Effect of Activating and Inactivating Mutations on the Phosphorylation and Trafficking of the Human Lutropin/Choriogonadotropin Receptor

Le Min and Mario Ascoli

Department of Pharmacology The University of Iowa College of Medicine Iowa City, Iowa 52242-1109


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The effects of several mutations of the human LH receptor (hLHR) on the phosphorylation, internalization, and turnover of the cell surface receptor were examined. Three gain-of-function mutations associated with Leydig cell hyperplasia (L457R and D578Y) and one associated with Leydig cell adenomas (D578H), one signaling-impaired mutation associated with Leydig cell hypoplasia (I625K), and two laboratory designed signaling-impaired mutations (D405N and Y546F) were used. The signaling-impaired mutations showed a reduction in human CG (hCG)-induced receptor phosphorylation and internalization. Mutation of the phosphorylation sites of these loss-of-function mutants had little or no additional effect on internalization. Cotransfection with G protein-coupled receptor kinase-2 (GRK2) rescued the hCG-induced phosphorylation and internalization of the signaling-impaired mutations but only if the phosphorylation sites were intact. Overexpression of arrestin-3 rescued the rate of internalization regardless of whether or not the phosphorylation sites were intact.

Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous studies have shown that, like many other G protein-coupled receptors (GPCRs), the binding of agonist to the rat LH receptor (rLHR) leads to the phosphorylation of the receptor (1) and that this process facilitates the internalization of the agonist-rLHR complex (2, 3) via clathrin-coated pits by a pathway that requires the participation of clathrin, a nonvisual arrestin and dynamin (3, 4, 5, 6). In agreement with this model, it has also been shown that activating mutations of the rLHR enhance the internalization of hCG whereas phosphorylation-deficient or inactivating mutations of the rLHR impair the internalization of hCG (2, 3, 7, 8, 9).

Since GPCR activation is an important determinant of the agonist-induced GPCR phosphorylation (reviewed in Refs. 10, 11), the effects of activating and inactivating mutations of the rLHR on the internalization of hCG could be directly due to the effects of these mutations on receptor activation or they could be more indirectly mediated by changes in receptor phosphorylation. This question has been difficult to address with the rLHR because the level of expression of the rLHR in transiently transfected cells is rather low. Although this low level of transient expression does not affect our ability to conduct internalization assays (3, 5, 6), the phosphorylation of the rLHR can only be studied in stably transfected cells (1, 2, 3, 12, 13). Thus, structure-function studies on the phosphorylation of the rLHR are very laborious because stably transfected cell lines with matched receptor numbers (at least ~100,000 receptors per cell) must be prepared for each mutant to be analyzed (2, 3, 12, 13). In addition, the use of cotransfection strategies that are helpful in understanding receptor phosphorylation and internalization cannot be readily accomplished using stably transfected cell lines.

In recent comparative studies on the human LH receptor (hLHR) and the rat LH receptor we noticed that the level of expression of the hLHR is much higher than that of the rLHR (~40,000 and ~400,000 receptors per cell, respectively) in 293 cells transiently transfected with optimal amounts of plasmid. Since these high levels of expression of the hLHR allow for the quantitation of receptor phosphorylation in transiently transfected cells, we reasoned that this experimental system would be more amenable to structure-function studies designed to address the involvement of receptor activation and phosphorylation in the process of internalization. In the studies presented herein we took advantage of these properties of the hLHR and analyzed several mutants of the hLHR to better understand the importance of receptor activation and phosphorylation in the trafficking of the hLHR.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Signaling Properties of hLHR Mutants
Three naturally occurring activating mutations of the hLHR, L457R, D578Y, and D578H, were chosen for these studies. The L457R and D578Y mutations are germ-line mutations that were initially found in boys with male-limited, gonadotropin-independent precocious puberty (14, 15). These point mutations are located in transmembrane helix 3 (TM3) and TM6, respectively. The D578H mutation of the hLHR is a somatic mutation that was recently identified in Leydig cell adenomas of three boys exhibiting precocious puberty (16). Heterologous cells expressing any of these three mutants display an elevated level of cAMP in the absence of agonist stimulation but respond to further agonist stimulation with a minimal increase or with no increase in cAMP accumulation (14, 16, 17).

Since many of the experiments described below necessitate robust surface expression, our choice of naturally occurring mutations that impair signaling was severely limited by the finding that many of these mutants are usually characterized by decreased expression and/or proper plasma membrane localization. We eventually settled on a point mutation in TM7 (I625K) that reduces cell surface expression only by approximately 50% (18). This mutant was initially identified in three brothers with a mild form of Leydig cell hypoplasia (18). When compared with cells expressing the wild-type hLHR (hLHR-wt), the agonist-induced cAMP response of cells expressing the I625K mutation (measured indirectly as an increase in the expression of a cAMP-driven reporter gene) is characterized by a rightward shift in the EC50 and a reduction in the maximal response. Because it is important to examine mutations that impair signaling, we also chose to analyze two laboratory-designed hLHR mutations (D405N in TM2 and Y546F in TM5) that, based on equivalent mutations of other GPCRs (7, 8, 19, 20, 21, 22), were predicted to result in an impairment in agonist-induced activation of the hLHR without affecting surface expression.

