Postendocytotic Trafficking of the Follicle-Stimulating Hormone (FSH)-FSH Receptor Complex
Hanumanthappa Krishnamurthy,
Hiroshi Kishi,
Mei Shi,
Colette Galet,
Ravi Sankar Bhaskaran,
Takashi Hirakawa and
Mario Ascoli
Department of Pharmacology, The University of Iowa, Iowa City, Iowa 52242-1109
Address all correspondence and requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2-319A BSB, 51 Newton Road, The University of Iowa, Iowa City, Iowa 52242-1109. E-mail: mario-ascoli{at}uiowa.edu.
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ABSTRACT
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Although the fates of the internalized hormone-receptor complexes formed by the lutropin/choriogonadotropin and the TSH receptors have been examined in some detail, much less is known about the fate of the internalized FSH-FSH receptor (FSHR) complex.
Using biochemical and imaging approaches we show here that the majority of the internalized FSH-FSHR complex accumulates in endosomes and subsequently recycles back to the cell surface where the bound, intact hormone dissociates back into the medium. Only small amounts of FSH and the FSHR are routed to a lysosomal degradation pathway, and the extent of FSH-induced down-regulation of the cell surface and total FSHR is minimal. This pathway was detected in heterologous (human kidney 293T) cells transfected with the rat (r) or human (h) FSHR as well as in a mouse Sertoli cell line (MSC-1) or a mouse granulosa cell line (KK-1) transfected with the rFSHR.
Additional experiments using a series of C-terminal deletions of the rFSHR and the hFSHR showed that the recycling of the internalized FSH-FSHR complex and the extent of hFSH-induced down-regulation is dictated by a short stretch of amino acids present at the extreme C-terminal end of the receptor.
We conclude that most of the internalized FSH-FSHR complex is recycled back to the cell surface, that this recycling pathway is highly dependent on amino acid residues present near the C terminus of the FSHR, and that it is an important determinant of the extent of down-regulation of the FSHR.
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INTRODUCTION
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THE FOLLITROPIN, LUTROPIN, and TSH receptors (FSHR, LHR, and TSHR) are the founding members of a subfamily of G protein-coupled receptors (GPCRs) that are characterized by the presence of a large extracellular domain containing several leucine-rich repeats and are now known as the leucine-rich repeat containing GPCRs (LGR) (1). Although the LGR family includes several orphan receptors (1) as well as the receptors for relaxin (2) and Leydig insulin-like peptide (3), the FSHR, LHR, and TSHR are also related to each other by the homologous structure of their ligands, which are collectively known as the glycoprotein hormones. The glycoprotein hormones, known to be the most complex hormones present in mammals, are noncovalently bound heterodimeric glycoproteins (4, 5).
As is the case with other GPCRs (reviewed in Refs. 6, 7, 8, 9), the hormone-induced activation of the glycoprotein hormone receptors results in their internalization and sorting to intracellular vesicular compartments (10, 11, 12, 13, 14, 15, 16, 17). Although there are a number of studies characterizing the internalization and intracellular sorting of the LHR and the TSHR (10, 11, 12, 13, 14, 15), available information on the fate of the FSH-FSHR complex is much more limited (16, 17, 18). For example, the complex formed by human (h) chorionic gonadotropin (CG) and the murine or porcine LHR is known to be internalized and routed to the lysosomes where both the hormone and the receptor are degraded (10, 11, 12, 13, 14), whereas a substantial portion of the internalized hCG-hLHR is recycled back to the cell surface (13, 19). In the case of the internalized TSH-hTSHR complex, the hormone is routed to the lysosomes and degraded, but the receptor is recycled back to the cell surface (15). Older studies on the fate of the FSH bound to rat Sertoli cells indicated that some of the bound FSH was internalized and degraded by a pathway that is sensitive to lysosomal inhibitors, but the fate of the receptor was not investigated (18). More recent studies have shown that the endogenous FSHR and TSHR display a polarized distribution in their target cells (20, 21) whereas the LHR does not (22). When expressed in MDCK cells, however, all three receptors sort to the basolateral surface (16, 17). In these cells the FSHR and the LHR (but not the TSHR) can mediate the transcytotic transfer of their respective ligands from the basolateral to the apical surface (16, 17). The structural features of the FSHR that mediate this basolateral localization and transcytosis are located in the C-terminal tail of the receptor.
In this paper we use a number of biochemical and imaging approaches to examine the fate of the internalized FSH-FSHR complex in several cell lines transfected with the rat (r) FSHR or the hFSHR.
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RESULTS
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Fate of the Internalized hFSH
The fate of the internalized [125I]hFSH was measured using a protocol that allows for measurement of hormone degradation in a manner that is independent of the rate of internalization (12, 23, 24). This protocol consisted of two incubations. During the first incubation, 293T cells transiently transfected with the wild-type myc-rFSHR (myc-rFSHR-wt) were allowed to bind and internalize [125I]hFSH at 37 C. The cells were then washed with a neutral buffer to remove the free [125I]hFSH and with an acidic buffer to remove the surface-bound [125I]hFSH. A second incubation (37 C) was then performed to allow the cells to process the internalized [125I]hFSH. This second incubation was done in medium without FSH or in medium containing an excess of FSH to prevent the reassociation of any undegraded [125I]hFSH that had recycled to the surface and was released from the cells into the medium. During this second incubation the medium was assayed for degraded and undegraded hormone released and the cells were assayed for [125I]hFSH that remained internalized and for [125I]hFSH that had returned to the cell surface. Because binding and internalization were stopped from occurring at the beginning of the second incubation (t = 0 in Fig. 1
), all the internalized, surface-bound, degraded, and undegraded [125I]hFSH found at different time points during the second incubation were derived from the [125I]hFSH that was internalized during the first incubation. As shown in Fig. 1
, A and B, more than 95% of the cell-associated radioactivity was located intracellularly at t = 0. As the second incubation progressed, however, there was a decline in the intracellular radioactivity and a rapid and transient increase in the radioactivity associated with the cell surface. Some of the internalized radioactivity was also released into the medium as degraded (trichloroacetic acid-soluble) or undegraded (trichloroacetic acid-insoluble) hormone. A comparison of the patterns of radioactivity found in cells reincubated in medium without FSH (Fig. 1A
) or with an excess of FSH (Fig. 1B
) reveal that the presence of FSH enhanced the loss of internalized hormone (compare solid squares in Fig. 1
, A and B) and the appearance of undegraded hormone in the medium (compare open diamonds in Fig. 1
, A and B) and it decreased the amount of hormone associated with the cell surface (compare solid circles in Fig. 1
, A and B). For example, at the end of the experiment 4060% of the internalized [125I]hFSH was returned to the medium in a undegraded form in cells that were reincubated with an excess of FSH (Fig. 1B
) whereas only 1020% of the internalized [125I]hFSH was returned to the medium in an undegraded form in cells that were reincubated without an excess of FSH (Fig. 1A
). This is quite different from the fate of the [125I]hCG internalized by 293T cells expressing the myc-rLHR-wt (Fig. 1
, C and D). In this case the majority of the internalized [125I]hCG is returned to the medium in a trichloroacetic acid-soluble form, and this occurs regardless of the presence or absence of an excess of hCG during the second incubation (compare Fig. 1
, C and D).

