Eur. J. Biochem. 256,631 -636 (1986) 10 FEBS 1986

Cycle of the vasoactive intestinal peptide and its binding site in a human adenocarcinoma cell line (HT 29) JosC LUIS, Jean-Marc MULLER, Brigitte ABADIE, Jean-Michel MARTIN, Jacques MARVALDI and Jacques PICHON Institut de Chimie Biologique, Universite de Provence. Unit(: associte au Centre National de la Recherche Scientifique 202, Marseille

(Received October 11, 1985/January 30, 1986) - EJB 85 1121

The disappearance of vasoactive-intestinal-peptide (VIP) binding sites at the cell surface of a cultured target cell, originating from a human colonic adenocarcinoma (HT 29 cell line), was studied, after preexposition of the cell to the peptide, as a function of time, VIP concentration and temperature. Maximum effect (60 - 8O0/o loss of binding capacity) was obtained after a 5-10 min exposure of the cells at 37°C with a VIP concentration of 100 nM. The t l jz of maximum disappearance was less than 2 min and the concentration of native VIP giving half-maximum decrease in lZ5I-VIP binding was 6 nM. The affinity of remaining binding sites for VIP was not affected compared to that of control cells (Kd = 0.3 nM). Disappearance of VIP binding sites was spccific sincc, with the same conditions of preincubation, the specific binding of '251-labeled epidermal growth factor to HT 29 cells was not modified. The phenomenon was reversible and 90% of binding capacity could be restored in less than 60 min by incubating cells in VIP-free medium. Correlatively we showed, by two independent experimental procedures, that lZ5I-VIP, initially bound to HT 29 cells, was maximally internalized after 10 min of incubation at 37"C. All the data strongly suggest that: (1) internalization of VIP is receptor-mediated; (2) upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells.

The regulation of the concentration of cell-surface re- ceptors is a general way for the cell to modulate its respon- siveness to hormones, growth factors or neurotransmitters. Loss of cell-surface receptor binding is generally obtained by exposure of target cells to the effector itself. This phenomenon occurs with a number of polypeptidic hormones including insulin [l], epidermal growth factor (EGF) [2], platelet-derived growth factor [3,4], human growth hormone [5], thyroliberin [6] and thyrotropin [7, 81. The reduced capacity of cells to bind the ligand was not attributable, in most cases, to a decreased affinity of the receptor for its ligand but to a reduc- tion in the number of cell surface receptors [l, 5 , 7, 9, 101. In several cases, the internalization of the ligand-receptors complexes, followed by the degradation of the receptor and of the ligand have been demonstrated [3,11- 131. These events have been proposed to represent the down-regulation of cellular receptors. The consequence of receptor down-regula- tion is generally a refractoriness of cells to agents acting at the cell-surface receptors. For example, the down-regulation of EGF receptors leads to a desensitization of the cells to the growth factor and to a reduced mitogenic response [14]. Likewise, in pathological states, such as some cases of obesity, large concentrations of insulin in the blood stream involve a decreased sensitivity of target tissues to the hormone [15, 161.

The down-regulation of /?-adrenergic receptors has been extensively studied and it appears from two recent reports [I 7,

Correspondence to J. Pichon, Institut de Chimie Biologique, Universite de Provence, 3 place V. Hugo, F-13331 Marseille cedex 3, France.

Ahhrrviations. VIP, vasoactive intestinal peptide; EGF, epi- dermal growth factor; DTSP, dithiobis(succinimidylpropionate); SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electropho- resis; BSA, bovine serum albumin; DME, Dubecco's modified Eagle's medium.

181 that one may distinguish between sequestration and down- regulation of j-receptors. Sequestered receptors are not accessible to ligands unable to diffuse through the plasmic membrane while they are still accessible to ligands able to cross the membrane. On the other hand, down-regulated re- ceptors are not accessible to either type of ligands [17]. Down- regulation of /?-receptors could also be due to a loss in total cellular content of receptors [18].

