Phosphorylation of the Lutropin/Choriogonadotropin Receptor Facilitates Uncoupling of the Receptor from Adenylyl Cyclase and Endocytosis of the Bound Hormone

Zheng Wang, Xuebo Liu and Mario Ascoli

Department of Pharmacology University of Iowa Iowa City, Iowa 52242-1109


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stably transfected cell lines expressing the wild-type rat LH/CG receptor (rLHR) or a full-length rLHR in which S635, T638, S639, S649, and S653 were simultaneously mutated to alanine residues (designated rLHR-5S/T->A) were used to probe the importance of receptor phosphorylation on the regulation of receptor functions. The mutant receptor binds hCG with high affinity and transduces the hormonal signal into increases in cAMP and inositol phosphate accumulation comparable in magnitude to those elicited by the wild-type receptor.

In contrast to cells expressing rLHR-wt, which respond to hCG or phorbol 12-myristate 13-acetate stimulation with an increase in rLHR phosphorylation, the phosphorylation of rLHR in cells expressing rLHR-5S/T->A is severely blunted. Likewise, the phorbol 12-myristate 13-acetate-induced desensitization of hCG-induced cAMP accumulation is drastically reduced in cells expressing rLHR-5S/T->A. In contrast, the hCG-induced desensitization of hCG-induced cAMP accumulation is delayed, but not abolished, in cells expressing rLHR-5S/T->A. Lastly, the rate of internalization of the receptor-bound hCG is slower in cells expressing rLHR-5S/T->A than in cells expressing rLHR-wt.

These results show that phosphorylation of rLHR is necessary, but not sufficient, for uncoupling of the receptor from adenylyl cyclase and for endocytosis of the receptor-bound hormone.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Like many other G protein-coupled receptors, the LH/CG receptor (LHR) and the FSH receptor (FSHR) become phosphorylated upon agonist stimulation (1, 2). In keeping with the knowledge that phosphorylation is an important event in the agonist-induced desensitization of many G protein-coupled receptors (reviewed in Refs. 3–6), we proposed that the phosphorylation of LHR and FSHR is also involved in the gonadotropin-induced desensitization of these receptors (1, 2). Other investigators, however, have presented evidence that is not consistent with this hypothesis (7, 8, 9).

In an ongoing series of experiments we set out to formally test the hypothesis that phosphorylation is involved in the desensitization of gonadotropin receptors. Using human kidney 293 cells stably transfected with the rat LHR (rLHR) complementary DNA (cDNA), we showed that rLHR becomes rapidly phosphorylated in serine residues when the cells are exposed to hCG or a phorbol ester [phorbol 12-myristate 13-acetate (PMA)], and that these two stimuli also induce desensitization of the hCG-responsive adenylyl cyclase (1, 10, 11, 12, 13). Further analysis of three progressive C-terminal truncations of the rLHR showed that most of the hCG- or PMA-induced phosphorylation occurs in one or more of four serine residues (S635, S639, S649, and S652) located in the C-terminal cytoplasmic tail of the rLHR (12, 13). Furthermore, our data showed that two C-terminally truncated species of rLHR (designated rLHR-t628 and rLHR-t631) that cannot be phosphorylated also display a delay in hCG-induced desensitization, a severe impairment in PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation (10, 13). In contrast, another C-terminally truncated species of rLHR (designated rLHR-t653) that can be phosphorylated behaved similarly to the wild-type rLHR with regard to hCG- or PMA-induced desensitization as well as hCG-induced receptor down-regulation (13).

The experiments presented herein were designed to determine whether the different functional properties of rLHR-t653, rLHR-t631, and rLHR-t628 described above are due to removal of the entire 632–653 region or to removal of the phosphorylation sites contained within this region. In addition, we wanted to determine whether rLHR phosphorylation is needed for the efficient endocytosis of the receptor-bound hormone. To this end we constructed and analyzed a full-length rLHR mutant in which the four serines and single threonine present in the 632–653 region were simultaneously mutated to alanines. This mutant was analyzed for hCG- and PMA-induced phosphorylation, uncoupling, and down-regulation as well as for its ability to mediate the endocytosis of the bound ligand.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Preparation and Functional Properties of rLHR-5S/T->A
Phosphoamino acid analysis and phosphorylation experiments involving three different C-terminally truncated species of the rLHR have identified four serine residues in the 632–653 region of the rLHR as the major locus of hCG- or PMA-induced phosphorylation (12, 13). To confirm and extend these observations, we constructed and analyzed a full-length receptor mutant in which these four residues (S635, S639, S642, and S649) were simultaneously mutated to alanines. Although we cannot detect phosphothreonine in the phosphorylated rLHR (12), the single threonine residue (T638) located in the 632–653 region was also mutated to alanine to account for the possibility that the phosphorylation of a single threonine residue may not be detected in the phosphoamino acid analysis. Thus, the new receptor mutant characterized here (designated rLHR-5S/T->A) is a full-length mutant in which S635, T638, S639, S649, and S652 were simultaneously mutated to alanines (Fig. 1Go).