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Fig. 1. Fate of the Internalized [125I]hFSH and [125I]hCG in Cells Transiently Transfected with the myc-rFSHR-wt or myc-rLHR-wt
293T cells transiently transfected with the myc-rFSHR-wt (A and B) or the myc-rLHR-wt (C and D) were incubated with a [125I]hFSH (A and B) or [125I]hCG (C and D) at 37 C as described in Materials and Methods. After washing to remove the free hormone, the surface-bound hormone was released by a brief exposure of the cells to an isotonic pH 3 buffer and the cells were placed in medium (t = 0 in the figure) without hormone (A and C) or in medium supplemented with an excess of eFSH (B) or hCG (D) as described in Materials and Methods. The cells were incubated at 37 C and, at the times indicated, the medium was removed and saved. The cells were washed with cold medium and they were briefly exposed again to the isotonic pH 3 buffer, thus releasing any internalized hormone that had recycled back to the surface. The acid-stripped cells were solubilized with NaOH. The radioactivity that remained associated with the cells after the acid elution (solid squares) and the radioactivity released by the acid treatment (solid circles) were subsequently quantitated in a -counter. The saved medium was used to determine the amount of degraded (open circles) and undegraded (open diamonds) [125I]hFSH or [125I]hCG released back into the medium as described in Materials and Methods. Three 35-mm wells were used for each time point. During the initial incubation (i.e. before t = 0) two of the wells contained 125I-labeled hormone only and the third also contained an excess of nonlabeled hCG or eFSH. The radioactivity associated with the third well was used to correct for nonspecific binding. Each point represents the mean ± SE of three independent transfections. The absence of an error bar indicates that the SEM is too small to be shown. Results are expressed as % of the total radioactivity present at t = 0 (10,00020,000 cpm/well in individual experiments).
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From the data presented in Fig. 1B
it can be estimated that the rates of recycling and degradation of the internalized [125I]hFSH are 0.06/min and 0.002/min. These correspond to half-times of 11 and 365 min, respectively.1
The integrity of the internalized [125I]hFSH released into the medium was further ascertained by polyacrylamide gel electrophoresis run at 4 C and in the absence of reducing agents. Under these conditions, the two glycoprotein hormone subunits remain associated unless the samples are boiled before electrophoresis (10, 25). The results presented in Fig. 2
show that the internalized [125I]hFSH that is released from the cells back into the medium comigrates with [125I]hFSH under conditions that preserve the heterodimer (Fig. 2
, lanes 13). Boiling the [125I]hFSH standard or the released radioactivity before electrophoresis results in the appearance of a doublet that migrates faster than the heterodimer (Fig. 2
, lanes 46). The lower band of this doublet is likely to be [125I]hFSH
because it comigrates with [125I]hCG
(data not shown). The identity of the upper band was not investigated. It could be hFSHß or it could simply represent the heterogeneous nature of hFSH
. In this respect it should also be noted that although the distribution of radioactivity between the two subunits of [125I]hFSH is not known, most of the 125I incorporated into [125I]hCG is incorporated into the
-subunit (25).

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Fig. 2. Nature of the Internalized [125I]hFSH Recycled Back into the Medium
293T cells transiently transfected with the myc-rFSHR-wt were allowed to internalize [125I]hFSH and processed as described in the legend to Fig. 1 . The cells were reincubated in medium containing an excess of eFSH for 90 min. Aliquots of the medium (containing 1,0002,000 cpm of the [125I]hFSH released from the cells) were diluted 2-fold with a 2-fold concentrated sodium dodecyl sulfate sample buffer without reducing agents. The samples were then boiled for 1 min or applied directly to a 10% polyacrylamide gel without boiling. SDS-PAGE was then performed at 4 C as described earlier (25 ). Under these conditions the heterodimer does not dissociate into subunits unless the samples are boiled before electrophoresis (10 25 ). The results of a representative experiment are shown.