The vasoactive intestinal peptide (VIP), a 28-amino-acids polypeptide of the VIP/secretin/glucagon family, possesses a wide variety of biological activities [19] at the central or peripheral levels. Complete understanding of the mechanism of VIP action necessitates an investigation of the tine structure of the receptor site and of the cycle of VIP and VIP receptors and their processing within the cell. It has been suggested that receptor-mediated endocytosis might be involved in the hepatic clearance of VIP [20]. The first results supporting the hypothesis of such a receptor-mediated endocytosis of VIP were obtained recently in an adenocarcinoma cell line (HT 29 cells) originating from human colon. These data demonstrated that lZ5I-VIP bound to its receptors was rapidly (less than 10 min) recovered in an intracellular compartment and then released in the incubation medium in a degraded form [21, 221. When HT 29 cells were pretreated with VIP at 37°C the VIP receptors were no longer detectable on the cell surface [23]. This suggests that VIP is internalized together with its receptor or in other words by receptor-mediated endocytosis. Moreover, two recent reports demonstrated that a prolonged exposure of mouse T cells (18 h in the presence of 100 nM VIP) [24] or HT 29 cells (3 h in the presence of 10 nM VIP) [25] resulted in a loss of cell-surface receptors and, in the case of HT 29 cells, resulted in an attenuated responsiveness of the adenylate cyclase [25].

In the present report, we have investigated in detail the cycle of VIP and VIP binding sites in HT 29 cells. The loss of

632

cell-surface receptors is very fast ( t , ,2 -= 2 min), dependent on time, temperature, peptide concentration and is reversible.

MATERIALS AND METHODS

Reagents

Porcine VIP, bovine serum albumin (BSA) were purchased from Sigma. Murine epidermal growth factor (EGF) was from Collaborative Research and '251-EGF (100 Ci/g) was obtained from Amersham. Dulbecco's modified Eagle's medi- um (DME medium) was from Gibco. Fetal calf serum was from Eurobio. Dulbecco phosphate-buffered saline (NaCI/Pi) was from Oxoid. Dithiobis(succinimidy1propionate) (DTSP) was from Pierce Chemical Company (Rockford, USA).

VIP was iodinated by the chloramine T method [26] to a specific activity of 250-300 Ci/g, i.e. an average of 0.4-0.5 atom '2sI/molecule [27].

Cell culture and maintenance

The human adenocarcinoma cell line (HT 29) [28], a gift of Dr Zweibaum (Paris, France), was routinely cultured in DME medium containing 4.5 g/l glucose and 10% fetal calf serum in a humidified atmosphere of 95% air, 5% C02. The culture medium was changed every day. Subcultures were obtained by harvesting growing cells with 0.53 mM EDTA containing 0.05% trypsin in NaCl/Pi.

HT 29 cells were seeded in 4-well or 24-well multidish plates (2 cm2/well) at a density of 2 x los cells/well. Sub- confluent monolayers containing approximatively 106 cells were used throughout the experiments.

I 2 ' l - VIP binding conditions

HT 29 cell monolayers were rinsed with NaC1/Pi contain- ing 0.1% bovine serum albumin (NaCI/P,/BSA) and in- cubated at 37 "C for indicated times in DME medium contain- ing 2% BSA in the presence of 10 nM VIP. Monolayers were rapidly washed twice with ice-cold NaCl/P,/BSA and then treated with 100 mM sodium acetate buffer pH 3.6 containing 150 mM NaCl for 8 rnin at 4°C. The cells were rinsed again twice with ice-chilled NaC1/Pi/BSA, incubated in presence of 0.5 nM 1251-VIP in the binding medium (DME medium, pH 7.3, containing 1 YO BSA, 0.1 YO bacitracin, 15 mM Hepes, 150 pM phenylmethylsulfonyl fluoride) for 3 h at 4°C and then washed three times with ice-cold NaCI/P,/BSA. In some experiments the low-pH treatment of the cell monolayers was omitted before 1251-VIP binding reaction was carried out.

Cross-linking of bound "I- VIP to HT 29 cells

Conditions for cross-linking experiments have been de- scribed elsewhere [23]. Briefly, monolayers of HT 29 cells (3 x lo6 cells) were incubated for 3 h at 4°C with 0.5 nM lZ5I-VIP. The cells were carefully washed with 60 mM Hepes pH 8 containing 0.15 M NaCl. The cross-linking reaction was performed in the presence of 2 mM DTSP at room tempera- ture and stopped 20 rnin later. The monolayers were lysed in 200 p1 SDS-containing sample buffer without reducing agent and submitted to electrophoresis in polyacrylamide gel (SDS- PAGE) according to the method of Laemmli [29].

10007

800.

600-

LOO.

