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Endocrinology, doi:10.1210/en.2002-0063
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Endocrinology Vol. 144, No. 10 4393-4402
Copyright © 2003 by The Endocrine Society

Regulation of Follitropin Receptor Cell Surface Residency by the Ubiquitin-Proteasome Pathway

Brian D. Cohen, Jason T. Bariteau, Lori M. Magenis and James A. Dias

Laboratory of Clinical and Experimental Endocrinology and Immunology (B.D.C., J.T.B., L.M.M., J.A.D.), Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, and Department of Biomedical Sciences (J.A.D.), State University of New York at Albany, Albany, New York 12208

Address all correspondence and requests for reprints to: James Dias, Laboratory of Clinical and Experimental Endocrinology and Immunology, Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, New York 12208. E-mail: james.dias{at}wadsworth.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Little is known of the normal physiological processes that govern the cell surface residency of the human follitropin receptor (hFSHR), a G protein-coupled receptor expressed in the ovary and testis. In the hFSHR, the third intracellular (3i) loop is considered to be pivotal in attenuation of ligand activation, particularly internalization. To gain a better understanding of these processes, we used a yeast-based interaction trap to identify cytoplasmic proteins in a human ovarian cDNA library that interacted with the hFSHR 3i loop. Among the cDNA identified, four encoded isoforms of ubiquitin. Immunoprecipitated hFSHR probed with an antiubiquitin antibody revealed that the receptor is ubiquitinated, although not exclusively on the 3i loop. Cell-surface hFSHR levels increased when expressed at nonpermissive temperature in a temperature-sensitive, ubiquitination-defective cell line. Similarly, after treatment with proteasome inhibitors, HEK293 cells stably transfected with an hFSHR expression plasmid showed an increase in follitropin binding. Proteasome inhibitors did not affect the rate of FSH internalization when receptors were saturated before internalization was measured. In contrast, internalization decreased when binding experiments were performed under nonequilibrium conditions. A mutant hFSHR-K555R, which removes the only lysine in the 3i loop available for ubiquitination, was still ubiquitinated, illustrating that, although the third loop enables and interaction with ubiquitin, it is not the sole site of ubiquitination. These observations are consistent with a role for ubiquitination in the regulation of hFSHR cell surface residency. Additionally, it can be inferred that a sequence in the 3i loop is involved in regulating receptor ubiquitination and internalization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLITROPIN IS ESSENTIAL for maintenance of normal ovarian folliculogenesis and testicular gametogenesis. The human follitropin receptor (hFSHR) is a G protein-coupled receptor (GPCR) expressed predominantly in the granulosa cells of the ovary and the Sertoli cells of the testis (1). The hFSHR is a member of a subclass of GPCR known collectively as the glycoprotein hormone receptors. This group includes the LH receptor and the TSH receptor. GPCR characteristically have seven transmembrane regions, resulting in the formation of four extracellular and four intracellular domains. In addition to the large extracellular domain of 350 residues and the C-terminal tail, three extracellular and three intracellular loops form between the membrane spanning regions (2). The intracellular domains of the FSHR have been implicated in multiple physiological functions, including basolateral targeting of the receptor in polarized cells (3), signal transduction (4, 5), and desensitization and internalization of the receptor to terminate signaling (6, 7).

As for other GPCRs, the process of FSHR desensitization includes phosphorylation of the ligand-bound receptor by intracellular kinases (8). Receptor phosphorylation has been demonstrated to occur on serine and threonine, but not tyrosine, residues (9) and has been localized by detailed mutational analysis to the first and third intracellular loops (10). To date, two kinases have been demonstrated to phosphorylate the FSHR, including the G protein-coupled receptor kinases (11) and protein kinase C (9).

Internalization of GPCR postactivation is a major aspect of the desensitization process, in which the receptor is removed and made inaccessible to subsequent hormone stimulation. Internalization is predominantly, although not exclusively, mediated by arrestin binding and phosphorylation of the receptor (12). Arrestin binds to the third intracellular loop of hFSHR to mediate association with the endocytotic machinery (11). Following internalization, the hormone receptor complex has been localized to the lysosome and lysosomotropic reagents inhibit FSH degradation (13). In addition to the lysosome, degradation of proteins intracellularly is also accomplished in part by a large multicatalytic complex known as the proteasome (14).

To target proteins for proteasome degradation, an 8.6-kDa protein, ubiquitin, is covalently attached to {epsilon}-amino lysines of substrate proteins. Generally, the process of ubiquitin targeting for proteasome degradation has been studied for cytosolic proteins. In recent years, it has come to light that membrane proteins are also ubiquitinated, e.g. the human GH receptor (15). GPCR shown more recently to be ubiquitinated include the yeast {alpha}-factor receptor (16), human µ and {delta} opioid receptors (17), and rhodopsin (18). It has been only recently appreciated that ubiquitination of GPCR is involved in agonist-dependent down-regulation as well as a quality-control role in the endoplasmic reticulum (17). During a search for regulators of FSHR activation, we discovered that the third intracellular loop of the hFSHR interacts with various isoforms of ubiquitin. This observation led to the discovery that hFSHR is ubiquitinated and that proteasome function regulates plasma membrane hFSHR residency. While this article was being prepared, a similar discovery was made for the ß2-adrenergic receptor (19).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of FSHR bait plasmids and screening in the yeast interaction trap
Growth and transformation of yeast for the interaction trap (also known as the two-hybrid screen) were performed as described in (20). The yeast strain RFY231 (MAT{alpha}, trp1, his3, ura3, leu2::6lexAop-LEU2) was transformed using the one-step transformation method (21), with the plasmid pSH18–34 ß-galactosidase reporter (kind gift of Dr. Steve Hanes, Wadsworth Center, Albany, NY) and the bait plasmid lexA:loop3, which contains the amino acids 531–559 of the hFSHR (CYIHIYLTVRNPNIVSSSSDTRIAKRMAM) fused to the carboxyl terminus of lexA (Table 1Go). FSHR numbering is based on the amino acid positions in the mature protein after the removal of the signal sequence. The lexA-bait fusions were generated by PCR amplification of the regions indicated, using the appropriate primers (as listed in Table 1Go) and the plasmid pCN1 (22) as a template. The PCR products were digested with EcoRI and XhoI (NEB, Beverly, MA) and inserted into the plasmid pJK202 (kind gift of Dr. Steve Hanes, Wadsworth Center).


