Functional Properties of Leptin Receptor Isoforms ... - Diabetes

Functional Properties of Leptin Receptor Isoforms Internalization and Degradation of Leptin and Ligand-Induced Receptor Downregulation Shigeo Uotani, Christian Bjørbæk, Jens Tornøe, and Jeffrey S. Flier

Long (ObRb) and short (ObRa) leptin receptor isoforms are thought to play essential roles in mediating leptin signaling and the transport and degradation of leptin, respectively. Although the capacity of these cloned receptor species to mediate signal transduction has been reported, there is no information on the ability of individual receptor species to mediate leptin internalization and degradation or to undergo ligand-induced downregulation. We therefore studied these parameters in Chinese hamster ovary (CHO) cells stably expressing either ObRa or ObRb isoforms of the leptin receptor. We determined that both ObRa and ObRb mediated internalization of 125I-labeled leptin by a temperature- and coated pit–dependent mechanism. Both ObRa and ObRb also mediated degradation of 125I-leptin by a lysosomal mechanism, and this was more efficiently mediated by ObRa in these cells. Neither leptin internalization nor degradation by ObRa was affected by mutation of the conserved Box 1 motif. By studying deletion mutants of ObRa, we found that efficient internalization was dependent on a motif located between amino acids 8 and 29 of the intracellular domain of ObRa. Exposure of cells expressing ObRa or ObRb to unlabeled leptin for 90 min at 37°C produced downregulation of available surface receptors, and this effect was of greater magnitude in cells expressing ObRb. Whereas CHO cells expressing the growth hormone receptor showed marked downregulation of ligand binding after exposure to dexamethasone (DEX) or phorbol myristic acid (PMA), PMA had no effect on expression of ObRa or ObRb, and DEX reduced binding to cells expressing ObRb by 15%. Thus, the two leptin receptor isoforms, ObRa and ObRb, mediate leptin internalization by a coated pit–dependent mechanism, leptin degradation by a lysosomal pathway, and ligand-induced receptor downregulation. The differential capacity of the two receptor isoforms may relate to the different roles of the receptor isoforms in the biology of leptin. Diabetes 48:279–286, 1999 From the Department of Medicine (S.U., C.B., J.T., J.S.F.), Division of Endocrinology, Beth Israel Deaconess Medical Center, Boston, Massachusetts; and Nagasaki University School of Medicine (S.U.), Nagaski, Japan. Address correspondence and reprint requests to Jeffrey S. Flier, MD, Department of Medicine, Division of Endocrinology, Beth Israel Deaconess Medical Center, 99 Brookline Ave., Boston, MA 02215. E-mail: [email protected]. Received for publication 5 June 1998 and accepted in revised form 19 October 1998. J.S.F. acts as a consultant for and has received a research grant from Eli Lilly. BBB, blood-brain barrier; BSA, bovine serum albumin; CNS, central nervous system; CSF, cerebrospinal fluid; DEX, dexamethasone; GHR, growth hormone receptor; K d , dissociation constant; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PMA, phorbol myristic acid; STAT, signal transducer and activator of transcription; TCA, trichloroacetic acid. DIABETES, VOL. 48, FEBRUARY 1999