200- Fi - m

120 67 M ~ ~ O - ~

1 1 A

6ool B

0 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .+ 0 5 10 15 20 2 5 30

Slice number

Fig. 1. Disappearance of' VIP-receptor complexes ut the HT 29 cell surface, demonsirated by covalent cross-linking. HT 29 cell monolayers were incubated with 0.5 nM 'zsI-VIP at 4°C for 180 min. After washing, cells were incubated with 2 mM DTSP for 20 min at 20°C. Cell lysates were analysed by SDS-PAGE run undcr non-reducing conditions. Gels (12.5%) were cut into 0.3-mm-thick slices and radio- activity in each slice was counted in a y spectrophotometer. (A) Control cells. (B) Cells incubated 10 min, at 37°C before performing the cross-linking reaction. Data shown correspond to one experiment representative of three

RESULTS

Fate of '251-VIP bound on HT 29 cell surfctce

In a preceding report [21], we have demonstrated, by acetic acid treatment, that 1251-VIP previously bound to HT 29 cell surface was rapidly internalized when these cells were in- cubated at 37 T. Cell-associated '251-VIP was initially located at the cell surface. After a 10-min incubation of the cell mono- layers at 37"C, there was a rapid loss (65%) of radioactivity from the cell surface. Simultaneously, a large proportion of radioactivity appeared intracellularly and then increased in the medium as a function of time. We have also demonstrated [21], by trichloracetic acid precipitation (15 rnin at 20"C), that '2sI-VIP was released in the medium in a degraded form (50 - 60%) while a rather low amount of degraded VIP was recovered intracellularly (10 - 30%) or at the cell surface (5 - 20%).

To confirm these data by another experimental approach, the following experiments were carried out. '2sI-VIP was initially bound at 4°C to the cell surface, then cells were incubated at 37°C or 4°C for 10 rnin and '2sI-VIP was cross- linked to its receptor with DTSP under the conditions already described [23]. Data from Fig.1 shows clearly that the labelling of the major polypeptide of M , 64000 was greatly reduced (68% of labelling reduction) in cells incubated at 37 'C (Fig. 1 B) comparative to cells incubated at 4'C (Fig. 1 A). This value is in good agreement with those obtained (65%) in experiments done with acetic-acid-treated HT 29 cells [21].

633

o l . . . . . . . . . . . . . . . . . . . . . 0 10 20 30 LO

Time hid

Fig.2. Time course of disappearance of VIP binding sites during pre- treatment of H T 29 cells wiifh native VIP. HT 29 cell monolayers were treated as described in Materials and Methods. The amount of 1251-VIP which binds to cells at time zero of incubation at 37°C (control) is taken as 100% of binding. Cells were treated (0) or not (+) with 100 mM acetate buffer, 150 mM NaCl, pH 3.6 for 8 rnin at 4"C, prior '251-V1P binding at 4 ' C for 180 min. Values are means of four different experiments

Fate of the HT 29 cell-surface VIP binding sites

To demonstrate that the loss of cell-surface-associated VIP, which occurs at 37 "C, was the result of disappearance of VIP binding sites at the cell surface, we have measured the VIP binding capacity of HT 29 cells previously exposed to native VIP. After preincubation at 37°C in the presence of 10 nM VIP, cell monolayers were carefully washed and then tested for their '251-VIP binding capacity at 4°C. The time course of disappearance of VIP binding sites is shown in Fig. 2 (closed symbols). A rapid loss of VIP binding sites (tl!2 < 2 min) was observed. Exposure of HT 29 cells for 5 rnin at 37°C in the presence of VIP resulted in a reduced binding capacity of 80%.

In order to demonstrate that the disappearance of VIP binding capacity is not only due to VIP receptor occupancy by native VIP, the cell monolayers were washed with a low- pH buffer to remove remaining surface-bound VIP prior to performing the ' 251-VIP binding reaction. A preliminary study was necessary in order to set up the optimal pH conditions which allowed a maximal removal of surface- bound VIP and, later on, a maximal VIP rebinding. These data are shown in Fig.3. In the presence of acetate buffer pH 3.6 containing 150 mM NaCl, 90% of the '251-VIP was removed from the cell surface. These cells were still able to rebind 125-VIP at 95% of the control. These conditions were selected and used to repeat the experiments described above. Under these new conditions, a rapid loss ( t l j 2 "N 2 min) of VIP binding was also observed (Fig.2; open symbols), but this decrease reached only 60% of binding of control cells instead of 80%. Hence, the occupancy of VIP receptors by remaining native VIP only accounts for about 20%. A similar percentage of disappearance of VIP binding capacity was obtained if remaining VIP binding sites were revealed using [3-'2'II-Tyr'o] VIP (data not shown).

