Molecular and Cellular Biochemistry 106: 31-39, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.

High-affinity binding sites for bombesin on mouse colonic mucosal membranes

Satya Narayan, Edwin Draviam, Srinivasan Rajaraman 1 and Pomila Singh Department of Surgery and 1 Department of Pathology, The University of Texas Medical Branch, Galveston, Texas 77550, USA

Received 29 October 1990; accepted 11 March 1991

Key words: gastrin releasing peptides, bombesin receptors, mouse colon mucosa

Abstract

Bombesin (BBS) has specific biological effects on colonic mucosal cells, but the presence of BBS receptors on colonic mucosa have not been described to-date. In the present study we examined the mouse colonic mu- cosal membranes for the presence of specific binding sites for BBS/gastrin releasing peptides (GRP), and characterized the binding kinetics and molecular weight of the specific binding proteins. The radiolabeled li- gand (125I-Tyr4-BBS), in the absence or presence of a 1000-fold excess of BBS, was used to establish the opti- mal binding assay conditions of time, pH and temperature for measuring the maximum number of specific binding sites for BBS related peptides. Under the optimal binding assay conditions, BBS displaced the bind- ing of ~25I-Tyra-BBS in a dose-related manner. A single class of high-affinity binding sites (Ka = 0.23 _ 0.02 riM) for BBS were measured, with a binding capacity of 27.3 _+ 4.6 fmoles/mg membrane protein. The binding sites were specific for binding BBS/GRP related peptides, since all structurally related peptides in- hibited the binding of 125I-Tyr4-BBS in a dose-dependent manner, while structurally unrelated peptides did not compete for the 125I-Tyr4-BBS binding sites. The relative binding affinity (RBA) of BBS/GRP related peptides was determined to be in the order of GRP (14-27) = GRP (18-27) > GRP (1-27) > neuromedin B > BBS. The BBS-receptor antagonists, [Leu13-~p-(CH2NH) Leu14]-BBS (LL-BBS) and D-Phe 6, BN(6-13) propylamide (D-Phe6,BN(6-13)-PA), inhibited the specific binding of ~25I-Tyr4-BBS to colonic mucosal membranes in a dose-dependent manner. The inhibitory potency of D-Phe6,BN[6-13]PA was significantly greater than that of LL-BBS. Molecular mass of the specific binding proteins for BBS/GRP was determined to be 70-80 KDa, by chemical cross-linking methods. The 70-80 KDa binding proteins were specific for bind- ing GRP-related peptides and were displaced in dose-dependent manner by increasing doses of BBS. These results thus suggest that the colonic mucosa may be yet another target for GRP related peptides.

Introduction

Bombesin (BBS), is a tetradecapeptide isolated from frog skin [10]. Its mammalian counterpart, gastrin releasing peptide (GRP) is a 27-amino acid peptide that shares a common C-terminus se- quence with BIBS [17]. BBS/GRP related peptides can elicit a variety of biological effects, including

regulation of normal [31, 38] and cancerous tissue growth [6, 24, 36], motility of gastrointestinal tract [7], stimulation of gastric and pancreatic secretion and release of a number of gastroenteropancreatic hormones [7, 14].

BBS/GRP immunoreactivity is found either in central or peripheral nervous system [22]. In pe- ripheral nervous system, BBS/GRP immunoreac-

32

Fig. 1. Photomicrograph of mucosal scrapings illustrating a homogenous population of mucosal epithelial cells. H and E, 500 x.

tivity is primarily confined to nerves in the gastroin- testinal (GI) tracts of mammals [8]. BBS/GRP nerve cell bodies are only displayed in the myenter- ic plexus of the stomach, small intestine and colon in rats and guinea pigs [5, 11]. Fibers from these plexus, containing BBS/GRP, are distributed throughout the GI tract and penetrate the mucosa in the stomach and colon but not the small intestine [5, 11].