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eptin, the product of the ob gene (1), is an adipocyte-derived hormone that plays a key role in the control of energy balance (2–5). Total deficiency of leptin due to a mutation in the leptin gene causes severe obesity in rodents and humans (1,6). Likewise, obesity results from leptin resistance, caused either by mutation within the leptin receptor gene, in rare instances, in rodents and humans (7–10) or by as yet unknown mechanisms in most cases of obesity in both species. Given this fact, an understanding of the functions of leptin receptor isoforms is a matter of considerable interest. Two major isoforms of the leptin receptor are the result of alternative splicing from a single gene (9). One isoform, ObRb, also referred to as the long form, appears to be the dominant signaling species of the receptor and is highly expressed in a limited number of sites, including key nuclei in the hypothalamus (9,11,12). Another isoform, ObRa, is more widely expressed (9,11,13) and is thought to play little or no role in signaling but to participate in leptin transport across the blood-brain barrier (BBB) (14,15) and in leptin degradation (16–18). Heretofore, virtually all studies of leptin receptor biology have focused upon the capacity of receptors to mediate signal transduction through both the Jak-STAT (signal transducer and activator of transcription) and mitogen-activated protein kinase pathways (11,19–21). This is a critical receptor function, as selective loss of the long form species in db/db mice results in a severe obesity phenotype with leptin resistance (8,9). Another aspect of receptor function that is likely to be of considerable importance for the understanding of leptin biology is the ability of the receptors to mediate leptin internalization and degradation and to undergo homologous and heterologous downregulation. Such processes are likely to be involved in the pathways of leptin transport and degradation, as well as in the process of leptin resistance accompanying hyperleptinemic states (22,23). The characterization of these processes in cells expressing either receptor isoform has not been reported and is the subject of this report. RESEARCH DESIGN AND METHODS Cell culture and transfections. Chinese hamster ovary (CHO) cells were cultured in Ham’s F12 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum, 50 U/ml penicillin G, and 50 µg/ml streptomycin under 5% CO2. COS cells were cultured in Dulbecco’s modified Eagle’s medium supplemented as above. cDNA for the short and long forms of the leptin receptor (ObRa and ObRb) were cloned into pcDNA3.1Zeo(–) (Invitrogen, Carlsbad, CA) as recently described by Bjørbæk et al. (21). CHO cells stably expressing ObRa or ObRb were generated by transfection using lipofectamine (Gibco), and cells resistant to Zeocin (Invitrogen) were selected over a 2-week period. Clones were 279

LEPTIN INTERNALIZATION AND DEGRADATION

screened by binding studies using 125I-labeled leptin. cDNA encoding the Box 1 mutant of ObRa was constructed as described by Bjørbæk et al. (21). A cDNA encoding the five amino acid deletion of the COOH-terminus of ObRa (ObRaC5) was constructed by first generating a polymerase chain reaction (PCR) product encompassing a unique EcoNI restriction enzyme site (codons 637–639) and the corresponding COOH-terminus of ObRa (codon 895). The downstream oligonucleotide primer was designed to include a stop code at codon 890 followed by a HinDIII site. The PCR product was then digested with EcoNI and HinDIII and subsequently ligated into the ObRa-pcDNA3.1Zeo(–) plasmid, which was digested with the same enzymes. A cDNA encoding the 26 amino acid deletion of ObRa (ObRa-C26) was made using a similar strategy by introducing a stop code at codon 879 in the downstream PCR primer. Both constructs were sequenced using standard techniques. COS and CHO cells were transiently transfected by lipofectamine using either wild-type or mutated ObRa cDNA constructs. CHO cells stably expressing the full-length rat growth hormone receptor (GHR) were provided by Dr. C. Carter-Su. Transient transfections were performed by standard techniques as described previously (21). Binding assays. For determination of equilibrium dissociation constants, CHO cells were incubated with 0.2 µCi 125I-labeled human leptin (Du Pont-NEN, Boston, MA) and indicated concentrations of unlabeled recombinant mouse leptin in Ham’s F12 medium containing 1 mg/ml of bovine serum albumin (BSA) (Sigma, St. Louis, MO) for 18 h at 4°C. Under these conditions, we obtained steady-state binding (data not shown). CHO cells expressing rat GHR were incubated with 100,000 cpm of 125I-labeled human growth hormone (Du PontNEN) in Ham’s F12 medium containing 1 mg/ml of BSA at 4°C for 2 h, in the presence or absence of 100 nmol/l unlabeled human growth hormone (Sigma). Cells were then washed three times with ice-cold phosphate-buffered saline (PBS) and dissolved in lysis buffer (1% Nonidet P-40, 0.5% Triton X-100, 1 mol/l NaOH). Radioactivity in the lysates was measured in a gamma-counter. Nonspecific binding was determined as the radioactivity bound in the presence of 100 nmol/l unlabeled leptin or growth hormone, or to cells transfected with empty vector. 125 I-leptin internalization. The internalization of leptin was determined by the acid-wash method (24,25). Confluent cells in 6-well plates were washed twice with ice-cold PBS and then incubated in 0.5 ml of binding buffer (Ham’s F12 medium with 1 mg/ml of BSA) with 100,000 cpm of 125I-leptin at 4°C for 2 h. After 2 h, cells were warmed to 37°C for periods from 5 to 15 min. At each time point, cells were washed and pelleted in ice-cold PBS and then resuspended in 0.5 ml of ice-cold PBS or ice-cold buffer (0.5 mol/l NaCl, 0.2 mol/l acetic acid, pH 2.0) and then incubated on ice for 5 min. Cells were then pelleted and resuspended again for 5 min in the same buffers, pelleted, and then lysed. Cell-associated radioactivity was counted in a gamma-counter. Values obtained after 2 h at 4°C were subtracted from all time points at 37°C. At each time point, percent internalization was determined as the percentage of specifically bound radioactivity that was acid resistant at each time point. The acid wash removed >95% of bound 125I-leptin, and cells exposed to acid wash retained >95% of the initial number of binding sites (data not shown). To determine the role of coated pits in leptin internalization, the protocol above was carried out in parallel in a hypertonic buffer (binding buffer supplemented with 0.3 mol/l sucrose) that is known to inhibit coated pit–mediated internalization (26). Degradation of 125I-leptin. Confluent cells in 6-well plates were washed with ice-cold binding buffer and preincubated with 0.5 ml of this buffer containing 100,000 cpm of 125I-leptin for 2 h at 4°C. After three washes in binding buffer, cells were incubated at 37°C in 0.5 ml of the same buffer for various times. At each time point, supernatants were mixed with 0.5 ml of 10% trichloroacetic acid (TCA) (27). After incubation on ice for 1 h, samples were centrifuged for 5 min at 10,000g. This procedure precipitated 98% of intact 125 I-leptin as determined by TCA precipitation of a known amount (cpm) of stock 125I-leptin and subsequent measurement of recovered radioactivity in the precipitate. Supernatants were counted to determine the TCA-soluble radioactivity (degraded ligand). Receptor downregulation. Confluent cells in 6-well plates were washed twice with binding buffer. Cells were then incubated with binding buffer containing 0–100 nmol/l unlabeled leptin for 90 min at 37°C. After acid wash, cells were washed three times with the binding buffer and incubated for an additional 2 h at 4°C with the same medium containing 100,000 cpm of 125I-leptin (28). Cells were washed three times, lysed, and the cell-associated radioactivity was determined in a gamma-counter.

