EXPERIMENTAL

CELL RESEARCH

200,467-472

(19%)

Regulation of Plasma Retinol Binding Protein Secretion in Human HepG2 Cells FRANCESCA Molecular

TOSETTI,’

NICOLETTA

Biology Laboratory,

FERRARI,

Zstituto Nazionale

ULRICH

INTRODUCTION

Retinol, one of the natural retinoids, is an essential nutrient involved in the control of cellular differentiation and proliferation. A great deal of data have been produced in more than 60 years of extensive studies on the properties of natural and synthetic retinoids as potent agents in cancer prevention and treatment [l, 21. Despite the encouraging developments in the therapeutic use of retinoids, the molecular details underlaying the biological action of these compounds are far from being completely elucidated. Retinoids are highly hydrophobic molecules which need specific carrier proteins to be transported in the body fluids and inside the cell. The plasma transport protein of retinol is the retinol binding protein (RBP). The role of RBP is to solubilize the hydrophobic retinal molecules and to deliver them to the peripheral tissues whom

CLAUDIO

BRIGATI,

AND GIORGIO VIDALI

per la Ricerca sul Cancro, Viale Benedetto XV, 10, 16132 Genova, Italy

Retinol binding protein (RBP) is the plasma transport protein of retinol. Mobilization of RBP from the liver stores is stimulated by retinol. During vitamin A deficiency, RBP secretion is specifically inhibited while its rate of biosynthesis is unaffected. As a consequence, RBP, as apoprotein, accumulates inside the endoplasmic reticulum (ER) of the hepatocyte, and a new elevated steady-state concentration is reached. We have studied the role of degradation on the regulation of RBP metabolism in retinol deficient HepG2 cells and determined the intracellular site where RBP degradation takes place. Pulse-chase experiments show that RBP half-life is ca.9 h in retinol-depleted cells. RBP degradation is slow and is insensitive to the treatment with NH&l, which inactivates lysosomal proteases and to the drug brefeldin A, which prevents protein export from the ER. The data obtained suggest that RBP degradation occurs, at least in part, in a pre-Golgi compartment. 2-Mercaptoethanol, at millimolar concentration, induces RBP secretion, suggesting a possible role for sulfhydryl-mediated #PO-RBP retention by resident ER proteins. 0 1992 Academic Press, Inc.

1To

PFEFFER,

reprint requestsshould be addressed. 467

[3]. The proteic moiety of the complex also contains conformational features so as to recognize a specific membrane bound receptor on the surface of the target cells [4]. The retinol-RBP complex associates in the plasma with one molecule of transthyretin (TTR), which prevents RBP filtration through the kidney glomeruli. Upon delivery of the retinol molecule to its membrane receptor, the RBP-TTR complex dissociates and RBP is filtered and degraded in the kidney [5]. Structural data exist which suggest that conformational changes are induced in the protein upon removal of the retinol molecule and that the preservation of the holo structure is fundamental for the RBP molecule to recognize both TTR and the membrane receptor [6,7]. RBP belongs to a family of proteins, named lipocalins, which share a conserved structural motif characterized by an eight-stranded antiparallel P-barrel and transport small hydrophobic molecules in the body fluids [S]. RBP is synthesized and secreted primarily by the hepatocytes. The newly synthesized RBP binds one retinol molecule in the endoplasmic reticulum [9] and the binding of the ligand triggers its transport through the classical route of secretion, from the Golgi apparatus to the secretory vesicles [lo]. During retinol deficiency, RBP secretion is blocked, while its rate of biosynthesis and the translatable level of RBP-specific mRNA in rat liver remain unaltered [ll]. As a consequence, apo-RBP concentration reaches an abnormal elevated value and accumulates within the endoplasmic reticulum [12, 131. To maintain the homeostasis, the hepatic cell must activate some degradative mechanism to dispose of the RBP excess that fails to be secreted. The experiments which are described in this work are intended to provide more insight into the molecular mechanisms controlling RBP secretion and catabolism during retinol deficiency. MATERIALS

AND METHODS

Chemicals. The following reagents were purchased from Sigma (St. Louis, MO): all-trans-retinal, the protease inhibitors leupeptin, pepstatin A, phenylmethylsulfonyl fluoride (PMSF). Brefeldin A (BFA) was a kind gift from Sandoz (Milano, Italy). Ammonium chloride was from Carlo Erba (Milano, Italy). [%]Methionine was from Amersham (Milano, Italy]. A polyclonal sheep antiserum against human RBP was purchased from PAA (Laborgesellschaft, Linz, Austria). 0014-4827/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

