Identification and Characterization of a Luteinizing Hormone/Chorionic Gonadotropin (LH/CG) Receptor Precursor in a Human Kidney Cell Line Stably Transfected with the Rat Luteal LH/CG Receptor Complementary DNA

R. William

Hipkin,

Jesus

Sanchez-YagQe*,

and Mario

Ascoli

Department of Pharmacology University of Iowa College of Medicine Iowa City, Iowa 52242

It is well established that the LH/CG receptor expressed in gonadal cells is an 85 to 92-kilodalton (kDa) glycoprotein. Additionally, however, a number of reports have noted the existence of other putative receptor species, but few attempts have been made to characterize these variant receptor species. A cell line [293L(wtl)] had previously been isolated which expresses large numbers of high affinity cell surface LH/CG receptors. Visualization of the LH/CG receptor species expressed in these cells and in rat luteal cells using ligand blots revealed 85 and 90-kDa LH/CG receptors, respectively, while immunoblots revealed another 68-kDa glycoprotein receptor in both cell types. The presence of both the 85 and 68-kDa receptor species was confirmed using immunoprecipitation and affinity purification of metabolically labeled 293L(wtl) cells. Enzymatic deglycosylations established that the 85kDa receptor is a sialoprotein, while the 68-kDa species contains exposed high mannose residues. Protease digestion before LH/CG receptor immunoprecipitations localized the 85-kDa receptor on the plasma membrane, while the 68-kDa receptor was shown to be located intracellularly. Pulse-chase experiments were then used to positively establish that the 68kDa receptor protein is actually a precursor of the 85-kDa LH/CG receptor species. (Molecular Endocrinology 6: 2210-2218, 1992)

exposure to elevated levels of LH/CG attenuates the cellular response in the face at continuous gonadotropin stimulation. Although this diminished sensitivity to LH/ CG can result from a gradual decrease in LH/CG receptor numbers (down-regulation), it can also occur very rapidly without any measurable reduction in the density of LH/CG receptors (uncoupling). To better understand these phenomena, it would be beneficial to establish a system in which to study cotranslational and posttranslational modifications of the LH/CG receptor. Such studies have been hampered by the relative scarcity of this receptor in gonadal tissues. In an attempt to overcome this constraint, embryonic kidney 293 cells were transfected with an expression vector encoding for the wildtype rat luteal LH/CG receptor (I), and a stable cell line [293L(wtl)] was isolated which expresses large numbers of high affinity cell surface LH/CG receptors (0.5 1 .O x lo6 LH/CG receptors/cell; Kd, -500 PM) (2). However, before their use for studies of potential posttranslational modifications of the receptor, it is necessary to more fully evaluate the physical characteristics of the LH/CG receptors expressed by the 293L(wtl) cells and to establish methods (such as immunoprecipitation) that can be used to ascertain whether covalent modifications indeed occur. A majority of studies using highly purified testicular or ovarian LH/CG receptor preparations have established that this receptor is a glycoprotein with a mol wt of 85,000-92,000 (3-5) It should be noted, however, that there have been a number of reports of additional putative LH/CG receptor species, ranging in size from 46-l 70 kilodaltons (kDa), but few systematic attempts have been made to positively identify and characterize these variant LH/CG receptor species (6-8). The studies reported herein use a number of biochemical approaches to identify and characterize the LH/CG receptors expressed by 293L(wtl) cells. These studies demonstrate that in addition to an 85-kDa LH/

INTRODUCTION Like other hormone receptors, the influence of LH/CG on cellular function is self-regulated, in that continuous 0888-8809/92/221 o-221 8$03.00/0 Molecular Endocrlnolcqy Copyrtght 0 1992 by The Endcane

Society

2210

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LH/CG

Receptor

Precursor

CG cell surface receptor, there exists a 68-kDa LH/CG receptor. Studies were initiated to conclusively establish the identity of this receptor variant in 293L(wtl) cells.

RESULTS identification of the LH/CG Receptor Species Expressed in Rat Corpus Luteum and 293L(wtl) Cells

Figure 1 depicts results from ligand blots of lectinpurified rat luteal and 293L(wtl) cell lysates. As previously reported, the rat luteal LH/CG receptor is approximately 90 kDa (4, 9) The 293L(wtl) cells, however, express a LH/CG receptor that is slightly smaller at 85 kDa, while the untransfected 293 cells do not synthesize a LH/CG receptor. This finding is consistent with immunochemical data presented in Fig. 2, in which the 85 and 90-kDa LH/CG receptor species are visualized with a polyclonal antibody raised against the purified

90 kDa 85 kDa

L _

EXCESSUNLABELLED hCG Fig. 1. Ligand Blots of the LH/CG Receptor Expressedin Rat Corpus Luteum, 293L(wtl), and 293 Cells Detergent extracts of rat corpus luteum, 293L(wtl), and 293 cells were partially purified with wheat germ-agglutinin agarose chromatography, electrophoresed under nonreducing conditions on a SDS-polyacrylamide gel (10 ng LH/CG receptor/mm gel), and electrophoretically transferred to PVDF membrane. The membrane strips were incubated overnight with 10 rig/ml [12Sl]hCG in the presence or absence of 50 IU unlabeled hCG, washed, and analyzed by autoradiography, as described in Materials and Methods.