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Because the fate of the [125I]hCG internalized by the rLHR and the hLHR is different (13, 19), we also compared the fates of the [125I]hFSH internalized by the rFSHR and the hFSHR. These experiments (Table 1
) show that there are no differences in the postendocytotic fate of [125I]hFSH in cells expressing the myc-rFSHR-wt or myc-hFSHR-wt. For comparison, the fate of the [125I]hCG internalized by the myc-rLHR-wt or the myc-hLHR-wt were examined at the same time (Table 1
). These experiments show that only the fate of the [125I]hCG internalized by the myc-rLHR-wt is unchanged by the presence of an excess of nonradioactive ligand during the second incubation. In cells expressing the myc-hLHR-wt, myc-rFSHR-wt-wt, or myc-hFSHR-wt, the presence of an excess of nonradioactive ligand during the second incubation resulted in an increase in the amount of internalized 125I-labeled hormone that is released in an undegraded form and a decrease in the amount of 125I-labeled hormone that is retained intracellularly. The differential effects of the presence of an excess of nonradioactive hormone during the second incubation are probably due to the higher binding affinity of hCG to the rLHR [dissociation constant (Kd) = 0.10.2 nM, see Ref. 13 ] when compared with the binding affinity of hCG to the hLHR (Kd = 12 nM, see Ref. 13) and the binding affinities of hFSH to the rFSHR (Kd = 12 nM, see Ref. 26) or to the hFSHR (Kd = 12 nM, our unpublished observations). In the presence of an excess of nonradioactive hormone in the medium, the 125I-labeled hormone that recycles back to the surface bound to the receptor would be in equilibrium with the nonradioactive hormone but the exchange of the recycled 125I-labeled hormone for nonradioactive hormone would be much more noticeable for the three receptors that have a lower affinity (i.e. the hLHR-wt, rFSHR-wt, and hFSHR-wt) than for the receptor with the high affinity (i.e. the rLHR-wt). Even in the presence of nonradioactive hormone, however, the amount of internalized [125I]hFSH that is released in an intact form from cells expressing either the myc-rFSHR-wt or myc-hFSHR-wt is higher than the amount of [125I]hCG that is released in an intact form from cells expressing the myc-hLHR-wt. Conversely, the amount of internalized [125I]hFSH that is degraded and released is lower than the amount of [125I]hCG that is degraded and released from cells expressing the myc-hLHR-wt.
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Table 1. Postendocytotic Fate of the Gonadotropin Hormones Internalized by the Rat and Human Gonadotropin Receptors
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To determine if the fate of the receptor-bound FSH was due to the use of a heterologous cell line (293T) for expression, we also expressed the myc-rFSHR-wt in a mouse Sertoli cell line (designated MSC-1, see Ref. 27) or a mouse granulosa cell line (designated KK-1, see Ref. 28). The data presented in Table 2
show that most of the [125I]hFSH internalized in these two cell types is also released back into the medium in an undegraded form rather than as degraded hormone.
Lastly, our data also show that the fates of the internalized [125I]hFSH and FSHR are independent of receptor density and of the method of expression (i.e. transient vs. stable). In 293T cells transiently transfected with the rFSHR-wt, the fate of the internalized [125I]hFSH does not change when the density of receptors is decreased by 1 order of magnitude (data not presented). In addition, in transiently transfected 293T cells the expression of the rFSHR is about 10 times higher than that of the hFSHR (data not shown), yet the fate of the internalized hormone (Fig. 1
and Table 1
) and the internalized receptors (Figs. 3
and 6
) are the same. The expression of the rFSHR in transiently transfected 293T cells is also about 10 times higher than in transiently transfected KK-1 cells, yet the fate of the internalized hormone is the same in both cell types (Table 2
). Lastly, in Table 2
the fate of the internalized [125I]hFSH was examined in 293T and KK-1 cells that were transiently transfected with the rFSHR-wt and in MSC-1 cells that were stably transfected with the same construct (see Materials and Methods),2 yet the fate of the internalized hormone is the same.

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Fig. 3. Subcellular Localization of the myc-rFSHR-wt, myc-hFSHR-wt, and Truncations Thereof
293T cells were cotransfected with the indicated myc-tagged receptors and Rab5a-GFP (left panels) or the indicated myc-tagged receptors and procathepsin D-GFP (right panels). The transfected cells were washed and incubated with (100 ng/ml) or without hFSH at 37 C for 90 min. The cells were fixed and the myc-tagged receptors (in red) were visualized using an anti-myc monoclonal antibody (9E10) and a CY5-conjugated antimouse antibody. The intrinsic fluorescence of Rab5a-GFP and procathepsin D-GFP is shown in green and colocalized components are shown in yellow. The cells were observed and analyzed using a Bio-Rad confocal microscope at the Central Microscopy Facility of The University of Iowa. The results of a representative experiment are shown.
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Fig. 6. Effect of C-Terminal Truncations of the myc-rFSHR or myc-hFSHR on the Fate of the Internalized [125I]hFSH
A, Amino acid sequences of the C-terminal tails of the rFSHR and hFSHR. The box at the left indicates the predicted end of transmembrane helix 7. Dashes indicate identical amino acids and dots indicate gaps introduced for optimal alignment. The C-terminal end of the different truncations prepared are indicated by the vertical lines. The gray shading highlights the residues identified here as being particularly important in determining the fate of the FSHR. The tyrosine and leucine residues previously identified (16 ) as important determinants of the basolateral sorting of the hFSHR are marked with an asterisk. B, 293T cells were transiently transfected with the myc-rFSHR-wt, myc-hFSHR-wt, or C-terminal truncations thereof. The fate of the internalized [125I]hFSH was measured as described in the legend to Fig. 1 except that only the 90-min time point was analyzed. An excess of eFSH was also present during the second incubation. Results are expressed as % of the total radioactivity present at t = 0 (10,00020,000 cpm/well in individual experiments), and they represent the mean ± SE of three independent transfections.
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We conclude that the fate of the [125I]hFSH internalized by target or nontarget cells expressing the myc-rFSHR-wt or the myc-hFSHR-wt is the same. Most of the internalized [125I]hFSH is recycled back to the medium as the intact hormone. Because 293T cells can be transfected with the economical calcium phosphate method, the rest of the experiments shown below were done using this cell type.
Fate of the FSHR
The fates of the myc-rFSHR-wt and hFSHR-wt during the endocytosis of the hormone were ascertained by confocal microscopy. For these experiments, 293T cells were transiently cotransfected with the myc-tagged receptors and Rab5a-green fluorescent protein (GFP) (a marker for early endosomes, see Ref. 29) or with the myc-tagged receptors and procathepsin D-GFP (a marker for lysosomes, see Ref. 30) and the subcellular localization of these molecules was determined by confocal imaging in cells that had been incubated with or without hFSH. The results presented in Fig. 3A
show that in the absence of hFSH the myc-tagged receptors are located mostly at the cell surface. Although there is some intracellular receptor as well (presumably representing the precursor form of the rFSHR, see Ref. 31), there is no colocalization of the intracellular receptor with either Rab5a-wt-GFP or procathepsin D-GFP. Also, as expected, Rab5a-GFP is localized to the plasma membrane and intracellular vesicles (i.e. endosomes, see Ref. 29), whereas procathepsin D-GFP is located only to intracellular vesicles (i.e. lysosomes, see Ref. 30). Incubation of cells with hFSH results in a redistribution of some of the cell surface myc-tagged receptors into intracellular compartments that contain Rab5a-GFP (left panels of Fig. 3A
), but there is no colocalization of the myc-rFSHR-wt with procathepsin D-GFP (right panels of Fig. 3A
). We conclude that most of the internalized rFSHR-wt and hFSHR-wt are sorted into endosomes but not lysosomes.