By affinity cross-linking of '251-VIP to its binding site on intact H T 29 cells preincubated 20 rnin at 37°C with 10 nM native VIP, we have already demonstrated that an important loss (75%) of VIP binding sites occurred comparatively to control cells [23]. This value is in good agreement with the present data.

The specificity of this phenomenon was demonstrated by preincubating cell inonolayers in the presence of 10 nM VIP

Fig. 3. Ejfiect q f p H on acidic extraction of VIP initially hound to HT 29 cells and on subsequent rebinding of '251-VIP. One set of cells was incubated with 0.5 nM "'I-VIP for 180 min at 4°C. Cells were then treated with 0.5 ml of 100 mM acetate buffer, 150 mM NaCl at dif- ferent pH (3.6-6.5) for 8 rnin at 4°C. Radioactivity present in the acid extract was counted and expressed as percentage of initial binding (hatched blocks). A second set of cells was pretreated with acetate buffer at different pH as described above. Cells were then washed twice with NaCl/P,/BSA at 4°C and tested for their capability to bind '2sI-V1P at 4°C for 180 min. Radioactivity bound to cells was expressed as percentage of '2sI-VIP bound to untreated cells (empty blocks)

Table 1. Efect qf native VIP pretreatment on '251-VIP or '251-EGF binding to HT 29 cells Cells were preincubated with 10 nM native VIP for 10 rnin at 3 7 T , washed twice with NaCI/Pi/BSA at 4"C, treated with acetate buffer pH 3.6, washed again twice with NaCI/Pi/BSA and tested for their capability to bind '2sI-VIP or '"1-EGF at 4°C for 180 rnin. Non- specific binding was assessed by measuring radioactivity bound to the cells in the presence of excess native peptidc (1 pM)

Preincubation VIP bound EGF bound conditions

Control 10622 f 1080 3053 k 100

+10 nM VIP 4992+ 300 3449 & 220

for 10 min at 37°C. Cells were then tested for their 1251-VIP or lZ5I-EGF binding capacity. The results are shown in Table 1 . The specific binding of '2sI-VIP was decreased by 53% while the specific '251-EGF binding was not affected.

Dose effect of native VIP in regulating cell-surface VIP binding sites

Cell monolayers were incubated in the presence of various concentrations of native VIP for different periods of time. The '2sI-VIP binding reaction was then carried out after low- pH treatment of monolayers. The initial rate of 1251-VIP binding decrease was directly related to the concentration of native peptide used during the preincubation step (Fig. 4 A). The maximal decrease was obtained rapidly (5 - 10 min) for high concentrations of VIP and in about 30 min for low con- centrations. Preincubation in the presence of 0.01 nM VIP did not cause any significant loss of cell-surface binding sites for time as long as 90 min. The binding capacity of HT 29 cells was decreased by 50% for a VIP concentration of 6 nM (Fig.4B).

634

A

100 -*

0 0.001 0.01 0.1 1 10 100 1000 VIP concentration InM)

s ,

0. 0 10 20 30 LO SO 60 70 80 90

Time (minl

Fig.4. ( A ) Time course of effect of native VIP concentrations and ( B ) dose effect of native VIP on 12'I-VIP binding capacity of pretreuted HT 29 cells. (A) Cells were pretreated with different concentrations of native VIP (lo-" to lo-' M) for the indicated time at 3 7 T , washed and treated with acetate buffer pH 3.6 before performing Iz5I-VIP binding as described in Materials and Methods. Native VIP concentrations are: A lo-' M; 0 lo-* M; H M; 0 lo-'' M; + lo-" M. (B) Cells were pretreated for 30 min at 37°C with increasing amount of native VIP. Other conditions are as in A

Table 2. Affinity and number of VIP binding sites in pretreated H T 29 cells Cells were treated as described in Table 1, with 1 nM or 100 nM native VIP during the preincubation step. 1251-VIP binding was performed in presence of increasing amount of unlabelled VIP. Scatchard analy- sis of the data gave the number of binding sites and Kd for VIP

