desensitization of neuromedin b receptors (nmb-r) on native and

8
THE JOURNAL OF BIO~ICAL CHEMISTRY Vol. 269, No. 16, Issue of April 22, pp. 11721-11728, 1994 Printed in USA Desensitization of Neuromedin B Receptors (NMB-R) on Native and NMB-R-transfected Cells Involves Down-regulation and Internalization* (Received for publication, January 6, 1994, and in revised form, February 9, 1994) Richard V. Benya*, Takashi Kusuis, Fukuko Shikados, James E Batteyg, and Robert T. JensenS From the $Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases and the EiLaboratorv of Biological Chemistn? DeveloDmental Therapeutics Program, National Cancer Institute, National Znstitutes if Health, Beihesda,-Maryland 20892 The receptor for neuromedin B (NMB-R),a mamma- lian bombesin-related peptide, is widely distributed in the central nervous system and gastrointestinal tract. While it is known that thisreceptor is coupled to phos- pholipase C, like many other phospholipase C-activating receptors, little is known about regulation of the NMB-R subsequent to agonist stimulation. We studied both na- tive NMB-R on C-6 rat glioblastoma cells and wild type NMB-R cloned from rat esophageal muscle which was stably transfected into Balb/3T3 fibroblasts. Both cell types rapidly increased [SHlinositol phosphates and [Ca2+I1 in response to 1 pm NMB, whereas preincubation with 3 11~ NMB for 3 h markedly attenuated the ability of 1 NMB, but not 1 pm endothelin-1,to alter either cell type’s biological activity. Prolonged exposure to 3 m NMB caused a rapid decrease in the number of NMB-R, with the maximal receptor down-regulationseen at 24 h due to NMB-R internalization. After maximal down- regulation, removal of agonist resulted in a rapid resto- ration of NMB-R to the cell surface of both cell types. NMB-R recovery at 6 h was blocked by monensin, an inhibitor of receptor recycling, but was not affected by cycloheximide, a protein synthesis inhibitor. Resensiti- zation to agonist paralleled the recovery ofNMB-R in both cell types, and resensitization likewise was blocked by monensin. Our data demonstrate that the NMB-R un- dergoes rapid homologous desensitization consequent to agonist stimulation, which is mediated by receptor down-regulation and which, in turn, is regulated by in- ternalization. During resensitization, NMB-R reappear- ance on the cell surface membrane is independent of protein synthesis and is due to a recycling from an in- tracellular site. Bombesin-like peptides are responsible for a wide range of actions including acting as a growth factor (1-3), regulating various central nervous system functions (4-6), and stimulat- ing the release of numerous gastrointestinal peptides (7,8) and have potent stimulatory effects on numerous gastrointestinal tissues including smooth muscle, pancreas, and stomach (9- 11). Two receptors for the mammalian homologues of bombesin have been cloned and include the gastrin-releasingpeptide re- ceptor (GRP-R)’ (12, 13) and the neuromedin B receptor (NMB-R) (14). Both receptors are widely distributed in the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked indicate this fact. “advertisement” in accordance with 18 U.S.C. Section 1734 solely to The abbreviations used are: GRP-R, gastrin-releasing peptide re- ceptor; NMB-R, neuromedin B receptor; Et, endothelin; DMEM, Dul- becco’s modified Eagle’s medium. central nervous system and peripheral tissues, and activation of either receptor is coupled to phospholipase C in all tissues studied (15-20). Similar to other G-protein-coupled receptors, recent studies by some (21,22), but not others (23,241, suggest that activation of at least the GRP-R may result in its desensitization, down- regulation, and internalization. Specifically, studies show that consequent to incubation with agonist, the GRP-R undergoes homologous desensitization in Swiss 3T3 fibroblasts and pan- creatic acinar cells as well as undergoes rapid internalization in a number of cell systems (21, 23-27). While it has been proposed in at least one study (22) that internalization of the GRP-R may mediate its down-regulation and possibly is linked to its desensitization, that relationship has not been studied in detail. The lack of information regarding these processes in receptors for bombesin-related peptides is similar to the case for most other phospholipase C-linked G-protein-coupled recep- tors. In contrast to p-adrenergicreceptors, the relationship between the ability of agonists to increase phospholipase C function and cause desensitization, down-regulation, and inter- nalization has not been studied extensively and is generally unclear. The cellular basis for agonist-induced changes in receptor function by causing desensitization, down-regulation, and in- ternalization of G protein-coupled receptors has been best stud- ied for the P,-adrenergic receptor. Upon agonist exposure, the p,-adrenergic receptorrapidly uncouples from its G-protein (28). P,-Adrenergic receptor activation is followed by seques- tration of the receptor, likely due to its internalization via coated pits (29). With continued exposure to agonist, the inter- nalized receptor undergoes degradation (28). Recent work has identified the structural components of the &adrenergic re- ceptor involved in mediating a number of these processes, as well as identified the importance of cAMP-mediated kinases and second messenger-independent G protein-coupled receptor kinases in mediating these changes (30-32). Much less is known about theprocesses involved in mediat- ing these changes in receptors that activate phospholipase C, and a number of results suggest that the relationships between desensitization, down-regulation, and internalization as pro- posed for the &-adrenergic receptor mayhave limited applica- bility to phospholipase C-linked receptors. First, the desensiti- zation of the P,-adrenergic receptor caused by exposure to low concentrations of agonists is believed to be mediated via protein kinase A (33). Most phospholipase C-activating receptors do not concomitantly increase proteinkinase A thus, this second mes- senger is likely not involved in the desensitization of many phospholipase C-linked receptors. Although some phospho- lipase C-linked receptors also activate adenylate cyclase, a re- cent study with the GRP receptor (34) questions the potential role of adenylate cyclase activation in mediating this receptor’s 11721

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Page 1: Desensitization of Neuromedin B Receptors (NMB-R) on Native and

THE JOURNAL OF B I O ~ I C A L CHEMISTRY Vol. 269, No. 16, Issue of April 22, pp. 11721-11728, 1994 Printed in U S A

Desensitization of Neuromedin B Receptors (NMB-R) on Native and NMB-R-transfected Cells Involves Down-regulation and Internalization*

(Received for publication, January 6, 1994, and in revised form, February 9, 1994)

Richard V. Benya*, Takashi Kusuis, Fukuko Shikados, James E Batteyg, and Robert T. JensenS From the $Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases and the EiLaboratorv of Biological Chemistn? DeveloDmental Therapeutics Program, National Cancer Institute, National Znstitutes if Health, Beihesda,-Maryland 20892