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  		TABLE 1. Primers used to construct FSHR-lexA bait proteins

 
To generate a loop 1-loop two concatener (L1L2 bait), we digested the PCR product from L1 with EcoRI and BamHI and inserted it into pJK202 to generate the plasmid L1LKR. The L2 PCR product was then digested with BamHI and NcoI and inserted into the L1LKR plasmid digested with the same enzymes. This fusion bait contains a glycyl-seryl linker (SGGGG SGGGG SGGSG A) between the loop sequences. Linkers of this type have been used previously to combine subunits of proteins such as the T-cell receptor (23). The bait strain was then transformed with a human ovarian cDNA library in the prey vector pJG4–5 (Origene Technologies, Bethesda, MD) using the protocol of Agatep et al. (24). The library contained 4.0 x 106 clones of lengths 0.4–6.1 kbp generated from normal ovaries. A pool of 4.2 x 106 independent yeast transformants was passed through multiple rounds of selection and screening for identification of specific interactions. Initial selection for interaction was done on synthetic medium deficient in leucine to test for expression of the integrated LEU2 reporter gene construct in RFY231. Colonies from this selection were then screened on X-gal containing plates to test for expression of the ß-galactosidase reporter plasmid pSH18–34. We then checked the survivors of this screen to verify that library plasmid expression was required for the positive phenotypes, by growing cells in the presence of glucose instead of galactose (which drives library plasmid expression). Growth on leucine-deficient plates in the presence of glucose indicated a false positive because library plasmid expression was therefore not required for the phenotype. Library plasmids from clones that passed all three initial screens were isolated using the Zymoprep yeast miniprep kit (Zymo Research, Orange, CA) and transformed into the Escherichia coli strain KC8 for recovery. The plasmids were recovered from E. coli using a spin miniprep (QIAGEN, Valencia, CA). Recovered plasmids were restriction digested with EcoRI and XhoI to verify the presence of a cDNA insert.

Nonspecific bait-mating assay
Library plasmids isolated as described above were transformed into the yeast EGY48 (MAT{alpha}, trp1, his3, ura3, leu2::6lexAop-LEU2) using the one-step transformation method. This strain was then mated with RFY206 (MATa, trp1{Delta}::hisG, his3{Delta}200, ura3–52, lys2{Delta}201, leu2–3) harboring the pSH18–34 reporter plasmid and with bait plasmids as indicated in Fig. 1Go. Diploids were isolated on complete synthetic medium lacking histidine, tryptophan, and uracil and were replica plated to test bait/prey specificity. Plasmids of clones that specifically interacted with the third intracellular loop were subsequently sequenced using a primer corresponding to the B42 activation domain of the plasmid pJG4–5 (nucleotides 793–811, TCTTGCTGAGTGGAGATGC).



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  		FIG. 1. Ubiquitination of hFSHR is not exclusive to K555. HEK293 cells were transiently transfected with equal amounts of WT and mutant cDNA in the presence of an additional vector encoding for 3X-FLAG-ubiquitin. Transiently transfected cells (both control and FSH treated) were solubilized in 1% Igepal, 0.4% deoxycholate buffer with 5 mM N-ethylmaleimide and were homogenized briefly. Unlysed cells were removed by centrifugation, and the extracts were precleared (as described in Materials and Methods) before incubation with either mAb 106.105 (1 µg/ml) or nonspecific isotype control IgG2B at the same dilution. After electrophoresis and transfer to PVDF, membranes were probed with an anti-FLAG mAb M2 (A) (Sigma) or mAb 106.105 (B) (0.5 µg/ml).

 
Construction and analysis of hFSHR K555R
PCR was used to introduce a mutation that changed hFSHR codon 555 from AAG to CCG (lysine to arginine) in plasmid pCN42. Plasmid pCN42 contains the hFSHR cDNA from plasmid pCN1 (22) in the mammalian expression vector pIRES-neo (BD Biosciences Clontech, Palo Alto, CA) (24A ). Plasmids encoding wild-type hFSHR and the K555R mutant were transiently transfected using Lipofectamine Plus (Life Technologies, Rockville, MD) into separate cultures of human embryonic kidney (HEK)-293 cells (2 µg of each plasmid) in the presence of an additional plasmid encoding for a FLAG-epitope tagged cDNA encoding for ubiquitin (1.5 µg). The 3X-FLAG ubiquitin vector was the kind gift of Dr. Jeff Benovic (Thomas Jefferson University, Philadelphia, PA) (25). To compare ubiquitination of wild-type (WT) and the K555R hFSHR, transiently transfected cells were lysed, and SDS-PAGE analysis and immunoprecipitation, followed by Western blotting for the FLAG epitope, was performed.