Because the internalized FSHR localizes only to endosomes, it is likely to remain in an undegraded form and perhaps recycle to the cell surface. This model would predict little or no change in the density of cell surface or total FSHR during the endocytosis of hFSH. To test these predictions, we determined the density of cell surface and total myc-rFSHR-wt in the cells during the endocytosis of the hormone. First, the levels of cell surface myc-rFSHR-wt were measured by performing [125I]hFSH binding assays in cells incubated with a saturating concentration of hFSH for increasing periods of time. Artificial reductions in receptors caused by occupancy were avoided by briefly treating the cells with an acidic buffer before the [125I]hFSH binding assays (see Materials and Methods). The density of cell surface receptors was also measured using streptavidin blots of the myc-rFSHR-wt immunoprecipitated from surface biotinylated cells that had also been incubated with a saturating concentration of hFSH for increasing periods of time. Figure 4
shows that both experimental approaches revealed only a modest decline (
30%) in the density of cell surface rFSHR.

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Fig. 4. Agonist-Induced Changes in the Density of the Cell Surface and Total myc-rFSHR-wt
293T cells transiently expressing the myc-rFSHR-wt were biotinylated and stimulated with a saturating concentration of hFSH (1000 ng/ml) and lysed immediately or after the indicated incubation at 37 C. Lysates were immunoprecipitated with the 9E10 antibody, and the cell surface (open squares) and total receptor (solid triangles) were visualized on Western blots using streptavidin or the 9E10 antibody covalently coupled to horseradish peroxidase, respectively (see Materials and Methods). In parallel experiments the cells were briefly treated with an isotonic pH 3 buffer that releases the surface-bound hormone, and the residual cell surface receptors were measured using [125I]hFSH binding (open circles) as indicated in Materials and Methods. Each point is the mean ± SEM of three independent transfections. The Western blots at the bottom show the appropriate areas of representative blots where immunoprecipitated receptors from surface biotinylated cells were detected using streptavidin coupled to horseradish peroxidase (surface receptors) or the 9E10 antibody coupled to horseradish peroxidase (total receptors).
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In parallel experiments we also measured the levels of total receptors (i.e. cell surface and internalized) by immunoprecipitation and Western blots. These experiments (Fig. 4
) also showed only small changes in the density of total receptors.
The experiments summarized in Fig. 4
included longer periods of time (i.e. up to 6 h) than those used for the trafficking assays (i.e. at most 2 h, as shown in Figs. 1
and 3
). These longer incubation times were examined because the rate of disappearance of the cell surface rFSHR or total rFSHR could be much slower than the rate of recycling or degradation of the internalized hormone.
The drop in the density of cell surface and total myc-rFSHR-wt indicates that some of the internalized receptor is indeed degraded during the endocytosis of hFSH, but the low magnitude of these changes is consistent with the notion that a steady state is reached in which most of the internalized myc-rFSHR-wt that accumulates in the endosomes is recycled back to the cell surface.
Fate of the FSH-FSHR Complex
To determine whether the internalized rFSHR and [125I]hFSH traffic together, we used a protocol similar to that used in Fig. 1
to follow the fate of the internalized [125I]hFSH. For these experiments (Fig. 5
), however, the cells were treated with a permeable cross-linker (to stabilize the [125I]hFSH-rFSHR complex) and lysed, and the myc-rFSHR was immunoprecipitated with the 9E10 antibody. The amount of [125I]hFSH coimmunoprecipitated with the receptor was then measured in the immunoprecipitates. Cells containing only internalized [125I]hFSH (t = 0 in Fig. 5
) were allowed to process the internalized hormone at 37 C, and they were then treated with acid, thus leaving only the internalized radioactivity before cross-linking and immunoprecipitation. Another group of cells was processed without treatment, thus including both the internalized and recycled [125I]hFSH before cross-linking and immunoprecipitation. Figure 5
shows that when the cells containing only the internalized [125I]hFSH were cross-linked and immunoprecipitated there was a rapid and pronounced decline in the fraction of [125I]hFSH that remains receptor bound. When the cells containing the internalized and recycled [125I]hFSH were analyzed, however, the decline in receptor-bound [125I]hFSH was slower and less pronounced. Because the recycled [125I]hFSH always represents only a small percentage of the total cell-associated radioactivity (cf. Fig. 1
) a comparison of the amount of receptor bound [125I]hFSH in both groups of cells allows us to conclude that most of the surface-bound [125I]hFSH is bound to the receptor and that the [125I]hFSH that remains internalized dissociates from the receptor rather quickly.

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Fig. 5. Fate of the Internalized [125I]hFSH-rFSHR Complex in Cells Transiently Transfected with the myc-rFSHR-wt
293T cells transiently expressing the myc-rFSHR-wt were allowed to internalize [125I]hFSH at 37 C as described in Materials and Methods and in the legend to Fig. 1 . After washing to remove the free hormone, the surface-bound hormone was released by a brief exposure of the cells to an isotonic pH 3 buffer (t = 0 in the figure), and the cells were placed in medium with an excess of eFSH as described in Materials and Methods and Fig. 1 . The cells were incubated at 37 C and, at the times indicated, the medium was removed and the cells were washed. Some cells were processed without any further treatment (open circles) whereas others were briefly exposed again to the isotonic pH 3 buffer to release any internalized hormone that had recycled back to the surface (open squares). Both groups of cells were cross-linked with a permeable cross-linker and lysed before immunoprecipitation with the 9E10 antibody. The immunoprecipitates were then counted to determine the amount of [125I]hFSH that was cross-linked to the receptor. Each point represents the mean ± SE of three independent transfections. Results are expressed as % of the total radioactivity immunoprecipitated present at t = 0. At this time point, 12 ± 3% (n = 3) of the cell-associated radioactivity was immunoprecipitated with the 9E10 antibody. This percentage is comparable to the 14.5 ± 0.3% of the [125I]hFSH that can be immunoprecipitated from cross-linked cells containing only surface-bound [125I]hFSH (see Materials and Methods for details).