The receptor for neuromedin B (NMB-R), a mamma- lian bombesin-related peptide, is widely distributed in the central nervous system and gastrointestinal tract. While it is known that this receptor is coupled to phos- pholipase C, like many other phospholipase C-activating receptors, little is known about regulation of the NMB-R subsequent to agonist stimulation. We studied both na- tive NMB-R on C-6 rat glioblastoma cells and wild type NMB-R cloned from rat esophageal muscle which was stably transfected into Balb/3T3 fibroblasts. Both cell types rapidly increased [SHlinositol phosphates and [Ca2+I1 in response to 1 pm NMB, whereas preincubation with 3 1 1 ~ NMB for 3 h markedly attenuated the ability of 1 NMB, but not 1 pm endothelin-1, to alter either cell type’s biological activity. Prolonged exposure to 3 m NMB caused a rapid decrease in the number of NMB-R, with the maximal receptor down-regulation seen at 24 h due to NMB-R internalization. After maximal down- regulation, removal of agonist resulted in a rapid resto- ration of NMB-R to the cell surface of both cell types. NMB-R recovery at 6 h was blocked by monensin, an inhibitor of receptor recycling, but was not affected by cycloheximide, a protein synthesis inhibitor. Resensiti- zation to agonist paralleled the recovery of NMB-R in both cell types, and resensitization likewise was blocked by monensin. Our data demonstrate that the NMB-R un- dergoes rapid homologous desensitization consequent to agonist stimulation, which is mediated by receptor down-regulation and which, in turn, is regulated by in- ternalization. During resensitization, NMB-R reappear- ance on the cell surface membrane is independent of protein synthesis and is due to a recycling from an in- tracellular site.

Bombesin-like peptides are responsible for a wide range of actions including acting as a growth factor (1-3), regulating various central nervous system functions (4-6), and stimulat- ing the release of numerous gastrointestinal peptides (7,8) and have potent stimulatory effects on numerous gastrointestinal tissues including smooth muscle, pancreas, and stomach (9- 11). Two receptors for the mammalian homologues of bombesin have been cloned and include the gastrin-releasing peptide re- ceptor (GRP-R)’ (12, 13) and the neuromedin B receptor (NMB-R) (14). Both receptors are widely distributed in the

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked

indicate this fact. “advertisement” in accordance with 18 U.S.C. Section 1734 solely to

The abbreviations used are: GRP-R, gastrin-releasing peptide re- ceptor; NMB-R, neuromedin B receptor; Et, endothelin; DMEM, Dul- becco’s modified Eagle’s medium.

central nervous system and peripheral tissues, and activation of either receptor is coupled to phospholipase C in all tissues studied (15-20).

Similar to other G-protein-coupled receptors, recent studies by some (21,22), but not others (23,241, suggest that activation of at least the GRP-R may result in its desensitization, down- regulation, and internalization. Specifically, studies show that consequent to incubation with agonist, the GRP-R undergoes homologous desensitization in Swiss 3T3 fibroblasts and pan- creatic acinar cells as well as undergoes rapid internalization in a number of cell systems (21, 23-27). While it has been proposed in at least one study (22) that internalization of the GRP-R may mediate its down-regulation and possibly is linked to its desensitization, that relationship has not been studied in detail. The lack of information regarding these processes in receptors for bombesin-related peptides is similar to the case for most other phospholipase C-linked G-protein-coupled recep- tors. In contrast to p-adrenergic receptors, the relationship between the ability of agonists to increase phospholipase C function and cause desensitization, down-regulation, and inter- nalization has not been studied extensively and is generally unclear.

The cellular basis for agonist-induced changes in receptor function by causing desensitization, down-regulation, and in- ternalization of G protein-coupled receptors has been best stud- ied for the P,-adrenergic receptor. Upon agonist exposure, the p,-adrenergic receptor rapidly uncouples from its G-protein (28). P,-Adrenergic receptor activation is followed by seques- tration of the receptor, likely due to its internalization via coated pits (29). With continued exposure to agonist, the inter- nalized receptor undergoes degradation (28). Recent work has identified the structural components of the &adrenergic re- ceptor involved in mediating a number of these processes, as well as identified the importance of cAMP-mediated kinases and second messenger-independent G protein-coupled receptor kinases in mediating these changes (30-32).

Much less is known about the processes involved in mediat- ing these changes in receptors that activate phospholipase C, and a number of results suggest that the relationships between desensitization, down-regulation, and internalization as pro- posed for the &-adrenergic receptor may have limited applica- bility to phospholipase C-linked receptors. First, the desensiti- zation of the P,-adrenergic receptor caused by exposure to low concentrations of agonists is believed to be mediated via protein kinase A (33). Most phospholipase C-activating receptors do not concomitantly increase protein kinase A thus, this second mes- senger is likely not involved in the desensitization of many phospholipase C-linked receptors. Although some phospho- lipase C-linked receptors also activate adenylate cyclase, a re- cent study with the GRP receptor (34) questions the potential role of adenylate cyclase activation in mediating this receptor’s

11721

Page 2: Desensitization of Neuromedin B Receptors (NMB-R) on Native and

11722 NMB-R Desensitization

desensitization, down-regulation, and internalization. Second, since P-adrenergic receptor kinase does not phosphorylate the qadrenergic receptor (35), but can phosphorylate the phos- pholipase C-coupled substance P receptor (361, the role of sec- ond messenger-independent G-protein-coupled receptor ki- nases mediating these receptor changes in phospholipase C-linked receptors is not universal. Third, the difference in kinetics of desensitization of adenylate- and some phospho- lipase C-coupled receptors suggest that different processes may be involved. Whereas &-adrenergic receptor desensitization is rapid (33), the time course for agonist-induced desensitization of various phospholipase C receptors is variable. For some mus- carinic cholinergic receptors coupled to phospholipase C, desen- sitization requires hours of exposure to agonist (37-39), whereas desensitization with others such as those for sub- stance P, histamine, or platelet-activating factor occurs within minutes (40-42). Fourth, with prolonged agonist incubations, many phospholipase C receptors undergo down-regulation and internalization; however, because activation of these receptors by agonists generally does not increase CAMP, activation of CAMP-mediated kinases should not play a role in mediating their down-regulation as it does for the &adrenergic receptor (33).

To address these issues, in the present study we determined the ability of agonists to cause desensitization, down-regula- tion, and internalization in cells containing native and stably transfected NMB receptors which are coupled only to phospho- lipase C .