466

TOSETTI

Cell culture. Human hepatocellular carcinoma HepG2 cells (ATCC HB 8065) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5 mM glutamine, 50 U/ml penicillinstreptomicin, 1% nonessential amino acids for minimum essential medium Eagle (Flow), and 10% fetal calf serum (PAA) or 10% delipidated fetal calf serum [14] (complete medium). Cells grown in the presence of retinol were maintained for at least three passages in complete medium containing delipidated serum and 1 pM retinol was added to the cells as a sterile dispersion in 0.15 M NaCl, 500 pg/ml bovine serum albumin [15]. Medium containing retinol was changed every 3 days. Metabolic labeling. Semiconfluent HepG2 cells were washed with PBS, detached in PBS-0.02% EDTA, and washed again in methionine-free medium. Since adhesion to the substrate has no influence on the rate of synthesis and secretion of RBP by HepG2 cells (data not shown; see also Ref. [15]) cells were labeled in suspension at the density of 2 X lOs/ml in a minimum amount of medium. After a 15min incubation in methionine-free medium containing 10% dialyzed FCS, 5 mM glutamine, 1% nonessential amino acids, cells were labeled with 200 &i/ml [s’S]methionine for 2 h or pulse labeled for 1 h and chased for the times indicated with cold methionine in the presence or absence of 3 pM retinol and drugs. When brefeldin A or NH&l was used, cells were preincubated in the presence of the drug for 1 h at 37°C in complete medium. BFA was used at the concentration of l-5 pglml and ammonium chloride at the concentration of 50 mM [ 161. The drugs were also present during the pulse and chase. Immunoprecipitation and electrophoresis. At the times indicated cells where collected by centrifugation, washed with PBS, and lysed in 1 ml of 70 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1% Triton X-100, 0.05% SDS (TTS buffer), 1 mM PMSF, 3 wg/ml aprotinin, 1 wg/ml leupeptin, 25 mM iodoacetamide. Cell lysates were first precleared with protein A-Sepharose (Pharmacia-LKB) for 1 h at ambient temperature. Immunoprecipitation was performed overnight at 4°C with 2 ~1 of antiserum to human RBP, 2 ~1 of rabbit anti-sheep serum, and 20 ~1 of protein A-Sepharose. Immunoprecipitates were washed three times in TTS buffer, washed once in 5 mM Tris-HCl, pH 7.5, eluted in Laemmli SDS sample buffer for 2 min at 100°C in the presence or absence of 5% 2-mercaptoethanol, and loaded on 15% SDS-polyacrylamide gels [17]. In the immunoprecipitation experiment performed to test the reactivity of the antibodies used with unfolded RBP (Fig. 3B), the protein purified from human plasma [ 181 was partially reduced with 1 mM 2-mercaptoethanol for 2 min at lOO”C, alkylated with 25 mM iodoacetamide for 15 min at ambient temperature, then diluted with PBS, and reacted with the antibodies conjugated to CNBr-activated Sepharose 4B (Pharmacia-LKB) overnight at 4’C. Densitometric analysis. Gels were treated with Amplify (Amersham), dried under vacuum, and exposed to Hyperfilm MP (Amersham) at -80°C. The autoradiograms were analyzed by densitometric scanning of the bands using an automated Ultroscan XL densitometer (Pharmacia-LKB). The densitometric scanning of the bands was conducted as a two-dimensional analysis. When not otherwise stated, the percentage of secreted RBP was calculated according to the formula % secreted RBP = (secreted RBP/intracellular RBP + secreted RBP) X 100 and, for the pulse and chase experiments, % secreted RBP at the end of chase = (secreted RBP at the end of chase/secreted RBP at the end of pulse) X 100. The same calculations were adopted for the intracellular protein. All data represent the mean of at least two independent experiments and the calculations were made on two different exposures of the same gel. REP purification. RBP was purified from human plasma according to the method of Berni et al. [18]. RESULTS

Retinol Induces RBP Secretion in HepG2 Cells HepG2 cells grown in medium containing 10% fetal calf serum (normal medium) were labeled with 200 &i/

ET

AL.