One additional mutant (designated 5S/A), in which five serine residues in the C-terminal tail of the hLHR (residues 657, 661, 670, 674, and 676) were simultaneously mutated to alanines, was constructed and analyzed. This mutant was predicted to be phosphorylation deficient based on the knowledge that four equivalent serines of the rLHR (residues 635, 639, 649, and 652) become phosphorylated upon agonist stimulation (2, 3, 12).1

Although the signaling properties of some of the hLHR mutants described above have been previously characterized (14, 16, 17, 18), it was important to document these properties under our experimental conditions. This was done by measuring cAMP accumulation in transiently transfected 293 cells incubated with increasing concentrations of hCG. For simplicity, however, the data presented in Table 1Go summarize only the response obtained with a concentration of hCG (3 nM) chosen to elicit maximal cAMP accumulation in cells expressing hLHR-wt (c.f. Fig. 1Go). To correct for the inherent variability associated with measuring the cAMP responses of transiently transfected cells, the basal and hCG-induced cAMP responses mediated by the different mutants were corrected by normalization to the cholera toxin-induced cAMP response measured in the same experiment (see columns labeled "Basal/Cholera Toxin" and "hCG/Cholera Toxin" in Table 1Go). Thus, instead of using the absolute levels of cAMP, the responsiveness of cells expressing the different mutants shown in Table 1Go should be compared by using these ratios. Lastly, all cells were also tested for 125I-hCG binding to ensure that variations in signaling were not due to variations in receptor expression. Table 1Go shows that the cell surface expression of all activating mutations (L457R, D578Y, and D578H) is comparable to that of hLHR-wt. When incubated in the absence of agonist, the basal levels of cAMP detected in cells expressing the three activating mutations are 15- to 30-fold higher than those detected in cells expressing an equivalent density of hLHR-wt (see "Basal/Cholera Toxin" in Table 1Go). These elevated levels of basal cAMP account for 25–50% of the amount of cAMP produced when cells expressing an equivalent density hLHR-wt are stimulated with a maximally effective concentration of hCG. A comparison of the hCG/cholera toxin ratio to the basal/cholera toxin ratio in Table 1Go shows that the cells expressing the activating mutants respond poorly to hCG stimulation (D578Y) or not at all (L457R or D578H). Full dose-response curves for cells expressing these mutants have been published by others (14, 16, 17) and are in general agreement with the data shown in Table 1Go.


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Table 1. cAMP Accumulation in 293 Cells Transiently Transfected with Several Mutants of the hLHR

 


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Figure 1. Representative Dose-Response Curves for the hCG-Induced cAMP Accumulation of Cells Transiently Transfected with the Laboratory-Designed Inactivating Mutations of the hLHR

293 cells plated in 35-mm wells were transiently transfected with hLHR-wt (squares), hLHR-D405N (triangles), or hLHR-Y546F (circles), and dose-response curves for the hCG-induced cAMP accumulation were performed and analyzed as described in Materials and Methods. The results of a representative experiment using duplicate wells for each concentration of hCG are shown. The EC50s for hCG were 0.1, 1.3, and 2.9 nM, and the maximal hCG responses were 3,900, 3,000, and 2,800 pmol/106 cells in the cells transfected with the hLHR-wt, -D405N, or -Y546F, respectively. 125I-hCG binding was 676, 611, and 689 fmol/106 cells, and the responses to 0.6 nM cholera toxin were 1,424, 1,420, and 1,304 pmol cAMP/106 cells in the cells transfected with the hLHR-wt, -D405N, or -Y546F, respectively. The vertical dashed line shows the concentration of hCG used in the experiments summarized in Table 1Go.

 
The cell surface expression of the two laboratory-designed mutations that are predicted to impair signaling (D405N and Y546F) are also comparable to that of hLHR-wt. The hCG/cholera toxin ratios summarized in Table 1Go show that cells expressing these mutants display a reduced cAMP responsiveness to a concentration of hCG (3 nM) that is maximally effective in cells expressing hLHR-wt. The representative dose- response curves presented in Fig. 1Go show that this reduction is due mostly to an approximately 10- to 30-fold increase in the EC50 for hCG displayed by cells expressing the D405N or Y546F mutants. The maximal hCG-induced cAMP response of cells expressing the D405N or Y546F mutants were only approximately 15 to 25% lower that those expressing a comparable density of hLHR-wt (Fig. 1Go). Cells expressing the naturally occurring mutation that impairs signaling (I625K) show an approximately 2-fold decrease in surface expression compared with cells expressing hLHR-wt as expected from published results (18). The reduced cAMP responsiveness to 3 nM hCG shown by the hCG/cholera toxin ratio in Table 1Go for cells expressing the I625K mutant is a reflection of a reduction in the maximal response and an increase in the EC50 as previously documented by other investigators (18). Although this reduced hCG response mediated by the I625K mutant should be interpreted with some caution (because of its reduced surface expression, see Table 1Go), we do not think that it can be solely explained by reduced surface expression because at these levels of receptor expression a 2- to 5-fold decrease in the expression of hLHR-wt has little or no effect on basal or hCG-stimulated cAMP accumulation (data not shown). Lastly, the basal cAMP and hCG-induced cAMP responses (as judged by the response ratios shown in Table 1Go) mediated by the 5S/A mutant were indistinguishable from those of cells expressing a comparable density of hLHR-wt.

Phosphorylation of hLHR Mutants
After labeling with 32P-orthophosphate, transiently transfected cells expressing the different mutants were stimulated with maximally effective concentrations of hCG or PMA for 15 min at 37 C. Since the concentrations of hCG needed to induce phosphorylation of the LHR closely resemble those needed for receptor occupancy, we used a saturating concentration of hCG (26 nM) in an attempt to fully saturate the receptors and optimize the phosphorylation signal during the short incubation period used [the dissociation constant (Kd) for hCG binding to the hLHR is 1–3 nM, see Ref. 6 ]. Stimulation of 293 cells stably expressing the rLHR-wt with 200 nM PMA has been previously shown to result in robust phosphorylation of rLHR (1), and it was included here as a positive control. Cell lysates were prepared, equalized for receptor expression (based on parallel binding assays performed in intact cells), immunoprecipitated with the 9E10 antibody, resolved on SDS gels, and visualized and quantitated with a PhosphorImager as described in Materials and Methods.

Stimulation of cells expressing hLHR-wt with hCG or PMA results in an approximately 3- and 4-fold increase in the incorporation of 32P into the receptor, respectively (Figs. 2Go and 3Go). In general agreement with our analysis of phosphorylation sites of the rLHR (2, 3, 12), we found that the simultaneous mutation of five serine residues clustered toward the C-terminal end of the hLHR (a mutant designated 5S/A) results in undetectable basal phosphorylation, an approximately 90% inhibition of phosphorylation induced by hCG, and a 70–80% inhibition of the PMA-induced phosphorylation (Figs. 2Go and 3Go). Thus, we can readily conclude that most of the hCG-induced phosphorylation of the hLHR occurs in one or more of these five residues. We can also conclude that the sites phosphorylated in response to PMA or hCG stimulation are overlapping but not identical.



