The mammalian bombesin-related peptides, gastrin-releasing peptide (GRP) ()and neuromedin B, are thought to have important roles in a number of diverse physiological and pathological processes. These peptides act to regulate numerous central nervous system functions including circadian rhythm (1) and body temperature(2) , stimulate the release of many gastrointestinal hormones(3, 4) , have profound effects on immune function(5) , and are potent stimulators of gastrointestinal tissues including the pancreas (6) , stomach(7) , and muscle(8) . Importantly, these peptides also function as potent growth factors both in normal tissues (9, 10) and tumors(9, 11, 12, 13) , as well as act as autocrine growth factors in some human small cell cancer cell lines(9) . Bombesin-related peptides mediate their effects at least in part by activating phospholipase C after binding to specific GRP-preferring receptors (GRP-R) and neuromedin B-preferring receptors(14, 15, 16) .

Consequent to agonist exposure, bombesin receptors become refractory to further stimulation by the same agonist (homologous desensitization). In addition to desensitization, upon agonist exposure, bombesin receptors also undergo internalization and down-regulation. Similar to other G protein-coupled receptors, both acute desensitization (i.e. within minutes of agonist exposure) and chronic desensitization (i.e. agonist exposure for hours) have been reported with the GRP-R(17, 18, 19, 20, 21, 22, 23) . However, the inter-relationship between these multiple receptor-modulatory processes (including receptor internalization, down-regulation, and desensitization as well as their mediators) is largely unknown not only for bombesin receptors but also for most phospholipase C-linked receptors. In contradistinction, much is known, especially for acute desensitization, about the mechanisms regulating adenylate cyclase-activating receptors, such as the -adrenergic receptor (AR).

Acute desensitization of the AR occurs as a consequence of receptor phosphorylation driven by both second messenger-dependent and second messenger-independent (i.e. -adrenergic receptor kinase I and II) kinases(24) , as well as due to receptor G protein uncoupling; however, internalization is not involved(25) . In contrast, with prolonged agonist exposure, chronic desensitization of the AR is completely independent of any known second messenger-dependent or -independent kinase and appears to involve receptor down-regulation(26) . The role of such kinases in regulating the acute or chronic desensitization of phospholipase C-linked receptors is largely unclear. Although second messenger-independent kinases have been shown to possess some degree of promiscuity, such that the -adrenergic receptor kinase can also phosphorylate the phospholipase C-activating substance P receptor in vitro(27) , other phospholipase C-linked receptors such as the -adrenergic receptors are not so phosphorylated(28) . Therefore, this cross-specificity may vary for different phospholipase C-linked receptors. Similarly, the role of second messenger-dependent kinases in regulating the desensitization of phospholipase C-linked receptors likewise is unclear. Although activation of both protein kinase C (PKC) and protein kinase A (PKA) can induce acute desensitization of the AR(29) , most phospholipase C-coupled receptors do not activate this latter kinase, making it unlikely that PKA regulates either the acute or chronic desensitization of these receptors. Furthermore, the role of PKC in mediating either the acute or chronic desensitization of phospholipase C-activating receptors is unclear. For instance, although some studies have shown that desensitization of phospholipase C-coupled muscarinic cholinergic receptors is PKC-mediated and apparently involves receptor phosphorylation(30) , other studies do not support these conclusions (31, 32, 33) . Indeed, when muscarinic cholinergic receptors, G-proteins, and PKC are reconstituted in vesicles, agonist-induced receptor phosphorylation is not observed(34) .

Some(22) , but not other (17, 23) studies, have demonstrated that PKC may play a role in mediating acute desensitization of the GRP-R. There is no data on the role of PKC in mediating chronic GRP receptor desensitization. It has been proposed that chronic desensitization may be mediated at least in part by receptor down-regulation and internalization(21) . In a recent study it was demonstrated that multiple serines and threonines located within the GRP-R carboxyl terminus, including a PKC consensus sequence, were important in regulating this receptor's internalization(35) . However, it is unknown whether these residues also are important in mediating chronic desensitization and down-regulation of the GRP-R. In this study, therefore, to gain insight into the possible importance of either the PKC consensus sequence or other serine/threonine residues in the GRP-R carboxyl terminus in mediating chronic desensitization and down-regulation, as well as into the possible inter-relationship of these processes, we have determined the ability of bombesin to induce these receptor changes in mutant GRP receptors.