Specific BBS binding sites have been demon- strated to be widely distributed throughout the GI tract [20, 32]. In the rat GI tract, BBS binding was localized to the circular muscle layer of the gastric fundus and antrum, submucosal layers of small in- testine, and longitudinal and circular muscles and submucosal layers of the colon [20]. In canine GI tract, specific BBS binding sites were found either on muscle layers of the stomach and ileum, and/or on neuronal elements in the myenteric plexus in the gastric fundus, antrum and small intestine. Though, in the above autoradiographic studies, specific binding of BBS was not apparent in the mucosal layers of the GI tract, specific binding sites for BBS are apparently present on several endocrine cells,

present in the mucosal layers of the GI tract. BBS directly stimulates the release of gastrin and several other GI hormones from cells/tissues in culture [1, 2, 12]. Aponte and colleagues [30], recently dem- onstrated significant stimulatory effects of BBS on release of peptide YY (PYY) from colonic mucosal cells, in vitro; this indicates a possible presence of BBS binding sites on PYY cells within the colonic mucosa, since PYY cells are known to be primarily localized within the colonic mucosa of most species [9, 18].

In the present studies we examined the colonic mucosal membranes for a possible presence of spe- cific binding sites for BBS/GRP related peptides, and further characterized the molecular weight and binding characteristics of the binding proteins.

Materials and methods

Chemicals

Tris-base, MgCI2, sucrose, 4-(2-hydroxyethyl)-l- piperazine ethane sulfonic acid (HEPES), EGTA,

bovine serum albumin (fraction V), bacitracin, soybean trypsin inhibitor, trasylol, phenylmethyl- sulfonylfluoride (PMSF), were purchased from Sigma Chemical company (St. Louis, MO). Bom- besin (BBS), Tyr4-BBS, neuromedin C, neurome- din B, GRP (1-27), GRP (14-27) were purchased from Peninsula Laboratories (Belmont, CA). BBS-receptor antagonists, [Leu~3-~-(CH2NH) Leu~4]-bombesin (LL-BBS) and D-Phe6,BN(6-13) PA, was the generous gift of Dr. David H. Coy, Peptide Research Laboratories, Tulane University School of Medicine, New Orleans, LA. 125I-Tyr 4- BBS was iodinated using immobilized lactoperoxi- dase and glucose oxidase procedure by our publish- ed procedure [27]. The specific activity of Tyr 4- BBS was 1235 Ci/mmol. 125I-GRP-(1-27) (specific activity 1812 Ci/mmol) was procured from Amer- sham Corporation (Arlington Heights, IL). EGS [ethylene glycolbis (succinimidyl succinate)] was purchased from Pierce Chemical Co., Rockford, IL.

Collection of tissue

Male BALB/c mice, 20-25 g body weight, were ob- tained from Harlan Sprague Dawley Inc., Indi- anapolis, IN, and fed purina chow and water ad lib- itum. The mice were maintained at 22°C for one week in dark/light cycles of 12 h each. They were killed by decapitation and the full length colon im- mediately excised, cut open along the length, cleaned and washed in ice-cold tissue collection buffer A (10 mM Tris, 137 mM NaC1, 5 mM KC1, 2mM CaC12, 2.5mM MgC12 and 0.25M sucrose, pH7.4) containing 0.1% bovine serum albumin (BSA), 0.1% soybean trypsin inhibitor, 0.1% ba- citracin and 0.1 mM PMSF. After washing the mu- cosal side thoroughly, the mucosa was scraped with the help of ice-cold glass slides, and the colonic mu- cosa immediately frozen in liquid nitrogen. Crude membranes were prepared from colonic mucosa as published previously [21], with a slight modifica- tion in the homogenizing buffer, which additionally contained 0.1 mM PMSF. In a few experiments the scraped mucosa was embedded in 2% agarose and fixed in 10% neutral formalin overnight. The fixed tissue block was processed routinely, embedded in

33

paraffin and 3/xm sections were stained with hema- toxylin and eosin. The preparation consisted en- tirely of mucosal epithelial cells without contam- ination with smooth muscle or neuronal cell population (Fig. 1).

