Nucleotides regulate the binding affinity of the recombinant type A cholecystokinin receptor in CHO K1 cells

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Regulatory Peptides 61 (1996) 87-93

Nucleotides regulate the binding affinity of the recombinant type A cholecystokinin receptor in CHO K1 cells G.T. Blevins, E.M.A. van de Westerlo, C.D. Logsdon, P.M. Blevins, J.A. Williams * Department of Physiology, University of Michigan, 7744 Medical Science 11, Ann Arbor, MI 48109-0622, USA Received 17 May 1995; revised 17 September 1995; accepted 5 October 1995

Abstract

Cholecystokinin (CCK) receptors on rat pancreatic acinar cells display two binding affinity states in the presence of adeninine and guanine triphosphates with the effect of ATP mediated by the enzyme nucleoside diphosphate kinase. To determine whether this behavior was intrinsic to a single receptor protein we studied the binding affinity of CHO cells stably transfected with a cloned rat CCK A receptor. ~25I-CCKbinding to intact cells at 37°C revealed two affinity states for CCK of K d values 20 pM and 2.4 nM. Membranes prepared from these cells displayed a single affinity state for CCK but two affinity states could be restored in the presence of GTP[3,S], ATP and ATP[-yS] but not AMP-PCP. ATP and ATP[TS] but not AMP-PCP were substrates for nucleoside diphosphate kinase present in CHO cell membranes and transferred their terminal phosphate to GDP. These findings indicate that the interconvertible affinity states of the CCK receptor are inherent in a single receptor protein and that nucleoside diphosphate kinase mediates the effect of ATP to regulate these two affinity states Keywords: Cholecystokinin receptor; Nucleoside diphosphate kinase; ATP; GTP

1. Introduction

Cholecystokinin (CCK) receptors are the predominant mediators of hormonally-stimulated exocrine pancreatic secretion and gall bladder contraction. These glycoprotein receptors belong to the seven transmembrane-spanning guanine nucleotide binding protein (G protein) coupled receptor superfamily [1,2]. Numerous studies have shown that the CCK receptor on intact pancreatic acinar cells exists in two agonist binding affinity states, occupancy of which effects the induction of different intracellular events and consequent biological effects [3-6]. On the other hand, CCK receptors on pancreatic membranes exhibit only a single binding affinity state [7-10]. In earlier studies we evaluated the difference in binding characteristics between CCK A receptors on intact pancreatic acini and isolated membranes by studying CCK binding to streptolysin O-permeabilized rat pancreatic acini, a model exhibiting binding characteristics similar to membranes [ 11 ]. Using this preparation we found that both ATP

* Corresponding author. Tel.: + 1 (313) 7644376. 0167-0115/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSD1 01 67-0 1 1 5 ( 9 5 ) 0 0 1 42-5

and GTP[7S] had the ability to induce two CCK binding affinity states. We subsequently formulated the hypothesis that the effect of ATP was mediated by the activity of the ubiquitous enzyme nucleoside diphosphate kinase (NDPK) which catalyzes the transfer of high energy phosphate groups between nucleoside triphosphates (mostly ATP) and nucleoside diphosphates, in this case GDP [12,13]. Both membrane and cytosol associated forms of this enzyme have been found in a number of cells and the activity of NDPK is believed to play an important role in a number of signal transduction systems [14-17]. Although it has been demonstrated that NDPK does not transfer phosphate groups directly to G protein bound GDP in vitro, NDPK may have the ability to channel formed GTP into signalcoupling G proteins, [18-20]. This enzymatic reaction is probably the major mechanism for maintenance of GTP levels in close proximity to guanine nucleotide binding proteins. This evidence strongly suggests that the activity of NDPK regulates the affinity of the CCK A receptor on pancreatic acini [21]. With the recent cloning of the CCK A receptor (1) it is now possible to determine if the nucleotide regulation of CCK binding affinity is mediated via a single receptor protein. Transient expression of CCK A