EXPERIMENTAL PROCEDURES

Materials

Mutant GRP-R Construction

Transfection and Maintenance of Cell Lines

Binding Studies

Receptor Down-regulation

Cell Membrane Preparation

Cross-linking of GRP-R

Measurement of Inositol Phosphates (IP)

Measurement of [Ca]

RESULTS

The three mutant GRP-R cell lines demonstrated similar dose-response curves for the ability of bombesin to inhibit binding of [I-Tyr]bombesin to that seen with the unaltered wild-type receptor (35) (data not shown). Specifically, half-maximal inhibition of binding (K) of [I-Tyr]bombesin with unlabeled bombesin was observed at 0.9 ± 0.3 nM for wild-type GRP-R, at 2.2 ± 1.0 nM for MGT-346, at 4.9 ± 0.8 nM for JF1, and at 1.0 ± 0.3 nM for TS360AA. All cell lines expressed approximately similar numbers of receptors (B, in fmol/10 cells), with wild-type GRP-R expressing 672 ± 133, MGT346 expressing 609 ± 115, JF1 expressing 897 ± 107, and TS360AA expressing 437 ± 88 (data not shown).

With agonist exposure, wild-type GRP-R was rapidly internalized(35) . The internalization of mutant-truncated receptor MGT346 and JF1 was significantly attenuated (Table 1). In contrast, removal of the protein kinase C consensus sequence in mutant TS360AA only partially attenuated internalization (Table 1).

We next determined the rate of GRP-R down-regulation. A previous study of the wild-type receptor revealed that GRP-R down-regulation was rapid, with exposure to 6 nM bombesin causing half-maximal decreases in receptor number by 3 h and maximal decreases by 6 h(18) . Similarly, in the present study, maximal GRP-R down-regulation was achieved after a 6-h exposure to 3 nM bombesin (Table 1). In contrast to wild-type GRP-R, mutant MGT346 underwent relatively little receptor down-regulation (Table 1, Fig. 1). That this attenuation of receptor down-regulation was not a nonspecific consequence of receptor truncation was demonstrated by mutant JF1. Replacement of the carboxyl terminus Ser and Thr in JF1 resulted in a similar attenuation of down-regulation as observed for the truncation mutant. In contrast to that which was observed for receptor internalization, removal of only the PKC consensus sequence in mutant TS360AA also markedly attenuated receptor down-regulation (Table 1, Fig. 1). These data suggest that receptor internalization and down-regulation are dissociable phenomena that are regulated by different carboxyl terminus residues in the GRP-R.

Figure 1: Effect of 3 nM bombesin preincubation on receptor number (B) on cells expressing wild-type GRP-R, MGT346, JF1, and TS360AA. Cells grown to confluence in F-175 flasks were exposed to 3 nM bombesin for the indicated times and subjected to bombesin competitive binding experiments with [I-Tyr]bombesin. B and K were determined using the least squares curve fitting program LIGAND(37) . There was no significant change in K for any of the cell types with bombesin preincubation. Data are expressed as the percentage of B expressed in bombesin pretreated cells as compared with untreated control cells processed in parallel. Each data point represents the mean ± S.E. of at least three separate experiments, with each value measured in duplicate in each experiment. Top, membranes from cells expressing wild-type GRP-R or mutant receptors TS360AA were incubated with 3 nM bombesin for the indicated times and were then subjected to cross-linking with I-GRP. Equal amounts of cell membrane protein were loaded at each time point per cell type. This gel is representative of two separate experiments.

To investigate further the potential role of second messenger-dependent kinase activation in GRP-R down-regulation as compared with its role in mediating internalization, we examined these processes in two mutant receptors that we have previously demonstrated fail to activate phospholipase C(41) . With substitution of arginine at position 139 with glycine (construct R139G) or alanine at position 263 with glutamic acid (construct A263E), the mutant GRP-Rs were found to retain high affinity binding but not to activate phospholipase C(41) . Internalization was markedly reduced in construct R139G and was partially reduced for construct A263E. Activation of second messenger-dependent kinases was clearly important for regulating down-regulation since this process was greatly attenuated for both of these mutant receptor constructs (Table 1).