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Figure 2. Phosphorylation of hLHR Mutants

293 cells plated in 100-mm dishes were transiently transfected with the indicated constructs to give equivalent expression of cell surface receptor as described in Table 1Go. The cells were labeled with 32P-orthophosphate for 3 h at 37 C and further incubated for 15 min with buffer only, 26 nM hCG, or 200 nM PMA at 37 C as indicated. Lysates were prepared and aliquots containing identical amounts of cell surface receptor were partially purified on a wheat germ agglutinin column and immunoprecipitated as described in Materials and Methods. The immunoprecipitates were resolved on SDS gels, and the radiolabeled bands were detected and captured in a digital format using a PhosphorImager. The results of a representative experiment displaying only the relevant portions of the gels are presented. The intensity of the bands should be compared only within each panel. They should not be compared among the different panels because we did not attempt to maintain constant exposure conditions.

 


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Figure 3. Quantitative Assessment of the Phosphorylation of hLHR Mutants

293 cells plated in 100-mm dishes were transiently transfected with the indicated constructs to give equivalent expression of cell surface receptor as described in Table 1Go. The cells were labeled with 32P-orthophosphate for 3 h at 37 C and further incubated for 15 min with buffer only, 26 nM hCG, or 200 nM PMA at 37 C as indicated. Lysates were prepared and identical amounts of cell surface receptor were analyzed exactly as described in the legend to Fig. 2Go. Each experiment included cells expressing the hLHR-wt that were incubated with buffer only and a maximum of six additional dishes consisting of two sets of transiently transfected cells (each expressing a different receptor construct and incubated with buffer only, hCG, or PMA). The magnitude of the phosphorylation signal measured in the two sets of cells was expressed as fold over the basal phosphorylation of the hLHR-wt included in the same experiment. Each bar represents the mean ± SEM of at least four independent transfections. a, Statistically different (P < 0.05) from wt basal. b, Statistically different (P < 0.05) from its own basal.

 
Cells expressing two of the three activating mutations (L457R and D578H) displayed an approximately 2-fold increase in agonist-independent receptor phosphorylation but the agonist-independent phosphorylation of the third activating mutation (D578Y) was not increased (compare the white bars for the activating mutations with the white bar for the hLHR-wt in Fig. 3Go). The addition of hCG to cells expressing any of these activating mutations resulted in little or no additional hLHR phosphorylation over that detected in the basal state (compare the shaded and white bars shown for each mutant in Fig. 3Go). The impairment in the ability of hCG to enhance the phosphorylation of the L457R, -D578Y, and –D578H mutants parallels the impairment in the hCG-induced cAMP response detected in cells expressing these mutants (c.f. Table 1Go). Lastly, the addition of PMA to cells expressing the L457R and D578Y mutants led to an increase in receptor phosphorylation over that seen in the basal state (compare the black and white bars shown for each mutant in Fig. 3Go), but cells expressing D578H failed to respond to PMA with an additional increase in phosphorylation above that detected in the basal state. The lack of correlation between constitutive activation and increased basal phosphorylation of the L457R, D578Y, and D578H mutants of the hLHR (Figs. 2Go and 3Go) agrees with recent findings reported for other GPCRs. For example, constitutively active mutants of the {alpha}1b- adrenergic (24) or the angiotensin II type 1 receptor (25) do not uniformly display an increase in agonist-independent phosphorylation. In addition, agonist binding fails to enhance the phosphorylation of some constitutively active mutants of these two GPCRs (24, 25).

As shown above, the magnitude of the effect of hCG on receptor phosphorylation is not particularly strong, and phosphorylation assays done in transiently transfected cells require robust expression of the different receptor constructs. Unfortunately, the relatively low levels of expression of the signaling-impaired I625K mutant (c.f. Table 1Go) precluded the use of this mutant in phosphorylation experiments. Therefore, the potential effects of signaling-impaired mutations on receptor phosphorylation could only be analyzed with the two laboratory-designed mutations that impair signaling (D405N and Y546F). Cells expressing these mutants displayed normal levels of agonist-independent receptor phosphorylation and an impairment in hCG-induced phosphorylation (compare the shaded and white bars shown for each mutant in Fig. 3Go) that paralleled the impairment in their ability to mediate a cAMP response (Table 1Go). Lastly, PMA was still able to enhance the phosphorylation of cells expressing any of the inactivating mutations (compare the black and white bars for each mutant in Fig. 3Go). Overall then, the properties of the basal and hCG-dependent phosphorylation of these mutations seem to parallel their activation properties (as measured by cAMP accumulation and shown in Table 1Go).

Agonist-Induced Internalization of hLHR Mutants
Since the methods used to measure the t1/2 of internalization are rather sensitive (see Materials and Methods) and this parameter is not dependent on the density of cell surface receptors (26), the t1/2 values of internalization of hCG could be reliably measured in cells expressing any of the seven mutants described above.

The data summarized in Table 2Go show that cells expressing the three activating mutations (L457R, D578Y, and D578H) internalize hCG with half-times that are 3- to 7-fold shorter than that measured in cells expressing hLHR-wt. Cells expressing the three signaling-impaired mutations (D405N, Y546F, and I625K) internalize hCG with half-times that are 5- to 7-fold longer than those measured in cells expressing hLHR-wt. Mutation of the phosphorylation sites of the hLHR (5S/A) lengthened the t1/2 of internalization of hCG less than 2-fold.