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Structural Determinants of the Postendocytotic Fate of the FSHR
To gain some information about the structural features of the FSHR that contribute to the fate of the internalized FSH-FSHR complex, we examined the fate of [125I]hFSH in cells transiently transfected with a series of C-terminal truncations of the myc-rFSHR or the myc-hFSHR (Fig. 6A
). The results presented in Fig. 6B
show that removal of the last five residues (see t1 mutants) of the human or rat FSHR had no effect on the fate of the internalized [125I]hFSH, whereas removal of the last eight residues (see t2 mutants) decreased the amount of internalized [125I]hFSH that is retained by the cells as undegraded hormone, and it increased the amount of internalized [125I]hFSH that is degraded and released into the medium. These changes were also accompanied by a decrease in the amount of internalized [125I]hFSHR that is recycled back to the surface and an increase in the amount of internalized [125I]hFSH retained by the cells. Further truncations that removed up to 42 residues from the C-terminal tail induced little or no additional change in the fate of the internalized [125I]hFSH over the changes detected by the removal of the last eight residues (compare the behavior of the t3, t4, t5, and t6 mutants with the t2 mutant).
Because we hypothesized that the recycling of the FSH-FSHR complex is an important determinant of the small magnitude of the hFSH-induced down-regulation of the FSHR (see above), we reasoned that FSHR truncations that promote the degradation of the internalized FSH should accumulate in lysosomes and promote down-regulation. These predictions were tested by confocal colocalization of the myc-rFSHR-t3 and myc-hFSHR-t3 (Fig. 3B
) and by comparing the extent of hFSH-induced down-regulation of cell surface receptors in cells expressing the truncated receptors and their full-length counterparts (Fig. 7
). The confocal analysis shows that, in contrast to the internalized wild-type receptors, which localize only to endosomes (Fig. 3A
), the internalized truncated receptors localize to endosomes and lysosomes (Fig. 3B
). The down-regulation of cell surface receptors was measured only using [125I]hFSH binding to intact cells because, as shown previously (cf. Fig. 4
), the results obtained using this assay are similar to those obtained by measuring the density of biotinylated receptors in surface-biotinylated cells. Figure 7
shows that the rerouting of the internalized truncated receptors to the lysosomes is accompanied by more extensive hFSH-induced down-regulation of the truncated receptors present at the cell surface.

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Fig. 7. Agonist-Induced Down-Regulation of the Cell Surface myc-rFSHR-wt, myc-hFSHR-wt, and Mutants Thereof
293T cells were transiently transfected with the indicated constructs to give equivalent levels of cell surface expression (24 ng of [125I]hFSH binding/106 cells). The cells were stimulated with a saturating concentration of hFSH (1000 ng/ml) and processed immediately or after a 6-h incubation at 37 C. The cells were briefly treated with an isotonic pH 3 buffer that releases the surface-bound hormone and then used to measure residual [125I]hFSH binding as indicated in Materials and Methods. Each bar shows the results (mean ± SEM) of three to four independent transfections.
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Lastly, because the activation of G proteins can provoke changes in the targeting of the FSHR (17), we examined the signaling properties of the two prototypical truncations of the FSHR (myc-rFSHR-t3 or myc-hFSHR-t3) that reroute the internalized [125I]hFSH to a degradation pathway. The data presented in Fig. 8
show that there is no association between changes in the routing of the internalized FSH to a degradation and the ability of cells to respond to hFSH with an increase in cAMP or inositol phosphate accumulation.

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Fig. 8. Signal Properties of the myc-rFSHR-wt, myc-hFSHR-wt, and Mutants Thereof
293T cells were transiently transfected with the indicated constructs to give equivalent levels of receptor expression ( 2 ng and 4 ng of [125I]hFSH binding/106 cells for cells expressing the rFSHR and hFSHR constructs, respectively) and incubated with a maximally effective concentration of hFSH (100 ng/ml for the cAMP assays and 500 ng/ml for the inositol phosphate assays) for the times indicated. The levels of cAMP and inositol phosphates were measured as described in Materials and Methods. Each point shows the results (mean ± SEM) of three independent transfections.
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DISCUSSION
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In the studies presented herein we used a number of biochemical and microscopic approaches to determine the fate of the internalized hFSH-FSHR complex. Collectively, our data provide evidence for the existence of a pathway whereby the majority of the internalized hFSH-FSHR complex accumulates in endosomes and subsequently recycles back to the cell surface where the bound, intact hormone dissociates back into the medium and can bind to the receptor again. The accumulation of the internalized FSHR in the endosomes was documented directly by confocal imaging. Recycling of the internalized hFSH-FSHR complex was directly demonstrated by cross-linking and immunoprecipitation of the hFSH-FSHR complex. The conclusion that the internalized receptor is recycled is further supported by the finding that the loss of cell surface and total FSHR that occurs during the internalization of hFSH is of relatively small magnitude. The accumulation of the internalized [125I]hFSH in a specific intracellular compartment was not documented directly. Because only a small percentage of the internalized [125I]hFSH is degraded and subsequently released into the medium, we can safely conclude that most of the internalized [125I]hFSH does not accumulate in lysosomes. The finding that most of the internalized [125I]hFSH is eventually found in the medium in an intact form clearly shows that most of the internalized [125I]hFSH is recycled back to the medium as intact [125I]hFSH. Therefore, it is reasonable to conclude that the internalized [125I]hFSH must recycle back to the cell surface from a prelysosomal compartment, presumably endosomes. The cross-linking/immunoprecipitation experiments also show that the [125I]hFSH that recycles back to the cell surface is bound to the rFSHR. This conclusion is also supported by the finding that the release of intact, internalized [125I]hFSH back into the medium is highly dependent on the presence of an excess of FSH in the incubation medium. This finding implies the presence of a reversible binding reaction whereby the surface-bound [125I]hFSH that is recycled back to the surface can dissociate into the medium and bind to the cells again. Under these conditions a futile cycle appears to ensue in which the recycled hormone that is surface bound is continuously internalized and recycled and the surface-bound and internalized radioactivity reaches an apparent steady state or changes very slowly. If an excess of FSH is present in the incubation medium, however, this excess FSH will bind to the cells and the dissociated, intact [125I]hFSH becomes detectable in the medium.