RESULTS

Characterization of stable cell lines expressing leptin receptor isoforms. Because of a lack of cell lines expressing endogenous leptin receptors, we established several independent lines of CHO cells stably expressing the mouse ObRa or ObRb leptin receptor isoforms. Binding studies with multiple clones were carried out at 4°C using 125I-labeled leptin with and 280

without 100 nmol/l unlabeled leptin. All clones expressing ObRa had approximately sixfold higher specific binding compared with clones expressing ObRb (Fig. 1). Only nonspecific binding was seen in mock-transfected cells. Total expression of ObRa in the cells was also higher as determined by Western blotting (data not shown). When equilibrium binding data from competition curves were subjected to Scatchard analysis (data not shown), a representative clone expressing ObRa had ~30,000 receptor sites per cell with a dissociation constant (Kd) of 0.2 nmol/l, and a representative clone expressing ObRb had ~1,500 receptors per cell with a dissociation constant (Kd) of 0.3 nmol/l. Similar dissociation constants for the long and short leptin receptor isoforms have been reported by others (7,29–31). Both ObRa and ObRb mediate internalization of 125I-leptin in stable CHO cell lines via a coated pit pathway. To identify receptor-mediated internalization of 125I-leptin, we analyzed the acid-resistant uptake of 125I-leptin in CHO cell lines at 37°C and 4°C. Leptin was internalized by both ObRa and ObRb receptor isoforms at 37°C (Fig. 2). Between 10 and 15% of specifically bound leptin was internalized after 15 min at 37°C by each isoform. With both receptors, incubation at 4°C (data not shown) or with hypertonic medium markedly reduced the amount of leptin internalized (Fig. 2), consistent with an active process that is mediated by a coated pit mechanism (26). Both ObRa and ObRb mediate degradation of 125I-leptin in stable CHO cell lines via a lysosomal pathway. Once internalized by a receptor-dependent mechanism, one fate of many peptide ligands is degradation by a lysosomal mechanism (32). To assess this, we preincubated CHO cells expressing ObRa or ObRb with 125I-leptin for 2 h at 4°C, under which condition minimal leptin is internalized, followed by washing