2211

nondenatured rat luteal receptor (Bugs). It should be noted, however, that in addition to the 85/90-kDa receptor proteins, the antibody reveals the presence of a 68-kDa protein. While this band can be easily seen on blots of lectin-purified 293L(wtl) and crude rat luteal material, it is only faintly visible on lectin-purified luteal blots and is not discernible on photographs. Previous studies have shown that the 85/90-kDa protein is glycosylated (Fig. 1) (3, 4, 9) Since the 68-kDa protein can be detected in lectin-purified 293L(wtl) and rat luteal cell lysates, it can be concluded that it also is a glycoprotein. lmmunoprecipitation experiments using [35S]methionine- or [35S]cysteine-labeled cells were also used to visualize the LH/CG receptor species expressed in 293L(wtl) cells. As shown in Fig. 3, immune (but not preimmune) antibody successfully immunoprecipitated both the 85- and 68-kDa receptor proteins from metabolically labeled 293L(wtl) cells, with the 68-kDa protein predominating. Indeed, the 85kDa protein is not always readily visible in immunoprecipitates of [35S] methionine-labeled cells (cf. Fig. 5). Since purification on wheat germ-agglutinin agarose before immunoprecipitation did not eliminate the 68-kDa protein, these data again indicate that this protein is glycosylated. Another option for visualization of the LH/CG receptor involves affinity purification of partially purified lysates from metabolically labeled 293L(wtl) cells (4). As can be seen in Fig. 4, the hCG-Affi-gel-10 resin did precipitate both the 85- and 68-kDa LH/CG receptor species, but with this strategy, the 85-kDa receptor protein was more predominant. Taken together these results show that the 293L(wtl) cells express two LH/CG receptor species. An 85-kDa protein can be detected in ligand blots (Fig. 1) and immunoblots (Fig. 2) as well as by immunoprecipitation (Fig. 3) or affinity purification (Fig. 4) of metabolically labeled cells. Using these latter procedures, we found that this receptor species is more readily detected in [35S]cysteine-labeled cells than in [35S]methioninelabeled cells (Fig. 3) presumably because of the greater abundance of cysteine residues in the LH/CG receptor (1). A 68-kDa protein is also easily detectable in immunoblots (Fig. 2) and in immunoprecipitates of metabolically labeled cells regardless of the amino acid used for labeling (Fig. 3). When methods based on hormone binding are used (i.e. ligand blots or affinity purifications), however, the results obtained depend on the amount of hormone used. In ligand blots, where a low concentration of hormone (10 rig/ml) is used, the 68kDa protein cannot be detected (Fig. 1). In affinity purifications, where a large concentration of hormone (100-200 Fg/ml) is used, the 68-kDa protein is detectable (Fig. 4). These findings can be easily reconciled if one assumes that the 68-kDa protein binds hCG with low affinity, and this affinity is lower than that of the 85kDa protein. Further Characterization of the Different LH/CG Receptor Species Expressed by 293L(wtl) Cells

While it has become apparent that a second, smaller LH/CG receptor species is expressed in luteal and

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MOL ENDO. 1992 2212

Vol6No.12

90 kDa 85 kDa

-

68 kDa

Fig. 2. Western Blots of the LH/CG Receptor Species Expressed in Rat Corpus Luteum, 293L(wtl), and 293 Cells Detergent lysates (crude and lectin-purified) were electrophoresed under nonreducing conditions on a SDS-polyacrylamide gel (10 ng LH/CG receptor/mm gel) and electrophoretically transferred to PVDF membrane. The membrane strips were incubated overnight with 3 pg/ml of a polyclonal antibody raised against the full-length rat luteal LH/CG receptor (Bugs IgG) or preimmune antibody (P.I. IgG), as described in Materials and Methods. Antibody binding was visualized as described in Materials and Methods. WGA, Wheat germ-agglutinin.

85 kDa 68 kDa -

85kDa 68kDa

b -

EXCESS hCG Fig. 4. Affinity Purification of LH/CG Receptor Species from [35S]Cysteine-Labeled 293L(wtl) Cells After metabolic labeling with [35S]cysteine, the LH/CG receptor species from k&in-purified detergent lysates were precipitated with 100 /LI packed hCG-Affi-gel-10 resin in the presence or absence of 100 IU crude hCG, as described in the Fig. 3. lmmunoprecipitation of LH/CG Receptor Species from [%]Methionine- or [%]Cysteine-Labeled 293L(wtl) Cells After metabolic labeling with [35S]methionine or [35S]cysteine, the LH/CG receptor species from detergent lysates (crude or lectin-purified) were immunoprecipitated with Bugs (Bugs IgG) or preimmune antibody (P.I. IgG), as described in Materials

and Methods.