Binding assay

To determine the specific binding sites for BBS/ GRP related peptides on mouse colonic mucosal membranes, 125I-Tyrg-BBS was used as the radiola- beted ligand. Aliquots of membrane suspension, in binding buffer (25mM HEPES, 5raM MgC12, 0.1 mM PMSF, 0.1% BSA, 0.1% bacitracin, 0.1% soybean trypsin inhibitor and 100 KIU of trasylol/ ml buffer pH7.4), containing 100-150txg mem- brane protein, were used as substrate in each tube. The optimal conditions of time, temperature and pH for measuring maximum number of specific binding sites for GRP related peptides on colonic mucosal membranes were initially established, and were determined to be - 30-40 min of incubation at 30°C; a pH of 7.2-7.5 was found to be optimal (data not shown). A 8-10 point binding assay was performed by incubating aliquots of membrane suspension with increasing concentrations (0.05 to i nM) of the radiolabeled ligand in the absence (to- tal binding) or presence (non-specific binding) of 1,000 x excess concentration of BBS. The reaction was terminated by adding excess ice-cold binding buffer, and the unbound ligand removed by centrif- ugation for 15 min at 30,000 g at 4 ° C. The pellet was washed two times with ice-cold buffer and the radioactivity in the pellet counted in a Beckman Minaxy 5000 counter ( - 7 8 % counting efficiency for 125I). The specific binding data thus obtained was analyzed by a Scatchard plot [25]. In order to determine the specificity of the binding sites, the relative binding affinity (RBA) of various peptides was determined as previously described [28]. Brief- ly, membrane aliquots were incubated with - 5 0 pM of 125I-Tyr4-BBS___ increasing concentrations (0.01 to 100nM) of unlabeled peptide, and the RBA of the competing peptide determined by the method of Bonne and Raynaud [3] as described previously [28].

34

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a~ E o a D

4

>.E

8

Q. 69

A

15

10.

5 -

.__._.-.-o

0~5 0's 0.'rs 1.b0

1251-Tyr4-bombesin (nM)

B

0 . 0 1 2 ~

0 0.5 1.5 2.5 3.5 fmoles specifically bound 1251-Tyr4°BBS

Fig. 2. Dose-dependent binding of 125I-Tyr4-BBS to mouse colonic mucosal membranes. Membrane aliquots (150/xg) were incubated with increasing concentrations of 12SI-Tyr4-BBS at 30 ° C for 30 min, in the presence or absence of 1,000 fold excess of unlabeled BBS. (A) Specific binding, at each concentration determined is shown. (B) A Scatchard plot of specific binding data shown in (A). Each point is the mean of three determinations from a representative of four similar experiments.

Molecular weight determination

To determine the apparent molecular weight of BBS/GRP binding proteins on mouse colonic mu- cosal membranes, the 125I-GRP bound receptor proteins were cross-linked with EGS. The binding of 125I-GRP to colonic mucosal membranes in the absence or presence of various concentrations of BBS was performed as described above under opti- mal binding assay conditions. At the end of the in- cubation, the tubes were centrifuged and the pel- lets washed three times with ice-cold buffer, devoid of BSA. The membrane pellet was resuspended in buffer, devoid of BSA, containing EGS (freshly made in dimethyl sulfoxide [DMSO]). The final concentration of EGS was 5 mM in < 1% total vol- ume of DMSO. The labeled membranes were in- cubated with EGS for 30 min at room temperature. The reaction was terminated by rapid centrifuga- tion at 4°C and membranes washed with excess binding buffer. The washed membrane pellets in the reaction tubes were solubilized in 0.05ml of electrophoresis sample buffer containing 6.25mM Tris-C1, 10% glycerol, 0.2% SDS and 0.001% bro- mophenol blue, in the absence or presence of 0.5% [5-mercaptoethanol. Samples were immediately heated to 100°C for 5 min and analyzed by one- dimensional SDS-polyacrylamide gel electropho-

resis using 9.5% acrylamide in the separating and 4% acrylamide in the stacking gel with 0.1% SDS, as per the method of Laemmli [15]. At the end of the electrophoresis, the gels were stained, des- tained, dried and exposed to X-ray films (Kodak X- OmatTMAR) for 7-14 days at - 7 0 ° C. The aut- oradiograms were scanned using a double-beam densitometer and the area under the peaks mea- sured with a Hewlett-Packard digitizer.