EXPERIMENTAL PROCEDURES Materials-Balb/3T3 fibroblasts were obtained from the American

Type Culture Collection (Rockville, M D ) and were then subject to clonal expansion to identify a line devoid of NMB-R or GRP-R as determined by binding and RNase protection assays. C-6 rat glioblastoma cells were also obtained from ATCC. Dulbecco's modified essential medium, fetal bovine serum, and aminoglycoside G-418 were all from Life Technolo- gies, Inc.; while cell culture dishware was from Costar. Bovine serum albumin (fraction V) and HEPES were obtained from Boehringer Mann- heim; soybean trypsin inhibitor, EGTA, trypsin, dithiothreitol, and bac- itracin were obtained from Sigma; glutamine was from the Media Sec- tion, National Institutes of Health; neuromedin B (NMB) and endothelin-1 (Et-1) were obtained from Peninsula Laboratories (Bel- mont, CA); disuccinimidyl suberate, rn-maleimidobenzoyl-N-hydroxy- succinimide ester, and IODO-GEN were from Pierce; Nalz6I was from Amersham; rny0-[2-~H]inositol(1&20 Ci/mmol) was from DuPont NEN; Dowex AGl-X8 anion exchange resin (100-200 mesh, formate form), bromphenol blue, and molecular weight standards were from Bio-Rad; Hydro-Fluor scintillation fluid was from J . T. Baker Co. (Phillipsburg, NJ); phosphate-buffered saline was from Biofluids (Ruckville, MD); glycerol was from Mallinckrodt; Coomassie Blue R-250 was from SchwardMann Biotech; and cycloheximide and monensin were from Calbiochem. Standard buffer consisted of 98 m NaCl, 6 m KC1,25 nw HEPES, 5 m pyruvate, 5 m fumarate, 5 m glutamate, and 0.1% soybean trypsin inhibitor.

fiansfection and Maintenance of Cell Lines-As described previously (18), Balb/3T3 cells expressing a stably transfected rat NMB-preferring bombesin receptor clone were generated from a NMB receptor clone isolated from a rat esophagus cDNA library and which was subcloned into a modified version of the pCD2 plasmid. Cells were passaged every 3-4 days at confluence, using 0.1% trypsin in 1 m EDTA. Rat glioblas- toma C-6 cells were maintained similarly and were passaged at conflu- ence. Both cell lines were cultured in DMEM containing 10% fetal bovine serum, with NMB-R-transfected cells additionally supplemented

Preparation of '251-[~-~~lNMB-1261-[~-~olI\TMB (2200 Ci/mmol) was prepared by adding 0.4 pg of IODO-GEN to 8 pg of [D-m'INMB with 2 pCi of Nalz6I in 20 pl of 0.5 M KPO, buffer (pH 7.4) as described previously (43). Briefly, after incubation at 22 "C for 6 min, 300 pl of 1.5 M dithiothreitol was added and the reaction mixture was incubated at 80 "C for 60 min. Free '"I was separated by applying the reaction mixture to a Sep-Pak cartridge (Waters Associates) which had been prepared by washing with 5 ml of methanol and 5 ml of 0.1% trifluoro- acetic acid; and the radiolabeled peptide was then eluted with 200-pl

with 280 &ml G-418.

sequential elutions ( ~ 1 0 ) of 60% acetonitrile/O.l% trifluoroacetic acid. Radiolabeled peptide was separated from unlabeled peptide by combin- ing the 3 elutions with the highest radioactivity and applying them to a reverse phase high performance liquid chromatograph (Waters &SO-

ciates, Model 204, equipped with a Rheodyne injector), using a 0.46 X 25 cm p-BondaPak column. The column was eluted with a linear gradient of acetonitrile and 0.1% trifluoroacetic acid from 16 to 64% acetonitrile in 60 min, with a flow rate of 1.0 d m i n . '261-[~-Tyro]NMB was stored with 1% (w/v) bovine serum albumin at -20 "C and was stable for at least 6 weeks.

Binding of '251-[~-~PlNMB to NMB-R-transfected and C-6 Cells- Binding studies using rat glioblastoma C-6 cells or NMB-R-transfected cells were performed by suspending disaggregated cells in binding buffer (standard buffer additionally containing 1 n w MgCl,, 0.5 m CaCl,, 2.2 m KHPO,, 2 m glutamine, 11 m glucose, 0.1% (wh) bacitracin, and 0.2% bovine serum albumin (w/v) (pH 7.4)). NMB-R- transfected cells were suspended at a concentration of 1 x lo6 celldml, whereas C-6 cells were suspended at a concentration of 15 x lo6 cells/ ml. Incubations contained 50 PM '251-[~'Tp'lNMB for 30 min at 22 "C, as previously described (18, 43). Nonsaturable binding of '261-[~- Tyr'INMB was the amount of radioactivity associated with C-6 cells or NMB-R-transfected cells when the incubation mixture contained 1 w NMB. Nonsaturable binding was <15% of total binding in all experi- ments, and all values in this paper are reported as saturable binding (i.e. total minus nonsaturable binding).

Measurement oflnositol Phosphates-%tal inositol phosphates were determined for either cell type by using a modification of the method of Berridge et al. (44) and as demonstrated previously (18,45). Cells were split 1:4 into 24-well plates and grown to confluence. Cells were then washed, and the media were replaced with DMEM without fetal bovine serum. For desensitization assays (see below), one plate further con- tained 3 m NMB and was processed in parallel with a second plate devoid of NMB. Both the control and NMB-incubated plates were then incubated at 37 "C for 24 h. During the final 6 h of this incubation, 100 pCi/ml rny0-[2-~HIinositol was added to both the NMB-pretreated and control flasks. Cells were then washed twice with ice-cold IP buffer (standard buffer additionally containing 10 m LiCl, 2 m CaCl,, 2% bovine serum albumin, and 1.2 m MgSO,), and then were exposed to either 1 w NMB or to 1 w endothelin-1 for variable lengths of time. Reactions were halted using ice-cold 1% HCl in methanol (v/v), and the inositol phosphates were isolated using a Dowex anion exchange col- umn. Free [3Hlinositol was removed by washing with 4 volumes of water; [3Hlglycerophosphoryl inositol was removed by washing with 4 volumes of 5 m disodium tetraborate in 60 m sodium formate; while [3Hlinositol phosphates were eluted using 100 nw formic acid in 1.0 m ammonium formate. Eluates were then assayed for their radioactivity after the addition of Hydro-Fluor scintillation fluid.

Measurement of [Ca2+li-Cells were mechanically disaggregated, re- suspended in binding buffer at a concentration of 2 x lo6 celldml con- taining 2 fura-WAM, and incubated at 22 "C for 45 min. ARer loading with fura-2, cells were washed three times in binding buffer. For meas- urement of [Ca2'],, 2-ml samples were placed in quartz cuvettes in a Delta PTI scan-1 spectrophotometer (PTI Instruments, Gaithersburg, MD). This instrument was modified so as to maintain an incubation temperature of 37 "C while continuously mixing the cuvette contents by means of a magnetic stirrer. Fluorescence was measured at 500 nm after excitation at 340 nm and at 380 nm. Autofluorescence of the unloaded cells was subtracted from all measurements, and [Ca2+li was calculated according to the method of Grynkiewicz et al. (46).