FIG. 1. 125I-leptin binding to CHO cells stably expressing ObRa and ObRb. Leptin binding competition was determined at 4°C in several independent CHO cell-line clones (numbers below diagram) expressing either ObRa or ObRb. Cells were cultured in 6-well plates and serum-starved for 18 h. Cells were cooled by washing in ice-cold binding medium and incubated for 2 h at 4°C with 125I-leptin in the absence ( ) or presence ( ) of 100 nmol/l unlabeled leptin. Data are expressed as means ± SE (n = 3 experiments). DIABETES, VOL. 48, FEBRUARY 1999

S. UOTANI AND ASSOCIATES

FIG. 2. Effects of hypertonia on 125I-leptin internalization. CHO cells expressing either ObRa or ObRb were preincubated with 125I-leptin at 4°C for 2 h and then warmed to 37°C for various times at which cells were washed with ice-cold PBS and then incubated in either PBS or in acidic buffer to determine total cell-associated and acid-resistant radioactivity, respectively. Internalized leptin was expressed as the percentage of specific cell-associated radioactivity that is acid-resistant at each time point. Values obtained after 2 h at 4°C were subtracted from all time points at 37°C. Nonspecific binding was determined by incubating parallel cultures with 100 nmol/l unlabeled leptin and was subtracted. , ObRa; , ObRb; ——, control buffer; -----, hypertonic buffer. Data are expressed as means ± SE (n = 3 experiments).

and warming cells to 37°C for various times. At each time point, TCA-soluble counts in the media were determined. As seen in Fig. 3A, no degraded leptin appears in the medium during the first 30 min. However, degraded leptin increases in the medium over the next 90 min in both ObRa- and ObRbexpressing cells. It is apparent that in these cells ObRa more efficiently targets leptin for degradation than does ObRb, with 37 vs. 11% degraded at 120 min. To assess the role of lysosomes in the receptor-mediated degradation of leptin, degradation was carried out in the presence and absence of lysosomal inhibitors, 10 mmol/l ammonium chloride, 10 mmol/l methylamine, or 50 µmol/l chloroquine. Degradation of leptin by both ObRa and ObRb was completely inhibited by each of these reagents, demonstrating that degradation is mediated by a lysosomal compartment in these cells (Fig. 3B). Leptin-mediated internalization and degradation via ObRa occur despite mutation of the Jak Box 1 site in the intracellular domain. We have previously shown that ObRa, which only has a predicted 34 amino acid long intracellular domain, has the capacity to activate Jak2 via a conserved Jak Box 1 motif in the juxtamembrane region of the receptor (21). To determine whether the Jak Box 1 site is required for internalization and degradation of leptin by this receptor, we mutated the site and determined the capacity of the wild-type and mutated receptors to mediate these processes. In these experiments, we used COS cells that were transiently transfected with receptor constructs. Both internalization (Fig. 4A) and degradation (Fig. 4B) of leptin DIABETES, VOL. 48, FEBRUARY 1999

FIG. 3. Degradation of 125I-leptin in transfected CHO cells and effects of lysosomal inhibitors. A: CHO cells expressing either ObRa ( ) or ObRb ( ) were preincubated with 125I-leptin for 2 h at 4°C. After washing, cells were incubated at 37°C for various times in the binding medium. At the desired times, the medium was removed and subjected to TCA precipitation. TCA-soluble radioactivity was taken as a measure of degradation of 125I-leptin. TCA-soluble radioactivity was expressed as a percentage of the initial cell-associated radioactivity. B: Cells expressing either ObRa ( ) or ObRb ( ) in 6-well plates were preincubated with 125I-leptin for 2 h at 4°C. After washing, cells were incubated at 37°C for 2 h without inhibitors or with 10 mmol/l ammonium chloride, 10 mmol/l methylamine, or 50 µmol/l chloroquine. The medium was then removed and subjected to TCA precipitation. The degradation of 125I-leptin was expressed as the percentage of the initial cell-associated radioactivity. Data are expressed as means ± SE (n = 3 experiments).