293L(wtl) cells, the identity of this protein and the reason(s) for its diminished size are addressed in the experiments described below. To determine whether the 68-kDa protein is a proteolytic fragment of the 85 kDa LH/CG receptor or simply an unrelated protein

Materials

and Methods.

expressed in the parental 293 cell line, another immunoprecipitation study was attempted using polyclonal antibodies raised against the first (anti-N) or last 14 (anti-C) amino acids in the LH/CG polypeptide in addition to Bugs and preimmune antibodies. As can be seen in Fig. 5, all of the immune antibodies were successful in immunoprecipitating the 68-kDa protein from the biosynthetically labeled 293L(wtl) cells, but not from the untransfected 293 cells. This experiment shows that the 68-kDa protein is expressed only in the cells

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LH/CG Receptor Precursor

CELL TYPE

68 kDa

lmmunoprecipitation of LH/CG Receptor Species from [%]Methionine-Labeled 293 and 293L(wtl) Cells After metabolic labeling with [%]methionine, the LH/CG receptor from lectin-purified detergent lysates was immunoprecipitated (as described in Materials and Methods) using antireceptor (Bugs IgG) or preimmune antibodies (P.I. IgG) as well as antibodies to the first 14 (anti-N IgG) and last 14 (antiC IgG) amino acids of the rat luteal LH/CG receptor.

Fig. 5.

transfected with the cDNA for the rat luteal LH/CG receptor and that the protein represents the intact polypeptide sequence of the receptor. The results presented in Fig. 5 (as well as Fig. 7) also serve to illustrate that nonspecific precipitation of other proteins can be a problem in some experiments. Subsequent improvements in technique resulted in a substantial reduction in these nonspecific precipitations, however (cf. Figs. 3, 8, and 9). Previous studies have shown that changes in the content of certain sugars, such as sialic acid, can cause seemingly inordinate shifts in the relative mol wt of several glycoproteins, including the LH/CG receptor (911). Since the 68-kDa protein is glycosylated, as judged by its ability to absorb to wheat germ-agglutinin-agarose (see above and Figs. 2, 3, and 5) the experiments presented in Figs. 6 and 7 were performed to ascertain whether differences in glycosylation patterns could be responsible for the diminished mol wt of the 68-kDa receptor relative to that of the 85kDa receptor. Western blots and immunoprecipitations were used in concert with glycosidase exposure to study the carbohydrate content of the 85 and 68-kDa receptors expressed in the 293L(wtl) cells as well as that of the 90kDa receptor expressed in rat luteal tissue. As can be seen in Fig. 6, incubation of partially purified cell lysates with neuraminidase or peptide N-glycosidase-F before immunoblotting showed that a difference in sialic acid content was responsible for the difference in size between the 90-kDa luteal and 85-kDa 293L(wtl) LH/CG receptors. Desialylation reduced the mol wt of both receptor species to approximately 78,000, while complete deglycosylation with peptide N-glycosidase-F re-

2213

duced the relative mol wt of the 90/85-kDa receptor further to 60,000, in agreement with previous findings (9, 11). Since the 68-kDa protein is readily discerned in immuoprecipitations of metabolically labeled cells, this method was used in an attempt to determine the carbohydrate nature of this receptor species (Fig. 7). The mol wt of the 68,000 receptor species was not affected by neuraminidase exposure, suggesting that this receptor species lacks terminal sialic acid residues. Complete deglycosylation with peptide N-glycosidase-F again decreased the mol wt to 60,000, which is in agreement with the Western blot data (Fig. 6). EndoglycosidaseH, which catalyzes the removal of exposed high mannose residues, was also effective in reducing the size of this band from 68,000 to approximately 60,000. Similar exposure of the 85kDa receptor to endoglycosidase-H was not deemed necessary after the demonstration of terminal sialic acid residues on that protein (Fig. 6). The presence of exposed high mannose residues strongly suggests that the 68-kDa protein is an immature form of the 85-kDa receptor (10, 12). Previous studies demonstrated that certain crude collagenase preparations contained an undefined Ca*+dependent protease, which “nicked” the LH/CG receptor protein without disrupting its ability to bind hCG (13). Susceptibility to this protease was, thus, used in an attempt to establish the location of the 85-kDa receptor as well as the 68-kDa species. The results presented in Fig. 8A show that incubation of intact [35S] methionine-labeled 293L(wtl) cells with the protease before immunoprecipitation results in degradation of the 85-kDa protein, and this degradation is absent when the cells are coincubated with EGTA (13). This confirms that, as expected, the 85-kDa receptor is located on the cell surface and is, therefore, accessible to the protease. It also supports earlier findings from crosslinking studies on intact cells describing a similarly sized LH/CG receptor species on the surface of cells transiently transfected with the cDNA for the rat luteal LH/ CG receptor (14). Figure 8A also demonstrates that the structure of the 68-kDa species was not affected by the protease in the intact cells. If, however, cells were solubilized before protease exposure, the 68-kDa species becomes very susceptible to proteolytic degradation (Fig. 88). This finding together with that illustrated in Fig. 8A would suggest that only inaccessibility to the protease affords the 68-kDa protein its resistance to degradation in the intact cell. Identification of the 66-kDa LH/CG Receptor Species as a Precursor of the 85kDa Receptor Confirmation that the 68-kDa receptor species represents the intact polypeptide sequence of the LH/CG receptor protein (Fig. 4) has high mannose moieties (Fig. 7), and is located in the intracellular compartment (Fig. 8) suggests that this protein is an immature form of the 85-kDa receptor. To the verify the relationship

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MOL 2214

ENDO.