NMB Receptor Down-regulation and Desensitization-Cells were split 1:2, and 48 h later were washed once in phosphate-buffered saline. One-half of the cells were resuspended in DMEM containing 3 m NMB, whereas the other half were resuspended in DMEM alone. At various time points, cells were harvested for binding experiments, and total cellular inositol phosphates or [Ca2+li was determined as described above. Analysis of the binding data using the least squares curve-fitting program LIGAND (47) permitted comparisons in mathematically de- rived receptor number (Bmm) and affinity (Ki) between NMB-pretreated and control cells. Down-regulation was expressed as the percent of control receptor number present on NMB-pretreated cells as compared to untreated control cells which were processed in parallel. Desensiti- zation was defined as that decrease in ability of 1 p NMB to alter cell activity and increase total cellular inositol phosphates or [CaZ+l, a t variable time points after preincubation with 3 m NMB.

NMB-R Internalization-Internalization of NMB-R was measured as that percentage of '251-[~-Ty#']NMB resistant to acid wash using the method of Haigler et al. (48) as described previously (18). Disaggregated cells were incubated with 75 PM '251-[~-"yr'lNMB for various times at

Page 3: Desensitization of Neuromedin B Receptors (NMB-R) on Native and

NMB-R Desensitization 11723

FIG. 1. Effect of preincubation with (NMB-R TRANSFECTED CELLSJ NMB on subsequent stimulation by NMB or endothelin-1 to increase ino- 1 1 NMB Stirnulation] sit01 phosphates. Cells were split 1:4 into 24-well plates and grown to conflu- ence, and the media were replaced with DMEM without serum. Cells were loaded with 100 pCi/ml my0-[2-~HIinositol for the final 6 h of incubation and then processed as described under "Experimental Proce- dures." Increases in total cellular inositol phosphates in C-6 cells were also deter- 3nMNMB - mined consequent to stimulation with 1 p~ endothelin-1 after preincubation with 3 n~ NMB (centerpanel ). For each experi- ment, each value was determined in quadruplicate, and the results are the means f S.E. of at least three separate 515 30 60 90 120515 30 60 90 120 515 30 60 90 120 experiments. TIME (minutes)

100-

4 "C or 37 "C in incubation buffer. ARer incubation, cell samples were added to 10 volumes of 0.2 M acetic acid (pH 2.5) containing 0.5 M NaCl for 5 min at 4 "C or to a similar volume of binding buffer. In all cases, parallel incubations were conducted in the presence of 1 p~ unlabeled NMB to determine changes in nonsaturable binding. Results are ex- pressed as the percentage of saturably bound '2sI-[~-QrolNMB that are internalized.

Cross-linking to NMB Receptors-Cell membranes were prepared by growing C-6 or NMB-R-transfected cells to confluence, washing once in binding buffer, and then resuspending in homogenization buffer (50 m~ Tris (pH 7.4), 0.2 mg/ml soybean trypsin inhibitor, 0.2 mg/ml benzami- dine). Cells were then homogenized on ice using a Polytron (Beckman Instruments) at speed 6 for 30 s. The homogenate was then centrifuged at 1500 rpm for 10 min in a Sorval RC-5B Superspeed centrifuge (Du- Pont NEN); the supernatant was removed and recentrifuged a t 20,000 rpm for 20 min. The pellet was resuspended in homogenization buffer and stored at -40 "C.

Cell homogenates were diluted with binding buffer to the concentra- tion of 0.25 and 1.5 mg of proteidml for NMB-R-transfected cells and for C-6 cells, respectively. 500-pl aliquots were preincubated with 0.5 n~ '261-[~-Tyro]NMB at 22 "C in 1.6-ml polypropylene tubes. After a 15-min incubation, the reaction mixture was centrifuged at 10,000 x g for 3 min. The pellet was washed twice in 1 ml of phosphate-buffered saline (4 "C) and resuspended in 200 p1 of cross-linking buffer (50 m~ HEPES (pH 7.5), 5 m~ MgCI,) containing 1 m~ m-maleimidobenzoyl-N- hy- droxysuccinimide as a cross-linking agent. After cross-linking at 22 "C for 30 min, the reaction was stopped by adding 25 pl of 1 M glycine. ARer 10 min on ice, the sample was centrifuged at 10,000 x g for 3 min. The supernatant was aspirated and the pellet was resuspended in 100 pl of 120 m~ Tris/HCl (pH 6.8). A 6-pl aliquot of the mixture was reserved to determine protein concentration. Cross-linked membranes were solubi- lized by adding 25 pl of gel loading buffer (0.4 M TridHC1 (pH 6.8), 20% SDS (w/v), 50% glycerol (v/v), 0.05% bromphenol blue (w/v), and 0.5 M dithiothreitol) at 22 "C for 60 min. After adjusting the protein concen- tration, 10 pg of proteidane of NMB-R-transfected cell membrane and 80 pg of proteidane of C-6 cell membrane were applied to a 3% (v/v) acrylamide, 0.1% (w/v) SDS stacking gel over a 10% (v/v) acrylamide, 0.1% (w/v) SDS separating gel that was 1.5 mm thick. Solubilized mem- branes were subjected to SDS-polyacrylamide gel electrophoresis using the Laemmli buffer system as previously described (49). Electrophoresis was camed out at 40 mAusing 25 m~ Tris, 0.2 M glycine, and 0.1% (w/v) SDS. Gels were stained with 0.1% (w/v) Coomassie Blue R-250 in 40% (v/v) ethanol and 10% (v/v) acetic acid and destained with 10% (v/v) ethanol and 7.5% (v/v) acetic acid. After overnight destaining, gels were equilibrated in 45% (vh) ethanol and 5% (vh) glycerol for 30 min and dried in a gel-slab drier (Hoefer Scientific Instruments, Model SE 540). Dried gels were exposed to storage phosphor screens for 3 days at 22 "C and processed using a PhosphorImager (Molecular Dynamics).

NMB Receptor Recovery-Cells were split 12 , and one-half of the cells were at the time of confluence exposed to 3 IIM NMB in DMEM for 24 h. Both the pretreated and the control cells were then washed twice in DMEM and then resuspended in DMEM. At various time points, cells were harvested for [3Hlinositol phosphate studies and binding experi- ments, while the binding data were analyzed using the least squares curve-fitting program LIGAND (47). To assess the mechanism regulat- ing recovery, additional experiments were performed with monensin (30 p ~ ) (50) or cycloheximide (0.1 m ~ ) (51) added to the cells after a 24-h incubation with 3 nM NMB and after washing twice in DMEM. Recovery

was expressed as the percent of control receptor number present on NMB-pretreated cells as compared to untreated cells processed in parallel.