123 ---E

SC

45

6

SC

SC

-DMEM-

CUltUW cmditiws

7

8 s

DMEM lO%retinol free serum

lO%serum

B RETJNOL indlnse w

pulse e

0

-

0.05

CSCSCS

0.2

1

___

escs

5

-

FIG. 1. RBP synthesis and secretion in HepG2 cells. (A) HepG2 cells grown in the presence of 10% fetal calf serum secrete less than 30% of RBP synthesized during 3 h 1abeJing with 200 PCi of [“S]methionine (lanes 3,4). Analogous distribution of RBP is seen in the same cells labeled in the absence of serum (lanes 5, 6) and in cells maintained in delipidated serum for three passages and labeled in the presence of dialyzed serum (lanes 7,8). Retinol added to the medium at the concentration of 3 PM induces the secretion of RBP (lanes 1,2). (B) RBP secretion is enhanced by the presence of retinol in a dose-dependent manner. Cells (3 X 106) grown in lipid-depleted medium were labeled with 200 &i/ml of [35S]methionine for 2 h and chased for the same time in the presence of increasing concentrations of retinol (ROH) from 0.05 to 5 PM. The values reported in the plot (C) represent the mean of two independent experiments. Calculation of secreted protein was made according to the formula indicated under Materials and Methods. c, cytosol; s, supernatant.

ml [35S]methionine for 3 h in the presence of 10% dialyzed fetal calf serum and cell lysates and medium were immunoprecipitated with an anti-human RBP serum. Under these conditions, about 30% of synthesized RBP is secreted by the cell (Fig. lA, lanes 3,4; in this calculation, the amount of RBP degraded during the continuous labeling is not considered). Analogous distribution of retained and secreted RBP is observed in cells grown in the same conditions and labeled in serum-free medium (Fig. lA, lanes 5, 6) and in cells grown for an ex-

REGULATION

469

OF RBP SECRETION

tended period of time in medium containing 10% delipidated serum (vitamin A depleted) (Fig. lA, lanes 7, 8). Thus, HepG2 cells constitutively secrete a certain amount of RBP when grown in retinol-depleted medium. Vitamin A, added to the cells during the labeling, enhances the secretion of its binding protein up to about 60% of synthesized RBP (Fig. lA, lanes 1, 2). In a different set of experiments HepG2 cells were grown in retinol-depleted medium (containing lipid-depleted serum) and RBP secretion in control delipidated cells was compared to the secretion of the protein from cells incubated in chase medium supplemented with retino1 at various concentrations (Figs. 1B and 1C). Cells were pulse-labeled for 2 h with 200 &i/ml [35S]methionine and chased for 2 h in the absence or presence of increasing concentrations of retinol. At the lowest concentration tested (0.05 FM), retinol stimulates RBP release from the cell and, under our experimental conditions, RBP secretion reaches a plateau at the 1 FM retinol concentration. The highest concentration tested (5 PM), routinely employed to study the induction of differentiation of several cell lines [19], does not produce any additional increase on RBP secretion. Our data are consistent with previously reported results on RBP release in culture medium, quantitated by radioimmunoassay, upon retinol administration in several responding cell lines or primary cultures of rat hepatocytes [15, 20, 21, 311. Kinetics of RBP Secretion and Degradation

No information is presently available on the fate of intracellular RBP which fails to be secreted by liver cells during vitamin A deficiency. This problem was approached by following the kinetics of RBP secretion and degradation in HepG2 cells maintained in medium depr&ed of retinol. HepG2 cells grown in delipidated medium were pulse labeled for 1 h with 200 uCilm1 135S1methionine-and chased in the presence or absence of3 PM retinol (Fig. 2). In the absence of retinol, the protein is detectable in the medium after 1 h of chase and its concentration progressively increases up to 20% of total synthesized RBP at 8 h of chase (Fig. 2C). When retinol is present during the chase, almost twice as much radioactively labeled RBP is found outside the cells. In the same figure, the total amount of protein still retained within the cell plus the amount secreted at the different times of chase is also plotted (Fig. 2A). In the presence of retinol, about 25% of the synthesized protein is lost during the 8-h chase, while in control cells a 40% loss is observed. The amount of RBP that escape degradation in cells chased in the presence of retinol (about 15% of initially synthesized RBP), compared to control cells, is comparable to the amount mobilized by the binding of retinol and secreted by the same cells (see Figs. 2A and 2C).