The ability of the three mutant receptor types to activate phospholipase C was similar to that observed with wild-type GRP-R. Bombesin caused 10-fold increase in [H]IP formation in cells expressing the wild-type receptor and each mutant (Table 2). Similarly, the concentration of bombesin causing a half-maximal increase in [H]IP did not differ (i.e. 3-5 nM) for the wild-type receptor and the three mutant clones (Fig. 2; Table 2).

Figure 2: Time course of the effect of 1 µM bombesin on total cellular [H]inositol phosphate generation in desensitized and control cells expressing wild-type GRP-R, MGT346, JF1, and TS360AA. Confluent cells were incubated in DMEM supplemented with 2% fetal bovine serum alone or additionally with 3 nM bombesin for 24 h. During the final 6 h of incubation, cells were further incubated with 100 µCi/ml myo-[2-H]inositol. After washing in IP buffer, cells were then exposed to 1 µM bombesin for the indicated times. Isolation of [H]inositol phosphates was performed as described under ``Experimental Procedures.'' Data is expressed as the percent maximal response recorded at t = 120 min in control cells. Each data point represents the mean ± S.E. of a minimum of three separate experiments, with each value measured in triplicate in each experiment.

We next determined whether there were differences between the three mutant GRP-R and wild-type receptor in undergoing desensitization in response to continued exposure to agonist. Incubation of wild type GRP-R with 3 nM bombesin for 24 h significantly decreased the ability of 1 µM bombesin to subsequently increase total cellular [H]IP (Fig. 3). After a 120-min exposure to 1 µM bombesin, control cells expressing wild-type GRP-R increased [H]IP over 10-fold, from 2076 ± 250 dpm to 22140 ± 410 dpm (Fig. 3). Preincubation with 3 nM bombesin, however, decreased the subsequent response to 1 µM bombesin by >70%, as cellular [H]IPs increased only 3-fold from 3380 ± 300 to 9040 ± 600 dpm (Fig. 3, topleftpanel; Table 2). In contrast, MGT346 was not affected by preincubation with 3 nM bombesin, as control cells increased [H]IPs from 2890 ± 150 to 29560 ± 680 dpm and bombesin-preincubated cells increased total cellular [H]IP from 3600 ± 310 to 27070 ± 750 dpm (Fig. 3, toprightpanel; Table 1and Table 2). Removal of the carboxyl terminus Ser and Thr similarly resulted in attenuation of GRP-R desensitization. Cells expressing mutant receptor JF1 not previously exposed to 3 nM bombesin increased total cellular [H]IPs when incubated with 1 µM bombesin for 120 min from 2640 ± 110 to 28260 ± 340 dpm, whereas cells expressing JF1 and previously exposed to 3 nM bombesin for 24 h increased [H]IPs from 3620 ± 220 to 26420 ± 280 dpm (89 ± 2% of control) (Fig. 3, bottomleftpanel; Table 1). That this attenuation in desensitization was specifically due to the presence of the carboxyl terminus PKC consensus sequence was revealed by the failure of cells expressing mutant receptor TS360AA to undergo agonist-induced desensitization. Specifically, control cells expressing TS360AA increased [H]IP from 3720 ± 270 to 32410 ± 440 dpm, whereas those preincubated with 3 nM bombesin increased [H]IP from 5500 ± 210 to 33330 ± 420 dpm (97 ± 2% of control) (Fig. 3, bottomrightpanel; Table 1and Table 2). These data support the contention that the distal carboxyl PKC consensus sequence is needed for GRP-R desensitization.

Figure 3: Effect of preincubation with 3 nM bombesin for 24 h on the dose-response curves for [H]inositol phosphate generation in cells expressing wild-type GRP-R, MGT346, JF1, and TS360AA. Confluent cells were incubated in DMEM supplemented with 2% fetal bovine serum and 100 µCi/ml myo-[2-H]inositol alone or with 3 nM bombesin for 24 h. After washing in IP buffer, cells were exposed to the indicated concentrations of bombesin for 60 min, with data expressed as the -fold increase to total cellular [H]inositol phosphates. Each data point represents the mean ± S.E. of a minimum of three separate experiments, with each value determined in duplicate in each experiment.