were the same in cells expressing wild-type and Jak Box 1–mutated ObRa. The mutation had no effect on total expression or on cell surface expression of ObRa, as determined by 125 I-leptin binding studies at 4°C and by Western blotting of total lysates (data not shown). 281


FIG. 5. Internalization of 125I-leptin by wild-type and truncated ObRa in CHO cells. 125I-leptin internalization in CHO cells transiently expressing either wild-type ObRa ( ) or ObRa with a 5 amino acid COOH-terminal deletion (ObRa-C5) ( ) or ObRa with a 26 amino acid COOH-terminal deletion (ObRa-C26) ( ). After the initial binding at 4°C, unbound tracer in the medium was removed. Cells were then incubated at 37°C for various times and subjected to acid wash as described in METHODS. Data were calculated as in Fig. 2 and are expressed as means ± SE (n = 3 experiments).

FIG. 4. Internalization and degradation of 125I-leptin in transfected COS cells expressing wild-type or Box 1 mutant ObRa. A: 125 I-leptin internalization in COS cells transiently expressing either wild-type ( ) or mutant ( ) ObRa was determined as described in Fig. 2. Data are expressed as means ± SE (n = 3 experiments). B: Degradation of 125 I-leptin in COS cells transiently expressing either wild-type ( ) or mutant ( ) ObRa was determined as described in Fig. 3. Data are expressed as means ± SE (n = 3 experiments).

Efficient 125I-leptin internalization by ObRa in CHO cells is dependent on a motif located between amino acids 8 and 29 of the intracellular domain of ObRa. Previous studies of membrane receptors have demonstrated the presence of specific motifs in the intracellular domains that are recognized by specific membrane-associated adaptor proteins and are required for efficient internalization (33,34). To identify such motifs in the 34 amino acid cytoplasmic domain of ObRa, we first made two deletion mutants of this isoform, as described in METHODS. Western blotting using antiObR antibodies of total lysates from transfected CHO or COS cells showed that expression levels of the mutants were the same as those of wild-type ObRa (data not shown). In addition, these truncations of ObRa did not affect specific binding of 125I-leptin at 4°C in transfected cells, suggesting similar cell surface expression (data not shown). We next stud282

ied ligand internalization in transiently transfected CHO cells. As shown in Fig. 5, deletion of the last five residues of ObRa (ObRa-C5) did not affect receptor-mediated internalization of 125I-leptin. However, deletion of 26 amino acids (ObRa-C26) reduced internalization ~60% at 5 min and ~40% at 10 min as compared with ObRa. These data therefore suggest that an internalization signal exists between amino acids 8 and 29 of the intracellular domain of ObRa. Leptin mediates downregulation of leptin receptor binding in stable CHO cell lines. To determine whether leptin exposure causes loss of available leptin binding sites on cells expressing ObRa and ObRb, cells were exposed to 0–100 nmol/l leptin for 90 min at 37°C and then acid washed to remove cell surface–bound leptin. This was followed by exposure to 125I-leptin, with and without 100 nmol/l unlabeled leptin, at 4°C for 2 h, to assess available binding sites. As seen in Fig. 6, exposure to leptin causes a downregulation of accessible leptin binding sites in cell lines expressing both ObRa and ObRb. Substantial downregulation of each isoform was seen after exposure to 0.1 nmol/l leptin, with binding reduced by 30 and 50% with ObRa and ObRb, respectively. With ObRaexpressing cells, the dose response to increasing leptin concentrations was flat above this level of leptin, whereas in cells expressing ObRb, leptin at increasing doses further suppressed leptin binding site availability, with a maximal suppression of 80% after exposure to 100 nmol/l leptin. Phorbol myristic acid and dexamethasone have differential effects on expression of GHRs versus ObRs. In stable CHO cell lines expressing GHRs, exposure to the phorbol ester phorbol myristic acid (PMA) and to the glucocorticoid dexamethasone (DEX) causes major loss of GHRs at the cell surface as determined through ligand binding (35,36). Because DIABETES, VOL. 48, FEBRUARY 1999


FIG. 7. Effects of PMA and DEX on 125I-leptin and 125I-GH binding to CHO cells. CHO cells were treated with 100 nmol/l PMA for 1 h or 100 nmol/l DEX for 24 h. 125I-leptin and 125 I-GH binding was performed as described in METHODS. Data are expressed as means ± SE (n = 3 experiments). , control; , DEX; , PMA.