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Table 2. Effect of Several Constructs on the Internalization of 125I-hCG Mediated by hLHR Mutants

 
Dynamin-K44A is a GTPase-deficient mutant of dynamin that behaves as a dominant-negative mutant and inhibits the internalization of many GPCRs, including the rLHR and hLHR (3, 5, 6, 27, 28, 29, 30). Overexpression of this construct lengthened the t1/2 of internalization of hCG mediated by the hLHR-wt and all mutants (Table 2Go). Arrestin-3(284–409), is a construct that encodes for the clathrin-binding domain of arrestin-3 and behaves as a dominant-negative inhibitor of nonvisual arrestin-mediated endocytosis (31). Overexpression of this construct also lengthened the t1/2 of internalization of hCG mediated by hLHR-wt, -5S/A, and all three activating mutants but had only a small effect on the already long t1/2 of internalization of hCG mediated by the signaling-impaired mutations (Table 2Go). It should also be noted that, in spite of the inhibitory effects of arrestin-2(284–309) on the t1/2 of internalization of hCG mediated by any of the activating mutations of the hLHR, the t1/2 of internalization of hCG measured in cells cotransfected with the activating mutants of the hLHR and arrestin-3(284–409) was similar (i.e. 11–31 min) to that detected in cells cotransfected with hLHR-wt and an empty vector (i.e. ~20 min). Conversely, arrestin-3 shortened the t1/2 of internalization of hCG mediated by hLHR-wt, -5S/A, and all three signaling-impaired mutations but had little or no effect on the already short t1/2 of internalization of hCG mediated by the activating mutations. The effects of arrestin-3 on the inactivating mutations were rather pronounced, and the t1/2 of internalization of hCG in cells co-transfected with the D405N, Y546F, or I625K mutants and arrestin-3 (i.e. 8–18 min) was similar to that detected in cells cotransfected with hLHR-wt and an empty vector (~20 min).

Since the D405N, Y546F, and I625K mutations impair receptor activation2 (Table 1Go), phosphorylation (Figs. 2Go and 3Go), and agonist internalization (Table 2Go), we cannot determine whether the impairment in internalization is due to the impairment in receptor activation or in the phosphorylation of the receptor. This is an important issue because the involvement of hLHR phosphorylation on internalization appears to be minimal as documented by the finding that the internalization of hCG is barely affected by mutation of five serine residues in the C-terminal tail of the hLHR (i.e. the 5S/A mutant in Table 2Go) that drastically impair phosphorylation (Figs. 2Go and 3Go) without impairing receptor activation (Table 1Go). An independent assessment of the relative importance of activation and phosphorylation in internalization was conducted by analyzing double mutants in which the phosphorylation sites of the D405N, Y546F, and I625K mutants were simultaneously mutated to alanine residues. Since the phosphorylation of signaling-impaired mutants can often be rescued by overexpression of one of the G protein-coupled receptor kinases (GRKs) (22, 33), we also analyzed the behavior of these mutants in cells cotransfected with GRK2.

The data presented in Fig. 4Go show that cotransfection with GRK2 enhances the hCG-promoted phosphorylation3 of the hLHR-wt, and it rescues the impairment in hCG-promoted phosphorylation displayed by the D405N and Y546F mutants. Figure 4Go also shows that mutation of the phosphorylation sites of hLHR-wt, hLHR-D405N, and hLHR-Y546F4 largely prevents phosphorylation even in cells cotransfected with GRK2. The results summarized in Fig. 5Go show that 1) mutation of the phosphorylation sites of hLHR-D405N, -Y546F, and -I625K5 had no measurable effect on the already slow t1/2 of internalization of hCG mediated by these mutants (compare white bars in the left and right panels of Fig. 5Go); 2) overexpression of GRK2 shortens the t1/2 of internalization of hCG mediated by D405N, Y546F, and I625K (compare white and black bars on the left panel of Fig. 5Go) to levels that are similar to those detected in cells expressing hLHR-wt; and 3) mutation of the phosphorylation sites of hLHR-D405N, -Y546F, and -I625K prevents the ability of GRK2 to rescue the slow rate of internalization of hCG mediated by these mutants (compare the white and black bars on the right panel of Fig. 5Go).



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Figure 4. Effects of GRK2 Cotransfection on the hCG-Induced Phosphorylation of hLHR-wt and Mutants Thereof

293 cells plated in 100-mm dishes were transiently transfected with the indicated constructs to give equivalent expression of cell surface receptor as described in Table 1Go. Wt-S/A, D405N-S/A, and Y546F-S/A denote constructs encoding for the wt, D405N, and Y546F receptors where Ser657, Ser661, Ser670, Ser674, and Ser676 were simultaneously mutated to alanine residues. Lanes labeled +GRK2 and -GRK2 are from cells that were cotransfected with the indicated receptor construct and 2 µg of GRK2 or pcDNA3.1, respectively. The amount of GRK2 transfected was optimized based on its effects on the hCG-induced phosphorylation of the hLHR-wt. The expression of the protein encoded by these constructs has been previously documented by Western blotting (34 39 ). The transiently transfected cells were labeled with 32P-orthophosphate for 3 h at 37 C and further incubated for 15 min with buffer only or 26 nM hCG at 37 C as indicated. Lysates were prepared, and identical amounts of cell surface receptor were immunoprecipitated without lectin purification as described in Materials and Methods. The immunoprecipitates were resolved on SDS gels and the radiolabeled bands were detected and captured in a digital format using a PhosphorImager. The results of a representative experiment displaying only the relevant portions of the gels are presented. The intensity of the bands should be compared only within each panel. They should not be compared among the different panels because we did not attempt to maintain constant exposure conditions.

 


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Figure 5. Effects of GRK2 Cotransfection on the Rates of Internalization of 125I-hCG Mediated by Inactivating Mutations of the hLHR

293 cells plated in 35-mm wells were transiently transfected with the indicated constructs, and the t1/2 of internalization of 125I-hCG was measured as described in Materials and Methods. The amount of hLHR constructs used (0.2 µg/well) was chosen to give equivalent expression of cell surface receptor as shown in Table 1Go. Columns labeled GRK2 and pcDNA3.1 are from cells that were cotransfected with the indicated receptor constructs and 0.2 µg of GRK2 or pcDNA3.1, respectively (also see legend to Fig. 4Go). The receptor constructs used in the right panel are the same as those used in the left panel except that those used in the right panel also had Ser657, Ser661, Ser670, Ser674, and Ser676 simultaneously mutated to alanine residues. Each bar represents the mean ± SEM of at least three independent transfections. The absence of an error bar indicates that the SEM is too small to be shown. a, Statistically different (P < 0.05) from cells cotransfected with pcDNA3.1.