Preincubation K.3 Number of conditions sites per cell

nM Without VIP 0.36 k 0.02 7400 f 350

1 nM VIP 0.39 k 0.05 4 840 k 460

100 nM VIP 0.27 f 0.04 2490k 90

Measurement of the affinity of' VIP receptor in VIP-pretreated H T 29 cells

The loss of cell surface VIP binding sites, that we have observed after exposure of HT 29 cells to VIP, could be only an apparent one due to an alteration in VIP receptor affinity for VIP. To test this possibility, HT 29 cell monolayers were pretreated with native VIP, washed with acetate buffer pH 3.6 and competitive experiments between ' 251-VIP and native VIP were performed. The Scatchard analysis of these results (Table 2) clearly demonstrated that, over the concentration range of native VIP used, there is a unique class of high- affinity binding sites with an apparent affinity constant which is unchanged whether or not the cells were pretreated with native VIP. Meanwhile, the number of cell-surface VIP bind- ing sites decreased by 35% or 66% in cells exposed to 1 nM or 100 nM VIP respectively. These results demonstrated that this decrease in '251-VIP binding of VIP-pretreated HT 29 cells is not the consequence of an alteration of VIP receptor affinity for its ligand, but is only due to a reduced number of binding sites available for VIP. It should be underlined that Kd values obtained in the present experiments were very close to the values obtained previously using monoiodinated VIP ~ 3 1 .

Ejject of temperature

We have investigated the effect of the temperature, during the preincubation step, on the loss of cell-surface VIP binding sites. HT 29 cell monolayers were exposed to 10 nM VIP at

. . . . . . . . . 0 30 60 90 120 Time lminl

Fig. 5. Ejyect of temperature pretreatment of 12sI-VIP binding capacity q f H T 29 cells. Cells were treated as in Fig.4A, except that preincuba- tion with lo- ' M native VIP was performed at: 4°C (+); 21 "C (0 ) ; 37°C (A). Data are means of two independent experiments

various temperatures and tested for their '251-VIP binding capacity at 4°C after low pH treatment. The time courses are shown in Fig. 5. In cells exposed to VIP at 21 "C, the initial rate of decrease in VIP binding was slower compared to those obtained in cells preincubated at 37"C, but the same steady state was reached after 20 min. When cells were exposed to VIP at 4°C no fall in VIP binding capacity was observed.

Recovery of VIP binding sites

In order to determine if VIP-induced reduction of cell- surface binding sites was a reversible phenomenon, VIP-pre- treated HT 29 cells were incubated in VIP-free medium containing 10% fetal calf serum at 37°C for different times, washed or not in acetate buffer pH 3.6 and tested for their 1251-VIP binding capacity. The results (Fig. 6) demonstrated that the reappearance of VIP binding sites was very fast ( t 1 , 2 z 15 min). The recovery of VIP binding capacity of these cells was 80 - 90% of control values after a 40-min incubation at 37°C in VIP-free medium. Similar data were obtained whether or not HT 29 cells were washed in acetate buffer pH 3.6 prior to testing the binding of 1251-VIP. When incuba- tion was performed in VIP-free medium containing 2% bovine serum albumin instead of fetal calf serum, the recovery was not modified (results not shown).

635

30 60 90 120 I50 180 Time [mid

Fig.6. Reexpression of VIP binding sites. HT 29 cells were pre- incubated with 10 nM native VIP for 10 min at 3 7 T , washed four times with NaCI/P,/BSA at 4°C and incubated for the indicated time at 37°C in DME medium supplemented with 10% fetal calf serum. Cells were then washed once at 4"C, treated (0) with acetate buffer pH 3.6 or not treated (+), washed twice with NaCI/Pi/BSA at 4°C and tested for their capability to bind '25T-VTP at 4°C for 180 min

DISCUSSION

In the present study we investigated the fate of 1251-VIP bound to the cell surface and the regulation of cell-surface VIP binding sites using the HT 29 human adenocarcinoma cell line as a model. Two main techniques were used for this purpose : the binding of radiolabelled ligand and the covalent cross-linking of ligand to its receptor with bifunctional re- agent.

After exposure of HT 29 cells at 37°C for 10 min, only 35% of 12'I-VIP which was initially bound to the cells at 4°C could be recovered from the cell surface by low-pH treatment of the cell monolayer [21]. The disappearance of cell-surface- bound 1251-VIP after incubation of the cells at 37"C, was further demonstrated by cross-linking experiments. The amount of the M , 64000 polypeptide, previously described as the high-affinity VIP binding site [23], was reduced by 68% in HT 29 cells exposed for 10 min at 37°C. The values obtained by the two experimental approaches were thus in very good agreement. Two important remarks can be drawn from these data.

a) The possibility that VIP bound to its receptor became resistant to low-pH solubilization after the exposure of VIP- receptor complexes at 37 "C is unlikely because a different experimental approach, namely affinity cross-linking which is pH-independent, gave similar results.

b) The affinity cross-linking of 1251-VIP to its receptor that we have developed with intact cells can be used as a quantitative assay to assess the amount of VIP bound to the HT 29 cell surface with a reasonably good yield (loo/, of the amount of 1251-VIP initially bound recovered after SDS- PAGE). This method compares well with the low-pH treat- ment technique.