FIG. 6. Downregulation of ObRa and ObRb. CHO cells stably expressing ObRa (A) or ObRb (B) were incubated in binding medium at 37°C together with various concentrations of unlabeled leptin. After 90 min, cells were washed with acidic buffer and incubated with 125I-leptin for 2 h at 4°C. The cell-associated radioactivity was collected in a lysis buffer. Nonspecific binding was determined by incubating parallel cultures with 100 nmol/l unlabeled leptin and was subtracted. Data are expressed as means ± SE (n = 3 experiments).

the ObRs and GHRs are in the same subgroup of the cytokine receptor family, we performed parallel experiments with CHO cells expressing GHR, ObRa, and ObRb. A 1-h exposure of cells to 100 nmol/l PMA caused a 68% reduction in GH binding, and a 24-h exposure of cells to 100 nmol/l DEX caused a 47% reduction of GH binding, as previously reported (35,37). In contrast, PMA had no effect upon leptin binding to cells expressing ObRa or ObRb. DEX had no effect on leptin binding to cells expressing ObRa, but it caused a small reduction in leptin binding to cells expressing ObRb (Fig. 7). DISCUSSION

The data presented here demonstrate that two leptin receptor isoforms, ObRa and ObRb, are capable of mediating leptin uptake into cells by a coated pit– dependent mechanism, after which leptin is degraded in the lysosomal compartment. DIABETES, VOL. 48, FEBRUARY 1999

Receptor-mediated endocytosis of a ligand, followed by targeting of endosomes to fuse with lysosomes where the ligand is degraded, is a common theme for membrane receptors (25,38), including members of the cytokine receptor family to which the leptin receptor belongs (32,39–43). Although both leptin receptor isoforms mediate these effects, we have noted some differences between the two isoforms. Whereas the initial rate of leptin internalization is similar by ObRb and ObRa, internalized leptin appears to be more effectively targeted to the lysosomal compartment for degradation by ObRa than by ObRb. Although this difference could be a consequence of unequal expression levels of the two receptor isoforms, it might also relate to the capacity of ObRa to participate in receptor-mediated leptin degradation when expressed in particular cellular contexts, such as cells of the kidney. To address this question, expression of leptin receptor isoforms in additional cell types will be required. By what mechanism is internalization and targeting of leptin receptor isoforms brought about? During the process of receptor-mediated endocytosis, a general process by which nutrients, hormones, and growth factors are efficiently transported into the cell (32), many receptors are selectively concentrated in clathrin-coated pits from which they are rapidly internalized and delivered to endosomes (32). Some receptors, such as the LDL receptor, are constitutively clustered in coated pits from which they internalize in the absence of ligand (44). Other receptors, such as the insulin receptor, go to coated pits and are internalized upon ligand binding (25). For some receptors, the signal for internalization may include activation of signal transduction pathways and/or receptor phosphorylation on tyrosine or serine residues (45–48). We have shown earlier that ObRa has the capacity to mediate activation of Jak activity in a transfection model, and that this is dependent upon an intact Jak Box 1 motif in the juxtamembrane domain (21). Thus, it is possible that the Jak Box 1 could be required for either of these processes. However, we 283