Immunoprecipitation
Transiently transfected HEK293 cells were left untreated or treated with 40 ng/ml human pituitary FSH for 15 min before harvest. The short incubation of FSH (as opposed to 1-h saturation in binding and internalization experiments described below) was designed to detect any early modification event after FSH binding. After pretreatment with FSH, cells were rinsed with 1x PBS, removed from the dish with PBS/EDTA, and centrifuged for 5 min at 1500 rpm in a tabletop centrifuge (Beckman, Richmond, CA). After aspirating off the supernatant, the cells were lysed in Igepal-DOC lysis buffer [1% Igepal CA-630, 0.4% deoxycholate, 10 mM Tris (pH 7.5), 6.6 mM EDTA, and 5 mM N-ethylmaleimide (Sigma, St. Louis, MO)] on ice for 10 min, followed by homogenization with a 20-sec pulse in an Omnimixer homogenizer at the highest setting. The homogenized material was centrifuged in a microcentrifuge for 10 min at maximum speed at 4 C, and the supernatant was transferred to a fresh tube, in which it was incubated with 100 µl of a 50% slurry of protein A-Sepharose (Upstate Biotechnology, Lake Placid, NY) for 30 min at 4 C with end-over-end rotation. The tubes were then spun at 13,000 rpm in a microfuge for 10 sec, and the precleared supernatant was incubated overnight with protein A purified monoclonal antibody (mAb) 106.105 (26), directed against hFSHR extracellular domain, or with a nonspecific isotype control, both at a dilution of 1 µg/ml. This dilution of purified antibody maximally precipitated hFSHR under these conditions. After overnight incubation with the antibodies, 100 µl of a 50% slurry of protein A-Sepharose were added to the extract/antibody mixture and rotated end-over-end for 2 h at 4 C. A cushion of 0.5x lysis buffer with 30% sucrose was then pipetted under the extract/protein A-Sepharose and centrifuged briefly. The pellet was then washed with lysis buffer and deionized water before the addition of Laemelli sample buffer containing 2-mercaptoethanol. The samples were then boiled, electrophoresed, and transferred to polyvinyl difluoride (PVDF) membrane for Western blot analysis as described below. Membranes were probed with an anti-FLAG monoclonal antibody-horseradish peroxidase conjugate (M2-HRP conjugate, Sigma) at a 1:1000 dilution.

SDS-PAGE and Western blot analysis
SDS-PAGE was performed as described previously (26). The HEK293 cell line was stably transfected with the pCN42 plasmid, resulting in a cell line expressing high levels of hFSHR (1 x 106 ± 0.2 receptors per cell) (24A ). HEK293-hFSHR cells were incubated in the presence of MG132 or vehicle for 1 h before the cells were washed with 1x PBS and then removed from the flask with PBS/EDTA. Cells were centrifuged, resuspended in 50 mM Tris (pH 7.5) and then sonicated for 2 x 15-sec pulses. The sonicated material was quantified by BCA assay (Pierce Chemical, Rockford, IL), and 5 µg of protein were loaded in each lane of a 7.5% SDS-PAGE gel. After electrophoresis, proteins were transferred from the gel to an Immobilon-P PVDF membrane (Millipore, Bedford, MA) using a semidry transfer apparatus (Bio-Rad, Hercules, CA). The membranes were then incubated in 5% nonfat milk in TBST [100 mM Tris HCl (pH 7.2), 0.9% NaCl, 0.5% Tween 20] overnight at 4 C to block nonspecific binding. After the overnight incubation, membranes were washed and incubated for 1 h at room temperature with primary antibody in milk/TBST. Antiubiquitin mAb P4D1 (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a dilution of 1:1000; anti hFSHR mAb 106.105 (26) was used at a dilution of 5 µg/10 ml. The membranes were washed twice for 30 min with 1x TBST and then incubated with either goat antirabbit or goat antimouse IgG peroxidase conjugate (Upstate Biotechnology) at a 1:5000 dilution in milk/TBST. After a 1-h incubation at room temperature, the membranes were washed again and then visualized with Supersignal West Pico ECL (Pierce Chemical) on x-ray film (Eastman Kodak, New Haven, CT).

Stable transfection of E36/ts20 Chinese hamster lung fibroblast cell lines
E36 and E36-ts20 Chinese hamster lung fibroblast cell lines were the kind gift of Dr. Alan Schwartz (Washington University, St. Louis, MO). The E36-ts20 cell line contains a temperature-sensitive mutation in the E1 ubiquitin activation enzyme. This causes ubiquitination to be inhibited when cells are grown at the nonpermissive temperature of 42 C. Cell lines were grown in 60-mm dishes at the permissive temperature of 30 C, as described in (27), to approximately 70% confluency and then transfected using Lipofectamine Plus and 2 µg plasmid pCN42. Stable transfectants were selected with 500 µg/ml G418 (Life Technologies). Receptor number in the stably transfected E36-hFSHR and ts20-hFSHR cell lines was 1.2 x 105 ± 0.1 and 3.73 x 105 ± 0.27, respectively.