RBP is Partly Degraded in a pre-Go@ Compartment

During vitamin A deficiency, most of newly synthesized RBP accumulates inside the endoplasmic reticulum (ER) [12, 131. To define RBP degradation kinetics in conditions which prevent RBP to escape from this compartment, we tried to interfere with the transit of RBP from the endoplasmic reticulum to the Golgi complex in the absence and in the presence of retinol. Therefore, we employed BFA, a pharmacological agent which inhibits the export of secretory and membrane proteins from the endoplasmic reticulum [16]. The treatment of HepG2 cells with 1 pug/ml BFA inhibits the secretion of RBP but not degradation (Figs. 2B and 2C). The kinetics of RBP degradation are actually enhanced by the treatment with BFA, 40% being lost within 1 h. This indicates that, at least within the first 8 h of chase, RBP degradation takes place in a pre-Golgi compartment. As expected, the addition of 3 PM retinol to the chase medium containing BFA, does not reverse the inhibition of RBP secretion caused by BFA, but induces RBP stabilization, which is particularly evident in the first hour of chase (Fig. 2C and 2B, respectively). To confirm these data, we tested the effects on RBP degradation of NH&l, a lysosomotropic agent which inhibits lysosomal degradation of proteins by raising the intralumenal pH and inactivating acid-dependent proteases [22]. In the presence of 50 mM NH&l, RBP degradation is not inhibited, as it proceedes approximately at the same rate as in control delipidated cells. At 8 h of chase, about 40% of initially synthesized RBP is lost in treated cells as in control cells, while the amount recovered in the culture medium is reduced compared to untreated cells (Figs. 2A and BC!). These data agree with the hypothesis that RBP degradation within 8 h occurs in a nonlysosomal compartment. Effects of 2-Mercaptoethanol

on RBP Secretion

To test whether RBP could be retained through a mechanism involving disulfide interchange reactions with resident ER proteins, as described for other proteins whose degradation occurs in the ER [23], pulse-labeled HepG2 cells were chased in medium containing 2-ME at two concentrations (1.4 and 7 mM) with or without 3 PM retinol (Fig. 3A). The results of this experiment can be summarized as follows: (a) At 1.4 mM concentration, 2-ME induces secretion of RBP to a level comparable to that induced by retinol (lanes 9,lO). (b) No further secretion is detected when retinol and 1.4 mM 2-ME are added simultaneously (lane 12). (c) At higher concentration (7 mM) 2-ME reduces RBP recovery from both cell lysates and supernatants (lanes 5, 11). The latter result, which can be in part prevented by retinol (lanes 7,13), may be due to increased RBP degradation, reduced antigenicity, or both. We have also excluded the possibility that the antibodies employed

470

TOSETTI

ET

AL.

60-

-A-

20 t

+BPA

-b-

04

I

0

I

2

time4 (h)

+BFA+ROH .

I

6

.

u

8

- +NH4cl

0

--.-

+BFA

+

+BFA+ROH

/

2

6

8

time4(h) FIG. 2. Kinetics of RBP secretion and degradation in HepG2 cells grown in retinol-depleted medium. HepG2 cells grown in lipid-depleted medium for at least three passages were pulse labeled for 1 h with 200 &i/ml of [%]methionine and chased under different culture conditions and RBP was immunoprecipitated with a specific antiserum. Cells were preincubated with the drugs for 1 h at 37°C before the labeling. Data were obtained from the densitometric scanning of the autoradiograms (not shown) as described under Materials and Methods and represent the mean of at least two independent experiments. In A and B, data are expressed as total recovered radioactivity (secreted + intracellular RBP) relative to radioactivity present at the end of pulse. In C, secreted RBP is calculated according to the formula described under Materials and Methods. (A) Control retinol deficient cells chased in the absence or presence of retinol (ROH) or 50 mM NH&l. (B) Brefeldin A (BFA)-treated cells chased in the absence or presence of retinol. Cells were preincubated with the drugs for 1 h at 37°C before labeling. (C) RBP secretion in retinol deficient cells labeled under different conditions.