demonstrate here that mutation of the Jak Box 1 has no effect on cell surface expression or on the rate at which ObRa mediates leptin internalization or degradation, suggesting that the signal mediating these processes is independent of this conserved motif in ObRa. Whether Jak-STAT signaling or phosphorylation of the receptor plays any role in these processes for the ObRb receptor isoform was not addressed here. Mutation of ObRb or coexpression of dominant-negative Jaks is required to address this issue. Efficient internalization may also require the presence of specific motifs in the intracellular domains of receptors, such as leucine-leucine or leucineisoleucine dipeptide motifs and tyrosine-based motifs, that are recognized by adaptor proteins that mediate the association with clathrin-coated pits (33,34,49). In the 34 amino acid long intracellular domain of ObRa, no tyrosine residues or known dipeptide motifs involved in internalization are present. However, peptide sequences that adopt a predicted -turn conformation have been reported to be involved in efficient internalization of a short form of the prolactin receptor (34). ChouFasman analysis of the primary structure of the intracellular domain of ObRa did predict a -turn motif between amino acids 10 and 23 of this domain (DNASTAR software, Madison, WI) (data not shown). Consistent with this region being a possible internalization motif, our studies of two deletion mutants of ObRa clearly identified a motif located between residues 8 and 29 as being required for maximal internalization in these cells. Because internalization still occurs at a significant, although reduced, rate, this suggests that other motifs, possibly located in the extreme membrane proximal region of ObRa, could also play a role in internalization of this receptor. Additional mutational analyses of both leptin receptor isoforms are required to further address these issues. We have found that the total and cell surface expression of ObRa was much higher than that of ObRb. This could possibly result from instability of the ObRb transcript or protein in these cells. However, since we and others have observed this result using various expression vectors in multiple cell lines (21,50), it is likely that the difference in expression is due to an intrinsic difference in the intracellular processing of the receptors. One possibility includes differences in recycling and degradation between the two receptors. For example, ObRa could be recycled while ObRb is degraded, both occurring in a constitutive and ligand-independent manner. The studies reported here are relevant to an understanding of the mechanism for leptin resistance in vivo. In most obese humans (22,23) and in animal models with obesity resulting from a high-fat diet or deficiency of brown adipose tissue (51), leptin levels in the blood are increased, and resistance to some or all of the actions of leptin appears to exist. The molecular basis for leptin resistance is therefore a matter of considerable importance. Substantial evidence suggests that the primary targets for leptin action reside within the brain. A key question in the understanding of leptin resistance is therefore whether the resistance occurs at the level of these target cells, in the process that delivers leptin to these sites of action, or at some other site. Several lines of evidence support the existence of a saturable system for leptin transport into the brain. First, studies with 125I-labeled leptin in intact rats reveal a saturable leptin uptake system with primary uptake into the choroid plexus and the region of the median eminence and arcuate nucleus (52). Second, studies of leptin levels in cerebrospinal fluid (CSF) of 284

normal individuals and those with anorexia nervosa (53) and obesity (54,55) reveal that leptin levels in CSF are 2–5% of those in blood, and the relationship between peripheral and CSF leptin is nonlinear, being saturated at levels commonly seen in obesity (54,55). Third, in rodents with peripheral leptin resistance due to diet-induced obesity or the NZO mouse (56), the response to centrally administered leptin may be intact, suggesting that the uptake step is rate limiting. Thus, the biochemical identity of the rate- limiting steps in leptin transport into sites of action in the brain requires investigation. It is likely that the process of leptin uptake into the central nervous system (CNS) is mediated by one or more species of the leptin receptor. The most likely species to be involved is the short form Ra, which was originally cloned from the choroid plexus (7), where it is abundantly expressed. We have recently identified this receptor species to be most highly expressed in cerebral microvessels (15), the site of the BBB, consistent with the hypothesis that this receptor is involved in leptin transport into the brain. Investigations of the potential mechanisms for receptor-mediated uptake, degradation, and clearance of leptin are therefore of interest. We have here determined the equilibrium dissociation constants (Kd) of ObRa and ObRb to be 0.2 and 0.3 nmol/l, respectively. These results are similar to those obtained by others of ObRa and ObRb using transfected cell systems (Kd = 0.4–0.7 nmol/l) (7,29–31). However, a lower affinity has been reported for endogenously expressed leptin binding sites on membrane samples from human brain microvessels (Kd = 5.2 nmol/l) (14). Although the latter study did not determine the identity of these binding sites, our studies of leptin receptor mRNA levels in rat brain microvessels point to the short form of the leptin receptor (15). The cloning of the short form of the leptin receptor from choroid plexus led to speculation that this site was important for receptor-mediated transport of leptin into the CNS via the CSF (7). However, leptin levels in the CSF are in the range of ~0.01 nmol/l (54,55), which are >30-fold lower than the Kd of ObRb. This suggests that leptin may not be transported to specific hypothalamic nuclei via the CSF, as the leptin concentration in the CSF would be too low to activate efficient signal transduction by ObRb. A more likely scenario may involve transport of intact leptin via ObRa across the endothelial cells of the BBB, followed by diffusion of leptin to specific hypothalamic neurons expressing ObRb. The expression of ObRa in the choroid plexus may play a role in degradation or transport of leptin out of the CSF. Using a defined transfection model, we observed that leptin exposure for 90 min depletes the pool of cell surface leptin receptors, an effect that is greater with ObRb than with ObRa. With ObRb, the dominant signaling form of the receptor, the leptin levels that produce downregulation in vitro are relevant to the physiological concentrations of leptin in vivo. Important sites of leptin action in the hypothalamus reside within the arcuate nucleus, and the status of the arcuate nucleus with respect to the BBB is not yet resolved (57). It is thus unknown at what concentration leptin reaches the specific neurons expressing ObRb within the hypothalamus. However, in order for leptin to activate sufficient signal transduction by ObRb, leptin concentrations need to be near the Kd of the receptor, which, based on the in vitro experiments, is in the range of 0.3–0.7 nmol/l. If peripheral levels of leptin found in normal and obese individuals (>5 nmol/l) (22,23) reach those key hypothalamic nuclei, major effects on recepDIABETES, VOL. 48, FEBRUARY 1999