All experiments comparing NMB receptor function in C-6 cells and NMB-R-transfected cells were performed in parallel on the same day. Consequently, all comparisons were made using the Student's t test, and values for p < 0.05 were considered to be significant. All data are expressed as means f S.E.

RESULTS

Binding of '251-[~-T'yr0]NMB to C-6 cells and to NMB-R-trans- fected cells was similar, demonstrating a much higher affinity for NMB than the related peptide GRP. NMB caused detectable inhibition at 0.1 IIM in both cell types and half-maximal inhi- bition (K,) at 1.9 f 1.1 IIM in C-6 cells and at 3.0 f 0.1 IIM in NMB-R-transfected cells. In both cell types, complete inhibition of '261-[~-Tyro]NMB binding was observed with 1 1.1~ NMB. In contradistinction, GRP displaced significantly less '261-[~- Tyr'INMB and was found to have an affinity for the NMB receptor of 520 f 30 IIM in C-6 cells and of 440 2 70 nM in NMB-R-transfected cells. C-6 cells also were found to possess endothelin type A receptors (data not shown). Detectable inhi- bition of lZ5I-Et-l was observed with 0.1 IIM endothelin-1, com- plete inhibition was observed with 1 PM endothelin-1, and half- maximal inhibition (K, ) was observed at 1.9 0.4 nM endothelin-1. In contrast, 1 p~ endothelin-3 displaced less than 50% of lZ5I-Et-l (data not shown).

In order to ascertain whether NMB receptor desensitization occurs, we determined the effect of preincubating both C-6 cells and NMB-R-transfected cells with 3 MI NMB for 24 h on the subsequent ability of either cell to generate inositol phosphates when stimulated with 1 1.1~ NMB. When stimulated with 1 p~ NMB, control C-6 and NMB-R-transfected cells rapidly in- creased total cellular inositol phosphates, with an increase from 890 f 30 dpm to 4,200 z 100 dpm in C-6 cells and from 8,200 4,100 dpm to 103,000 2 11,200 dpm in NMB-R-trans- fected cells (Fig. 1, left and right panels). For both C-6 and NMB-R-transfected cells, preincubation for 24 h with 3 nM NMB, the concentration that half-maximally inhibited lZ5I-[~- Tyr'INMB binding to both C-6 and NMB-R-transfected cells, markedly attenuated the ability of either cell type to respond to additional 1 p~ NMB. After 3 IIM NMB preincubation for 24 h, C-6 cells increased inositol phosphates from a basal value of 920 2 50 dpm to 2,100 100 dpm, while NMB-R-transfected cells increased inositol phosphates from 15,400 2 5,100 dpm to 28,300 2 1,600 dpm with the addition of 1 1.1~ NMB after 120 min (Fig. 1, left and right panels). Thus, the ability of C-6 cells to respond to maximal concentrations of NMB was attenuated by 64 f 7%; while the ability of NMB-R-transfected cells was attenuated by 86 f 9%. To ascertain whether this desensitiza- tion was homologous or heterologous in nature, we next deter-

Page 4: Desensitization of Neuromedin B Receptors (NMB-R) on Native and

11724 NMB-R Desensitization

0 100 0 50 100 TIME (seconds)

FIG. 2. Effect of preincubation with NMB on subsequent stimu- lation of changes in [Ca2+l, by NMB or endothelin in 6-6 cells. C-6 cells were incubated in DMEM alone or with 3 nM NMB for 3 h and were exposed to 2 pn4 fura-2lAM for the final 30 min of the incubation. Cells were washed and fluorescence was determined immediately as de- scribed under "Experimental Procedures." These tracings are repre- sentative of at least three separate experiments.

mined whether stimulation of increases in [3Hlinositol phos- phate by endothelin-1 was altered (Fig. 1, center panel). In control C-6 cells, 1 p~ increased [3Hlinositol phosphates from 420 2 50 dpm to 2,200 k 100 dpm; whereas in C-6 cells, prein- cubated with 3 nM NMB, the response was unchanged with 1 p~ endothelin-1 increasing [3H]inositol phosphates from 800 20 dpm to 2,600 2 100 dpm (Fig. 1, center panel 1.

Further evidence for homologous desensitization was ob- tained by evaluating the ability of C-6 cells to increase cytosolic calcium when pretreated with 3 n~ NMB (Fig. 2). In control cells, 1 p~ NMB increased [Ca"], from 77 2 10 nM to 228 k 22 nM (Fig. 2, left panel, top tracing). After preincubating C-6 cells with 3 m NMB for as short as 3 h, basal [Ca2+Ii was not significantly different from control cells (75 r 8 m). However, subsequent exposure to 1 NMB markedly attenuated the maximal increase in [Ca2+l, (128 2 12 m) without altering the rate of [Ca"], increase (Fig. 2, left panel, lower tracing). In contrast, preincubating C-6 cells with 3 nM NMB had no effect on the ability of 1 PM endothelin-1 to increase [Ca"], (Fig. 2, right panel). Collectively, these data demonstrate that NMB receptor down-regulation is associated with marked homolo- gous desensitization of this receptor's intracellular signaling pathways.

To ascertain whether the desensitization observed was due to an alteration in NMB receptor binding, we next determined the effect of preincubating both C-6 cells (Fig. 3, left panel) and NMB-R-transfected cells (Fig. 3, right panel) with half-maxi- mal concentrations of NMB (3 nM) on NMB receptor number and affinity. For both cell types, there was a decrease in recep- tor number. Maximal binding of '251-[~-Tyro]NMB to cells ex- posed to 3 n~ NMB rapidly decreased with time. To define whether this decrease was due to a decrease in receptor affinity or number, the NMB dose inhibition curve at different times was analyzed using a least squares curve-fitting program (47). At no point in time was receptor affinity for NMB significantly altered, including after 24 h of preincubation with 3 nM NMB (K, = 4.2 2 0.8 n~ control transfected cells uersus 3.8 2 0.6 m pretreated transfected cells) (data not shown). The initial rate of NMB receptor down-regulation was rapid, with maximal receptor down-regulation achieved between 3 and 6 h after exposure to 3 nM NMB. To further confirm that the decrease in pharmacologically derived receptor number (Bmm) in fact rep- resented an actual decrease in NMB receptors, cell membranes of either cell type were isolated at various time points after exposure to 3 m NMB and were subject to receptor cross- linking studies (Fig. 3, insets). Again, a rapid decrease in cross- linked NMB receptor was observed in membranes of both C-6 cells and NMB-R-transfected cells, with maximal down-regu- lation achieved after approximately a 3-h exposure to 3 m NMB in both cell types.