The data we have obtained in these studies and those from a previous report from this laboratory [21] showed that VIP bound to its receptor was internalized very rapidly when cells were incubated at 37 "C.

During the present investigation, we have questioned whether or not VIP internalization was mediated by receptor endocytosis and whether the VIP receptor could be down- regulated. The results presented here demonstrated clearly that, after short-time exposures of HT 29 cells to native VIP at 37 "C, an important decrease in 1251-VIP binding occurred

(down to 40% of control), even under conditions where the remaining native VIP was removed by low-pH treatment of cells prior testing their ' "I-VIP binding capacity. The kinetics of VIP binding site disappearance was very fast (t1,2 FZ 2 min) and was in agreement with the kinetics of 1251-VIP internaliza- tion [21]. In a previous report from this laboratory [23], it has been demonstrated that after a 20-min incubation at 37 "C of HT 29 cells in the presence of 10 nM native VIP, followed by affinity cross-linking of '251-VIP to its receptor, the amount of labelled M,-64000 polypeptide was decreased by 75% compared to control cells. These data are confirmed by the present results and demonstrate again the accuracy of the cross-linking experimental approach in such kind of studies. Taken together, these results support a mechanism of VIP internalization mediated by receptor endocytosis.

The phenomenon of disappearance of cell-surface VIP binding sites subsequent to the exposure of HT 29 cells to VIP at 37 "C was characterized in this report by several criteria.

a) The decrease in VIP binding capacity of cells exposed to VIP was not due to an alteration in VIP receptor affinity for the ligand, but to a decrease in the number of cell-surface VIP binding sites.

b) The extent of the loss of cell-surface VIP binding sites was dependent on native VIP concentration used during the preincubation step. The concentration of VIP necessary to induce the half-disappearance of VIP binding sites was found to be 6 nM.

c) The preincubation of cells with VIP under conditions where 53% of VIP binding sites disappeared from the cell surface did not affect the binding capacity of '251-EGF to its receptor.

d) The loss of cell-surface VIP binding sites was temperature-dependent.

e) The disappearance of VIP binding sites was almost fully reversible upon removal of VIP: 80 - 90% of VIP binding sites were recovered compared to the control. The reappearance of VIP binding sites was very fast (tl,2 z 15 min), compared to other receptor systems such as EGF receptor [30] or insulin receptor [31].

The fulfilment of these criteria are generally considered to be sufficient to conclude that a receptor is down-regulated [32] or at least sequestered [17, 331.

We have at present no experimental way to demonstrate whether the total cellular concentration of VIP binding sites is modified under our experimental conditions. Therefore, the term down-regulation, which we use hereafter must be taken in the broadest sense, that is disappearance of cell-surface receptors by internalization.

The concentration of VIP required for half-maximal down-regulation of VIP receptors was about 6 nM, whereas the concentration needed for half-maximal occupancy of VIP binding sites was 0.5 nM [23]. On the other hand, the rapid recovery of VIP binding capacity upon removal of native VIP from previously down-regulated HT 29 cells supports the idea of a recycling of intact VIP receptors. Therefore, constant recycling of the receptor could account for the discrepancy observed between the two concentrations of VIP.

The down-regulation of VIP receptor in HT 29 cells has been recently reported [25]. In these experiments, where only one time (3 h) of preincubation with VIP was used, the down- regulation of VIP receptors was associated with a re- fractoriness of the cells. They became unable to synthesize cyclic AMP upon subsequent VIP stimulation. Our data demonstrated that in fact, down-regulation of VIP receptor could be obtained for much shorter times of preincubation.

636

This observation raised the question of whether or not short- time down-regulated HT 29 cells were desensitized. This point is now under investigation.

We are aware that in none of our experiments have we demonstrated directly that the VIP receptor was internalized, but all our data strongly support the idea that VIP internaliza- tion was mediated by receptor endocytosis and that VIP re- ceptors, probably sequestered within the cell, were recycled back to the surface.

We thank Mr F. Giannellini for his expert technical assistance. This work was supported in part by the Associationpour le Dkveloppe- ment de la recherche sur le Cancer (ARC grant 6187) and by the Znstitut National de la SantP et de la Recherche Mkdicale (INSERM grant 847006).

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