Binding assays with E36/ts20 cell lines
Human pituitary FSH (hFSH) was radiolabeled as described previously (28) to an average specific activity of 23 µCi/µg. The ts20-hFSHR and E36-hFSHR cells were plated at a density of 450,000 cells/well in 35-mm plates. Parallel dishes were incubated at 30 C and 42 C for 2 h before radiolabeled hormone was added. Medium was removed, and serum-free medium containing 20 ng/ml 125I-FSH was added to each well in the presence or absence of 1 µg/ml unlabeled recombinant single-chain FSH (29). Hormone was allowed to bind for 1 h at the indicated temperature before cultures were placed on ice and washed twice with ice-cold 1x PBS, and cell-surface hFSH was eluted with ice-cold elution buffer (0.5 M NaCl, 0.2 M acetic acid) for 10 min on ice. Eluate was removed to a glass tube, and samples were counted with a {gamma} counter (Wallac, Turku, Finland).

HEK293-hFSHR-binding assays
The HEK293-hFSHR cells were grown in Eagle’s MEM supplemented with 10% FBS and 600 µg/ml G418 (Life Technologies) seeded at 150,000 cells/well in 24-well dishes. For some experiments medium was replaced with fresh medium containing 5 µg/ml cycloheximide or an equal volume of dimethylsulfoxide (DMSO) vehicle (0.1% vol/vol) 16 h before treatment with proteasome inhibitors. Medium was then removed and replaced with fresh medium containing proteasome inhibitors (proteasome inhibitors I + II and MG132, Calbiochem, La Jolla, CA) or an equal volume of DMSO vehicle (in the continued presence of cycloheximide where indicated) and allowed to continue incubation at 37 C for 1 h. After the preincubation, the medium was removed and serum-free medium containing 20 ng/ml 125I-FSH was added to each well in the presence or absence of 1 µg/ml unlabeled recombinant single-chain FSH (30). Hormone was allowed to bind for 1 h in the continued presence (where indicated) of cycloheximide and proteasome inhibitors or DMSO. Cell-surface hFSH was eluted and measured as described above.

Internalization measurement under nonequilibrium binding conditions
Internalization was measured by binding assays performed as described above but with 125I-FSH removed at various times (0, 5, 10, 15, 30, 35, 40, and 50 min) so as to measure cell-surface and cell-associated counts per minute. Cell-surface radiolabeled FSH was eluted as described above. After the eluate was removed for counting, the cells were washed with 1x PBS and then solubilized in 2 M NaOH for 1 h at room temperature to allow measurement of cell-associated counts per minute. Data were analyzed using spreadsheets provided by Dr. H. Steven Wiley (Pacific Northwest National Laboratory, Richland, WA) (31). Ke, the endocytotic rate constant, was calculated from the slope of the line generated by least squares regression of the line generated by graphing the cell-associated counts per minute against the integral of the cell surface binding, a calculation that includes time and counts per minute specifically bound. The half-time (t1/2) for internalization was calculated using the formula 0.692/Ke = t1/2.

Internalization measurement under equilibrium-binding conditions
Equilibrium-binding internalization assays were performed by incubating HEK293-hFSHR with 20 ng/ml 125I-FSH in serum-free media in the presence or absence of 1 µg/ml unlabeled recombinant FSH for 1 h at 37 C. Unbound radiolabeled hormone was removed, fresh buffer was replaced, and the cells were incubated at 37 C. At each time point, cells were removed from the incubator, placed on ice, washed with 1x PBS, and incubated on ice in elution buffer for 10 min eluted cell-surface FSH. After the eluate was removed for counting, the cells were washed with 1x PBS and then solubilized in 2 M NaOH for 1 h at room temperature to allow measurement of cell-associated counts per minute.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Multiple cDNA were isolated from a pool of 4.2 x 106 independent clones that was screened for proteins that interacted with the third intracellular loop of the hFSHR. Comparison of these sequences to the nr database (including GenBank, EMBL, DDBJ, and PDB sequences) by BLAST (32) revealed that the cDNA identified included four independent isoforms of ubiquitin. Several of these isoforms are fusion proteins with C-terminal extensions following the ubiquitin sequence (Table 2Go). Other isoforms are proteins with several repeats of the ubiquitin sequence in single polypeptides.


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  		TABLE 2. Forms of ubiquitin identified to interact with hFSHR loop 3

 
To determine whether the interaction detected in the genetic screen was specific, the ubiquitin isoforms were subjected to a mating assay with four other baits. Each bait represented one of the four intracellular domains of the follitropin receptor, with the exception of the concatener of the first and second intracellular domains. When tested for specificity in the nonspecific bait-mating assay, these proteins interacted with the third intracellular loop and not with bait proteins encoding for other intracellular domains of the hFSHR (data not shown). Although some breakthrough growth was seen for other baits, the third intracellular loop bait grows only in the presence of galactose, which is required for library plasmid expression, and therefore is a requirement for interaction specific growth.

Attachment of ubiquitin to target proteins is through the {epsilon}-amino groups of lysine side chains. In the third intracellular loop of hFSHR, which was used as the bait protein, there is a single lysine residue (K555). Mutation of the lysine residue to arginine removes the required side chain for ubiquitination of this residue while maintaining the charge of the residue. This mutation was incorporated into the primary sequence of full-length hFSHR. HEK-293 cells were transiently transfected with either WT or K555R mutant hFSHR cDNA together with a FLAG-epitope-tagged ubiquitin expression vector. hFSHR isoforms were immunoprecipitated with a monoclonal antibody directed against the extracellular domain of the hFSHR [mAb 106.105 (26)], and the immunoprecipitated receptor was probed with an anti-FLAG mAb to detect ubiquitination. As seen in Fig. 1AGo, ubiquitination of both WT and K555R mutant hFSHR can be detected. The FLAG epitope-tagged ubiquitin was detected in immunoprecipitated hFSHR samples near the molecular weight of the FSH receptor (Fig. 1BGo, anti-hFSHR immunoblot) in both the presence and absence of follitropin treatment. The approximate change in Mr of ubiquitinated hFSHR (Fig. 1AGo) relative to total hFSHR (Fig. 1BGo) is consistent with monoubiquitination, which was also observed for CXCR4, another GPCR that is ubiquitinated (25).