~ M B - R TRANSFECTED CELL:

-1 I , l"L-LhLL 1 2 3 4 5 6 / 2 4 . 1 2 3 4 5 6 2 4

TIME [hours) FIG. 3. Down-regulation of NMB receptors from C-6 cells (left)

or NMB-R-transfected cells (right). Confluent C-6 cells or NMB-R- transfected cells were incubated with 3 n~ NMB in DMEM for the times indicated and were then resuspended in binding buffer containing 50 PM 1251-[~TyrolNMB alone or with varying concentrations of unlabeled NMB. Scatchard analysis of the competitive binding data demonstrated no change in receptor affinity with the decrease in receptor number (BmJ Receptor number is expressed as the percentage of receptors present on control cells processed in parallel. Basal NMB receptor num- ber was 28 t 2 fmol/106 cells on C-6 cells and was 1489 t 221 fmol/106 cells on NMB-R-transfected cells. Inset, membranes from C-6 cells (left panel) or from NMB-R-transfected cells (rzght panel) were incubated with 3 n~ NMB for the indicated times and were then subjected to cross-linking to 1251-[~-TyrolNMB. Equal amounts of cell membrane pro- tein were loaded at each time point per cell type. This gel is represent- ative of two separate experiments.

To evaluate further whether this decrease in NMB receptor number was specific for NMB receptors and similar in the 2 cell types, binding to the NMB receptors and endothelin receptors on C-6 cells and NMB receptors on transfected cells was com- pared (data not shown). After 3 h of preincubation with 3 nM NMB, neither C-6 cells nor NMB-R-transfected cells revealed significant alterations in K, from control cells while both cell lines expressed significantly less receptors Specifically, for C-6 cells, the B,= decreased from 29 2 1 fmoV10' cells to 8 2 1 fmoY10' cells after a 3-h preincubation with 3 rm NMB; while the K, remained unchanged (3.3 2 1.0 m to 2.9 2 0.8 m). Similarly, for NMB-R-transfected cells, the B,,, decreased from 1765 2 155 fmoV10' cells to 436 * 34 fmol/106 cells; while the K, again remained unchanged (2.2 r 0.3 nM to 2.3 2 0.3 I"). There was no change in EtA receptor affinity for endothelin-1 after exposure to 3 nM NMB as compared to control cells (K, = 2.3 -c 0.2 nM control uersus 2.5 2 0.3 n~ pretreated), nor was there any significant alteration in Et, receptor number (Bmm = 1280 r 141 fmoV106 cells control versus 1288 2 209 fmoV10' cells pretreated), demonstrating that the down-regulation was spe- cific for NMB receptors.

We next determined whether NMB receptor internalization occurred in both cell types and might contribute to the rapid receptor down-regulation. Acid stripping studies to remove sur- face-bound ligand with both C-6 cells (Fig. 4, left panel) and on NMB-R-transfected cells (Fig. 4, right panel gave a similar result. In both cell types, NMB receptor internalization was temperature-dependent, with >85% of receptors internalized after a 90-min exposure to radiolabeled ligand at 37 "C, but less than 10% internalized at 4 "C. These data demonstrate that both cell types rapidly internalized bound ligand.

We did not possess a membrane-permeable ligand with which to measure total receptor number and therefore were not able to relate internalization directly to down-regulation. How- ever, we assessed this relationship by determining the ability of maximally down-regulated NMB receptors to recycle to the cell surface. After preincubating either C-6 tells (Fig. 5, left p a w l )

Page 5: Desensitization of Neuromedin B Receptors (NMB-R) on Native and

NMB-R Desensitization 11725

1 0 0 4 t [NMB.R TRANSFECTED CELLSI I 1

4°C

510 20 40 60 90 5 1 0 20 40 60 90

TIME (minutes) FIG. 4. Time and temperature dependence of internalization of

'mI-[D-TyrolNMB in C-6 cells (lefl) or in NMB-R transfected cells (right). C-6 or NMR-R-transfected cells were incuhatrd with 75 PY 1251-1~~-~01NMB for the indicated times. and the aliquots were exposrd to 0.2 M acetic acid in 0.5 M NaCl (pH 2.5) to remove surface-hound ligand. Internalized ligand was the proportion of saturahly hound counts not removed by exposure to acid wash. Results are expressed as the proportion of total saturahly bound ligand at any time point that was not removed by acid exposure. For each experiment, each value was determined in triplicate, with each point representing the mean t S.E. of at least three separate experiments.

: $ 1 20 v / i

I S . < . . # {t I

1 3 6 ' 18 4E-F 18 a /-". 24 TIME (hours)

FIG. 5. Recovery of NMB receptors ( E t & in C-6 cells (Iep) and NMB-R-transfected cells (right) after 24-h exposure to 3 nM NMR. C-6 crlls and NMB-It-transfected crlls were exposed to 3 nM NMI3 in I)MEM for 24 h. washed, disaggregated, and then rrsuspended in DMISM containing 50 PM t2sI-[~)-Tyr"lNMB with and without varying cnncrntrations of unlabeled ligand. Scatchard analysis of the binding data permitted determination of the receptor numher (I?,,,-), which was expressed as the percentage of receptors present on control cells pro- cessed in parallel. For each experiment, each value was determined in duplicate, with each point representing the mean = S.E. of at least three separate experiments.

or NMB-R-transfected cells (Fig. 5, right panel) with 3 nM NMB for 24 h followed by exposure to peptide-free media, there was a rapid return in NMR receptors. Twenty-four h after removal of the peptide, 55 t 10% of NMB receptors on C-6 cells and 75 r 14% of NMB receptors on NMB-R transfected cells were pharmacologically evident, as compared to untreated control cells. The rate of receptor return to the cell surface was similar for both cell types, with half-maximal receptors returned at approximately 6 h and maximal receptors returned at approxi- mately 24 h after removal of NMB (Fig. 5).