could not recognize partially unfolded RBP molecules generated in the presence of 2-ME. RBP purified from human plasma was reduced in the presence of 1 mA4 2-ME. The amount of RBP immunoprecipitated by the antibodies conjugated to CNBr-activated Sepharose 4B was similar in reduced and nonreduced samples (Fig. 3B). Thus, RBP degradation is enhanced in cells chased in the presence of 7 mikf 2-ME. Moreover, the simultaneous presence of retinol seems to protect intracellular RBP from degradation. RBP Folding and Secretion As secretory or membrane proteins not properly folded are generally retained inside the ER [24], we investigated if intracellularly retained and degraded RBP is present in a nonnative conformation, compared to secreted RBP. Since disulfide bonds formation can be considered a probe for protein folding [25], we analyzed RBP mobility in SDS polyacrylamide gels under reducing and nonreducing conditions. The RBP molecule contains six cysteine residues. Three disulfide bonds,

which are required for ligand binding activity, are established in the mature secreted protein [26]. We observed that reduced RBP has a slower mobility in SDS gels, as compared to that of the unreduced protein. The presence of different concentrations of 2-ME generates multiple bands: between the fully oxidized form (three disulfide bonds, M, 21,000) and the fully reduced form (M, 24,000), two other intermediate bands originate by partial reduction of the native protein, probably corresponding to the presence of one and two disulfide bonds, respectively. At the concentration of about 1 mkf, all four forms are present in solution (see Fig. 3, lane 3). Addition of retinol does not alter the pattern of RBP reduction by 2-ME (data not shown). In in uiuo experiments, none of the slower migrating partially reduced forms can be detected in the immunoprecipitated intracellular protein, even in cells labeled with a short lomin pulse (data not shown). DISCUSSION RBP secretion by the liver cell is a highly regulated process, and one of the most relevant effects related to

REGULATION

OF

A 1234561 2-m ROH

.

.

-

1.4

1 .

2 .

.

+

I

1.4

I

3

4

+ -

+ +

8

9

10

. -

. +

1.4 -

11 12

13

I -

I +

1.4 +

B

MW (kD) 31

5 -

2.ME IlmM At,'

21 14

FIG. 3. RBP secretion is induced in the presence of reducing agents. (A) Retinol deficient HepG2 cells were labeled with 200 aCi/ ml [%]methionine for 2 h and chased for the same time in the presence of 1.4 or 7 mM 2-mercaptoethanol(2-ME) (lanes 4,10, and 511 respectively) or in the presence of 2-ME and 3 pM retinol (ROH) (lanes 6, 7,12, 13). Lane 1, intracellular RBP after the pulse. Lane 2, control cytosol after 2 h. Lane 3, cytosol after 2 h of chase plus retinol. Lanes 8,9, control secreted RBP after 2 h in the absence (8) or presence (9) of retinol. c, cytosol; s, supernatant. (B) Partially reduced RBP purified from human plasma (lane 4), as unreduced RBP (lane 2) is immunoprecipitated by the antibodies (Ab) used in the previously described experiments. Control unreduced (lane 1) and reduced RBP (lane 3). The recovery of immunoprecipitated RBP is low, due to the small amount of conjugated antibodies used compared to RBP concentration in the reaction mixture. Control antibodies conjugated to CNBr-activated Sepharose 4B-treated like samples in lanes 2, 4 (lane 5). Coomassie blue stain of the SDS gel.

vitamin A deficiency is the blocking of RBP secretion from the hepatocyte. In retinol deficient cells, the amount of RBP associated with the Golgi-rich fraction decreases and RBP is found almost exclusively associated with the rough and smooth endoplasmic reticulum fraction [ 12,131. The critical step in RBP secretion, probably controlled by retinol, is therefore the transport from the endoplasmic reticulum to the Golgi complex [32]. The main goal of our work was to design an experimental model to study the fate of intracellular RBP retained inside the cell in conditions of retinol deprivation and to define the role of retinol in modulating the kinetics of RBP degradation and secretion. In agreement with other reports [20], we have shown that HepG2 cells grown in medium containing 10% fetal calf serum or 10% delipidated serum, secrete RBP and that the secretion can be enhanced by retinol in a dosedependent manner (Fig. 1). Pulse-chase experiments conducted on delipidated HepG2 cells (Fig. 2) show that RBP secretion is a slow process: the protein can, in fact, be detected in the chase medium at 1 h of chase and the release proceedes increasing until 8 h of chase. The amount of RBP secreted in the presence of retinol after