Vol6No.12

1992

CORPUS

LUTEUM

293L(wtl)

78 kDa -

60 kDa

-

19O

Ql’ & 6%

\96

\9O

,96 Q\'

&

6%

,96 Q\'

,96

,96 &

6”

Ql’

Fig. 6. Effect of Glycosidase

Digestion on the 85/90-kDa LH/CG Receptor Species from Corpus Luteum and 293L(wtl) Cells Lectin-purified detergent extracts of rat corpus luteum and 293L(wtl) cells were incubated at 37 C with no additions for 8 h, 100 mu/ml neuraminidase (NEURAMIN.) for 60 min, or 20 U/ml peptide N-glycosidase-F (PGNase F) for 8 h. The extracts were then electrophoresed under nonreducing conditions on a SDS-polyacrylamide gel (10 ng LH/CG receptor/mm gel) and electrophoretically

transferred to PVDF membrane. The membrane strips were incubated overnight with antireceptor antibody (Bugs IgG) or preimmune antibody (P.I. IgG), as described in Materials and Methods and Fig. 2.

between these two receptor species, pulse-chase experiments were initiated. 293L(wtl) cells were biosynthetically labeled with [35S]cysteine for 2 h (pulsed), the labeling medium was replaced, the cells were incubated for a further 0, 2, 4, or 6 h (chased), and the LH/CG receptors were immunoprecipitated. As shown in Fig. 9, the 68-kDa protein band was predominant immediately after the 2-h labeling with [35S]cysteine. At different times after the onset of the chase, the intensity of the 68-kDa receptor species gradually diminished, and the intensity of the 85kDa receptor protein band gradually increased. Thus, it is clear that the 68-kDa protein is a precursor of the 85kDa protein. It should also be noted that another 165kDa receptor protein was evident immediately after the pulse labeling of the cells. Studies have been initiated to positively identify this receptor species.

DISCUSSION

We have shown that 293L(wtl) cells, a transfected cell line expressing large number of high affinity cell surface LH/CG receptors (2) actually synthesize at least two forms of the LH/CG receptor protein. The studies we have presented here suggest that the receptor species responsible for high affinity cell surface binding of hCG is an 85-kDa glycoprotein (Figs. 1, 2, and 8A). This finding is consistent with the size of the LH/CG receptor deduced from previous studies on normal and malignant (i.e. MA-IO) gonadal cells (3, 4,

9, 13). In fact, our data show that the small difference in mol wt between the 85-kDa receptor species in 293L(wtl) cells and that found in normal gonadal tissues is due to differences in sialic acid content (Fig. 6). Indeed, much larger changes in mobility on sodium dodecyl sulfate (SDS)-polyacrylamide gels can result from relatively small changes in the carbohydrate content of glycoproteins (lo), including the LH/CG receptor (9, 11). The studies presented here also show that 293L(wtl) cells express a 68-kDa precursor of the mature 85-kDa LH/CG receptor. We identified this protein as a precursor because 1) it represents the intact polypeptide structure of the mature receptor (Fig. 5); 2) it contains exposed high mannose residue (Fig. 7); 3) it is located intracellularly, rather than on the cell surface (Fig. 8B); and 4) pulse-chase experiments show a precursorproduct relationship, whereby the 68-kDa protein is converted to the 85-kDa protein (Fig. 9). Although the presence of similar immature receptor forms containing high mannose residues has been noted for other cell surface receptors (10,12), this is the first demonstration of a similar precursor for the LH/CG receptor. The relative abundance of the 85- and 68-kDa proteins has proven difficult to quantitate. For example, using immunoblots, it appears that the 85-kDa protein is the most prevalent (Fig. 2). In fact, in some experiments, the 68-kDa protein was difficult to detect with this methodology (Fig. 6). On the other hand, the 68kDa protein is definitely the most prevalent species detected using immunoprecipitations (Figs. 3 and 5). This apparent discrepancy can be explained by assum-

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LH/CG Receptor Precursor

2215

d6* kDa -60

P.I. IgG

-

kDa

BUGS IgG

Fig. 7. Effect of Glycosidase Digestion on the 68-kDa LH/CG Receptor Species lmmunoprecipitated from [?S]Methionine-Labeled 293L(wtl) Cells After metabolic labeling with [35S]methionine, the LH/CG receptor from lectin-purified detergent lysates was immunoprecipitated from 293L(wtl) cells using antireceptor (Bugs IgG) or preimmune antibodies (P.I. IgG). The immunoprecipitates (in sample buffer with reducing agents) were diluted 20-fold to decrease the SDS concentration to 0.1% before incubation at 37 C with no additions, 100 mU endoglycosidase-H (END0 H), or 20 U peptide N-glycosidase-F (PGNase F) overnight, or with 100 mU neuraminidase (NEURAMIN.) for 60 min, as described in Materials and Methods and Fig. 3.