88

G.T. Blevins et al./ Regulatory Peptides 61 (1996) 87-93

receptors in COS cells resulted in a single CCK binding affinity of 1.9 nM when assessed using ]25I-BH-CCK8 [22] although lower affinity states were revealed by analyzing the binding of the CCK antagonist [3H]L-364,718. By contrast, when rat CCK A receptors were stably expressed in CHO cells two distinct agonist affinity states of 30 pM and 2 nM could be determined [23] which are quite comparable to similar measurements on rat pancreatic acini. We report here that the recombinant CCK A receptor in CHO cells exists in two affinity states, while similar to pancreatic membranes, only a single binding affinity state is observed in CHO cell membranes. Moreover, the affinity of this receptor in CHO cell membranes is regulated by ATP similar to our previous findings in permeabilized rat pancreatic acini and rat pancreatic membranes. These findings suggest that the interconvertible affinity states of the CCK receptor observed in pancreatic acini are an inherent characteristic of this receptor and further suggests that NDPK activity mediates the effect of nucleotides to induce two CCK binding affinity states.

2. Materials and methods

Synthetic cholecystokinin octapeptide (CCK8) was a gift from the Squibb Institute (Princeton, NJ). The following were purchased: ATP, guanosine 5'-0-(3thiotriphosphate) ( G T P [ y S ] ) , adenosine 5'-0-(3thiotriphosphate) (ATP[yS]), and adenylyl (/3,y-methylene)diphosphonate (AMP-PCP) from Boehringer-Mannheim (Indianapolis, IN); Bio-Rad protein assay reagent from Bio-Rad (Richmond, CA), [L25I]Bolton-Hunter labeled CCK8 (125I-BH-CCKS) from New England Nuclear (Boston, MA). Unless otherwise noted, all other chemicals were from Sigma Chemical (St. Louis, MO).

2.1. Expression of C C K A receptor in CHO cells and preparation of cell membranes The full length cDNA encoding the r a t C C K A receptor (1) was kindly provided by S.A. Wank (NIH, Bethesda, MD) and stably transfected into CHO-K1 cells as previously reported [23]. Routinely, CHO K1 cells were cultured in DMEM media supplemented with 5% FBS at 37°C in a humidified atmosphere of 5% CO 2. Cell membranes were prepared by homogenization of CHO cells in 9 volumes of 50 mM Tris-Cl, 1 mM benzamidine, pH 7.4, using 20 strokes of a motor driven teflon homogenizer with a tight pestle. The homogenate was centrifuged at 17,000 × g for 35 min. Pelleted membranes were resuspended in fresh homogenization buffer and again centrifuged at 17,000 × g for 35 min. Pelleted membranes were then resuspended in binding buffer (20 mM Hepes, 5 mM MgCI:, 1 mM EGTA, 100 mM NaC1, at pH 6.5) and a small aliquot was removed for measurement of the protein concentration. The remainder was supplemented

with 5 m g / m l BSA, 0.5 m g / m l bacitracin, 0.2 m g / m l SBTI and stored in small aliquots at - 8 0 ° C until used for measurement of either CCK binding or NDPK activity.

2.2. Measurement of nucleoside diphosphate kinase activity in CHO cell membranes Nucleoside diphosphate kinase activity in CHO cell membranes was measured as previously described [21], using a modification of the method of Kimura and Shimada [24]. Briefly, membranes were incubated in 100 /xl of a solution containing, 50 mM Tris-HC1 (pH 7.0), 1 mM theophylline, 5 mM MgC12, 1.0 p,M [8-3H]GDP, and nucleotides at 24°C, for 8 min. For measurement of the kinetic characteristics for ATP, 400 /zM GDP was added to all tubes. Reactions were terminated by the addition of 10 ~1 of 0.1 M EDTA and 10 /xl 100 mM GTP. Membranes were pelleted by centrifugation of reaction mixtures and a 10 /zl aliquot of each supernatant was spotted on polyethyleneimine-cellulose sheets and developed for 14 cm in 2 N formic acid/1.2 M LiC12 at 4°C. The reaction products were located under a UV lamp, scraped off and extracted with 750/xl 0.7 M MgC12, 20 mM Tris-HC1, pH 7.4, for 1 h at room temperature. The extraction mixture was then centrifuged at 1000 × g for 3 min and a 500/xl aliquot of supernatant was removed, added to 5 ml scintillation fluid and radioactivity determined by scintillation counting.