The pathways leading to the degradation of the internalized [125I]hFSH or the decline in the levels of cell surface FSHR are difficult to investigate because of the small amount of hormone that becomes degraded and the small magnitude of receptor down-regulation. Because other investigators have previously reported that the receptor-mediated degradation of FSH that occurs in rat Sertoli cells can be inhibited by lysosomotropic agents (18), it is safe to conclude that the degradation products of hFSH detected in the medium are a consequence of the lysosomal localization and degradation of a small portion of the internalized FSH. It is not known whether the lack of detection of the FSHR in lysosomes is due to the sensitivity of the assays used or because the internalized receptor does not accumulate in lysosomes. Alternatively, it is possible that the internalized FSHR accumulates in the lysosomes but the N-terminal myc epitope is rapidly removed. More experiments are needed to answer this question because lysosomal and nonlysosomal pathways for degradation of endocytosed receptors have been described (32, 33, 34, 35).
Three distinct postendocytotic fates can be assigned to ligand-receptor complexes that are internalized by receptor-mediated endocytosis (reviewed in Refs. 9 , 36, 37, 38, 39, 40). The first, which is best documented for the low-density lipoprotein-low-density lipoprotein-receptor complex (36), involves the acidic pH-induced dissociation of the ligand-receptor complex in an endosomal compartment and subsequent sorting of the ligand to a lysosomal degradation pathway and the recycling of the receptor back to the cell surface. In a second pathway, which has been extensively documented for the epidermal growth factor/epidermal growth factor-receptor complex and the complex formed by hCG and the rodent or porcine LHR, the ligand-receptor complex remains associated during postendocytotic trafficking, and it is routed to the lysosomes where both the ligand and the receptor are degraded (9, 11, 13, 14, 15). In the third pathway, which is best documented for the transferrin-transferrin receptor complex (41, 42), the ligand-receptor complex also remains associated during postendocytotic trafficking but it recycles back to the membrane from an endosomal compartment. Curiously the three glycoprotein hormone receptors follow each of the three pathways described above. We show here that both components of the FSH-FHSR are recycled back to the cell surface. The internalized TSH-TSHR complex is segregated in such a way that the internalized TSH is routed to a lysosomal degradation pathway but the TSHR recycles back to the surface (15). Lastly, both components of the internalized hCG-porcine LHR or hCG-rodent LHR complex are routed to a lysosomal degradation pathway (11, 13, 14, 15). The fate of the hCG-hLHR complex appears to be intermediate between the fate of the FSH-FSHR complex and the fate of the hCG-rodent LHR complex (13, 19). When the postendocytotic fate of the internalized hCG is assayed in cells expressing the hLHR under conditions that prevent the reassociation of the undegraded hCG released, there is a fairly even distribution in the amount of internalized hCG that is released back into the medium in degraded and undegraded forms (Table 1
).
Most investigators agree that the fate of internalized GPCRs is dictated by their C-terminal tails, but an exact definition of the motifs involved is not always available (43, 44, 45, 46, 47). The importance of the C-terminal tail of the LHR and the TSHR in dictating their postendocytotic fates has already been documented (15, 19, 48), and the regions of the LHR that dictate its intracellular fate are among the best characterized for all GPCRs (19, 48). The importance of the C-terminal tail of the FSH in determining the fate of the internalized FSH-FSHR complex has been previously documented by the finding that grafting the C-terminal tail of the rLHR (a receptor that does not recycle, see above) into the rFSHR redirects some of the internalized FSH into a degradation pathway (19). The data presented here show that a truncation that removes the extreme C-terminal pentapetide (i.e. the t1 truncation in Fig. 6
) has no effect on the postendocytotic fate of the FHS-FSHR complex, whereas truncations that remove the last eight residues or more (i.e. the t2, t3, t4, t5, and t6 truncations) reroute a substantial portion of the internalized FSH-FSHR complex to a degradation pathway. Because only truncations were used in this analysis, it is not possible to be more specific about the residues responsible for recycling. It could be one or more of the three residues located between the t1 and the t2 truncations or one or more of the eight residues removed by the t2 truncations. Because only five of the eight residues removed by the t2 truncation are conserved between the hFSHR and the rFSHR (shaded in gray in Fig. 6A
), it is safe to conclude, however, that one or more of these five conserved residues (instead of the three nonconserved residues) is involved in recycling. Further definition of the exact residues involved in the recycling of the internalized FSHR will require additional mutagenesis studies involving individual point mutations rather than truncations as done here. It is interesting to note, however, that the Leu (see asterisk on Fig. 6A
) present in the short sequence identified here was previously identified as one of two residues that participate in the basolateral sorting of the hFSHR (16). The other residue identified as being important for basolateral sorting (16) is an upstream Tyr (also marked with an asterisk on Fig. 6A
) that was removed in the t3 mutants examined here. This Tyr does not appear to play a role in recycling as judged by the similar behavior of the t2 and t3 mutants (Fig. 6B
).
Other than their common location near the C terminus, the few amino acid sequences involved in GPCR recycling have, so far, revealed no obvious similarities in primary structure (see Fig. 6A
and Refs. 19 , 45 , and 48). It may be reasonable to assume then, that their involvement in recycling is likely to be related to their ability to support certain forms of secondary or tertiary structure in the C-terminal tail of these receptors.