ing that the ability of the antibody to recognize these two proteins is dependent on their conformation (i.e. in soluble form as opposed to attached to a blotting membrane). As noted above (see Resulrs), methods based on hormone recognition identify only the 85kDa species (Fig. 1) unless very large quantities of hCG are used (Fig. 4). This finding agrees with data showing that there is only one class of high affinity (Kd, 200-500 PM) receptors in detergent extracts of 293L(wtl) cells (15) (our unpublished observations) and suggest that the 68-kDa species binds hCG with very low affinity. The reason for the low binding affinity of the 68-kDa species is not known. Since it represents a partially glycosylated form of the mature receptor, the low binding affinity could be a consequence of the incomplete glycosylation. This interpretation would be in agreement with data of Dufau and co-workers (9, 16) who, using glycosidase digestions and site-directed mutagenesis, concluded that glycosylation of the LH/CG receptor is needed for high affinity binding. On the other hand, Ji and co-workers (17) and Petaja-Repo and co-workers (11) used glycosidase digestions and inhibition of glycosylation in intact cells to reach the opposite conclusion. Thus, until this controversy is resolved, we cannot address the possibility that the incomplete glycosylation of the 68-kDa LH/CG receptor species reported here is responsible for its low binding affinity. Other interpre-

tations, such as incomplete or incorrect folding of the polypeptide chain, are also possible. A 68-kDa LH/CG receptor protein was also identified in immunoblots of rat corpora luteum (Fig. 2). It was not possible to conclusively show that this protein is also a precursor of the 90-kDa receptor present in this tissue, however, because of the experimental limitations encountered when working with normal gonadal cells. In this respect, it is also important to note that previous reports from this laboratory failed to identify this low mol wt species in immunoblots from rat corpora luteum (4). We believe that this discrepancy relates to the enhanced sensitivity of the methods used here as opposed to those used previously (4). For example, we now use a blotting membrane that has a higher proteinbinding capacity (i.e. polyvinylidine difluoride (PVDF) rather than nitrocellulose) and a more sensitive second antibody detection system (alkaline phosphatase rather than horseradish peroxidase). It is possible that immature LH/CG receptor species may have been isolated in earlier studies, but were thought to be either receptor subunits (18) or possibly proteolytic fragments (19, 20) of what was subsequently shown to be the mature 85 to 92-kDa LH/CG receptor species (3, 4). Vuhai-Luuthi and co-workers (8) recently described a 68-kDa receptor species in the porcine testis without establishing its identity. This re-

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MOL 2216

ENDO.

1992

Vol6

CONDITIONS OF PROTEASE EXPOSURE

0 MIN

15 MIN

45 MIN

45 MIN

CHASE

TIME

Oh

2h I

4h I

No. 12

6h I

165 kDa +

85 kDa -m 68 kDa -m 65 kDa 68 ktla

-t -

DURATION OF PROTEASE EXPOSURE

68 kDa

0 MN

,

15 MN

I)

B Fig. 8. The Effect of Protease Incubation of Intact (A) or Solubilized (B) [%]Methionine-Labeled 293L(wtl) Cells on Subsequent lmmunoprecipitation of the LH/CG Receptor Species A, After metabolic labeling with [35S]methionine, intact 293L(wtl) cells were incubated with collagenase type I (250 pg/ml) for the indicated times, as described in Materials and Methods. The cells were then lysed and immunoprecipitated, as described in Materials and Methods and Fig. 3. B, After metabolic labeling with [%]methionine, 293L(wtl) cells were detergent solubilized and incubated with collagenase type I (250 *g/ml) for the indicated times, as described in A. The lysate was then immunoprecipitated, as described in Materials and Methods and Fig. 3.

species did not appear to bind hCG, because it could not be isolated (from extracts that had been preincubated with hCG) using an anti-hCG affinity matrix. A protein corresponding in size to the mature receptor, however, could be isolated using this same procedure (8) lmmunoprecipitation of the LH/CG receptor from a relatively crude cellular lysate of 293L(wtl) cells, which

ceptor

Fig. 9. Pulse-Chase Labeling and Subsequent Immunoprecipitation of LH/CG Receptor Species from 293L(wtl) Cells After metabolic labeling with 200 pCi/ml [35S]cysteine for 2 h (pulsed), 293L(wtl) cells were further incubated in complete medium (chased) for 0, 2, 4, or 6 h. At these times, the cells were lysed and immunoprecipitated, as described in Materials and Methods.

express large numbers of LH/CG receptors, establishes this system as an ideal experimental paradigm to study both the maturation and potential posttranslational modifications of the receptor. In the present study, we have, for the first time, identified and characterized a precursor of the LH/CG receptor. Current experiments are aimed at determining whether the rate of conversion of the immature to the mature form of the LH/CG receptor is under hormonal control and if this step is involved in controlling the sensitivity of the cells to the hormone. Moreover, this system is ideally suited to study certain covalent modifications of the LH/CG receptor (such as acylation or phosphorylation; see Refs. 21 and 22), which have been shown to affect the function of other G-protein-coupled receptors.