To determine whether the initial component of these reap- pearing NMB receptors reflected de nouo synthesis or their recycling, we compared the effects of monensin, which has been shown to inhibit receptor recycling in a number of cell systems ( 5 0 , 5 2 4 6 ) and of cycloheximide, a protein synthesis inhibitor, on both C-6 and NMB-R-transfected cells. Although monensin alone did not affect NMB receptor number on either C-6 cells (Fig. 6, top panel) or on NMB-R-transfected cells (Fig. 6, bottom panel ), its presence markedly attenuated the recovery of NMB

.c UJ I1

I-

e

T=O. No addltlons m4 No addillons

With Monensln i : With Cyclohexamlde [ms/ '

~

Monensin Alone+; C clohexamide ALne

No additions - ' "L" ..A T=O. c ' . . I :IASH

e l No addittons

With Monensin j

Wtth Cyclohexamlde = I I 1-p INMR F1 T R A W r r C T F D

'0 ?; ?? 40 50 60 70 80 90100

(:rLi , "1- .A

~I

FIG. 6. Effect of monensin and cycloheximide on NMR receptor recycling in C-6 cells ( t o p ) and NMR-R-trnnRfected celln (hot- torn). C-6 cells and NMI3-R-transfc.rtrd wlls wrrc' rxpnwd to .'$ nw NVI3 for 24 h so as to maximnlly down-regulate NMR rrcrptor numher f t = 0 1 . Recovery of NMB recrptor binding was drtrrminrd 6 h aftPr t h r crlls were washed and the I).MEM containmg 3 n\c NM13 had hrcn rrplacrd with DMEM alone ( I = 61. Monensin alonr (30 p \ c ~ had no rffrct on NMH receptor numher. hut, whrn prrsrnt during t h r 6-h rrcovry prrirxi, I t totally blocked the recyclinR of rrcrptors to the crll surfncr. Cyclohrx- imide alone f 10 p ~ ) had no effrct on N.MB recrptor numhrr. and, whrn present during the 6-h recovey period, it alno had no rffrct on N.MB receptor recycling to the surface of rithrr C-6 crlls or NMR-R-trans- fected cells ( 1 = 6 ) . Scatchard analysls of thr hinding d a h permitted determination of the receptor numher fHmm,), which was rxpreswd as the percentage of receptors present on control crlls prrmssrd in paral- lel. For each rxprrimrnt. each valur was drtrrminrd in duplicatr. with each point representing thr mran 2 S.E. of at least thrrr srparntr experiments.

receptors in either cell type. Specifically, only 8 t 2"? of control NMR receptors in C-6 cells and 18 t 3c; in NMR-R-transfected cells remained after 24 h of incubation with 3 nM NMR. Six h after removal of the NMB, 45 t 2'7 of control receptors in C-6 cells and 52 t 1 8 in NMR-R-transfected cells could hc detected. With the addition of 30 VM monensin during the 6-h recovery period, only 9 t 2%- of control NMR receptors on C-6 cells and 30 t 4% of NMB receptors on NMR-R-transfected cells could be detected. In contradistinction, cycloheximide alone did not al- ter NMB receptor number by itself in C-6 cells (Fig. 6, f o p panel) or in NMR-R-transfected cells (Fig. 6. botforn pnnrf ), nor did it alter the rate ofNMR receptor recovery in either cell type. These data demonstrate that the increase in NMR receptor number after maximal desensitization is due to a recycling of internalized NMR receptors rather than dr nooo synthesis of receptors.

To relate the possible receptor down-regulation and internal- ization to desensitization, we studied the effect of receptor re- cycling on NMR receptor function. After 24 h of exposure to 3 nM NMB, and with NMR receptors maximally down-regulated. C-6 cells only increased basal inositol phosphates from 1,100 t 120 dpm to 1,800 t 200 dpm whereas NMR-R-transfected cells only increased basal inositol phosphates from 18,300 t 2.800 dpm to 25,100 t 4,800 dpm (Fig. 7). After a 6-h recovery from continuous exposure to 3 nM N,MB, there was a partial resto- ration in the ability of C-6 cells to respond to 1 p~ NMR, with basal inositol phosphates increasing from 860 t 40 dpm to 3000 2 60 dpm (Fig. 7, fefc panr l ) ; R similar recovery was noted in

Page 6: Desensitization of Neuromedin B Receptors (NMB-R) on Native and

11726 NMB-R Desensitization

L C - 6 1

‘”“““L /

5 15 30 60 90 120 5

Y I

.k 6 hr - Rkcovery Monensm wlth

15 30 60 90 120

TIME (minutes) FIG. 7. Ability of monensin to alter the effect of 1 NMB on

increasing cellular inositol phosphates in C-6 cells (left) or NMB-R-transfected cells (right) during NMB receptor recy- cling. Cells were split 1:4 and grown to confluence in 24-well plates, and media were replaced with DMEM alone (CONTROL) or were supplemented with 3 m NMB for 24 h (maximal down-regulation). After complete down-regulation of the NMB receptor on both cell types, cells were washed of peptide and were either resuspended in DMEM alone for 6 h (6-h Recovery) or with 30 pm monensin (6-h Recovery with Monensin). Cells were loaded with 100 mCi/ml my0-[2-~H]inositol for the final 6 h of incubation and then processed as described under “Ex- perimental Procedures.” Both control C-6 cells (left panel) and control NMB-R-transfected cells (right panel) rapidly increased cellular inosi- tol phosphates in response to 1 NMB, from 930 2 50 dpm and 6,700 f 2,000 dpm to 4,200 t 120 dpm and 107,000 t 15,300 dpm. For each experiment, each value was determined in quadruplicate, and the re- sults are the means t S.E. of at least three separate experiments.

NMB-R-transfected cells as inositol phosphates increased from 16,300 2 1,200 dpm to 72,800 2 9,100 dpm (Fig. 7, rightpanel). This recovery in inositol phosphate responsiveness (65% of con- trol in C-6 cells and 56% of control in NMB-R-transfected cells) parallels the degree of NMB receptor recycling to the cell sur- face at this time point (see Fig. 5) . For both C-6 and NMB-R- transfected cells, however, co-incubation with 30 VM monensin blocked the recovery in inositol phosphate stimulation observed a t t = 6 h, correlating with this receptor’s ability to block the reappearance of NMB receptors on the cell surface (Fig. 7).

DISCUSSION

In the present study, we demonstrate that for both rat C-6 glioblastoma cells expressing native NMB-R and for Balb/3T3 cells stably transfected with the cDNA for the NMB-R derived from rat esophageal muscularis mucosa (14, 571, agonist expo- sure to NMB caused NMB receptor down-regulation, internal- ization, and desensitization. In the case of the C-6 glioblastoma cells, these processes were shown to be homologous in nature. The evidence that both cell types demonstrated down-regula- tion was that with exposure to NMB, there was a rapid time- dependent decrease in receptor number whether assessed by binding studies or cross-linking studies. That this was not a nonspecific effect on cell function was demonstrated by the fact that there was no change in Et, receptors on C-6 glioblastoma cells after incubation with 3 rm NMB, whereas the number of NMB receptors decreased by 70%. The evidence for internal- ization was that both cell types demonstrated a rapid increase in the amount of radiolabeled agonist, ‘251-[TyroJNMB, that was not acid-strippable. Numerous studies have demonstrated with various ligands that internalization is a temperature-depend- ent process (18, 19, 25). In the present study, incubations at 4 “C for up to 90 min caused no increase in non-acid-strippable ligand, whereas with incubations at 37 “C, r90% of the ligand by this time existed in a non-acid-strippable state. The evi- dence that both cell types underwent desensitization is that