2.3. Binding of ~25I-BH-CCK8 to CHO cells Binding of 125I-BH-CCK8 to CHO cells was performed at 37°C, in Hepes-Ringers buffer solution prepared as previously described [25]. In brief, aliquots of CHO cells containing approx. 1 - 107 cells were placed in 25 cm 2 cell culture flasks with 5 pM 125I-BH-CCK8 and graded concentrations of CCK8 ranging from 10 pM to 0.1 /xM. The gas phase for all incubations was 100% 0 2. Ninety minutes after beginning the incubation 3 1-ml aliquots of CHO cell suspension were removed from each flask, layered over 2 ml ice-cold 0.9% NaCI, centrifuged at 200 x g for 3 min, washed twice with ice-cold 0.9% NaCI and bound radioactivity was measured by y-counting. Protein concentrations of the CHO cells was measured and binding is expressed relative to protein concentration.

2.4. Binding of lesI-BH-CCK8 to CHO cell membranes Binding experiments were performed in 1.5 ml microcentrifuge tubes, in a total volume of 0.3 ml, at 30°C. Incubation tubes contained binding buffer (20 mM Hepes, 5 mM MgC12, 1 mM EGTA, 100 mM NaCI, supplemented with 5 m g / m l BSA. 0.5 m g / m l bacitracin, and 0.2 m g / m l SBTI, pH 6.5), 25 pM 125I-BH-CCK8, unlabeled CCK8 at concentrations ranging from 10 pM to 100 nM, and 100 /xg membranes along with the specified nucleotide or

G.T. Blevins et aL/ Regulatory Peptides 61 (1996) 87-93 nucleotide analogue. Binding reactions were begun by the addition of membrane protein and were ended after 90 min (the time to reach steady state at 30°C), by centrifugation at 12,000 × g in a microcentrifuge for 2 min. The supernatants were discarded and pellets washed twice with 0.3 ml ice-cold incubation buffer containing no added proteins and radioactivity associated with the pellets was measured. Binding isotherms were analyzed using a personal computer version (Elsevier Biosoft) of the L I G A N D nonlinear least-squares curve fitting program of Munson and Rodbard [26]. Goodness of fit to 1-site and 2-site models were tested and whether a significant difference existed was determined by the program.

2.5. SDS-PAGE and Western transfers One-dimensional gel electrophoresis was performed as described by Laemmli [27]. Sixteen /xg of the samples were loaded per lane on 12% SDS-PAGE rain±gels and run at 200 V. After gel electrophoresis, proteins were transferred to nitrocellulose membranes according to Towbin [28] using g l y c i n e / T r i s / m e t h a n o l transfer buffer containing 0.1% SDS to enhance the transfer from the gel at 30 V overnight. Nitrocellulose membranes were blocked with Tris-buffered saline (pH 7.6) containing 5% bovine serum albumin and 0.2% Tween 20 for 1 h. Membranes were then washed and probed with anti-NDPK [29] antisera at a concentration of 1:5000 for 2 h. Membranes were washed again then incubated with horseradish peroxidase (HRP)labeled goat anti-rabbit antibody for 90 rain. Membranes were then washed five more times before being visualized by enhanced chemiluminescence following the manufacturers procedures, by exposing the membranes to Dupont Reflection Autoradiography Films.

2.6. Statistical procedures Experimental measurements were made in duplicate and data is expressed as the means ___S.E. of n experiments. Statistically significant differences were determined by A N O V A with Newman-Keuls multiple range test; P < 0.05 was considered significant.

89

3. Results 3.1. Binding characteristics of in CHO cells

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Binding studies were performed to determine whether the recombinant CCK receptor expressed in the nonnative environment of the CHO cell exhibited the functional binding characteristics of the native CCK receptor. The C H O - C C K A cells utilized in these experiments showed high specific binding and analysis of binding competition data by unlabeled CCK-8 indicated two binding affinity states (Fig. 1) as previously described in pancreatic acinar cells [2-6]. Affinities for the high- and low-affinity states were 19.6 + 5.5 pM and 2 . 4 + 0.6 nM (n = 4), respectively, with approx. 1.3% of the bound ligand appearing in the high-affinity state (BMAxH = 15.3 + 2.8 f m o l / m g protein, BMAXL= 1 181 + 103 f m o l / m g protein). The number of binding sites is almost certainly over estimated due to ligand internalization [25]. In contrast to our findings in intact CHO cells, the recombinant C C K A receptor on C H O cell membranes existed in only a single binding affinity state (Fig. 2A, Table 1), exhibiting a K D of 198.4 + 24.8 pM and a maximal binding capacity of 271.8 _+ 62.4 f m o l / m g protein.