MATERIALS Hormones

AND and

METHODS

Supplies

Purified hCG (CR-125) was kindly provided by the National Hormone and Pituitary Agency of the NIDDK. lodination of hCG was performed using lactoperoxidase, as previously described (23). PVDF membrane and all electrophoresis and blotting reagents were purchased from Bio-Rad (Richmond, CA). Peptide N-glycosidase-F and endoglycosidase-H were purchased from Boehringer Mannheim (Indianapolis, IN), while collagenase type I (code CLS; 199 U/mg) was obtained from Cooper Biomedical/Worthington (Malvern, PA). Neuraminidase and protein-A-agarose were obtained from Sigma (St. Louis, MO). Specialized culture medium (cysteineand methionine-free Dulbecco’s Modified Eagle’s Medium), Tran3%-Label (70% [35S]methionine and 20% [35S]cysteine), and [35S]cysteine were purchased from ICN Biomedicals (Irvine, CA). Kodak XAR film (Eastman Kodak, Rochester, NY) was used for ligand blots and immunoprecipitations. Tissue culture plasticware and supplies were obtained from Corning

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LH/CG

Receptor

Precursor

2217

(Corning, NY) and Gibco (Grand Island, NY), respectively. All other reagents were purchased from commonly used suppliers and were of the best grade available (for details, see Ref. 4).

wt standards were dyed with Coomassie calibrate each gel. The gels were incubated salicylate, dried, and exposed to preflashed for l-l 4 days (3).

blue and used to with 1 M sodium Kodak XAR film

Cells Human embryonic kidney 293 cells (CRL 1573) were obtained from the American Type Culture Coliection (Rockville, MD) and maintained in Dulbecco’s Modified Eaale’s Medium with 10 mM HEPES, 50 pg/ml gentamicin, and-lo% heat-inactivated newborn calf serum, pH 7.4. The preparation of clonal stable transfectants expressing the wild-type rat luteal LH/CG receptor [293L(wtl) cells] has been previously described (2). Cells were plated on day 0 and used on day 3 or 4, as described previously (2). Metabolic Detergent

Labeling Lysates

of 293L(wtl)

Cells

and

Preparation

of

Experimental cultures of 293L(wtl) cells were maintained in six-well (35-mm) elates. The medium was removed from the wells and replaced with 1 ml methionine-free medium (augmented as described above, but with 1% heat-inactivated newborn calf serum) containing 100 &i/ml Tran?S-Label or cysteine-free medium containing 100 or 200 &i/ml [?S]cysteine. Unless otherwise stated, the cells were then incubated for 6 h at 37 C under 5% C02, cooled on ice, and incubated overnight at 6 C. The cells were then washed twice with icecold buffer A (0.15 M NaCl and 20 mM HEPES, pH 7.4) and subsequently scraped into buffer A containing protease inhibitors (1 mM phenylmethylsulfonylfluoride, 1 PM pepstatin-A, 1 PM leupeptin, and 1 mM EDTA). The cells were pelleted by centrifugation, lysed by vortexing in buffer A containing 0.5% Nonidet P-40 and the aforementioned protease inhibitors (buffer B), and centrifuged at 100,000 x g for 30 min. In some cases, labeled and unlabeled detergent extracts were also prepared, using buffer B containing 10% glycerol to help preserve hCG-binding activity (24). These extracts were used in all experiments using ligand blots or affinity purifications. The cellular lysates were then partially purified using wheat germ agglutinin agarose chromatography, as described previously (4) except that 3 mM triacetylchitotriose was used to elute the glycoproteins from the lectins. Rat luteal cell lysates were prepared as previously described (4). The crude or lectinpurified lysates from luteal and unlabeled 293L(wtl) cells were then assayed for [‘251]hCG-binding activity, as previously described (4) and stored frozen at -20 C until needed. lmmunoprecipitation 293L(wtl) Cells

of Metabolically

Labeled

293 and

[?S]Methionineand [35S]cysteine-labeled cell lysates were supplemented with SDS (final concentration, 0.5%) and precleared by incubation at 6 C for 1 h with 150 pg normal rabbit serum immunoglobulin G (IgG) prebound to 50 ~1 packed and washed protein-A-agarose. After preclearing, the lysate corresponding to two 35mm wells was incubated for 2 h at 6 C with either 150 fig immune or preimmune IqG prebound to 50 ~1 packed and washed protein-A-agarose.-A number of polyclonal IgGs were used, including anti-LH/CG receptor antibody 145 (4), herein termed Buqs, as well as antibodies aqainst synthetic peptides representing the first (anti-N) or last 14 (anti-C) amino acids in the rat LH/CG receptor polypeptide (25). After incubation, protein-A was washed repeatedly, as previously described (3) and the bound material was eluted with 150 ~1 sample buffer [2% (wt/vol) SDS, 10% glycerol, 20 mM EGTA, 0.5 mg/ml bromophenol blue, and 62.5 mM Tris, pH 6.81 containing reducing agents (5% p-mercaptoethanol and 50 mM dithiothreitol) and stored frozen at -20 C until use. The samples and mol wt standards were electrophoresed on 7.5% SDS-polyacrylamide gels, as previously described (3). The mol