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Figure 1. Intracellular Regions of the rLHR

The amino acid sequence of the three cytoplasmic loops and the C-terminal cytoplasmic tail of the rLHR is shown (for review, see Ref. 43). The five residues (S635, T638, S639, S642, and S649) that were simultaneously mutated to alanines are indicated. L628, R631, and Q653 are also indicated as a point of reference, as they represent the C-terminal end of three truncated forms of rLHR that were previously characterized. Serine 659, another potential phosphorylation site (see Discussion), and two additional serines (365 and 617) that have been ruled out as potential phosphorylation sites (12, 13) are also indicated.

 
After transfection of 293 cells with the cDNA encoding for rLHR-5S/T->A, several clonal cell lines were obtained expressing different numbers of cell surface receptors. One of these cell lines that expressed a high density of mutant receptors [designated 293L(5S/T->A-2); binding capacity, 237,000 ± 13,000 molecules of hCG/cell] and a cell line that expressed a comparable density wild-type rLHR [designated 293L(wt-17); binding capacity, 232,000 ± 15,000 molecules of hCG/cell] were chosen for further analysis. The Kd values for the binding of hCG to 293L(wt-17) and 293L(5S/T->A-2) cells were 301 ± 1 and 365 ± 60 pM, respectively.

As shown in Table 1Go, the basal levels of cAMP, the EC50 for cAMP accumulation, and the maximal cAMP response to hCG are somewhat higher in 293L(5S/T->A-2) cells than in 293L(wt-17) cells. To correct for the possibility of clonal variation we also measured the cAMP response to cholera toxin and calculated a ratio for the maximal cAMP response to hCG and the maximal cAMP response to cholera toxin. This ratio is somewhat lower in 293L(5S/T->A-2) cells than in 293L(wt-17). As this response ratio is comparable to those previously reported for other cell lines expressing rLHR-wt (12, 13), we conclude that the 5S/T->A mutation of rLHR has only a minimal effect on the transduction of the cAMP signal. The same can be said for the effects of this mutation on the transduction of the inositol phosphate signal. When corrected for the inositol response induced by NaF, the hCG-induced inositol phosphate response is comparable in 293L(wt-17) and 293L(5S/T->A-2) cells (data not shown).


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Table 1. Human CG- and Cholera Toxin-Stimulated cAMP Accumulation in Stably Transfected 293 Cells Expressing rLHR-wt or rLHR-5S/T-->A

 
Human CG and PMA-Induced Phosphorylation
The data presented in Fig. 2AGo show that basal, hCG-induced, and PMA-induced phosphorylations of rLHR are severely impaired in cells expressing rLHR-5S/T->A. Longer exposures and densitometic scanning of the autoradiograms indicated that phosphorylation is not completely abolished, however. In two independent experiments quantitated by densitometry, hCG-induced phosphorylation was reduced by 92 ± 2%, and PMA-induced phosphorylation was reduced by 89 ± 1%. These findings are consistent with previous data showing that phosphorylation is largely preserved in a C-terminal truncation of rLHR at residue 653, but is completely abolished when truncations are performed at residue 631 or 628 (12, 13). The reduced phosphorylation in cells expressing rLHR-5S/T->A cannot be explained by differential immunoprecipitation of rLHR-wt and rLHR-5S/T->A, as both receptors can be readily detected in immunoprecipitates of [35S]cysteine/methionine-labeled cells (Fig. 2BGo). As previously reported, immunoprecipitates from cells metabolically labeled with [35S]cysteine/methionine reveal not only the cell surface 85-kDa rLHR, but also a 68-kDa immature intracellular precursor of the rLHR (12, 13, 14).