3.2. Effect of nucleotides on binding of CCK to total CHO cell membranes We have previously found that ATP is capable of inducing two binding affinity states on permeabilized rat pancreatic acini and rat pancreatic membranes, on both of which the 'native' CCK receptor is found [11,21]. We evaluated the ability of nucleotides to regulate the binding of mSI-BH-CCK8 to the recombinant CCK receptor in CHO cell membranes. All nucleotides examined were capable of inhibiting I/SI-BH-CCK8 binding; however, in contrast to our previous observations in permeabilized acini [11] none of the nucleotides tested could inhibit binding to near nonspecific levels (Fig. 3). Of the nucleotides tested, the most potent at inhibiting ~25I-BHCCK8 binding was G T P [ y S ] with an ID50 of 13 nM while

Table 1 Nucleotides that serve as substrates for NDPK induce two measurable CCK binding affinity states on total membranes from CHO cells transfected with the CCK receptor Treatment Control (9) 1 mM ATP (6) 1 mM ATP[yS] (6) 100 nM GTP[3,S] (5) 100 p.M AMP-PCP(3)

KDH (pM)

198.4 ± 24.8 95.6 ± 17.3 121.7 ± 24.2 117.8 ± 27.3 176.9 ± 23.1

KDL (nM) 2.6 ± 0.7 3.7 + 2.2 3.9 ± 0.8 -

BMAXH(fmol/mg) 271.8 + 62.4 54.3 ± 29.0 a 44.7 ± 17.7 a 38.5 + 14.9 a 463.5 + 130.4

All values are the mean _+S,E. for the number of experiments in parentheses. a Significantly(P < 0.05) different than Control.

BMAXL(fmol/mg) 174.4 ± 49.0 151.4 ± 55.6 85.9 _-4_-9.2 -

BMAX total (fmol/mg) 271.8 ± 62.4 228.7 + 78.0 196.2 + 59.2 124.5 ± 13.5 a 463.5 ± 130.4

90

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Fig. 1. Representative experiment demonstrating competitive inhibition of 125I-BH-CCK8 binding to CHO cells by CCK8. (Inset) Scatchard plot of specific CCK binding. The line in the main figure was fit by eye, whereas the two lines in the inset are the lines for high- and low-afrmity components as calculated by nonlinear least-squares fitting of the data.

ATP[TS] and ATP inhibited binding with IDs0s of 53 nM and 55 /xM, respectively. In C H O cell membranes the C C K receptor also existed in a single binding affinity state that could be converted to two binding affinity states by ATP (Fig. 2B, Table 1) Additionally, as previously observed with the native C C K A receptor, other nucleotides that serve as substrates for N D P K could induce two measurable C C K binding affinity states o f the recombinant C C K A receptor on CHO cell membranes (Table 1). In the presence of ATP or A T P [ 7 S ] the receptor was converted to two binding affinity states with no change in the total number of measurable receptors. However, in the presence of GTP[TS] similar affinity states were also observed but with a significant ( P < 0.05) decrease in the total number of measurable receptors (Table 1). W e have previously found that the nonhydrolyzable analogue of ATP, A M P - P C P did not serve as a substrate for N D P K and also could not induce two binding affinity states of the native C C K receptor on pancreatic membranes [21]. Our findings in the present study are similar in

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Fig. 3. Inhibition of specific '25I-BH-CCK8 binding CHO cell membranes by GTP[yS], ATP[TS] and ATP. CHO membranes were incubated at 37°C for 90 min with 25 pM ~25I-BH-CCK8and the designated nucleotide or nucleotide analogue. Membranes were washed twice and radioactivity associated with membranes determined. Nonspecific binding determined in the presence of 100 nM CCK8 has been subtracted. Data are means + S.E. of 3 or 4 experiments performed in duplicate.

that A M P - P C P did not induce two binding affinity states of the recombinant C C K receptor.