Protein was measured by the method of Lowry and colleagues [16] by using crystalline BSA (Frac- tion V) as standard.

Results

Binding of 1251-Tyr4-BBS to colonic mucosal membranes

To determine the optimal conditions for binding of 125I-Tyra-BBS to colonic mucosal membranes, we determined the time and temperature dependency of the binding. At temperatures < 30 ° C, specific binding of 125I-Tyr4-BBS did not attain maximum levels even after 2 h of incubation. At 30 ° C, on the other hand, maximum specific binding was observ- ed at 30-40 rain of incubation, after which point it demonstrated a slight decline. At 37 ° C, specific

binding was once again submaximal at all time points. A temperature of 30 ° C and incubation time of 30 min was thus considered optimal for mea- suring maximum number of specific binding sites for 12SI-Tyr4-BBS on mouse colonic mucosal mem- branes (data not shown). Under the optimal bind- ing assay conditions, a dose-dependent binding of 125I-Tyr4-BBS to mucosal membranes was defined, and results are shown in Fig. 2A. The specific bind- ing increased linearly with increasing concentra- tions of 125I-Tyr4-BBS, and was >50% of total binding at all concentrations of ~25I-Tyr4-BBS used in the present studies. The specific binding data from Fig. 2A 'was re-analyzed by Scatchard analysis using non-linear regression analysis of the binding data by the least squares method, using the pro- gram 'Ligand' of Munson and Rodbard as modified by G.A. McPherson (Elsevier - Biosoft, 1985) (Fig. 2B). A single class of high-affinity binding sites for 125I-Tyr4-BBS was evident on mouse colon- ic mucosal membranes, with an equilibrium dis- sociation constant (Kd) of 0.23 + 0.02 nM (n = 4), and a binding capacity of 27.3 _+ 4.6 (n = 4) fmoles/ mg mucosal membrane protein.

Specificity of 125I-Tyr4-BBS binding to mouse colonic mucosal membranes

To determine the specificity of the BBS/GRP bind- ing sites, a variety of structurally related and un- related peptides were tested for their ability to in- hibit the binding of ~25I-Tyr4-BBS to mucosal membranes. Based on the molecular concentration of the peptides required for 50% inhibition of the specific binding of 125I-Tyr4-BBS to mucosal mem- branes, the relative binding affinity of BBS/GRP analogues was calculated as previously published [28]. The RBA of the BBS/GRP related peptides, for displacing the binding of 125I-Tyrg-BBS, was in the order of GRP (14-27)* = GRP(18-27)* > GRP (1-27) > neuromedin B > BBS (* = p < 0.05 BBS vs. others) (Fig. 3). All the BBS/GRP related pep- tides examined, inhibited the binding of 125I-Tyr4- BBS in a dose-dependent manner (Fig. 3). All un- related peptides tested (VIP, insulin, CCK-8, gas- trin), did not inhibit the specific binding of

35

125I-Tyr4-BBS to colonic mucosal membranes sig- nificantly (data not shown).

The specificity of ~25I-Tyr4-BBS binding to mouse colonic mucosal membranes was further character- ized by determining the inhibitory effects of two BBS-receptor antagonists, LL-BBS and D-Phe6, - BN(6-13)PA. Both the antagonists inhibited the 125I-Tyr4-BBS binding in a dose-related manner, and the inhibitory potency of D-Phe6,BN(6-13)PA was significantly greater than that of LL-BBS (p < 0.05) (Fig. 4).

Molecular weight of 125I-GRP binding proteins on mouse colonic mucosal membranes

The molecular weight of 125I-GRP binding proteins on the colonic mucosal membranes was deter- mined by the cross-linking procedure described un- der Methods. The autoradiograph of 125I-GRP, cross-linked with 5mM EGS, and subjected to electrophoresis under reducing conditions is shown in Fig. 5. The autoradiograph demonstrates the presence of a single band with an apparent mole- cular mass of 70-80 KDa. BBS displaced the bind- ing of 125I-GRP to the 70-80 KDa binding proteins in a dose-related manner (Fig. 5). Under both re- ducing and non-reducing conditions, the 70- 80 KDa binding proteins for 125I-GRP were observ- ed, indicating absence of sub-units linked by dis- ulfide bonds.