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Figure 2. Human CG and PMA-Induced Phosphorylation of rLHR-wt and rLHR-5S/T->A

Immunoprecipitates were prepared from cells that had been metabolically labeled with 32P for 3 h (A) or [35S]Translabel for 24 h. In A, the 32P-prelabeled cells were also incubated with buffer only, 1000 ng/ml hCG for 5 min, or 200 nM PMA for 30 min before immunoprecipitation. These concentrations and incubation times were chosen to elicit a maximal response (1, 12, 13). In both panels, equivalent amounts of cellular protein were used for immunoprecipitation. The results presented are from a densitometric scan of a representative autoradiogram.

 
Agonist and PMA-Induced Desensitization
The results summarized in Fig. 3AGo show that preincubation of 293L(wt-17) cells with hCG results in a time-dependent decrease in cAMP responsiveness to a subsequent challenge with hCG (desensitization). The results summarized in Fig. 3BGo show that this loss of responsiveness cannot be accounted for by a decrease in cell surface receptors (down-regulation) because these decline at a much slower rate. As this loss of responsiveness also cannot be explained by changes in the functional properties of Gs or adenylyl cyclase (11, 13, 15, 16, 17, 18), these results operationally define a type of desensitization (which we call uncoupling) that must be due to a change in the functional properties of the rLHR rather than to a change in the density of cell surface rLHR.



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Figure 3. Human CG-Induced Desensitization and Down-Regulation of rLHR-wt and rLHR-5S/T->A

A, 293L(wt-17) or 293L(5S/T->A-2) cells were preincubated with 100 ng/ml hCG at 37 C for the indicated times. The free and bound hCG were then removed (see Materials and Methods), and the cells were divided into two groups and incubated without or with 100 ng/ml hCG for 15 min at 37 C. At the end of this incubation, the medium was aspirated, and the cells were used to determine the intracellular cAMP content. The amount of cAMP accumulated in the group of cells incubated without hCG was then subtracted from that present in the cells incubated with hCG, and the results were expressed as a percentage of the cAMP response determined under identical conditions but using cells that were preincubated without hCG. Each point represents the average ± SEM of three independent experiments. B, 293L(wt-17) or 293L(5S/T->A-2) cells were preincubated with 100 ng/ml hCG at 37 C for the indicated times. The free and bound hCG were then removed (see Materials and Methods), and the cell surface LHR were measured during an overnight incubation (at 4 C) with 100 ng/ml [125I]hCG. The results were then expressed as a percentage of the [125I]hCG bound to cells treated identically but preincubated without hCG. Each point represents the average ± SEM of three independent experiments.

 
The results presented in Fig. 3Go also show that although hCG-induced down-regulation of rLHR is preserved in the phosphorylation-negative mutant, hCG-induced uncoupling is delayed. The delay in uncoupling detected in cells expressing rLHR-5S/T->A agrees well with previous data obtained for receptor truncations. Thus, cells expressing rLHR-t653, a rLHR truncation that retains the phosphorylation sites mutated here, uncouple normally when incubated with hCG (12, 13). In contrast, cells expressing rLHR-t631 or rLHR-t628, two rLHR truncations that remove the phosphorylation sites mutated here, show a delay in uncoupling indistinguishable from that shown in Fig. 3AGo for cells expressing rLHR-5S/T->A (10, 12, 13). The delay in the time course of uncoupling detected in cells expressing rLHR-5S/T->A is obvious at early time points. During a 15-min preincubation with a saturating concentration of hCG, 293L(wt-17) cells lose 40–60% of their ability to respond to a subsequent challenge with any concentration of hCG, whereas the ability of 293L(5S/T->A-2) cells to respond to a subsequent challenge with any concentration of hCG is not impaired (Fig. 4Go).



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Figure 4. Effect of Prior Exposure to hCG on the cAMP Response Induced by Increasing Concentrations of hCG

293L(wt-17) and 293L(5S/T->A) cells were initially divided into two groups and preincubated without (open bars) or with 100 ng/ml hCG (dark bars) for 15 min to induce receptor uncoupling. At the end of this preincubation, the free and receptor-bound hormone were removed (see Materials and Methods), and each group of cells was divided into four subgroups. One subgroup of cells was further incubated without hormone for 15 min at 37 C to determine the residual levels of cAMP present after the preincubation. The other three subgroups were restimulated with increasing concentrations of hCG (as indicated on the abscissa) for 15 min at 37 C. The bars shown represent the levels of intracellular cAMP measured at the end of the restimulation period after correction for residual levels of cAMP. Basal levels of cAMP were 4.4 ± 0.4 and 23.1 ± 3.3 pmol/106 cells for 293L(wt-17) cells preincubated without or with hCG, respectively. The corresponding values for 293L(5S/T->A-2) cells were 0.7 ± 0.1 and 7.4 ± 0.8 pmol/106 cells. Each bar represents the average ± SEM of three independent experiments. The numbers in parentheses above each pair of bars depict the cAMP response in cells preincubated with hCG expressed as a percentage of the response in cells preincubated in the absence of hCG.