3.3. Detection o f N D P K in CHO cell membranes by activity and immunoblotting The divalent cation-dependent phosphotransferase nucleoside diphosphate kinase catalyzes the transfer of highenergy y-phosphates from nucleoside triphosphates to nucleoside diphosphates. One such transphosphorylation reaction utilizes G D P and ATP, yielding GTP and A D P as the products. Nucleoside diphosphate kinase in CHO cell membranes transferred y-phosphates from ATP to G D P with a maximal velocity of 3.4 _+ 0.6 pmol m i n - l (/xg protein) - l . Similar experiments utilizing ATP[TS] demonstrated that C H O cell membranes catalyze the transfer of the thiophosphate group from ATP[TS] to GDP forming GTP[TS] with a maximal velocity of 91.9_+ l l . 1 fmol m i n - l ( / x g p r o t e i n ) - ]. To further confirm the presence of N D P K in C H O cell membranes immunoblots were performed using an antiserum raised against rat pancreatic N D P K - a (Fig. 4). This antiserum identified a protein in membranes from C H O cells, AR42J cells and rat pancreas with an apparent molecular mass of 18 kDa, the molecular mass of NDPK.

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Fig. 2. Representative experiments demonstrating competitive inhibition of 125I-BH-CCK8binding to CHO cell membranes in the absence (A) or presence (B) of l mM ATP. Nonspecific binding determined in the presence of 100 nM CCK8 has been subtracted. (Inset) Scatchard plots of specific CCK binding. Straight lines are the best fit for a single binding site (A) or 2 sites (B) as calculated from nonlinear least squares analysis of the data points.

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4. Immunoblot of CHO cell and pancreatic membranes (C, CHO cell membranes; A, AR4-2J cell membranes; T, total rat pancreatic membranes). Membranes (50/xg) were run on a 5-15% gradient SDS-PAGE, transferred to nitrocellulose as described and probed with an antiserum (553) raised against NDPK. Fig.

G.T. Blevins et a l . / Regulatory Peptides 61 (1996) 87-93

4. Discussion

The C C K A receptor is a unique molecule with the ability to exist in multiple interconvertible CCK binding affinity states [2-6,11,22]. We have previously demonstrated that the binding affinity of the CCK receptor on permeabilized rat pancreatic acini or pancreatic membranes was modulated by ATP and GTP[yS] [11,21]. Those studies provided evidence indicating that ATP regulates CCK receptor affinity via a mechanism involving the enzyme nucleoside diphosphate kinase. In the present study we have found that NDPK activity is present in CHO cells and that transfer of phosphate groups from ATP to GDP occurs at a rate similar to that observed in rat pancreatic acinar cell membranes, suggesting that CHO cell NDPK is a comparable enzyme. Additionally, we have demonstrated the presence of this enzyme in CHO cells by immunoblotting using an antiserum produced against rat pancreatic NDPK-a [29]. In these immunoblots the antiserum clearly detected proteins of similar molecular masses (18 kDa) in membranes from CHO cells, AR42J cells and rat pancreas, further evidence that NDPK is present in the membrane fraction along with the CCK receptor. The data in the present study supports our previous findings that the intracellular mechanism governing the induction of two CCK binding affinity states by nucleoside triphosphates containing bases other than guanine is most likely NDPK [21,29]. The multiple interconvertible affinity states of the pancreatic acinar cell C C K g receptor are believed to be regulated via interaction of this receptor with one or more heterotrimeric G proteins including Gq [1,3,4,6,12]. However, in contrast to findings in pancreatic membranes in which most nucleotides tested could inhibit ~25I-BH-CCK8 binding to nonspecific levels, in CHO cell membranes none of the nucleotides examined could inhibit J25I-BHCCK8 binding to nonspecific levels. This may indicate that in CHO cell membranes some of the C C K g receptor is not coupled to G proteins. We previously found that AMP-PCP, a nonhydrolyzable ATP analogue was unable to induce two CCK binding affinity states [21]. As in the former study, the failure of a nonhydrolyzable ATP analogue to induce two CCK binding affinity states suggests that the nucleotide effects are not merely the result of nucleotides binding to G proteins or the CCK receptor. Additionally, this finding suggests that hydrolysis of these nucleotides is necessary for the induction of multiple CCK binding affinity states. Furthermore, though PKC mediated phosphorylation is associated with heterologous desensitization of the CCK A receptor in pancreatic acini [30,31], we have previously demonstrated, using broad-spectrum protein kinase inhibitors, that phosphorylation is not important for the regulation of CCK A receptor affinity [21]. The evidence is most consistent with NDPK exerting its activity in an indirect manner, influencing CCK receptor-G protein interaction through modulation of local GTP concentrations. In