For other GPCRs, ubiquitination is involved in the regulation of internalization (19). To determine whether the same is true for hFSHR, a cell line was obtained that contains a temperature-sensitive mutation in the ubiquitin E1 activation enzyme (ts20, gift of Alan Schwartz). Both this cell line and the parental control cell line (E36) were stably transfected with plasmid pCN42 encoding for the hFSHR. FSH binding was about 20% higher (P = 0.0457) to ts20-hFSHR cells grown at 42 C (the nonpermissive temperature at which the E1-ubiquitin activation enzyme mutant is inhibited) than to the same cells grown at 30 C (permissive temperature for the E1-ubiquitin activation enzyme mutant) (Fig. 2Go). A similar effect was detected in another isolate of the ts20 cell line tested in a similar manner (data not shown). In contrast, binding to the E36-hFSHR cells, which do not have the E1-ubiquitin activation enzyme mutation, was similar at each temperature.



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  		FIG. 2. Inhibition of the E1 ubiquitin conjugation enzyme alters cell surface hFSHR levels. The E36-ts20 cell line has a temperature-sensitive mutation in the E1 ubiquitin conjugation enzyme preventing ubiquitination of target proteins in cells grown at the nonpermissive temperature (42 C). Both E36-hFSHR and ts20-hFSHR cells were grown at 30 C before being moved to 42 C for 2 h or continuing at 30 C for 2 h. After preincubation, 125I-FSH was added in the presence or absence of 1 µg/ml scFSH for an additional hour. Cell surface binding was measured after the 1 h FSH incubation in two separate experiments performed in triplicate. The difference in binding between the ts20-hFSHR at 30 C and 42 C was determined with the Prism software package (GraphPad Software, San Diego, CA) using an unpaired t test (*, P = 0.0457). In contrast, no significant difference was measured in the binding to the E36-hFSHR cell line at the permissive and nonpermissive temperatures.

 
Proteasome inhibitors were used to determine the role of the ubiquitin/proteasome pathway in hFSHR turnover and degradation in mammalian cells. The hypothesis was that if ubiquitination targeted hFSHR to the proteasome, then proteasome inhibitors would increase hFSHR protein levels of HEK293 cells stably transfected with hFSHR cDNA. The inhibitors tested were all cell-permeable and inhibited the chymotrypsin-like activity of the proteasome (33). Figure 3Go shows a representative experiment using the inhibitor MG132. MG132 increased the levels of hFSHR as visualized by Western blot analysis of whole-cell extracts of HEK293-hFSHR cells grown in the presence or absence of the inhibitor. This inhibitor increased FSH receptors and caused about a 30% increase in cell-surface 125I-FSH binding in a dose-dependent manner (Fig. 4Go). In cells treated with MG132, increased binding did not alter the EC50 of 125I-FSH displacement by unlabeled FSH, suggesting that there was no difference in the dissociation constant, which is proportional to the EC50 (Fig. 5Go). There was no change in dissociation constant (KD) for FSH binding to broken cells that were previously frozen and thawed (data not shown), a pattern consistent with the hypothesis that the effect of MG132 is due to its effect on protein turnover, rather than a change in the affinity constant of FSH for its receptor. Taken together these results demonstrate that inhibition of the proteasome increases the level of hFSHR protein and that a hallmark of the effect is higher levels of cell-surface binding.



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  		FIG. 3. Total hFSHR levels are increased by inhibition of proteasome degradation. Cells treated for 1 h with 30 µM MG132 were harvested and analyzed by SDS-PAGE and Western blot as described in Materials and Methods. Coomassie blue staining of a parallel gel shows that equal amounts of protein were loaded. Immunoblotting with mAb 106.105 (anti-hFSHR, 5 µg/10 ml) or mAb P4D1 at a dilution of 1:1000 (antiubiquitin, Santa Cruz Biotechnology) shows increased hFSHR and increased total ubiquitinated protein, respectively.

 


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  		FIG. 4. Incremental concentrations of MG132 correlate with higher binding of hFSH to target cells. HEK293-hFSHR cells were preincubated for 1 h with increasing doses of MG132 as indicated, all in an equal volume of vehicle (DMSO). Incubation with 125I-FSH and measurement of cell surface binding were performed as described in Materials and Methods. Error bars represent the SE from three experiments. Data were analyzed with the Prism software package (GraphPad Software) using a one-way ANOVA followed by Newman-Keuls multiple comparison test [a, significantly different from control (P < 0.001); b, significantly different from 10 µM MG132 treatment (P < 0.001)].

 


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  		FIG. 5. Preincubation of cells with MG132 results in increased binding of hFSH without alteration of the affinity constant. HEK293-hFSHR cells were treated for 1 h with 30 µM MG132 before the addition of radiolabeled hFSH with increasing amounts of unlabeled hormone. Results are expressed as a percentage of maximal binding to control cells. Total binding was increased in the presence of MG132 [a, binding significantly different from the corresponding point without MG132 treatment (P < 0.001); b, same but P < 0.05)]. Comparison by t test of best-fit variables from the nonlinear regression curve determined that the maximum binding was significantly different (P < 0.001), but no significant difference was measured in the logEC50 (1.106 ± 0.037).