RBP

SECRETION

471

8 h approximately doubles compared to control cells. The half-life of the protein is about 9 h, and it remains unaltered if retinol is present in the chase medium (Fig. 2). The data obtained indicate that most of the RBP synthesized under conditions of retinol deficiency is retained intracellularly and a part is eventually degraded. An alternative degradative pathway, independent from the lysosomal one, has been recently described which allows the removal from the ER of misfolded or unassembled secretory and membrane proteins which fail to be secreted [28]. This homeostatic cellular response has been interpreted as the consequence of a quality control mechanism, which prevents the export from the ER of proteins which relevant biological functions in anomalous conformations. For example, apolipoprotein B (apoB), a component of the lipid metabolism whose secretion is dependent on lipid availability, is degraded at the level of the ER [29]. Our data show that RBP synthesized by retinol deficient HepG2 cells is in part degraded in a pre-Golgi compartment. This assumption is based on the failure of both BFA, a drug which inhibits the egress of proteins from the ER, and NH&l, a compound which inhibits lysosomal proteases, to inhibit RBP degradation. Under these conditions RBP degradation occurs as in control cells (Fig. 2). Retention inside the ER caused by BFA is not per se coupled to degradation [28]: thus we believe that RBP degradation in the presence of BFA represents a mechanism to dispose of newly synthesized apoRBP. Similarly, upon treatment of delipidated HepG2 cells with NH&l the kinetics of RBP degradation is not affected until 8 h of chase (Fig. 2). This result indicates that about 40% of synthesized RBP is degraded in a compartment other than lysosomes. Molecular dynamics simulations data have suggested that apo-RBP has a different conformation from that of the holoprotein and that RBP in the apo form cannot perform most of its functional roles, like the recognition of a membrane receptor on target cells [7]. Therefore, it is possible that, in the absence of retinol, apo-RBP could be recognized as not properly folded by the molecular machinery, which controls the exit of proteins from the ER, and thus be degraded. We have also addressed the question as how RBP is retained inside the ER, testing the hypothesis that the intracellular transport of RBP could be regulated by a recently proposed mechanism involving disulfide interchange reactions with some resident ER protein, like BiP (binding protein) or PDT (protein disulfide isomerase) or some other unknown protein [23]. In that work, the responsiveness of secretory proteins to the presence of reducing agents which disrupt -SH-mediated interactions, has been indicated as a probe to establish if a protein is retained inside the ER by this mechanism. The results obtained on the effects of 2-ME at 1.4 mM concentration on RBP secretion (Fig. 3) are indicative

472

TOSETTI

that this mechanism plays a role in the regulation of RBP secretion. However, the higher 2-ME concentration employed in our experiments (7 m&f), which has been proved to be effective and not toxic to the cells [23], causes a more pronounced disappearance of intracellular RBP and, at the same time, a reduction of RBP secretion, as compared to control cells. The addition of retinol to the chase medium together with 7 mM 2-ME, protects RBP from degradation and partially restores RBP secretion. Having excluded the possibility that the antibodies employed in this experiment did not react with unfolded RBP, we think that the presence of the reducing agent in the chase medium could induce an alteration in RBP folding that results in an enhanced susceptibility to pre-Golgi degradation. The binding of retinol to apo-RBP might protect intracellular RBP by competing with the molecular factor(s) responsible of apo-RBP retention and degradation. However, in contrast with the results obtained with 2-ME, when the presence of full or incomplete disulfide bonding was analyzed in RBP synthesized by retinol deficient cells, no folding intermediates were found (data not shown). Although structural abnormalities could be responsible of the intracellular retention of RBP synthesized in retinol deficient cells, with our methodology it is not possible to detect any difference in the folding of RBP synthesized in the presence or absence of retinol. Most probably, minor changes in the conformation of apo-RBP, observed in vitro upon retino1 removal [ 71, may contribute to the block of apo-RBP transport through the secretory pathway. Further experiments are needed to identify the conformational features on apo-RBP that signal RBP retention and degradation to the cell. We thank M. Rocco, A. Rubartelli, and R. Sitia for helpful discussions, P. Perfumo and F. Campelli for technical assistance, M. Isola for photographical work, and Sandoz (Milano, Italy) for kindly providing BFA. This work was supported by grants from the Consiglio Nazionale delle Ricerche (CNR) Nr.91.00398.CT04 to N.F., from the Minister0 della Sanita’ Nr.12418003 and Nr.12419008, and from the Associazione Italiana per la Ricerca sul Cancro (AIRC) Nr.13453027 to G.V.

REFERENCES

ET

A. B., and Goodman, Press, New York. 4.

Sivaprasadarao, 255,561-569.