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MATERIALS AND METHODS
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Plasmids and Cells
A full-length cDNA encoding the rFSHR-wt in pcDNAI/Neo has been described (49). A full-length cDNA encoding the hFSHR-wt was generously donated by Ares Serono. The rFHSR-wt and hFSHR-wt were epitope tagged by introducing the myc epitope between the predicted C terminus of the signal peptide and the predicted N terminus of the mature receptor as described earlier for the rat and human LHR (50, 51) and subcloned into pcDNA3.1. Truncations of the myc-rFSHR and myc-hFSHR were prepared using standard PCR strategies. Previously described myc-rLHR-wt (50) and myc-hLHR-wt (51) constructs in pcDNA1/neo or pcDNA3.1 were used for expression of these receptors. An expression vector for Rab5a-GFP was generously donated by Dr. Phil Stahl (Washington University, St. Louis, MO). A procathepsin D-GFP construct was generously donated by Dr. Jonathan M. Backer (Albert Einstein College of Medicine, New York, NY).
Human kidney 293T cells are a derivative of 293 cells that express the Simian virus (SV)40T antigen (52) and were provided to us by Dr. Marlene Hosey (Northwestern University, Chicago, IL). A transformed mouse Sertoli cell line (designated MSC-1; see Ref. 27) and a transformed mouse granulosa cell line (designated KK-1; see Ref. 28) were provided by Dr. Michael Griswold (Washington State University, Pullman, WA) and Dr. Ilpo Huhtaniemi (Imperial College, London), respectively.
293T cells were plated in 35-mm wells that had been gelatin coated. They were transfected when 7080% confluent using 12 µg of plasmid DNA and the calcium phosphate method of Chen and Okayama (53). After an overnight incubation, the cells were washed and used 24 h later. KK-1 cells were plated in 35-mm wells and transfected using 12 µl Lipofectamine and 2 µg of plasmid DNA according to the manufacturers instructions. MSC-1 cells were transfected with the myc-rFSHR-wt using Fugene according to the manufacturers instructions. A nonclonal population of stably transfected cells was obtained by selection with 700 µg/ml G418.
Fate of the Internalized Hormones
This was measured using modifications of procedures previously used with [125I]hCG (12, 13, 23, 24). Transfected cells were washed with assay medium (Waymouths MB752/1 modified to contain 20 mM HEPES and 1 mg/ml albumin, pH 7.4) and then incubated with subsaturating concentrations of [125I]hCG (10 ng/ml) or [125I]hFSH (40 ng/ml) for 30120 min at 37 C. The concentrations of 125I-labeled hormone and length of the initial incubation were chosen mostly to accumulate enough intracellular radioactivity (10,00020,000 cpm) for the measurements described below. At the end of this initial incubation (labeled t = 0 in the figures), the cells were placed on ice and washed two to three times with 2-ml portions of cold wash medium (Hanks balanced salt solution with 1 mg/ml albumin). The surface-bound hormone was then released by incubating the cells in 1 ml of cold 50 mM glycine, 150 mM NaCl, pH 3, for 24 min (10). This buffer was removed and the cells were washed once more with the same acidic buffer and then once with cold assay medium. The cells were then placed back in 1 ml of warm assay medium with no hormone or containing 500 ng/ml of crude hCG or 500 ng/ml equine (e) FSH (to prevent the reassociation of any undegraded [125I]hCG or [125I]hFSH released from the cells back into the medium), and a second incubation at 37 C was conducted to allow the cells to process the hormone that had been internalized during the first incubation. At different times during the second incubation, the dishes were placed on ice, the medium was saved, and the cells were washed once with 2 ml of cold wash medium. The assay and wash media washes were combined and precipitated with 10% trichloroacetic acid to determine the amounts of degraded and undegraded hormone released (10). The cells were then incubated in 1 ml of cold 50 mM glycine, 150 mM NaCl, pH 3, for 24 min to release and quantitate any internalized hormone that had recycled to the surface, and the cells were solubilized with 1N NaOH to determine the amount of hormone that remained internalized.
In some experiments (see Fig. 5
) we also determined whether the cell-associated [125I]hFSH was free or receptor bound. This was done by cross-linking the cells with a permeable cross-linker (to stabilize the hormone-receptor complex) followed by lysis and immunoprecipitation of the myc-tagged receptor with the 9E10 antibody. Briefly, transiently transfected cells were allowed to internalize [125I]hFSH as described above, and the surface-bound [125I]hFSH was removed with the pH 3 buffer described in the preceding paragraph. At this point (t = 0 in Fig. 5
) the cells were placed back in warm medium containing an excess of nonradioactive FSH (see above) and incubated for another 30 or 90 min to allow them to process the internalized [125I]hFSH. At these two time points, the cells were washed to remove any released hormone and they were divided into two groups. One group was treated with acid again to remove any internalized hormone that had recycled back to the surface (thus leaving only the internalized hormone) and the other was not. This second group contains the internalized hormone as well as any hormone that recycled back to the surface. Both groups of cells were then treated with a permeable cross-linker to stabilize the [125I]hFSH-FSHR complex (see Refs. 48 , 54 , and 55) and the cells were lysed as described elsewhere (54). Aliquots of the lysates were counted to determine the total [125I]hFSH present, and other aliquots were used to immunoprecipitate the myc-tagged receptors with a monoclonal anti-myc antibody (9E10) as described previously (48, 55). The receptor immunoprecipitates were then used to determine the [125I]hFSH that was cross-linked to the FSHR.
To quantitate the efficiency of cross-linking and immunoprecipitation, we used the method described above to cross-link and immunoprecipitate the [125I]hFSH associated with cells that had been cotransfected with the myc-rFSHR-wt and dynamin-K44A and incubated with a saturating concentration of [125I]hFSH. Under these conditions all of the [125I]hFSH associated with the cells is bound to the cell surface rFSHR (48, 55) and 14.5 ± 0.3% (n = 3) of the [125I]hFSH solubilized from the cross-linked cells could be immunoprecipitated with the 9E10 antibody.
Confocal Microscopy
293T cells were plated in eight-chamber coverslip culture vessels coated with polylysine (BioCoat from Becton Dickinson and Co., Franklin Lakes, NJ). They were cotransfected (in a total volume of 400 µl) with 100 ng of myc-tagged receptors and 10 ng of Rab5a-wt-GFP or procathepsin D-GFP using the methods described above. Two days after transfection the cells were incubated with or without hFSH (100 ng/ml) for the indicated times at 37 C. The myc-tagged receptors were visualized with the 9E10 monoclonal antibody followed by a CY5-conjugated secondary antibody, and the Rab5a-wt-GFP or procathepsin D-GFP was visualized by the intrinsic fluorescence of the fused GFP as described previously (19, 48, 54). Confocal imaging was performed using a Bio-Rad confocal microscope (Bio-Rad Laboratories, Inc., Hercules, CA) at the Central Microscopy Facility of The University of Iowa as described previously (19, 48, 54).