To determine whether the alterations in receptor desensitization were accompanied by changes in agonist affinity, [H]IP dose-response curves in the presence and absence of 3 nM bombesin preincubation were generated for all four cell types (Fig. 2). There was no change in potency of bombesin in increasing [H]IP in either the wild-type or the three mutant GRP receptors after desensitization (Fig. 2, Table 2). These data demonstrate that desensitization is accomplished by a reduction only in agonist efficacy without affecting agonist potency.

Since GRP-R-induced activation of phospholipase C increases [Ca](6, 15, 16) , we next determined whether the agonist-induced desensitization observed with [H]IP formation also could be observed with [Ca]. Cells expressing wild-type GRP-R increased [Ca] from 103 ± 4 nM to 299 ± 4 nM when stimulated with 1 µM bombesin ([Ca] = 196 ± 4; Table 2, Fig. 4). However, when these cells were preincubated with 3 nM bombesin for 24 h, the ability of 1 µM bombesin to increase [Ca] was attenuated by 64% ([Ca] = 71 ± 3 nM) (Fig. 4, topleftpanel; Table 2). In contrast, the ability of mutant receptors MGT346, JF1, and TS360AA to increase [Ca] was not significantly attenuated by preincubation with 3 nM bombesin for 24 h (Fig. 4, Table 2). These data confirm that the distal carboxyl-terminal PKC consensus sequence of the GRP-R specifically is required for mediating agonist-induced desensitization of the increase in [Ca] and [H]IP.

Figure 4: Effect of preincubation with 3 nM bombesin for 24 h on the subsequent stimulation in [Ca] by 1 µM bombesin on cells expressing wild type GRP-R, MGT346, JF1, and TS360AA. Cells were incubated in DMEM supplemented with 2% fetal bovine serum alone or with 3 nM bombesin for 24 h and were then incubated with 2 µM fura-2/AM for 30 min. Cells were washed twice and change in fluorescence immediately determined consequent to stimulation with 1 µM bombesin. These tracings are representative of at least three separate experiments.

DISCUSSION

In the present study, we investigated the possible role of PKC or other kinases in mediating chronic desensitization and down-regulation of the GRP-R by acting upon various serines and threonines located within this receptor's carboxyl terminus. We elected to investigate the GRP-R carboxyl terminus for a number of reasons. Most importantly, studies involving the prototypical AR have demonstrated the importance of this receptor's carboxyl terminus in mediating desensitization through both second messenger-dependent and independent kinases, which phosphorylate multiple serines and threonines in this region(24) . The few studies extant for phospholipase C-coupled receptors, including those for epidermal growth factor (42, 43) and platelet-derived growth factor (PDGF)(44) , demonstrate that these receptors also are phosphorylated in the carboxyl terminus consequent to binding agonist and that phosphorylation effects their ability to activate phospholipase C. In contrast, a recent study of the seven transmembrane-spanning phospholipase C-coupled M muscarinic cholinergic receptor revealed that although the threonine residues in the carboxyl terminus were important for mediating down-regulation, they were of minimal importance in regulating agonist-induced desensitization(45) . This suggests that additional processes or kinases may mediate desensitization, as opposed to down-regulation, of the M muscarinic cholinergic receptor. For many phospholipase C-linked receptors, including those for epidermal growth factor(42, 43) , platelet-derived growth factor(44) , and the muscarinic cholinergic receptors(34, 46, 47) , activation of PKC appears to partially mediate receptor desensitization. However, other studies of muscarinic cholinergic receptors (31, 32, 33) and studies of the phospholipase C-activating thrombin(48) , somatostatin(49) , and platelet-activating factor receptors (50) suggest that PKC may not be important in mediating desensitization. Within the carboxyl terminus of the GRP-R exists a PKC consensus sequence(35, 51) . Although an earlier study of this receptor suggested that multiple serines and threonines played a major role in regulating GRP-R internalization, the PKC consensus sequence per se was only partially involved in regulating this process(35) . Thus, while earlier investigations have implicated the carboxyl terminus in regulating the desensitization of some receptors, the specific role of PKC in mediating the desensitization of phospholipase C-activating receptors in general is not clear.