Purification of the LH/CG Labeled 293L(wtl) Cells

Receptor from [%]CysteineUsing hCG-Affi-Gel

Lectin-ourified lvsates of f?Slcvsteine-labeled 2931wtl) cells were prepared’as described above, except that dells’were plated in loo-mm plates. The lysates were applied to washed hCG-Affi-gel-10 resin (prepared according to manufacturer’s instructions) in the presence or absence of 100 IU crude hCG (100 IJI packed hCG-Affi-gel/lOO-mm plate). The lysates were then incubated at 6 C for 3 h, centrifuged, and washed repeatedly (3). The precipitated material was eluted by incubation for-1 5 min on ice, with vor-texing in 150 ~1 sample buffer containing reducing agents (see above), and analyzed by SDSpolyacrylamide gel electrophoresis and autoradiography. Proteolytic

Degradation

of the LH/CG

Receptor

293L(wtl) cells were labeled with [?S]methionine as described above, except that the cells were then immediately incubated for 0, 15, or 45 min (four 35mm wells/time point) in medium containing 250 pg/ml of a type I collagenase preparation that has been shown to contain an unidentified Ca”-dependent protease that will “nick” the LH/CG receptor without interfering with hCG binding (13). Some cells were incubated for 45 min with the collagenase solution plus 3 mM EGTA, which has been shown to inhibit the activity of this protease (13). At the end of the incubation period, intact cells or lysates were cooled on ice, and EGTA was added to 3 mM to inhibit further protease activity. The intact cells were washed twice with buffer A, solubilized if required, and immunoprecipitated, as described above. In other experiments, the metabolically labeled cells were solubilized (as described above) before incubation with collagenase for 15 min. At the end of the incubation period, intact cells or lysates were cooled on ice, and EGTA was added (3 mM) to inhibit further protease activity. Western

and Ligand

Blots

High mol wt standards and crude or lectin-purified preparations of 293L(wtl) or luteal cells in sample buffer with no reducing agents were applied to 7.5% SDS-polyacrylamide gels at a density of 10 ng LH/CG receptor/mm gel. An equivalent amount of lysate from the parental 293 cells was used in some experiments as a negative control. In studies addressing the carbohydrate content of the LH/CG receptor, lysates were first incubated with no additions for 8 h, 100 mu/ml neuraminidase for 1 h, or 20 U/ml peptide N-glycosidase-F for 8 h in the presence of the aforementioned protease inhibitors as well as 5 mM N-ethylmaleimide and 3 mM EGTA. The proteins were then electrophoretically transferred to PVDF membrane, as previously described (4). PVDF membrane was used rather than nitrocellulose due to its increased protein-bindinq capacity. The membrane was cut into 3-mm strips and washed twice (5 min/wash) with buffer C (20 mM Tris and 500 mM NaCI, pH 7.5) and then with buffer C plus 0.2% Tween-20 (buffer D). The strips were blocked with Blotto (5% Carnation instant nonfat drv milk, 0.25% aelatin. 0.2% Tween-20. and 10% glycerol in buffer C). Thestrips .used for Western blots were then incubated overnight at room temperature with Blotto containing 3 fig/ml Bugs or preimmune rabbit serum IgG. The membrane strips were washed three times with buffer C and 1% Nonidet P-40 (buffer E) and twice with buffer C at 5 min/ wash. The strips were then incubated for 1 h with a 1:3000 dilution of an alkaline phosphatase-linked goat antirabbit antibody in buffer C containing 3% BSA and 10% glycerol (buffer F). The second antibody was replaced with fresh buffer F, and

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MOL 2218

ENDO.

1992

the strips were washed twice for 10 min with buffer E and, finally, twice with buffer C. The blots were visualized using 5 bromo-4-chloro-3-indoyl phosphatep-toluidine/p-nitro blue tetrazolium color development reagents according to the supplier’s instructions. Membrane strips used for ligand blots were processed as described for Western blots, except that the Blotto did not contain gelatin, and the strips were incubated overnight in Blotto containing 10 rig/ml [‘2”l]hCG with or without crude hCG (50 lU/ml). After this incubation period, the strips were washed four times with buffer E and twice with buffer C (5 min/wash), then dried and exposed overnight at -70 C to Kodak XAR film with Cronex Plus intensifying screens (DuPont, Wilmington, DE).