 
In previous experiments we had also shown that the hCG-induced down-regulation was normal in cell lines expressing rLHR-t653, but it was faster in cells expressing rLHR-t631 or rLHR-t628 (13). The data presented in Fig. 3BGo clearly show that the enhanced down-regulation detected in rLHR-t631 and rLHR-t628 is not due to the deletion of phosphorylation sites, because the hCG-induced down-regulation is similar in cells expressing rLHR-wt and those expressing rLHR-5S/T->A. Thus, we must now conclude that other amino acids in the 632–653 region are important to the process of down-regulation.

The addition of PMA to 293 cells transfected with rLHR-wt also leads to receptor phosphorylation and desensitization of the cAMP response to hCG that is due to uncoupling rather than receptor down-regulation (1, 12, 13). In contrast to the hCG-induced uncoupling, which results in a reduction in the cAMP response to all concentrations of hCG tested (5–200 ng/ml; as illustrated in Fig. 4Go), the PMA-induced uncoupling leads to a reduction in the cAMP response only when the cells are rechallenged by low concentrations of hCG (i.e. <5 ng/ml; see Fig. 5Go). As the concentrations of hCG used in the rechallenge increase, the inhibitory effect of PMA wanes to the point where there is no effect or even an increase in the cAMP response (Fig. 5Go). The data presented in Fig. 5Go also show that, in keeping with the impairment in PMA-induced phosphorylation, 293L(5S/T->A-2) cells display a substantial impairment in the magnitude of PMA-induced uncoupling. This finding is consistent with previous data for receptor truncations, which showed that a reduction in PMA-induced uncoupling correlates with the removal of the phosphorylation sites mutated here (13).



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Figure 5. Effect of Prior Exposure to PMA on the cAMP Response Induced by Increasing Concentrations of hCG

293L(wt-17) and 293L(5S/T->A-2) cells were divided into two groups and preincubated without (open bars) or with 200 nM PMA (dark bars) for 30 min. At the end of this preincubation, each group of cells was divided into six subgroups. One subgroup of cells was further incubated without hormone for 15 min at 37 C, and the other five subgroups were further incubated with increasing concentrations of hCG (as indicated on the abscissa) for 15 min at 37 C. The bars shown represent the levels of intracellular cAMP measured at the end of this incubation after subtraction of the amount of cAMP present in the cells incubated without hCG. Basal levels of cAMP were 0.9 ± 0.2 and 0.4 ± 0.1 pmol/106 cells for 293L(wt-17) cells preincubated without or with PMA, respectively. The corresponding values for 293L(5S/T->A-2) cells were 1.4 ± 0.1 and 0.7 ± 0.1 pmol/106 cells. Each bar represents the average ± SEM of three independent experiments. The numbers in parentheses shown above each pair of bars depict the cAMP response in cells preincubated with PMA expressed as a percentage of the response in cells preincubated in the absence of PMA.

 
Receptor-Mediated Endocytosis
Although it is clear that the receptor-bound [125I]hCG is internalized and degraded when it binds to the LHR expressed in gonadal or transfected cells (19, 20, 21), little is known about the regulation of this process. Since it is known, however, that progressive truncations of the C-terminal cytoplasmic tail that remove the four serine residues identified as phosphorylation sites also enhance the rate of hormone internalization (20), the availability of a rLHR-5S/T->A allows us to determine whether the increased internalization rate of the truncated receptors is due to the removal of phosphorylation sites.