5. 6.

Peterson,

7. 8. 9. 10.

Lippman, S. M., Kessler, J. F., and Cancer Treat. Rep. 71,493-5X.

2.

Lippman, vich, M. 555-560. Goodman,

3.

Received Revised

D. S. (1984)

July 30,199l version received

in The

January

Retinoids

29, 1992

M. B., Roberts,

Eur.

J. Clin.

J. B. C. (1988) Znuest.

Academic Biochem.

J.

1,437-444.

Aqvist, J., Sandblom, P., Jones, T. A., Newcomer, M. E., van Gunsteren, W. F., and Tapia, 0. (1986) J. Mol. Biol. 192, 593604. Cowan, S. W., Newcomer, M. E., and Jones, T. A. (1990) Proteins 8, 44-61. Godovac-Zimmerman, J. (1988) Trends Biockm. Sci. 13,64-66. Hendriks, H. F., Elhanany, E., Brower, A., de Leeuw, A. M., and Knook, D. L. (1988) Hepatology 8, 276-285. Smith, J. E., Deen, D. D., Sklan, D., and Goodman, D. S. (1980) J. Lipid Res. 2 1, 229-237.

Rask, L., Valtersson, C., Anundi, H., Dallner, G., and Peterson, P. A. (1983) 102.

13.

Harrison, E. H., Smith, J. E., and Goodman, D. S. (1980) Biochin. Biophys. Acta 628,489-497. Cham, B. E., and Knowles, B. R. (1976) J. Lipid Res. 17, 176181.

14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

30.

@porn,

P. A. (1971)

Findlay,

2, pp. 41-88,

12.

F. L. (1987)

S. M., Lee, J. S., Lotan, R., Hittelman, W., WargoJ., and Hong, W. K. (1990) J. Natl. Cancer Inst. 82,

A., and

Vol.

Soprano, D. R., Smith, Chem. 257, 7693-7697.

28. Meyskens,

D. S., Eds.),

11.

29. 1.

AL.

31. 32.

J. E., and Goodman,

D. S. (1982)

J. Biol.

Kvist, S., Eriksson, U., Exp. Cell Res. 143,91-

Smith, J. E., Borek, C., and Goodman, D. S. (1978) Cell 15, 865-873. Bonifacino, J. S., Suzuki, C. K., and Klausner, R. D. (1990) Nature 247,79-82. Laemmli, U. K. (1970) Nature 227,680~685. Berni, R., Ottonello, S., and Monaco, H. L. (1985) Anal. Biothem. 150,273-277. Davies, B. H., Pratt, B. M., and Madri, J. A. (1987) J. Biol. Chem. 262, 10,280-10,286. Marinari, L., Lenich, C. M., and Ross, A. C. (1987) J. Lipid Res. 28,941-948. Dixon, J. L., and Goodman, D. S. (1987) J. Cell. Physiol. 130, 14-20. Lippincott-Schwartz, J., Bonifacino, J. S., Yuan, L. C., and Klausner, R. D. (1988) Cell 54,209-220. Alberini, C. M., Bet, P., Milstein, C., and Sitia, R. (1990) Nature 347,485-487. Pelham, H. R. B. (1989) Annu. Reu. Cell Biol. 5, l-23. Creighton, T. E. (1978) Prog. Biophys. Mol. Biol. 33, 231-297. Raz, A., Shiratori, T., and Goodman, D. S. (1970) J. Biol. Chem. 245, 1903-1912. Knowles, B. B., Howe, C. C., and Aden, D. P. (1980) Science 209,497-499. Klausner, R. D., and Sitia, R. (1990) Cell 62, 611-614. Davis, R. A., Thrift, R. N., Wu, C. C., and Howell, K. E. (1990) J. Biol. Chem. 265, 10,005-10,011. Hurtley, S. M., and Helenius, A. (1989) Annu. Reu. Cell Biol. 5, 277-307. Ronne, H., Ocklind, C., Wiman, K., Rask, L., Obrink, B., and Peterson, P. A. (1983) J. Cell Biol. 96, 907-910. Goodman, D. S. (1987) Harvey Lect. 81, 111-132.

Regulation of plasma retinol binding protein secretion in human HepG2 cells.

Retinol binding protein (RBP) is the plasma transport protein of retinol. Mobilization of RBP from the liver stores is stimulated by retinol. During v...
1MB Sizes 0 Downloads 0 Views