Discussion

Specific binding sites for BBS/GRP related pep- tides are apparently widely distributed along the GI tract [20, 32]. Specific binding of BBS was not demonstrated by autoradiographic studies in the canine colon [33], but was demonstrated in the lon- gitudinal and circular muscular layers and submu- cosal layers of the rat colon [20]. In the present study, we observed the presence of specific binding sites for BBS on histologically proven mouse colon- ic mucosa, which demonstrated a high binding af- finity and a low capacity for binding the BBS relat- ed peptides. It appears likely that some or all of the

36

~.=__ m 20-

. o

~--~ o

~- 6 0 t / /

r - C J c - - ~ A

80 I1) 13_

1 0 0 I i 12 11 10 9 8

Peptide concentration (-Log M)

Fig. 3. Inhibition of binding of 125I-Tyr4-BBS to mouse colonic mucosal membranes by various peptides. Mucosal membranes were incubated with 12SI-Tyr4-BBS for 30 min at 30 ° C in the ab- sence or presence of increasing concentrations of various pep- tides. The % inhibition of specific binding of 125I-Tyr4-BBS in re- lation to the concentration of the peptide hormone used is presented. Each data point is the mean of two observations in the same experiment, and the data is representative of two simi- lar experiments. O = BBS, • = GRP (1-27), A = GRP (14- 27), • = neuromedin B, [] = GRP (14-27).

BBS binding sites measured in the present studies are present on the endocrine (PYY containing) cells, since Aponte and colleagues [1] recently demonstrated a significant release of PYY from isolated colonic mucosal cells, in vitro, in response to BBS. BBS, was also reported to stimulate the growth of colonic mucosa in suckling rats [23], in- dicating a possible presence of BBS receptors on cell populations in the colonic mucosa that respond to growth effects of BBS. The presence of only a small number of BBS binding sites on a relatively small percentage of the colonic mucosal cells, may have precluded the ability to detect these sites aut- oradiographically in previous studies [20]. Simi- larly, even though it is well-known that BBS acts directly on G cells in culture to release gastrin [12, 30], (indicating the presence of BBS receptors on the G cells, which was confirmed in preliminary studies [29, 32]), some of the above mentioned aut- oradiographic studies [20] did not demonstrate the presence of BBS receptors/binding sites on the an- tral mucosal cells.

_>,

° ~ . ~

rt

2O

80- ~ o ~

Ioo- -// 12 1 '1 1'0 ~)

Peptide concentration (-Log M)

A

i

8 7

Fig. 4. Percent inhibition of specific binding of 125I-Tyr4-BBS by increasing concentrations of BBS-receptor antagonists. Colonic mucosal membranes were incubated with a25I-Tyr4-BBS in the absence of presence of increasing concentrations of the BBS- receptor antagonists as described in Materials and methods. The log-dose inhibition afforded by the increasing concentrations of BBS and the antagonists, towards specific binding of lzSI-Tyr4- BBS, is shown. Each data point is the mean of two observations within one experiment, and is representative of two similar ex- periments. © = BBS, A = Leula-ap-(CH2NH) Leu14-bombesin, and • = D-Phe6,BN(6-13)propylamide.

BBS binding to the colonic mucosal membranes in the present studies was apparently specific for BBS related peptides, and was not displaced by a number of unrelated peptides examined (including CCK, gastrin, PYY, VIP and insulin). All the BBS/ GRP related peptides examined inhibited the bind- ing of BBS to colonic mucosal membranes in a dose-dependent manner, and the relative rank or- der of potency for these peptides was GRP (14- 27) = GRP(18-27) > GRP (1-27) > neuromedin B >BBS. These results indicate that the BBS/ GRP binding sites on the mouse colonic mucosal membranes can bind GRP related peptides, that share the C-terminal amino-acid residues. The elongation of the N-terminal end did not signif- icantly affect the binding affinity of the GRP relat- ed peptides for binding the colonic mucosal mem- branes. Our results thus agree with the observations of Gorgosky and colleagues [13], that more than 7 but no more than 9 C-terminal amino-

acids appear to be required for rendering a high binding affinity to the GRP related peptides.