The data summarized in Fig. 6Go show that the internalization of [125I]hCG is slower in 293L(5S/T->A-2) cells than in 293L(wt-17) cells. Additional experiments in which the rate constants for endocytosis (ke) were measured in these two cell lines indicated that [125I]hCG is internalized with a half-life of 74 ± 4 min (n = 3) in 293L(wt-17) cells and 103 ± 8 min (n = 3) in 293L(5S/T->A-2) cells. Thus, we can now conclude that the enhanced rate of internalization previously detected in C-terminally truncated forms of rLHR is not due to the removal of phosphorylation sites. In fact, our data suggest that the agonist-induced phosphorylation of rLHR facilitates its internalization.



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Figure 6. Internalization and Degradation of the Receptor-Bound [125I]hCG in 293L(wt-17) and 293L(5S/T->A-2) Cells

Cells were preincubated with 40 ng/ml [125I]hCG for 15 min at 37 C. At the end of this incubation (time 0 in the figure), the free hormone was removed by washing, the cells were placed in warm fresh medium without hormone, and the incubation was continued at 37 C. At the times indicated, the cells were placed on ice and used to determine the amount of surface-bound, internalized, and degraded hormone as described in Materials and Methods. The bottom panels show the same data, except that the internalized and degraded radioactivity were manually combined to simplify the data into two, rather than three, components. Each point represents the average ± SEM of three independent experiments. The initial counts per min bound were 58,000 ± 1,000 and 38,000 ± 4,000 for 293L(wt-17) and 293L(5S/T->A-2) cells, respectively.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous studies from this and other laboratories have shown that the hCG- or PMA-induced desensitization of hCG-stimulated cAMP synthesis may be due to changes in the functional properties of the rLHR and/or to changes in the density of cell surface rLHR, rather than to a change in the functional properties of Gs or adenylyl cyclase (11, 13, 15, 16, 17, 18). Under the experimental conditions employed herein, the hCG- or PMA-induced desensitization detected in transfected 293 cells appear to be due to changes in the functional properties of the rLHR (a phenomenon that we call uncoupling), rather than to changes in the density of cell surface rLHR (a phenomenon that we call down-regulation) for the following reasons. First, within the range of receptor expression used in these experiments, the maximal cAMP response to hCG is basically independent of receptor number (13). Second, when PMA is used as a stimulus, desensitization occurs in the absence of receptor down-regulation (13), and when hCG is used as a stimulus, the time course of down-regulation is much slower than that of desensitization (13). Thus, this experimental paradigm should allow us to determine the molecular basis of the hCG- or PMA-induced uncoupling of the rLHR from adenylyl cyclase.

As only phosphoserine is detectable in the phosphorylated rLHR (12), there are only seven potential phosphorylation sites present in the intracellular regions of this receptor. Two of these (S365 and S617) can be readily excluded by the finding that receptor phosphorylation is completely abolished in two downstream truncations (rLHR-t628 and rLHR-t631) that retain these two residues (12, 13). Studies with these two truncations and an additional truncation at residue 653 where phosphorylation is minimally affected (12, 13) have indicated that the four serines mutated here (S635, S639, S649, and S653) are the major loci of phosphorylation. A small decrease in the phosphorylation signal detected in rLRH-t653 (12, 13) led us to suspect that S659 may represent an additional phosphorylation site. The fact that the phosphorylation of 5S/T->A is severely impaired, but not lost, confirms the conclusion that S635, S639, S649, and/or S653 represent the major loci of phosphorylation and further indicates that S659 represents the residue responsible for the residual phosphorylation detected in this mutant.

The time course of hCG-induced uncoupling in cells expressing rLHR-wt suggests the existence of two phases (13). These consist of a fast phase that occurs within 5 min of hCG addition and leads to a 40–60% reduction in hCG-stimulated cAMP synthesis and a slower phase that ensues for the next 50–60 min and leads to a further 20–40% reduction in hCG-stimulated cAMP synthesis. The removal (12, 13) or mutation of phosphorylation sites delays the early phase of hCG-induced uncoupling, but has little or no effect on the slow phase. We would argue that this delay in the onset of uncoupling is caused by the phosphorylation of S635, S639, S649, and/or S652, and that the phosphorylation of S659 (discussed above) is functionally silent. Thus, the functional properties of the agonist-induced uncoupling of rLHR-t631 and rLHR-t628 (both of which show no residual phosphorylation and lack S635, S639, S649, S652, and S659) (12, 13) are identical to those of rLHR-5S/T->A, in which only S635, S639, S649, and S652 were mutated and show minimal residual phosphorylation. Clearly then, agonist-induced uncoupling is delayed regardless of the presence or absence of S659. The converse is also true, in that the removal of S659 without removing S635, S639, S649, and S652 (which was accomplished in rLHR-t653) results in a receptor that displays a minimal reduction in phosphorylation and a normal time course and magnitude of agonist-induced uncoupling (12, 13). Taken together, our results indicate that phosphorylation is necessary, but not alone sufficient, for the hCG-induced uncoupling of rLHR.