91

view of the ability of ATP to induce two binding affinity states of the CCK receptor in CHO cell membranes it is expected that GTP[TS], a nonhydrolyzable guanine nucleotide would have the same capacity. Nonhydrolyzable guanine nucleotides are known to induce functional uncoupling of agonist-receptor-G protein ternary complexes, subsequently modulating the affinity of a number of G protein-coupled receptors which exist in interconvertible binding affinity states [32-34]. These phenomena are believed to occur as a result of the persistent G protein stimulation that results from the ability of nonhydrolyzable guanine nucleotide analogues to resist the inherent GTPase activity of G proteins. The ability of GTP[3~S] to decrease the total number of measurable CCK receptors in a recombinant system further supports the paradigm that the affinity state of peptide hormone receptors does depend upon receptor-G protein interactions [35]. Others have studied the effects of GTP[3,S] on adrenergic and muscarinic receptors both of which have the ability to exist in interconvertible binding affinity states. In the presence of GTP['y S] these receptors exhibit a shift of some percentage of these receptors from high affinity to low affinity, with no decrease in the total measurable number of receptors. Taking into consideration the ability of CCK receptors solubilized free of G proteins to bind agonist with an affinity similar to the low affinity state of this receptor, the present study supports the model we have previously [11] suggested, that GTP[~/S] elicits a decrease in measurable CCK receptors through induction of a third CCK agonist binding affinity state too low to measure. That such a reduction of binding is also seen for bombesin receptors [11] suggests that this may be a general finding for polypeptide receptors. In view of the ability of hydrolyzable nucleotides to induce two binding affinity states in CHO cell membranes from which the preponderance of endogenous GDP is lost, this study once again raises the question of whether NDPK can transfer phosphate groups from ATP to G protein bound GDP, a phenomenon that has been suggested in other systems [36,37]. Recent in vitro evidence suggests that this is not the case [20,38]. However, the in vitro evidence does not address modulation of the GDP/GTP ratio at the inner surface of -the plasma membrane by NDPK which could result in channeling of newly formed GTP into G proteins. Considering the concept of compartmentalization, if NDPK and CCK receptors coexist in microcompartments along with G proteins NDPK might act by impacting the GTP concentration within these compartments. Furthermore, in a different system others have demonstrated that exogenously added GTP is less potent than GTP formed endogenously by NDPK for inhibition of equilibrium binding of [3H]FMLP to formyi peptide, again suggesting that compartmentalization plays a role [39]. In summary, nucleotides capable of serving as substrates for NDPK induce two measurable CCK binding affinity states on membranes from CHO cells transfected

92

G.T. Blevins et aL / Regulatory Peptides 61 (1996) 87-93

with the C C K A receptor. The data presented in the present study demonstrates that NDPK is present in CHO cells, catalyzes the transfer of high energy phosphate groups from nucleoside triphosphates to GDP resulting in the formation of GTP and this process plays a role in the regulation of the affinity of the recombinant C C K A receptor. Thus the different affinity states of the CCK receptor observed on pancreatic acinar cells are most certainly due to a single receptor molecule interacting with nucleotide binding proteins.

Acknowledgements This work was supported by NIH grant DK41225 to J.W. and by the Michigan Gastrointestinal Peptide Center (DK34933). G.B. was supported by NIH Training Grant T32 DK07367.

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