after incubation with 3 rm NMB, the ability of a maximally effective concentration of NMB to stimulate increases in phos- phoinositides was markedly reduced compared to control cells. Similarly, in C-6 glioblastoma cells after preincubation with 3 IMI NMB, the ability of a maximally effective concentration of NMB ( i e . 1 p d to stimulate increases in [Ca2+Ii was reduced by >50%. The fact that this desensitization was homologous in nature and not simply due to a decreased ability of the cell to respond was demonstrated by the failure of NMB to influence the ability of Et-1 to interact with Et, receptors on C-6 glio- blastoma cells or to stimulate increases in either [Ca2+], or phosphoinositides. For each of the processes of down-regula- tion, internalization, and desensitization, both the kinetics and stoichiometry of the changes seen in native NMB-Rs on C-6 glioblastoma cells and in the NMB-R-transfected 3T3 cells were similar, demonstrating that these changes are not unique to the cell type or due to interaction with small numbers of another receptor subtype. Thus, the kinetics and stoichiometry of de- sensitization, down-regulation, and internalization are intrin- sic properties of the NMB-R.

The main conclusion of the present study is that it is likely that NMB-R desensitization is mediated significantly by the down-regulation of cell surface receptors, which in turn is due to NMB-R internalization after exposure to agonists. Down- regulation and desensitization of a number of phospholipase C-linked receptors such as those for muscarinic cholinergic agents, endothelins, substance P, and cholecystokinin have been reported (40,5842). In the case of cholecystokinin recep- tors (58), certain subtypes of muscarinic cholinergic receptors (59) and receptors for platelet-activating factor (60), the de- crease in agonist potency with agonist incubation is secondary to a decrease in membrane-associated receptor number, whereas in other tissues such as muscarinic cholinergic recep- tors on pancreatic acini, this change is mediated by a decrease in receptor affinity without a change in receptor number (61, 62). In our study, agonist exposure decreased NMB-R receptor number without changing NMB-R affinity. The decrease was receptor-specific, furthermore, because no change in the phos- pholipase C-coupled Et, receptor was seen. That the internal- ization was likely mediating the down-regulation was sug- gested by the similarity in kinetics of the down-regulation and internalization processes. Additionally, a recycling of internal- ized NMB-R to the cell surface was associated with a reversal of the down-regulatory processes. The monocarboxylic acid cat- ion ionophore, monensin, has been shown to inhibit the recy- cling of receptors for insulin, low density lipoproteins, epider- mal growth factor, transfenin, cholecystokinin, and asialoglycoproteins (50, 52-56). When monensin alone was added in the present studies, it had no effect on the number or affinity of NMB receptors. This result is similar to that seen with monensin with insulin receptors on IM-9 lymphocytes and U-937 monocytes (52) and is in contrast to that observed for receptors for low density lipoproteins on fibroblasts (53), epi- dermal growth factor on hepatocytes (54), for transferrin on K562 cells (55), and for asialoglycoproteins on hepatoma cells (56). This suggests that ongoing agonist-independent recycling of the NMB receptor either does not occur or is very slow in both cell types studied. Monensin, however, inhibited the reversal of the down-regulation, supporting the conclusion that the reap- pearance of the cell surface NMB receptors was due to a recy- cling. In some cell systems with prolonged receptor down-regu- lation subsequent to agonist stimulation, such as is seen for the muscarinic cholinergic receptors, the reappearance of down- regulated receptors requires protein synthesis because it is blocked if cycloheximide is included (63, 64). In contrast with NMB-R, cycloheximide had no effect, demonstrating that no synthesis of new receptors was required for their reappearance

Page 7: Desensitization of Neuromedin B Receptors (NMB-R) on Native and

NMB-R Desensitization 11727

on the cell surface. The lack of effect of cycloheximide on the NMB-R reappearance on the cell surface, the ability of the recycling inhibitor monensin to block reappearance and the fact that with the transfected cells S O % of the control agonist pretreatment level of NMB-R reappeared on the cell surface strongly suggests there is minimal NMB-R degradation during the agonist incubation times used in the present study, and that NMB-R reappearance represents a recycling from an in- tracellular site where the receptors had translocated during down-regulation. That the down-regulation, in turn, mediated the desensitization is suggested by a number of results. First, both the down-regulation and desensitization were homologous in nature and thus linked only to activation of the NMB-R. Secondly, the recycling of internalized NMB receptors and re- sultant increase in cell surface NMB receptors closely followed the reversal of the desensitization process. Specifically, after a 6-h recovery period following maximal NMB-R down-regula- tion and desensitization, for both native and transfected NMB-R, approximately 40 to 50% of the cell surface receptors had reappeared and a 40 to 50% recovery in the ability of NMB to stimulate C3H]IP was observed. Thirdly, the coupling of these processes was further supported by the fact that monensin not only blocked the reversal of the down-regulation during a 6-h recovery period, but it also blocked the recovery from the de- sensitization over this time period.

The conclusions for the mechanism of NMB receptor-medi- ated desensitization in the present study agree with previous recent studies on the other receptor for bombesin-related pep- tides, the GRP receptor (22, 65). From binding studies it has been proposed that the GRP receptor undergoes recycling from intracellular sources (22) . In detailed studies relating changes in binding and the ability of bombesin to cause rapid desensi- tization to stimulation of enzyme secretion in guinea pig pan- creatic acini (22), it was proposed that this process is mediated by a rapid loss of unoccupied GRP receptors and that when the active receptors decreased below 50%, desensitization occurs. Similar to our studies, the resensitization of GRP-R in pancre- atic acini (22) was found to not be affected by cycloheximide and it was proposed that possible recycling of internalized inactive GRP receptors might explain the resensitization. This conclu- sion is supported by an additional recent study on Swiss 3T3 cells (27) which demonstrate a close temporal correlation be- tween the recovery after desensitization of the mitogenic effects of GRP and the return of cell surface GRP receptors. Similar to our results with the NMB-R, each of these studies suggests that there exists a close relationship between loss of cell surface GRP receptors and the extent of subsequent agonist respon- siveness, suggesting similarities in agonist regulation for the family of receptors mediating the actions of bombesin-related peptides in mammals.

In conclusion, we have demonstrated that NMB receptors undergo agonist-induced homologous desensitization by a proc- ess that is driven by receptor down-regulation. Receptor down- regulation is, in turn, primarily mediated by rapid receptor internalization while the number of NMB receptors present on the cell membrane is closely related to agonist potency (i.e. desensitization). Furthermore, this coupling of receptor num- ber to ability to alter cell behavior was found to exist during NMB receptor recycling after maximal receptor down-regula- tion.

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