 
To complement these data, we also rendered the activating mutations (i.e. L457R, D578Y, and D578H) phosphorylation deficient by mutation of their phosphorylation sites and examined the internalization of hCG mediated by these double mutants. Figure 6Go shows that mutation of the phosphorylation sites of hLHR-L435R, -D578Y, and -D578H lengthens the t1/2 of internalization of hCG approximately 3-fold, bringing it to values that are comparable to the t1/2 of internalization mediated by hLHR-wt.



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Figure 6. Rates of Internalization of 125I-hCG Mediated by Activating Mutations of the hLHR

293 cells were transiently transfected with the indicated constructs, and the t1/2 of internalization of 125I-hCG was measured as described in Materials and Methods. The amounts of hLHR constructs used (0.2 µg/well) were chosen to give equivalent expression of cell surface receptor as shown in Table 1Go. The receptor constructs denoted by the black bars are the same as those denoted by the white bars, except that those denoted by the black bars also had Ser657, Ser661, Ser670, Ser674, and Ser676 simultaneously mutated to alanine residues. Each bar represents the mean ± SEM of at least three independent transfections. a, Statistically different (P < 0.05) from mutant with intact phosphorylation sites.

 
Agonist-Independent Turnover of hLHR Mutants
The last series of experiments were designed to ascertain the effect of the mutations described above on the turnover of the cell surface hLHR in cells that had not been exposed to agonist. This goal was accomplished by following the fate of the hLHR after bio-tinylation of the cell surface proteins (35, 36). As shown in Fig. 7AGo, streptavidin blots of 9E10 immunoprecipitates of lysates obtained from biotinylated cells result in the visualization of a prominent (~85 kDa) band that is present in 293 cells transfected with hLHR-wt but absent from untransfected cells or from cells transfected with the empty expression vector. This band represents the mature, cell surface LHR as shown previously (37, 38).



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Figure 7. Biotinylation of the Cell Surface hLHR and Mutants Thereof

Panel A, 293 cells were transiently transfected with hLHR-wt or pcDNA3.1 or left untransfected as indicated. The cell surface proteins were covalently modified with biotin, and lysates were prepared, immunoprecipitated with the 9E10 antibody, and resolved on SDS gels. After electrophoretic blotting the biotinylated proteins were visualized using horseradish peroxidase-labeled streptavidin and the enhanced chemiluminescence (ECL) system as described in Materials and Methods. The results of a representative experiment are shown. Panel B, 293 cells were transiently transfected with the indicated constructs. The cell surface proteins were biotinylated, and the biotinylated proteins immunoprecipitated by the 9E10 antibody were detected as described above immediately after biotinylation (t = 0) or after a 4-h incubation of biotinylated cells at 37 C. The results of a representative experiment displaying only the relevant portion of the blot are shown.

 
Cells transfected with each of the mutants described above were then biotinylated and processed immediately after biotinylation or at the end of a 4-h incubation at 37 C in the absence of agonist. Cell lysates were prepared and the levels of biotinylated cell surface LHR were quantitated using streptavidin overlays of 9E10 immunoprecipitates as described in Materials and Methods. The results of a representative experiment showing only the appropriate areas of the blots are shown in Fig. 7BGo and the quantitation of several experiments is shown in Fig. 8Go. During the 4-h incubation period used here, there is little or no change in the complement of surface LHR in cells expressing the hLHR-wt or the 5S/A mutant. The density of surface receptors in cells expressing the three activating mutations decreased by 25–75% during the same time period, whereas the density of surface receptors in cells expressing the signaling-impaired mutations decreased by 10–40%. Thus, the faster turnover of the L435R, D578Y, and D578H mutants is not a property uniquely associated with constitutive activation.



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Figure 8. Agonist-Independent Turnover of the Cell Surface hLHR and Mutants Thereof

293 cells were transiently transfected with the indicated constructs to give equivalent expression of cell surface receptor as shown in Table 1Go. The cell surface proteins were biotinylated, the cells were lysed, and the lysates were immunoprecipitated with the 9E10 antibody. The immunoprecipitates were resolved on SDS gels, and the biotinylated proteins were detected as described in the legend to Fig. 6Go (also see Materials and Methods). Immunoprecipitates were prepared and analyzed immediately after biotinylation (t = 0) or after a 4-h incubation of biotinylated cells at 37 C. The ECL signal from the blots was visualized and quantitated using a Fluo-S MAX system (Bio-Rad Laboratories, Inc.). For each construct the signal detected at t = 4 h was expressed as % of the signal detected at t = 0 h. Each bar represents the mean ± SEM of at least three independent transfections. a, Statistically different (P < 0.05) from cells expressing hLHR-wt.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our laboratory has previously conducted extensive experiments on the internalization of hCG mediated by the rLHR and mutants thereof, and such studies reveal many interesting similarities with those reported here with the hLHR. The first similarity is that the receptor residues that are phosphorylated in response to agonist stimulation appear to be fairly conserved between the hLHR and the rLHR. Thus, the mutation of equivalent (see footnote 1) serine residues in the C-terminal tail of these two receptors (Ser635, Ser639, Ser649, and Ser652 in the rLHR and Ser657, Ser661, Ser670, Ser674, and Ser676 in the hLHR) largely prevents agonist- induced phosphorylation ( Figs. 2–4GoGoGo and Ref. 2). Second, the mutation of these phosphorylation sites lengthens the t1/2 of internalization of hCG mediated by the hLHR or the rLHR 1.5- to 2.5-fold (Table 2Go and Refs. 2, 3). It should be noted that although the magnitude of this change is roughly the same, the absolute value of the t1/2 of internalization of the phosphorylation-deficient mutant of the rLHR is approximately 10 times longer than that of the equivalent mutant of the hLHR simply because the t1/2 of internalization of the rLHR-wt is also several fold longer than that of the hLHR-wt (6). Third, equivalent mutations of the hLHR (D405N in TM3 and Y546F in TM6) and the rLHR (D383N in TM3 and Y524F in TM6) that impair agonist-induced activation of the receptor lengthen the t1/2 of internalization of hCG (Tables 1Go and 2Go and Refs. 7, 8), whereas equivalent mutations of the hLHR (L435R in TM3 and D578Y in TM6) and the rLHR (L435R in TM3 and D556Y in TM6) that induce agonist-independent receptor activation shorten the t1/2 of internalization of hCG (Tables 1Go and 2Go and Refs. 8, 9). Fourth, overexpression of a dominant-negative mutants of dynamin or a dominant-negative mutant of the nonvisual arrestins lengthen the t1/2 of internalization of hCG mediated by the hLHR and the rLHR (Table 2Go and Refs. 3, 5, 6), whereas overexpression of the nonvisual arrestins shortens the t1/2 of internalization of hCG mediated by the hLHR and the rLHR (Table 2Go and Refs. 3, 5, 6, 26, 39). Our studies also highlight two major differences between the hLHR and the rLHR. First, the t1/2 of internalization of hCG mediated by the rLHR-wt is much slower (~120 min) than that mediated by the hLHR-wt (~20 min). We have already shown that this difference can be fully accounted for by seven noncontiguous residues that are not phosphorylated and are present in topologically distinct domains (the second and third intracellular loops and the juxtamembrane region of the C-terminal tail) of the receptor (6). The second difference is on the effects of GRK2 on internalization. Overexpression of this kinase shortens the t1/2 of internalization of hCG mediated by the rLHR-wt approximately 2-fold (39), but it has only a minimal effect on the t1/2 of internalization of hCG mediated by the hLHR-wt (Fig. 5Go, left panel).