Receptor Down-Regulation
The density of cell surface receptors was measured using [125I]hFSH binding to intact transiently transected cells or by immunoprecipitation of the myc-tagged receptors from cells that had been biotinylated under conditions that label only the cell surface (51).
The biotinylated cells were placed on ice and lysed immediately (t = 0 samples) or incubated in 1 ml of warm assay medium containing a saturating concentration of hFSH (1000 ng/ml) at 37 C for the times indicated before lysis. The cells were lysed as described previously (54), the myc-tagged receptors were immunoprecipitated with a monoclonal anti-myc antibody (9E10), and the immunoprecipitates were resolved on sodium dodecyl sulfate gels and electrophoretically transferred to polyvinylidene difluoride membranes as described elsewhere (48, 54, 55, 56). The biotinylated receptors were detected in the immunoprecipitates using streptavidin conjugated to horseradish peroxidase and visualized and quantitated using the Super Signal West FEMTO Maximum Sensitivity system of detection from Pierce Chemical Co. (Rockford, IL) and a Kodak digital imaging system (Eastman Kodak Co., Rochester, NY) as described elsewhere (51, 54). This digital image capture system is set up to alert us when image saturation occurs and to prevent us from measuring the intensity of such images. For the binding assays the cells were washed twice with assay medium, and some cells were saved on ice and processed immediately (t = 0 samples), while others were incubated in 1 ml of warm assay medium containing a saturating concentration of hFSH as described above. At this point the cells were placed on ice and washed two to three times with 2-ml portions of cold wash medium. The surface-bound hormone was then released by incubating the cells in 1 ml of cold 50 mM glycine, 150 mM NaCl, pH 3, for 24 min as described above. This buffer was removed and the cells were washed once more with the same acid buffer and then twice with cold assay medium. The binding of [125I]hFSH was then measured as described earlier.
Total (i.e. cell surface and intracellular) receptors were measured as described above for the biotinylated receptors except that the immunoprecipitated receptors were detected using the 9E10 antibody conjugated to horseradish peroxidase instead of streptavidin.
Second Messenger Assays
Transiently transfected cells were washed and placed in 1 ml of warm assay medium. After a 15-min preincubation (at 37 C), duplicate wells were incubated with a maximally effective concentration of hFSH (100 ng/ml). Total cAMP (i.e. cells + medium) was extracted at the times indicated and measured by RIA as described elsewhere (57, 58, 59, 60). For the inositol phosphate assays the cells were placed in medium containing 2 µCi/ml of [2-3H]myo-inositol (New England Nuclear, Boston, MA) during the last 24 h of the posttransfection incubation. Before the assay the cells were washed and placed in 1 ml of warm assay medium containing 20 mM LiCl. After a 15-min preincubation (at 37 C), duplicate wells were incubated with a maximally effective concentration of hFSH (500 ng/ml). The medium was then aspirated at the times indicated, and the total inositol phosphates present in the cells were extracted and quantitated as described previously (60).
Hormones and Supplies
Purified hFSH (AFP-5720D, prepared from human pituitaries) and purified hCG (CR-127) were purchased from the National Hormone and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases. Purified recombinant hCG and hFSH were provided by Ares Serono (Randolph, MA). Partially purified eFSH was kindly donated by Dr. George Bousfield (Wichita State University, Wichita, KS), and crude hCG was purchased from Sigma Chemical Co. (St. Louis, MO). [125I]hFSH and [125I]hCG were prepared using the purified hormones as previously described (61). The 9E10 monoclonal antibody was prepared by the Hybridoma Facility of the Cancer Center of the University of Iowa. The 9E10 monoclonal antibody coupled to horseradish peroxidase was purchased from Roche Clinical Laboratories (Indianapolis, IN). The CY5-coupled secondary antibody was from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Lipofectamine was from Invitrogen (San Diego, CA) and Fugene from Roche Clinical Laboratories. Cell culture supplies and reagents were obtained from Corning, Inc. (Corning, NY) and Life Technologies, Inc. (Gaithersburg, MD), respectively. All other chemicals were obtained from commonly used suppliers.
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ACKNOWLEDGMENTS
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We wish to thank Ares Serono (Randolph, MA) for purified recombinant hFSH and hCG and the plasmids encoding for the human gonadotropin receptors. We also thank Dr. George Bousfield (Wichita State University, Wichita, KS) for partially purified equine FSH, Dr. Marlene Hosey (Northwestern University, Chicago, IL) for 293T cells, Dr. Michael Griswold (Washington State University, Pullman, WA) for MSC-1 cells, Dr. Ilpo Huhtaniemi (Imperial College, London) for KK-1 cells, Dr. Phil Stahl (Washington University, St. Louis, MO) for the Rab5a-GFP expression vector, and Dr. Jonathan M. Backer (Albert Einstein College of Medicine, New York, NY) for the procathepsin D-GFP expression vector. Lastly we thank Dr. Deborah Segaloff for reading the manuscript.
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FOOTNOTES
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This work was supported by NIH Grant HD-28962 (to M.A.) The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa (supported by NIH Grant DK-25295) are also gratefully acknowledged. M.S. was supported by a training grant from the NIH (DK-07759), and T.H. was partially supported by a fellowship from the Lalor Foundation.
Abbreviations: CG, Chorionic gonadotropin; e, equine; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; FSHR, FSH receptor; h, human; LHR, LH receptor; r, rat; TSHR, TSH receptor; wt, wild-type.
1 Although the rate constant for recycling can be readily estimated from the data presented in Fig. 1
, the rate constant for degradation should be considered to be only an estimate because this rate is too slow to be accurately measured during the time course of the experiments presented here. 
2 Transiently transfected MSC-1 cells could not be used for these experiments because of the low levels of 125I-hFSH binding attained. 
Received for publication April 3, 2003.
Accepted for publication July 24, 2003.
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