 
The source of increased hFSHR in the MG132-treated cells could have occurred via multiple phenomena, including an increase in the rate of receptor synthesis. To test the hypothesis that MG132 increased the rate of hFSHR synthesis, HEK293-hFSHR cells were treated with cycloheximide overnight and then incubated in the presence or absence of MG132 for 1 h before measuring cell surface binding (Fig. 6Go). MG132 resulted in increased binding of 125I-hFSH binding to HEK293-hFSHR cells, compared with control-treated cells. In cells that were treated overnight with cycloheximide, a 35% decrease in binding was observed. However, proteasome inhibition of cycloheximide-treated cells resulted in an increase in binding similar to MG132-treated cells that did not receive cycloheximide.



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  		FIG. 6. Increased follitropin receptor levels following proteasome inhibition is not due to alteration of the rate of receptor synthesis. HEK293-hFSHR cells were treated overnight with 5 µg/ml cycloheximide or an equal volume of vehicle (DMSO). The cells were then treated for 2 h with 30 µg/ml MG132 or DMSO in the continued presence or absence of cycloheximide. During the last hour of the treatment, 125I-hFSH was added and allowed to bind to cell surface receptors. Data are expressed as a percentage of control cells (- cycloheximide, - MG132). Specific binding to control cells was 31835 ± 1112 cpm/well (±SE) with a nonspecific binding amount of 1294 ± 33 cpm/well. Data were analyzed with the Prism software package (GraphPad Software) using a one-way ANOVA followed by Newman-Keuls multiple comparison test [*, significantly different from same MG132 treatment (or control) without cycloheximide, P < 0.01; **, significantly different from same cycloheximide treatment without MG132, P < 0.001)].

 
In addition to MG132, other specific inhibitors of the proteasome were also tested to see whether they had similar effects on the level of FSH binding to the HEK293-hFSHR cell line. Figure 7Go shows the effect of multiple proteasome inhibitors, both alone and in combination. Surprisingly, proteasome inhibitors I and II had little effect individually on FSH binding. When these inhibitors were combined with MG132, FSH binding was significantly greater than was the binding to cells treated with MG132 alone. It was not possible to treat with higher doses of these proteasome inhibitors, to try and achieve inhibition with them individually, because of loss of cell viability at the higher doses (data not shown).



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  		FIG. 7. Combination of proteasome inhibitors has a synergistic effect on hFSHR levels in HEK293-hFSHR cells. Binding to HEK293-hFSHR cells was performed as described in Materials and Methods. Inhibitors at the concentrations indicated were added simultaneously and preincubated for 1 h before the onset of binding. One-way ANOVA followed by Newman-Keuls multiple comparison test was performed to characterize differences in binding by the various treatments [a, significantly different from control, P < 0.05; b, significantly different from proteosome inhibitor I (PSI) alone, P < 0.001; c, significantly different from proteosome inhibitor II (PSII) alone, P < 0.001; d, significantly different from MG132 alone, P < 0.001].

 
One way in which to address the mechanism of altered hFSHR levels is to measure the rate of disappearance of ligand (and presumably receptor as well) from the cell surface in a pulse-chase internalization paradigm. This approach has been used previously to determine the internalization rate of FSH into primary cultures of Sertoli cells (34). Cells were loaded with radiolabeled FSH, and then free hormone was removed. The amount of cell-surface-bound FSH was measured by acid elution. In agreement with the data in Fig. 3Go, MG132 caused an increase in the total hormone bound (Fig. 8Go). However, up to 2 h after free ligand was removed, there is no difference in the rate of internalization of FSH using this approach.



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  		FIG. 8. Internalization of hFSH is not altered in the presence of MG132 as measured under equilibrium-binding conditions. HEK293-hFSHR cells were preincubated with 30 µM MG132 for 1 h before incubation with radiolabeled hFSH for 1 h. Free hormone was then removed and disappearance of cell surface hormone was monitored. Although initial binding is increased by MG132, the disappearance of cell surface hormone takes place at the same rate as evidenced by the similar slopes of the lines on the graph.

 
The endocytotic rate constant is the slope of the line generated by graphing the cell-associated hormone (hormone resistant to elution from cells at low pH) against the integral of the cell-surface hormone at each time point (Fig. 9AGo) (31). Figure 9Go, B and C, shows the changes in cell-surface and cell-associated 125I-hFSH, respectively, as a function of time. These data were used to generate the graph in Fig. 9AGo and is shown to confirm the increase in cell-surface binding and the decrease in cell-associated ligand in the presence of MG132 relative to control-treated cells. The Ke was measured to be 0.0065 ± 0.0005 min-1 and 0.003 ± 0.001 min-1 for the control and MG132 treated, respectively. The t1/2 for internalization was calculated from the Ke as described in Materials and Methods to be 107.1 ± 8.2 min for control to 259.5 ± 86.5 min for MG132-treated cells.