It was interesting to note that GRP (14-27) and GRP(18-27), C-terminal tetradecapeptides of GRP (1-27), were the most potent homologues for inhibiting the binding of 125I-Tyr4-BBS to the colon- ic mucosal membranes. In previous studies we have similarly observed that the RBA of GRP homo- logues to rat antral membranes was in the order of GRP (14-27) > B B S > GRP (1-27) [29], and re- ported that GRP (14-27) was significantly more po- tent than BBS and GRP (1-27) in stimulating the release of gastrin from perfused rat stomach [14]; in these studies the relative potency of GRP(18-27) was not examined. GRP (14-27) was the most po- tent peptide for displacing binding of 125I-Tyr4-BBS to mouse colon cancer (MC-26) cells [21]. The binding affinity of GRP (14-27) for BBS/GRP re- ceptors on rat pituitary cells is also significantly higher than that of either BBS or GRP (1-27) [37].

The BBS receptor antagonists used in the pre- sent study, significantly inhibited the binding of BBS to mouse colonic mucosal membranes, fur- ther indicating that the specific binding sites, mea- sured in the present studies, for BBS/GRP related peptides are probably functional receptors and share the physico-chemical characteristics of BBS receptors described to-date on other gastroentero- pancreatic tissues. Of the two BBS receptor antag- onists used in the present study, D-Phe 6, BN(6-13) PA was the more potent antagonist with an EDs0 dose of 0 .6nM (n = 2), which was significantly lower than the EDs0 dose of LL-BBS (10.2 riM, n = 2) used in the present study. In a recent study, Wang and colleagues [35] similarly reported D- Phe6,BN(6-13)-PA to be the most potent BBS re- ceptor antagonist available to-date towards inhib- iting both the BBS-stimulated amylase release in guinea pig pancreatic acinar cells, (EDs0 dose of 1.6 nM) and the growth of Swiss 3T3 cells (EDs0 of 0.8 nM). The molecular weight of BBS binding proteins on mouse colonic mucosal membranes (70 to 80 KDa) was once again similar to that described on several other cell types, including MC-26 cells, pancreatic cancer (AR42J) and 3T3 cells [21, 27, 39].

In the present studies we observed the presence

37

Fig. 5. Autoradiographic profile of 125I-GRP binding to GRP/ BBS binding sites on mouse colonic mucosal membranes. Mem- branes were labeled with 0.1 nM 125I-GRP + increasing concen- trations of BBS and cross-linked with 5 mM EGS as given under Methods. Solubilized proteins from the membranes were sub- jected to SDS-PAGE under reducing conditions and analyzed autoradiographically. The position of the standard molecular weight marker proteins is indicated. Lane a = total binding of 125I-GRP in the absence of BBS. BBS concentrations (nM) in Lanes b-f are as follows: b = 0.01nM, c= 0.1nM,d = lnM, e = 10 nM, f = 100 nM. A similar autoradiographic profile was obtained under non-reducing conditions (data not shown).

of only one class of high affinity binding sites on mouse colonic mucosal membranes. Similarly in several other tissues/cells, a single class of high af- finity binding sites, with equilibrium dissociation constant values ranging from 0.7-2.1 nM have been described [4, 19, 26, 39]. However, in mouse colon cancer cells, we recently reported the presence of two classes of relatively high affinity binding sites for BBS/GRP related peptides [21]; this indicates the possibility that yet another sub-type of BBS

38

binding sites is expressed on neoplastic transforma- tion of colonic mucosal cells.

Acknowledgements

Supported by grants from the National Institutes of Health (PO1 DK 35608 and CA 38651).

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