Acknowledgments We wish and Drs. reading Segaloff antibodies.

to thank Bruce D’Souza for his technical assistance, Susan Sierke and Deborah Segaloff for their critical of the manuscript. We would also like to thank Dr. for the gift of the 293L(wtl) cells and anti-LH/CG

Received August 18, 1992. Revision received September 15, 1992. Accepted September 15, 1992. Address requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, University of Iowa College of Medicine, Iowa City, Iowa 52242. This work was supported by a grant from the NIH (CA40629) and funds from the Roy J. Carver Charitable Trust. The services and facilities provided by the University of Iowa Diabetes and Endocrinology Research Center (Grant DK25295) are also gratefully acknowledged. Ayudante Ley de Reforma Universitaria in the Department of Biochemistry and Molecular Biology at the University of Salamanca (Spain) and initially supported by a NATO postdoctoral fellowship and more recently by an International Research Fellowship (TW-04655) from the Fogarty International Center, NIH. l

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luteinizing hormone/human choriogonadotropin. J Biol Chem 264:4636-4641 8. Vuhai-Luuthi MT, Jolivet A, Jallal B, Salesse R, Bidart JM, Houllier A, Guiochon-Mantel A, Garnier J, Milgrom E 1990 Monoclonal antibodies against luteinizing hormone receptor. lmmunochemical characterization of the receptor. Endocrinology 127:2090-2098 9. Minegishi T, Delgado C, Dufau ML 1989 Phosphorylation and glycosylation of the luteinizing hormone receptor. Proc Natl Acad Sci. USA 86:1470-1474 10. Cummings RD, Kornfeld S, Schneider WJ, Hobgood KK, Tolleshaug H, Brown MS, Goldstein JL 1983 Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor. J Biol Chem 258:15261-l 5273 11. Petaja-Repo UE, Merz WE, Rajaniemi HJ 1991 Significance of the glycan moiety of the rat ovarian luteinizing hormone/chorionic gonadotropin (CG) receptor and human CG for receptor-hormone interaction. Endocrinology 128:1209-1217 12. Hedo JA, Kahn R, Hayashi M, Yamada KM, Kasuga M 1983 Biosynthesis and qlycosylation of the insulin receptor. Evidence for a single pojypeptide precursor of the two major subunits. J Biol Chem 258:10020-l 0026 13. Ascoli M, Segaloff DL 1986 Effects of collagenase on the structure of the lutropin/choriogonadotropin receptor. J Biol Chem 261:3807-3815 14. Wang H, Ascoli M, Segaloff DL 1991 Multiple luteinizing hormone/chorionic gonadotropin receptor messenger ribonucleic acid transcripts. Endocrinology 129:133-l 38 15. Xie Y-B, Wang H, Segaloff DL 1990 Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity. J Biol Chem 265:21411-21414 16. Zhang R, Tsai-Morris CH, Kitamura M, Buczko E, Dufau ML 1991 Changes in binding activity of luteinizing hormone receptors by site directed mutagenesis of potential glycosylation sites. Biochem Biophys Res Commun 181:804-808 17. Ji I, Slaughter RG, Ji TH 1990 N-Linked oligosacharides are not required for hormone binding of the lutropin receptor in a Leydig tumor cell line and rat granulosa cells. Endocrinology 127:494-406 18. Bruch RC, Thotakura NR, Bahl OP 1986 The rat ovarian lutropin receptor: purification, hormone binding properties, and subunit composition. J Biol Chem 261:94959460 19. Kusuda S, Dufau M 1986 Purification and characterization of the rat ovarian receptor for luteinizing hormone. J Biol Chem 261:16161-16168 20. Minegishi T, Kusuda S, Dufau ML 1987 Purification and characterization of Leydig cell luteinizing hormone receptor. J Biol Chem 262:17138-17143 21. O’Dowd BF, Hnatowich M, Caron MG, Lefkowitz RJ, Bouvier M 1989 Palmitoylation of the human &adrenergic receptor. Mutation of cys341 in the carboxyl tail leads to an uncoupled nonpalmytoylated form of the receptor. J Biol Chem 264:7564-7569 22. Hausdorff WP, Bouvier M, O’Dowd BF, Irons GP, Caron MG, Lefkowitz RJ 1989 Phosphorylation sites on two domains of the &adrenergic receptor are involved in distinct pathways of recepto; desensitization. J Biol Chem 264:12657-l 2665 23. Ascoli M, Puett D 1978 Gonadotropin binding and stimulation of steroidogenesis in Leydig tumor cells. Proc Natl Acad Sci USA 75:99-102 24. Ascoli M 1983 An improved method for the solubilization of stable gonadotropin receptors. Endocrinology 113:2129-2134 25. Rodriguez MC, Segaloff DL 1990 The orientation of the lutropin/choriogonadotropin receptor as revealed by sitespecific antibodies. Endocrinology 127:674-681

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CG receptor complementary DNA.

It is well established that the LH/CG receptor expressed in gonadal cells is an 85- to 92-kilodalton (kDa) glycoprotein. Additionally, however, a numb...
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