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  		FIG. 9. Internalization measured under nonequilibrium-binding conditions is decreased by MG132 treatment. A, Internalization rates were measured by preincubation of cells with 30 µM MG132 or vehicle, followed by incubation with radiolabeled hFSH. Free hormone was removed, and cell-surface (B) and cell-associated (C) 125I-FSH was measured as described in Materials and Methods. Calculations of cell-associated molecules and the integral of cell-surface molecules with respect to time were performed using a Microsoft Excel spreadsheet kindly provided by Dr. H. Steven Wiley. Experiments were performed twice in triplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The responsiveness of gonadal cells to follitropin stimulation is critical for normal reproductive function. Following the initial binding event, signal transduction ensues. Signal transduction is both enabled and attenuated by cytoplasmic proteins, some of which are known (2, 35). Identification of cytoplasmic proteins that interact with the hFSHR cytoplasmic domains is an important goal in efforts to shed light both on normal receptor physiology and the effects of clinically isolated mutations that alter receptor function such as the D550G-activating mutation on intracellular loop 3 (36). The use of the yeast interaction trap two-hybrid system to identify interacting proteins has been well established (20). We chose to identify proteins from a human ovarian cDNA library that interacted with the third intracellular loop of the hFSHR. This domain of the hFSHR is the putative site of G protein coupling (4) as well as arrestin binding and receptor phosphorylation (10) that desensitizes the receptor. A goal of this study was to determine whether other proteins bind to this domain before arrestin-directed phosphorylation; these could include novel kinases, trafficking-regulating proteins, or signal transduction proteins.

Unexpectedly it was found that multiple isoforms of ubiquitin interacted with the hFSHR third intracellular loop. The interaction is specific: The library prey clones isolated interacted only with the hFSHR L3 bait and not with baits composed of other hFSHR intracellular cytoplasmic protein domains, even though the other intracellular loops each contain at least one lysine that could act as an ubiquitin acceptor site. As its name suggests, ubiquitin is present in every cell type, although some differences exist in the isoforms expressed. Importantly, UbiC, which we isolated as an interacting partner in this study, has been previously detected in human granulosa cells (37).

The lysine residue in the loop3 portion of the bait protein (corresponding to K555 in the full-length WT sequence) could act as an acceptor for ubiquitin in the Saccharomyces cerevisiae two-hybrid host strain. However, in full-length hFSHR, K555 is not required for ubiquitination of the receptor (Fig. 1AGo); ubiquitination not only can still be detected in immunoprecipitated hFSHR-K555R but actually appears to be increased. This does not mean that in WT receptor that K555 is not a target site for ubiquitination. In other proteins it has been shown that ubiquitination can be promiscuous (38); the absence of the normal target lysine results in the ubiquitination of an alternate lysine residue. The ubiquitination of alternate lysine residues of hFSHR-K555R is somewhat surprising because ubiquitin did not interact with the other intracellular loops in the yeast two-hybrid assay (data not shown). This implies that the third intracellular loop may play a role not just as a potential acceptor of ubiquitin but also as a recruitment site for an ubiquitin ligase or some other protein that itself recruits the ubiquitin ligase. This would be consistent with the interaction trap results; an intermediary such as an E3 ubiquitin ligase might serve as adapter between bait and prey in the nucleus. Another possible factor could be arrestin. Arrestin has been shown to be ubiquitinated and to interact with Mdm2, an E3 ubiquitin ligase to promote the ubiquitination of the ß2-adrenergic receptor (19). Sequences required for arrestin or the E3-ligase to bind to the third loop would be present in the full-length K555R mutant receptor, which could then lead to lysine residues in the other intracellular loops being ubiquitinated. However, in the intracellular loop baits, that signal would be absent except in the third intracellular loop bait.

The detection of any interaction by the yeast two-hybrid system and/or immunoprecipitation provides a scorecard of the players involved, but it does not necessarily shed light on the physiological role of the interaction. To gain more insight into the physiological role of the ubiquitin/proteasome pathway in hFSHR regulation, ubiquitination was ablated using a temperature sensitive mutant of the E1-activation enzyme. The use of the temperature-sensitive mutant E36-ts20 allows for temporary cessation of ubiquitination, whereas a complete block of ubiquitination would be lethal. Based on the maximal binding, the concentration of receptors is clearly different for each cell line. For reasons that are unclear, binding was higher in all independently cloned ts20 cell lines tested, compared with E36 lines. Because these were difficult lines to make stable for expression of FSHR, this difference in binding was not explored further. Because the binding experiments used a concentration of radiolabeled FSH that is in excess of the KD, the total binding observed is a good indication of the relative levels of receptor in each cell line. The important comparison between the two hamster lung fibroblast lines is the temperature effect, which is compared within each cell line. The relative levels of receptors would not be expected to affect the results, and this is borne out by the fact that these data were consistent with the proteasome inhibition data. Although this provides evidence for a role for ubiquitination in hFSHR cell surface residency, this static approach does not identify whether it is the ubiquitination of hFSHR or some regulatory protein that results in the increased cell-surface receptor.

Similarly to findings with the µ and {delta} opioid receptors and the ß2-adrenergic receptor, proteasome inhibitors increase the level of hFSHR protein (19, 39). The approximate maximal binding of MG132-treated cells is 30% higher than for untreated cells. However, a true maximal binding cannot be calculated using this method because the experiments were conducted at 37 C at which internalization is taking place. However, the EC50 is not significantly different between the treated and untreated cells, from which we can infer that there is no difference in the KD of the hormone-receptor complex with MG132 treatment because EC50 is proportional to KD. This increase in receptor protein must be due to the inhibition of proteasome-mediated degradation because other proteasome inhibitors used in concert with MG132 add to the MG132 increase in cell-surface binding.

To evaluate whether an increase in cell surface expression of the receptor and receptor ubiquitination and proteasome degradation is a physiologically relevant pathway, it is desirable to use cell lines that are derived from native cell types in which the receptor is normally expressed. A line of simian virus 40 transformed rat Sertoli cells that was stably transfected with hFSHR and stable clones selected that expressed FSHR at physiological levels (approximately 6000 receptors per cell) was used for this purpose. MG132 treatment of this Sertoli cell line evidenced an 1.85-fold increase of cell surface receptor expression determined by 125I-FSH binding (data not shown).