tor expression would be expected based on the data reported here. Increasing evidence suggests that, whereas leptin action in the CNS is the primary and most important site of leptin action, peripheral actions of leptin, most likely exerted through ObRb, also exist (58–60). Thus, leptin-induced receptor downregulation may also be relevant to leptin resistance at sites of peripheral action. Note that the model system used here (e.g., transfected cells) would not detect any additional effects of leptin to alter receptor expression through effects on transcription of the leptin receptor gene, or effects on mRNA stability. Unfortunately, we were not able to identify clones with similar expression levels of ObRa and ObRb, an observation that has also been reported by others (50). It should therefore be noted that because of these differences in the expression of ObRa and ObRb in our stable cell lines, i.e., 30,000 vs. 1,500 receptor sites per cell, it is possible that the higher level of ObRa expression could saturate some cellular components that are used by both receptors. This could result in misleading conclusions regarding quantitative differences between ObRa and ObRb with regard to downregulation, and to internalization and degradation of leptin. Whether the observed differences between ObRa and ObRb with respect to these parameters are due to the receptors themselves or are secondary to differences in expression levels, these studies establish that both receptors mediate ligand internalization and degradation via a coated pit and lysosomal mechanism and undergo ligand-induced downregulation. One well-characterized mechanism by which hormone sensitivity can be modified involves the ability of other hormones or agents that activate distinct signaling pathways to modify receptor expression. This was recently studied in CHO cells stably expressing GHRs (35–37). The activation of protein kinase C pathways with PMA was shown to cause a rapid decline in GHRs at the cell surface (35,36). Although ObRs are closely related to GHRs, no such effect was evident in cells expressing ObRa or ObRb. Another potentially important cross-regulation involves DEX. As reported earlier (35,36), DEX caused a major time-dependent loss of GHRs, an effect that may in part explain the ability of excess glucocorticoids to induce resistance to GH in vivo (61). Because several lines of evidence suggest that glucocorticoids have a major ability to influence the actions of leptin in vivo (62), we performed similar studies in CHO cells expressing ObRs. In contrast to the GH system in the same cells, the effect of DEX on expression of ObRb was minimal (15% reduction), and no effect on expression of ObRa was seen. Thus, any effect of glucocorticoids to modify leptin sensitivity in vivo is likely to involve another site and mechanism of action. In conclusion, we have provided an initial characterization of the ability of the two major leptin receptor isoforms, ObRa and ObRb, to mediate leptin uptake and degradation and to undergo regulated expression at the cell surface in response to leptin, PMA, and DEX. These studies form the basis for further studies aimed at characterizing the role of leptin receptor isoforms in leptin transport, degradation, and signaling in vivo. ACKNOWLEDGMENTS

This work was supported by National Institutes of Health Grant DK-R37-28082 and by a grant from Eli Lilly, Indianapolis, Indiana (both to J.S.F.). DIABETES, VOL. 48, FEBRUARY 1999

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