When considered together these results clearly show that the rLHR and the hLHR internalize hCG by a pathway that is facilitated by agonist-induced activation and phosphorylation of the receptor and requires the participation of the nonvisual arrestins and dynamin. More importantly, our ability to analyze activation, internalization, and phosphorylation of the hLHR in transiently transfected cells (as shown in this paper) has now allowed us to conduct more extensive structure-function studies that provided novel mechanistic information about the relative importance of receptor activation and phosphorylation to the process of internalization. A summary of the most relevant data regarding this issue is presented in Table 3Go to facilitate the following discussion. The overall importance of receptor activation and phosphorylation can be readily documented by the finding that three distant mutations (i.e. D405N in TM2, Y546F in TM5, and I625K in TM7) of the hLHR that impair hCG-induced receptor activation and hCG-induced phosphorylation lengthen the t1/2 of agonist internalization to values (100–150 min) that are close to the t1/2 of internalization of hCG mediated by the hLHR-wt in cells in which endocytosis has been blocked with a dominant- negative mutant of dynamin (~160 min). The results summarized in Table 3Go also show that 1) phosphorylation is relatively unimportant to internalization if activation is normal because the 5S/A mutant internalizes hCG with a t1/2 (~30 min) that is only slightly longer than that of hLHR-wt (~20 min); and 2) activation is relatively unimportant to internalization if phosphorylation is normal because the long t1/2 of internalization of hCG displayed by the signaling-impaired mutants can be shortened to 30–50 min when the phosphorylation of these mutants is rescued by cotransfection with GRK2. When considered together, these results are consistent with a model whereby hCG-induced activation and phosphorylation of the hLHR play redundant roles in internalization. Ultimately, however, it is obvious that in the absence of phosphorylation or receptor activation, the long t1/2 of internalization of hCG can be readily rescued by overexpression of arrestin-3 (Table 2Go). Thus, the data presented here also document the paramount importance of the interaction of the hLHR with a nonvisual arrestin to the process of internalization.


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Table 3. Summary of the Relative Importance of Receptor Activation and Phosphorylation on the Agonist-Induced Internalization of the hLHR

 
The large number of studies conducted with the ß2-adrenergic receptor (ß2-AR) have resulted in a model that highlights the importance of the ß2-AR phosphorylation to the process of internalization. In this model the main role of the agonist-induced activation of the ß2-AR is to promote the GRK-catalyzed phosphorylation of the receptor (reviewed in Refs. 28, 29, 30). This phosphorylation of the ß2-AR in turn enhances the affinity of the receptor for a nonvisual arrestin leading to the formation of a binary complex between the phosphorylated ß2-AR and a nonvisual arrestin. Since the nonvisual arrestins bind clathrin with high affinity, this complex targets the receptor to clathrin-coated pits for internalization (28, 29, 30). The results presented here indicate that the model derived from the study of the ß2-AR is generally applicable to the agonist-induced internalization of the hLHR. A salient difference, however, is that, instead of highlighting the importance of receptor phosphorylation, our data suggest that the agonist-induced activation and phosphorylation of the hLHR play redundant roles in internalization. In addition, recent studies performed with other GPCRs suggest that generalizations about the relative roles of GPCR activation and phosphorylation to the process of internalization cannot be readily made. For example, phosphorylation of the PTH (40) and the FSH receptors (22) seem to be less important than activation in the process of internalization, whereas the phosphorylation of the µ-opioid receptor seems to be more important than activation in the process of internalization (41).

The reasons behind the ability of activating mutations of the hLHR (L457R, D578Y, and D578H) to internalize hCG faster than hLHR-wt (Table 2Go) remain elusive. As shown here these mutants cannot be further activated or phosphorylated by hCG (Table 1Go and Figs. 1Go and 2Go). The involvement of receptor phosphorylation in the internalization of hCG can still be documented by mutation of the phosphorylation sites of these activating mutants, however (Fig. 6Go). Thus, when occupied by hCG, these three activating mutations seem to assume a conformation that bypasses many of the requirements (i.e. activation, phosphorylation, and/or arrestin binding) needed for the internalization of agonist.