The delay in the time course of hCG-induced uncoupling (as opposed to a change in magnitude) observed in the phosphorylation-deficient mutants of rLHR is not peculiar to this receptor, nor unexpected from previous reports on the ß2-adrenergic receptor (22). In fact, the removal (by truncation) or mutation of the C-terminal phosphorylation sites of the ß2-adrenergic receptor also results in a delay in the time course of agonist-induced desensitization, but has little or no effect on the magnitude of desensitization detected upon prolonged incubation with agonist (22).

In contrast to the data discussed above for hCG-induced uncoupling, the magnitude of PMA-induced uncoupling is severely impaired by the removal (13) or mutation of phosphorylation sites. We would again argue that this decrease in the magnitude of PMA-induced uncoupling is caused by the phosphorylation of S635, S639, S649, and/or S652, and that the phosphorylation of S659 (discussed above) is functionally silent. Thus, there is little or no PMA-induced phosphorylation or uncoupling in cells expressing rLHR-t631 and rLHR-t628 (both of which lack S635, S639, S649, S652, and S659) (12, 13) or in cells expressing rLHR-5S/T->A, in which only S635, S639, S649, and S652 were mutated. In contrast, the removal of S659 without removing S635, S639, S649, and S652 (which was accomplished in rLHR-t653) results in a receptor that displays a minimal reduction in PMA-induced phosphorylation and a normal magnitude of PMA-induced uncoupling (12, 13). Taken together our data argue that the PMA-induced phosphorylation of this locus is the most important (if not the only) posttranslational modification involved in the PMA-induced uncoupling of the rLHR.

The data presented here also show that the hCG-induced phosphorylation of the rLHR at residues S635, S639, S649, and/or S652 is needed for the efficient endocytosis of the receptor-bound hormone. This is the only case where previous data obtained with rLHR truncations that remove or maintain phosphorylation sites do not match the data obtained with the simultaneous mutation of the phosphorylation sites. Thus, rLHR-t653 (phosphorylation-positive truncation) and rLHR-t631 (phosphorylation-negative truncation) were previously shown to exhibit a faster rate of internalization than rLHR-wt (20), whereas as shown here, rLHR-5S/T->A exhibits a slower rate of internalization. These results are also interesting because of recent reports of the possible importance of activation and/or phosphorylation of G protein-coupled receptors in the process of internalization. There is a growing body of evidence indicating that the activation of G protein-coupled receptors in general (23, 24, 25, 26, 27) is needed for the efficient internalization of the hormone-receptor complex. Likewise, although initial experiments argued against a role for phosphorylation on the sequestration of ß2-adrenergic receptors (22, 28, 29), emerging models for this (30, 31, 32) as well as the M2 muscarinic receptor (33) indicate that agonist-induced phosphorylation facilitates the agonist-induced sequestration of these two receptors.

We have previously reported that the rate of endocytosis of the antagonist-occupied mouse LHR is slower than the rate of endocytosis of the agonist-occupied mouse LHR (26) and that two mutations of the rLHR that impair signal transduction also slow down the endocytosis of the bound agonist in transfected 293 cells (27). Although we interpreted these data to indicate that an agonist-induced conformational change in the LHR and/or a physical association of the LHR with its cognate G protein(s) were needed for efficient endocytosis of the bound ligand, the data presented here argue that we should also consider the possibility that LHR phosphorylation is an important component of this process.