Most studies focusing on GRP-R desensitization have examined the effects of incubations with agonists after relatively short periods of exposure (acute desensitization, occurring within minutes of added agonist). While some investigators have failed to implicate a role for PKC in mediating acute desensitization(17, 23) , others have concluded that in at least certain cell types PKC does partially mediate bombesin-induced acute desensitization(22) . In contrast, however, relatively little is known about the potential mediators of GRP-R desensitization consequent to long periods of exposure to bombesin (chronic desensitization, occurring hours to days after the addition of agonist). In this paper we demonstrate that the GRP-R carboxyl terminus PKC consensus sequence is critical for mediating the chronic desensitization seen with the GRP-R. When this consensus sequence was mutated by changing the threonine at position 360 and the serine at position 361 to alanines, the mutant receptor TS360AA demonstrated minimal ability to undergo desensitization. This decreased ability to undergo desensitization was not due to an impairment in receptor affinity because this receptor had an identical affinity to the wild type receptor. Furthermore, this decrease was not due to an alteration in G protein-receptor coupling because the TS360AA mutant had an identical EC for stimulating [H]IP as wild-type GRP-R, and maximally effective concentrations of agonists caused a similar change in mobilization of cellular calcium in both the mutant and the wild-type receptor. These results stand in contrast to those of a similar study of the M muscarinic cholinergic receptor where similar carboxyl-terminal receptor alterations decreased receptor down-regulation but had minimal effects on chronic desensitization(45) . These results also demonstrate that the importance of the carboxyl terminus PKC consensus sequences in mediating chronic desensitization varies with the different phospholipase C-coupled receptors.

It has been suggested that G protein-coupled receptor kinases may mediate the acute desensitization of a number of different phospholipase C-coupled receptors, similar to that shown to be the case for the AR(52) . The role of G-protein receptor kinases in causing chronic desensitization is unknown. One study (26) of the AR concluded that neither second messenger-dependent (PKA or PKC) nor -independent (-adrenergic receptor kinase) kinases were likely involved in mediating agonist-induced chronic desensitization of this receptor because ARs with the consensus sequences for these kinases mutated demonstrated unaltered chronic desensitization. In contrast, whereas our results support a role for PKC in mediating chronic GRP-R desensitization, they do not provide support for the involvement of any G-protein receptor kinase in mediating chronic desensitization of the GRP-R. Removal of all of the GRP-R carboxyl terminus serines and threonines, including the PKC consensus sequence, did not impair desensitization more than removing the PKC consensus sequence alone. Similarly, removal of most of the GRP-R carboxyl terminus did not impair desensitization more than removing the PKC consensus sequence alone. These results suggest that the carboxyl terminus PKC consensus sequence is what is critical for regulating GRP-R desensitization and provide no support for the involvement of G-protein receptor kinases in regulating the desensitization of this receptor consequent to prolonged agonist exposure. However, it remains possible that some G-protein receptor kinase still could be involved in mediating a component of this desensitization process under different experimental conditions. Conversely, it is possible that the current mutants studied do not allow for G-protein receptor kinase involvement to be detected. Activation of the distal PKC consensus sequence, for example, could be needed to activate the G-protein receptor kinases in this receptor. Alternatively, potentiating interactions between activation of the distal PKC consensus sequence and G-protein receptor kinases acting on other serines and threonines in the carboxyl terminus, similar to the effects observed between PKA and -adrenergic receptor kinase in causing acute desensitization of the AR, might occur but would not be detected using the GRP-R mutants discussed herein.