In summary, the studies presented herein and elsewhere (2, 3, 7, 8, 9) show a remarkable association between the activation and the internalization of the LHR, and the new studies presented here imply that the activation and phosphorylation of the hLHR play redundant roles in the process of internalization. The intimate association between receptor activation and internalization suggests that internalization is part of the process of activation or, conversely, that it represents a short feedback loop involved in the termination of hormone action. Either scenario argues for continued research to fully understand the molecular and cellular basis of this pathway.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Cells
A full-length cDNA encoding for the hLHR (42) was generously provided by Ares Serono. Conventional PCR strategies were used to introduce a small oligonucleotide (5'-GAACAAAAGCTTATTTCTGAAGAAGACTTG-3') encoding the myc epitope (EQKLISEEDL, see Ref. 43) between the predicted C terminus of the signal peptide (Ala24) and the predicted N terminus of the mature receptor (Leu25). This modified receptor was subcloned into pcDNA 3.1 for expression and used as a template for all the mutations used here. All mutants were also constructed using standard PCR strategies, and their identity was verified by automated DNA sequencing (performed by the DNA core of The Diabetes and Endocrinology Research Center of the University of Iowa). Preliminary experiments comparing the behavior of the hLHR-wt with and without the myc epitope showed that the addition of this epitope has little or no effect on receptor expression, hormone binding, agonist-induced activation (as measured by cAMP accumulation), or agonist-induced internalization of the hLHR. In addition, myc tagging of the closely related rLHR in the equivalent location was previously shown to have little or no effect on these properties of the rLHR (8, 44).

Vectors encoding for GRK2 (45) arrestin-3 and arrestin-3(284–409) (31) were generously provided by Dr. Jeff Benovic (Thomas Jefferson University, Philadelphia, PA). An expression vector for dynamin-K44A (27) was generously provided by Dr. Sandra Schmid (Scripps Research Institute, La Jolla, CA). All of these were subcloned into pcDNA3.1. The expression of these constructs in our experimental system has been previously documented (34, 39).

Human embryonic kidney (293) cells were maintained in DMEM containing 10 mM HEPES, 10% newborn calf serum, and 50 µg/ml gentamicin, pH 7.4. Transient transfections were done using the calcium phosphate method of Chen and Okayama (46). Cells were plated in 100-mm dishes that had been coated with gelatin and transfected (using 5 µg of plasmid for the 100-mm dishes or 0.2 µg of plasmid for the 35-mm wells) when 70–80% confluent. After an overnight incubation, the cells were washed, trypsinized, and, depending on the assay to be performed, plated in gelatin-coated dishes or wells and incubated for an additional 24 h before use.

Binding, Internalization, and cAMP Assays
The expression of the different receptor constructs was ascertained by measuring the binding of a saturating (13 nM) concentration of 125I-hCG (the Kd for hCG binding to the hLHR is 1–3 nM; see Ref. 6) to intact cells. All binding assays were done during a 1-h incubation at room temperature using transfected cells plated in gelatin-coated 35-mm wells (see above). They were all corrected for nonspecific binding, which was measured in the presence of 50 IU/ml of partially purified hCG (3,000 IU/mg). The methods used to measure the internalization of 125I-hCG have been described previously (5, 26). Determinations of the rates of internalization were done using at least five different data points collected at 3- to 10-min intervals (depending on the construct transfected) after the addition of a concentration of 125I-hCG (3 nM) equivalent to the Kd. The endocytotic rate constant (ke) was calculated from the slope of the line obtained by plotting the internalized radioactivity against the integral of the surface-bound radioactivity (6, 26, 47). The half-time of internalization is defined as 0.693/ke.

Hormonal responsiveness was assessed by measuring cAMP accumulation in intact transfected cells plated in gelatin-coated 35-mm wells (see above). Total cAMP was measured at the end of a 2-h incubation (37 C) in medium devoid of phosphodiesterase inhibitors6 and supplemented with buffer only or with concentrations of hCG (3 nM) or cholera toxin (0.6 nM) that are known to be maximally effective in transiently transfected cells expressing hLHR-wt (14, 48). Dose-response curves were generated by incubating transiently transfected cells with increasing concentrations of hCG as shown in Fig. 1Go. The parameters that describe these dose responses (i.e. EC50 and maximal response) were calculated from these data as described elsewhere (7, 12, 13).

Phosphorylation Assays
Cells were plated in 100-mm dishes that had been coated with gelatin and transfected when 70–80% confluent. After an overnight incubation, the cells were washed, trypsinized, and plated in gelatin-coated 100-mm dishes and in gelatin-coated 35-mm wells and incubated in DMEM containing 10 mM HEPES, 1% BSA, and 50 µg/ml gentamicin, pH 7.4, for 24 h.

The cells plated in 35-mm wells were used to assess cell surface receptor expression by 125I-hCG as described above. The cells plated in the 100-mm dishes were used for phosphorylation assays as follows. The medium was aspirated and the cells were incubated for 3 h at 37 C in DMEM devoid of phosphate but containing 10 mM HEPES, 1% BSA, 50 µg/ml gentamicin, and 200 µCi/ml 32P-orthophosphate. At this point the cells received buffer only, hCG (final concentration, 26 nM), or PMA (final concentration, 200 nM) and the incubation was continued for 15 min at 37 C. These concentrations and times were chosen based on the maximal effects of hCG and PMA on the phosphorylation of the rLHR (1, 2, 3, 13). Cells were placed on ice and washed once with buffer A (0.15 M NaCl, 20 mM HEPES, 5 mM EDTA, 3 mM EGTA, 50 mM ß-glycerophosphate, 10 mM NaF, 100 µM sodium orthovanadate, 1 mM phenylmethyl sulfonyl fluoride, 1 µM leupeptin, 0.08 µg/ml okadaic acid, 1 nM cypermethrin, and 1 µM pepstatin A, pH 7.4). After addition of 1 ml of lysis buffer (1% NP40, 4 mg/ml dodecyl-ß-D-maltoside. 0.8 mg/ml cholesteryl hemisuccinate in buffer A) the dishes were rocked on ice for 30 min. The lysate was clarified by centrifugation, and aliquots containing the same amount of receptor (calculated from the binding experiments done in parallel as described above) were partially purified on a wheat germ agglutinin column as described before (37). The eluant from the wheat germ agglutinin column was incubated with a monoclonal antibody to the myc epitope (9E10) that had been preabsorbed to agarose-conjugated protein G (see below) for 90–120 min at 4 C. After extensive washing the material absorbed to the beads was eluted by vigorous mixing of the beads in SDS sample buffer for 15 min at room temperature. The eluted material was then resolved on SDS gels, the gels were visualized and quantitated using a PhosphorImager, and the images were captured in a digital format for presentation.