In summary, the data presented herein clearly show that phosphorylation of the rLHR at S635, S639, S642, and/or S649 is necessary for hCG- and PMA-induced uncoupling and for the efficient endocytosis of the receptor-bound hormone. Additional studies are currently underway to determine the phosphorylation state and functional properties of additional mutants of the LHR in which S635, S639, S642, and S649 as well as S659 and T638 are individually mutated to alanine. Although we predict that the S659A or T638A mutations would have little or no impact on phosphorylation, uncoupling, or internalization, the analysis of the other four mutants will allow us to more accurately determine the identity of the phosphorylation sites and their impact on function. Additional experiments are also being performed to determine whether other posttranslational modifications of the rLHR are involved in hCG-induced uncoupling and to determine whether the previously described transduction-deficient mutants of rLHR do not internalize efficiently simply because they are not phosphorylated in response to hCG binding.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Cells
The cloning of the rat luteal LH/CG receptor cDNA and the template plasmid containing the full-length coding region plus portions of the 5'- and 3'-untranslated regions of the wild-type rLHR cDNA have been previously described (34). The mutant rLHR-5S/T->A, in which S635, T638, S639, S649, and S652 were simultaneously mutated to alanines in the full-length LH/CG receptor, was created by PCR with overlap extension (35) to alter the nucleotides coding for these residues. The sequence of the entire region of each mutant cDNA generated by PCR was verified by dideoxy sequencing (36). The mutant and wild-type rLHR cDNAs were subcloned into the eukaryotic expression vector pcDNAI/Neo (Invitrogen, San Diego, CA) for transfection.

The origin and handling of the parental human embryonic kidney (293) cells and the methods used for transfection and isolation of clonal cell lines stably transfected with the wild-type or mutant rLHR cDNAs have been described in detail previously (12, 27, 37, 38).

Other Methods
Metabolic labeling of cells and subsequent immunoprecipitation of the rLHR were achieved as previously described (1, 2, 12, 13, 14). Autoradiograms of the dried gels were obtained using intensifying screens, and the autoradiograms were scanned using a Bio-Rad Molecular Imaging System (Bio-Rad Laboratories, Richmond, CA). All of the autoradiograms shown here are reproductions from the scanned images. Equilibrium binding parameters for hCG were measured during an overnight incubation (4 C) of intact cells with a fixed concentration of [125I]hCG and increasing concentrations of hCG as described previously (37, 38). Concentration-response curves for the hCG-induced increases in cAMP and inositol phosphate accumulation were performed during a 30- and 60-min incubations at 37 C, respectively. These were analyzed as described previously (12, 13). Measurements of hCG-induced desensitization were performed after incubation of cells with a fixed concentration of hormone (100 ng/ml) for increasing periods of time. After removal of the free and bound hormone, the cAMP response of the cells was assessed during a 15-min incubation with the indicated concentrations of hCG (12, 13). PMA-induced desensitization was measured after incubating the cells with 200 nM PMA for 30 min. The cAMP response of the cells was then assessed during a 15-min incubation with the indicated concentrations of hCG (12, 13). The methodology used to asses the endocytosis of hCG and to measure the rates of internalization have also been described (27, 39, 40). For these experiments, the surface-bound hormone was removed after a brief exposure of the cells to an isotonic pH 3 buffer. The radioactivity that remains cell associated was considered to be internalized hormone. Degraded hormone was measured by solubility in trichloroacetic acid.

Hormones and Supplies
Purified hCG (CR-127) was obtained from the National Hormone and Pituitary Agency of the NIDDK. [125I]hCG was prepared as described previously (41), to give a specific radioactivity of 25,000–30,000 cpm/ng. [32P]orthophosphate was obtained from DuPont-New England Nuclear (Boston, MA). Methionine/cysteine- and phosphate-free DMEM as well as Tran35S-Label were purchased from ICN Biomedicals (Irvine, CA). Nonidet P-40, protease inhibitors, N,N',N'-triacetylchitotriose, protein A-agarose, fibronectin-like engineered polymer, and BSA were obtained from Sigma Chemical Co. (St. Louis, MO). Okadaic acid and cypermethrin were purchased from LC Laboratories (Woburn, MA). Wheat germ agglutinin was obtained from Vector Laboratories (Burlingame, CA), and Geneticin was obtained from Life Technologies (Grand Island, NY). The rabbit antibody to the rLHR (Bugs) has been previously described (42). Cell culture supplies were obtained from Corning (Corning, NY) and Life Technologies, respectively. All other materials were obtained from commonly used suppliers.


    ACKNOWLEDGMENTS
 
We thank JoEllen Fabritz for assistance with the cell culture, and Drs. Bill Hipkin and Deborah Segaloff for helpful discussions.


    FOOTNOTES
 
Address requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2–512 BSB, University of Iowa, Iowa City, Iowa 52242-1109.

This work was supported by a grant from the NIH (CA-40629). 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.

Received for publication October 3, 1996. Revision received November 13, 1996. Accepted for publication November 14, 1996.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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