The current study also provides some important insights into the possible importance of receptor down-regulation and internalization in mediating chronic GRP-R desensitization. With the AR, results obtained using mutant receptors and various specific inhibitors of the different receptor-mediated processes have demonstrated that acute desensitization is not mediated by down-regulation or internalization(53) . Chronic AR desensitization also is independent of internalization, but likely occurs by receptor down-regulation(26) . Although most studies of desensitization have focused on acute phenomena, the few studies of chronic desensitization of 7 transmembrane-spanning G-protein-coupled receptors have provided conflicting data regarding the role of down-regulation. For example, desensitization of the adrenergic receptor consequent to 24 h of exposure to agonist was not found to be due to receptor down-regulation(54) . In contrast, chronic desensitization of the dopamine D1 receptor was associated with its down-regulation(55) . In the present study we found that with all mutant GRP-Rs that the effects on receptor desensitization and down-regulation were identical. Although this result does not establish that GRP-R desensitization is regulated via its down-regulation, it does suggest that they are mediated by similar processes and raises the possibility that they are coupled. Earlier studies of the GRP-R, however, have not uniformly linked the chronic desensitization of this receptor with its down-regulation. For the GRP-R as well as for the closely related neuromedin B receptor, two studies utilizing kinetic analyses of binding and changes in biological activity have concluded that chronic desensitization is mediated by receptor down-regulation(20, 56) . These observations are in agreement with another study of GRP-R expressed by Swiss 3T3 cells where it was shown that chronic desensitization was mediated by receptor down-regulation(21) . Yet these findings stand in contrast to a study performed on GRP-R expressed by HIT-T15 cells (22) where stoichiometric and quantitative differences between desensitization and down-regulation led the authors to conclude that GRP-R desensitization was only partially mediated by receptor down-regulation. Thus, our findings confirm the link between GRP-R down-regulation and chronic desensitization and further provide mechanistic evidence that these processes are likely mediated by PKC.

One study suggests that GRP-R internalization mediates chronic desensitization and down-regulation(20) . However, a recent study (57) using fluorescent-labeled antibodies against the GRP-R, as well as our results in the present and a previous study (18) using an acid stripping methodology, demonstrate that internalization is a rapid process. In contrast, the desensitization and down-regulation of the GRP-R observed in these studies takes hours, demonstrating that these processes are kinetically well separated from receptor internalization. Our study clearly demonstrates that internalization is at least partially mediated by different intracellular processes than those mediating chronic down-regulation or desensitization. The mutant receptor TS360AA, which lacks the distal carboxyl-terminal PKC consensus sequence, internalized 79% of receptors as compared with wild-type GRP-R, but underwent minimal chronic desensitization and down-regulation. Similarly, one mutant GRP receptor, which did not activate phospholipase C (A263E) yet underwent 50% of maximal internalization, failed to undergo down-regulation. These results suggest that PKC activation is involved in mediating only 50% of the internalization, but is essential for any down-regulation and chronic desensitization of the GRP-R. The mediation of these processes by different mechanisms is supported further by the effect of removing all serines and threonines in the GRP-R carboxyl terminus in addition to the PKC consensus sequence in mutant receptor JF1. This construct resulted in almost a complete loss of internalization, but failed to cause additional decreases in down-regulation or desensitization as compared with the PKC deletion mutant. This result supports the importance of additional sites besides the carboxyl-terminal PKC consensus sequence in mediating maximal internalization but not chronic desensitization or down-regulation.

In conclusion, this study provides definitive evidence that chronic desensitization of the GRP-R is regulated by a PKC consensus sequence located within the carboxyl terminus of this receptor and provides no evidence that other serines and threonines in this receptor's carboxyl terminus contribute to chronic desensitization. These data also suggests that chronic desensitization of the GRP-R secondary to prolonged agonist exposure is coupled to receptor down-regulation and that down-regulation is itself also regulated by PKC. In contrast, and in conjunction with our earlier study(35) , internalization and down-regulation or chronic desensitization of the GRP-R are not coupled but are mediated at least in part by different cellular mechanisms.

FOOTNOTES

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: National Institutes of Health, Bldg. 10 Rm. 9C103, 10 Center Dr. MSC 1804, Bethesda, MD 20892-1804. Tel.: 301-496-4201; Fax: 301-402-0600.

()

The abbreviations used are: GRP, gastrin-releasing peptide; GRP-R, GRP receptor; PKA, protein kinase A; PKC, protein kinase C; AR, adrenergic receptor; G protein, guanine nucleotide binding protein; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate.

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