In more recent experiments we have been able to omit the wheat germ agglutinin column and use crude lysates for immunoprecipitation. This protocol is less laborious and more economical but it often reveals faint bands of a mol wt similar to that of the hLHR in the negative controls (c.f. Fig. 4Go). These are nonspecific bands, however, that can also be detected in untransfected cells or in cells transfected with pcDNA3.1.

Prebinding of the 9E10 antibody to the protein G agarose beads was accomplished by incubating 50 µl of a 20–25x dilution of concentrated antibody (a concentrated supernatant from cultured 9E10 cells) with 25 µl of a 50% slurry of protein G agarose (purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C. The beads were then washed by centrifugation and used as described above.

Analysis of the Turnover of the Free hLHR by Surface Biotinylation
Transfected cells plated in gelatin-coated 35-mm wells (see above) were washed four times with ice-cold PBS (10 mM sodium phosphate, 150 mM NaCl, pH 8) and then biotinylated during two consecutive 15 min incubations (at room temperature) with freshly prepared 0.5 mg/ml solutions of sulfosuccinimidyl-6-(biotinamido)hexanoate (from Vector Laboratories, Inc., Burlingame, CA) in the same buffer. The cells were then washed once with DMEM containing 10 mM HEPES, 10% newborn calf serum, and 50 µg/ml gentamicin, pH 7.4, and twice with PBS (35, 36). Some cells were saved on ice and processed immediately (t = 0 samples), while others were incubated in warm DMEM containing 10 mM HEPES, 10% newborn calf serum, and 50 µg/ml gentamicin, pH 7.4, for 4 h. At the indicated times the cells were placed on ice, lysed, and immunoprecipitated with the 9E10 antibody prebound to protein G agarose. Lysates and immunoprecipitations were prepared as described in Phosphorylation Assays, except that okadaic acid and cypermethrin were not included in the solutions and the lysates were not purified on wheat germ agglutinin before immunoprecipitation. Also, no attempt was made to normalize the amount of receptor immunoprecipitated among the different constructs used. The immunoprecipitates were resolved on SDS gels as described above, and the resolved proteins were electrophoretically transferred to polyvinylidene fluoride membranes as described elsewhere (49). After blocking (49), the blots were incubated for 1 h with 100 ng/ml of horse-radish peroxidase-conjugated streptavidin (from Vector Laboratories, Inc.), and the proteins were finally visualized using the Super Signal West Femto Maximum Sensitivity system of detection from Pierce Chemical Co. (Madison, WI). The blots were visualized and quantitated using a Fluo-S MAX system (Bio-Rad Laboratories, Inc., Hercules, CA). These images were also captured in a digital format for presentation (c.f. Fig. 7Go).

Hormones and Supplies
Human embryonic kidney (293) cells and the 9E10 hybridoma cell line were obtained from the American Type Culture Collection (Manassas, VA). Purified hCG (CR-127, ~13,000 IU/mg) was kindly provided by Dr. A. Parlow and the National Hormone and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases. 125I-hCG was prepared as described elsewhere (50). Partially purified hCG (~3,000 IU/mg) was purchased from Sigma (St. Louis, MO), and it was used only for the determination of nonspecific binding (see above). 125I-cAMP and cell culture medium were obtained from the Iodination Core and the Media and Cell Production Core, respectively, of the Diabetes and Endocrinology Research Center of the University of Iowa. Concentrated supernatant from the 9E10 cells was prepared by the Hybridoma Facility of the Cancer Center of the University of Iowa. Other 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.


    ACKNOWLEDGMENTS
 
We thank Drs. Kazuto Nakamura and Xuebo Liu for suggestions with different technical aspects of this project. We also wish to thank Dr. Jeffrey Benovic (Thomas Jefferson University, Philadelphia, PA) and Dr. Sandra Schmid (Scripps Research Institute, La Jolla, CA) for reagents and Dr. Deborah Segaloff (University of Iowa College of Medicine, Iowa City, IA) for critically reading this manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, The University of Iowa 2–319B BSB, 51 Newton Road, Iowa City, IA 52242-1109. E-mail: mario-ascoli{at}uiowa.edu

This work was supported by a grant from the NIH: CA-40629 to MA. The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa were supported by NIH grant DK-25295.

1 Note that the mature rLHR and hLHR highly homologous proteins are composed of 674 and 677 residues, respectively (23 ). The different numbers assigned to equivalent residues are artificially caused by differences in the numbering of amino acids. Since the N terminus of the mature hLHR is not known, residue number 1 is taken to be the methionine present at the N terminus of the signal peptide. In contrast, since the N terminus of the mature rLHR is known, this residue (which corresponds to residue 23 of the hLHR) is taken to be residue number 1. Back

2 Note that although we use cAMP as a measurement of receptor activation we do not imply that cAMP per se is necessary for internalization. In fact, a role for cAMP in this process has already been excluded (32 ). Back

3 In agreement with previous results (22 33 34 ), we found that GRK2 cotransfections did not enhance receptor phosphorylation in cells incubated without hCG. Back

4 Mutation of the phosphorylation sites of the activating or inactivating mutations of the hLHR had little or no effect on their signaling properties (not shown). Back

5 As noted above, the low level of expression of the I625K mutant made it difficult to reliably measure phosphorylation. Throughout the rest of this paper we assume that the properties of the phosphorylation of the I625K mutant are similar to those of the D405N and Y546F mutants. Back

6 Experiments performed in medium with isobutylmethylxanthine (a phosphodiesterase inhibitor) gave qualitatively similar results. Back

Received for publication June 7, 2000. Revision received July 25, 2000. Accepted for publication August 11, 2000.


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 DISCUSSION
 MATERIALS AND METHODS
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