The increase in cell-surface hFSHR by inhibition of ubiquitination or proteasome inhibition could have been the result of multiple phenomena, including: 1) change in the rate of internalization of ligand-occupied receptor; 2) change in the rate of hFSHR recycling postinternalization; 3) change in the steady-state, ligand-independent turnover of the hFSHR protein on the membrane; 4) change in transport from the Golgi to the membrane; or 5) change in the rate of synthesis of hFSHR protein or stability of hFSHR mRNA. Treatment overnight with cycloheximide tested the hypothesis that the effect of MG132 was occurring at the level of protein synthesis. Inhibition of protein synthesis overnight resulted in a decrease, but not a complete abrogation of 125I-hFSH binding, consistent with a long half-life of unoccupied receptor. A similar result was seen in studies of the ß2-adrenergic receptor, in which a 30% decrease was observed following overnight cycloheximide treatment in the absence of ligand (40). The ability of MG132 to increase cell-surface hFSHR levels was not affected by the cycloheximide treatment. These data lead one to reject the hypothesis that MG132 affects FSHR protein synthesis.

Two different approaches were used to measure MG132-induced differences in the rate of internalization. The first approach measured internalization rates by measuring cell-surface and cell-associated 125I-hFSH under conditions in which FSH binding had not reached equilibrium. The second approach was more like a pulse-chase experiment in which 125I-hFSH was added, binding was allowed to reach equilibrium, and free hormone was removed before observing the disappearance from the cell surface.

Previous determinations of the internalization rate of rat FSHR in HEK293 cells were performed under nonequilibrium conditions, and the endocytotic rate constant was calculated using the approach of Wiley and Cunningham (41). An alternate paradigm derived (31) is better suited to address the question of how proteasome inhibitors affect the internalization of hFSHR under nonequilibrium conditions. This integral, originally calculated by Lund et al. (31), takes into account the increase in cell-surface binding over the course of the measurements. Using this approach, the endocytotic rate constant, Ke, is the slope of the line generated by graphing the cell associated 125I-FSH molecules against the integral of cell-surface 125I-FSH molecules accumulated over time. MG132 treatment results in a 2-fold decrease in the Ke of hFSHR (relative to control) and consequently increases the t1/2 of internalization from 107.1 ± 8.2 min for control to 259.5 ± 86.5 min for MG132-treated cells. The t1/2 for hFSHR internalization is longer than previous determinations of the internalization rate of rFSHR in HEK293 cells (11), a difference that may be a product of using a different approach to calculating Ke, a different species of receptor, different receptor density, or a combination of these factors.

In contrast, based on the rate of disappearance of 125I-FSH from the cell surface of primary Sertoli cells (equilibrium binding followed by internalization), it was determined, that at 70 min, 50% of the hormone was no longer acid dissociable (34). Following this approach, it does not appear that there is any difference in the rate of internalization because of MG132 treatment. In the present study, using HEK293-hFSHR cell line, greater than 2 h was required to reach 50% disappearance of radiolabeled hormone from the cell surface, based on the measurement of the of internalization following equilibrium binding. Delayed internalization of the ß2-adrenergic receptor because of MG132 treatment is arrestin dependent (19), consistent with our findings with the hFSHR. The lack of effect of MG132 on the rate of internalization of hFSHR measured after equilibrium binding is suggestive of a modification of the pathway that is arrestin independent [i.e. the residual internalization seen in the presence of a dominant-negative arrestin isoform (7)].

It was predicted that the K555R mutant would lose its ubiquitination. However, the opposite was seen; it was more highly ubiquitinated.

Future studies will address identification of the site(s) of ubiquitination of hFSHR. Identification of this site will allow for the expression of mutants of hFSHR that are ubiquitination defective, and obviate the need to use proteosome inhibitors. In addition, identification of the amino acids that enable ubiquitin interaction with the FSHR 3i loop, will test the hypothesis that the hFSHR 3i loop is essential for ubiquitination/internalization.


   Acknowledgments
 
We thank Dr. Steve Hanes (Wadsworth Center) for sharing plasmids and assistance with the interaction trap, Dr. Alan Schwartz (Washington University) for providing the E36 and E36-ts20 cell lines, Dr. Jeff Benovic (Thomas Jefferson University) for providing the 3X-FLAG-ubiquitin construct, and Dr. H. Steven Wiley (Pacific Northwest National Laboratory) for providing spreadsheet templates for internalization rate calculation. Additionally, we thank Dr. Cheryl Nechamen for technical assistance with immunoprecipitations and the Wadsworth Center Molecular Genetics Core Facility for DNA sequencing and oligonucleotide synthesis.


   Footnotes
 
This work was supported by NIH Grants 2R01HD18407 (to J.A.D.), 1F32HD08537 (to B.D.C.), and NSF9987844 (to L.M.M.). J.T.B. was a recipient of an Endocrine Society Summer Research Award.

Abbreviations: DMSO, Dimethylsulfoxide; HEK, human embryonic kidney; hFSH, human pituitary FSH; hFSHR, human follitropin receptor; GPCR, G protein-coupled receptor; 3i, third intracellular loop; KD, dissociation constant; Ke, endocytotic rate constant; mAb, monoclonal antibody; PVDF, polyvinyl difluoride; t1/2, half-time; TBST, Tris-buffered saline and Tween 20; WT, wild-type.

Received November 20, 2002.

Accepted for publication June 24, 2003.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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