T.; et al. Soluble forms of the interleukin-6 signal-transducing receptor component gp130 in human serum possessing a potential to inhibit signals through membrane-anchored gpl30. Blood 82:1120-1126; 1993. 32. Noiva, R.; Freedman, R. B.; Lennarz, W. J. Peptide binding to protein disulfide isomerase occurs at a site distinct from the active sites. J. Biol. Chem. 268:19210-19217; 1993. 33. Papa, V.; Russo, P.; Gliozzo, B.; Gold_fine,I. D.; Vigneri, R.; Pezzino, V. An intact and functional soluble form of the insulin receptor is secreted by cultured ceils. Endocrinology 133:1369-1376; 1993. 34. Rubin, L. A.; Nelson, D, L. The soluble interleukin-2 receptor: Biology, function, and clinical application. Ann. Intern. Med. 113: 619-627; 1990. 35. Rutledge, E. A.; Root, B. J.; Lucas, J. J.; Enns, C. A. Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor. Blood 83:580-586; 1994. 36. Scemama, J.; Zahidi, A.; Fourmy, D.; et al. Interaction of ~2~I-Tyr4bombesin with specific receptors on normal human pancreatic membranes. Regnl. Pept. 13:125-132; 1986. 37. Shandelya, S. M.; Kuppusamy, P.; Herskowitz, A.; Weisfeldt, M. L.; Zweier, J. L. Soluble complement receptor type 1 inhibits

S O L U B L E G R P B I N D I N G P R O T E I N S IN SWISS 3T3 CELLS

38.

39. 40. 41. 42.

43. 44.

45. 46.

the complement pathway and prevents contractile failure in the postischemic heart. Evidence that complement activation is required for neutrophil-mediated reperfusion injury. Circulation 88:28122826; 1993. Sharoni, Y.; Viallet, J.; Trepel, J. B.; SausviUe, E. A. Effect of guanine and adenine nucleotides on bombesin-stimulated phospholipase c activity in membranes from Swiss 3T3 and small cell lung carcinoma cells. Cancer Res. 50:5257-5262; 1990. Shii, K.; Baba, S.; Yokono, K.; Roth, R. A. Covalent linkage of 125Iinsulin to a cytosolic insulin-degrading enzyme. J. Biol. Chem. 260: 6503-6506; 1985. Sinnett-Smith, J.; Zachary, I.; Rozengurt, E. Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking. J Cell. Biochem. 38:237-249; 1988. Swope, S. L.; Schonbrunn, A. The biphasic stimulation of insulin secretion by bombesin involves both cytosolic free calcium and protein kinase c. Biochem. J. 253:193-202; 1988. Terlecky, S. R.; Chiang, H. L.; Olson, T. S.; Dice, J. F. Protein and peptide binding and stimulation of in vitro lysosomal proteolysis by the 73-kDa heat shock cognate protein. J. Biol. Chem. 267:92029209; 1992. Tiesman, J.; Hart, C. E. Identification of a soluble receptor for platelet-derived growth factor in cell-conditioned medium and human plasma. J. Biol. Chem. 268:9621-9628; 1993. Towbin, H.; Stacheline, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354; 1979. Trepel, J. B.; Moyer, J. D.; Cuttitta, F.; et al. A novel bombesin receptor antagonist inhibits autocrine signals in a small cell lung carcinoma cell line. Biochem. J. 255:403-410; 1988. Vigna, S. R.; Giraud, A.; Soil, A. H.; Walsh, J. H.; Mantyh, P. W.

1001

47. 48.

49. 50. 51.

52. 53. 54. 55.

Characterization of bombesin receptors on canine antral gastrin cells. Regul. Pept. 19:144A; 1987. Vigna, S. R.; Giraud, A. S.; Mantyh, P. W.; Soll, A. H.; Walsh, J. H. Characterization of bombesin receptors on canine antral gastrin cells. Peptides 11:259-264; 1990. Vigna, S. R.; Mantyh, C. R.; Giraud, A. S.; Soll, A. H.; Walsh, J. H.; Mantyh, P. W. Localization of specific binding sites for bombesin in the canine gastrointestinal tract. Gastroenterology 93:12871295; 1987. Wada, E.; Way, J.; Shapira, H.; et al. cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor. Neuron 6:421--430; 1991. Westendorf, J. M.; Schonbrunn, A. Characterization of bombesin receptors in a rat pituitary cell line. J. Biol. Chem. 258:7527-7535; 1983. Willey, K. P.; Hunt, N.; Abend, N.; Northemann, W.; Ivell, R.; Leidenberger, F. Serum unmasks the binding of thyroid-stimulating hormone to endogenous and transfected receptors: Evidence for a soluble form of the receptor in human thyroid. J. Endocrinol. 139: 317-328; 1993. Younes, M.; Wank, S. A.; Vinayek, R.; Jensen, R. T.; Gardner, J. D. Regulation of bombesin receptors on pancreatic acini by cholecystokinin. Am. J. Physiol. 256:G291-G298; 1989. Zachary, I.; Rozengurt, E. High-affinity receptors for peptides of the bombesin family in Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA 82: 7616-7620; 1985. Zachary, I.; Rozengurt, E. Identification of a receptor for peptides of the bombesin family in Swiss 3T3 cells by affinity cross linking. J. Biol. Chem. 262:3947-3950; 1987. Zachary, I.; Sinnett-Smith, J. W.; Rozengurt, E. 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. J. Cell Biol. 102:2211-2222; 1986.