Role of G Protein-Coupled Receptor Kinases on the Agonist-Induced Phosphorylation and Internalization of the Follitropin Receptor

Maria de Fatima M. Lazari1, Xuebo Liu, Kazuto Nakamura, Jeffrey L. Benovic and Mario Ascoli

Department of Pharmacology (M. de F.M.L., X.L., K.N., M.A.) The University of Iowa College of Medicine Iowa City, Iowa 52242
Department of Microbiology and Immunology (J.L.B.) Kimmel Cancer Institute Thomas Jefferson University Philadelphia, Pennsylvania 19107


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4{alpha}, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or G{alpha}t, a scavenger of Gß{gamma}. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and G{alpha}t inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Of the consequences of agonist-induced activation of G protein-coupled receptors (GPCRs) is the phosphorylation of the receptor by a family of kinases called G protein-coupled receptor kinases (also known as GRKS, reviewed in Refs. 1, 2, 3). GPCR phosphorylation increases the affinity of these receptors for a family of proteins called arrestins, and the binary complex formed by the phosphorylated GPCR and a nonvisual arrrestin serves as a common molecular intermediate in at least two processes, uncoupling and coated pit-mediated internalization, that are involved in the desensitization/resensitization of cellular responses (1, 2, 3).

While this paradigm has been derived mostly from the study of the ß2-adrenergic receptor, recent studies have now shown that it is not fully applicable to all GPCRs. For example, the coated pit-mediated internalization of the µ-opioid receptor is stimulated by some, but not all, agonists (4, 5). The removal of the phosphorylation sites of the {delta}-opioid receptor (6) or the mutation of the phosphorylation sites of the PTH receptor (7) does not affect the agonist-induced internalization of these receptors. Moreover, in the absence of receptor phosphorylation, the overexpression of nonvisual arrestins can rescue the agonist-induced internalization of the ß2-adrenergic receptor (8) and the rat FSH receptor (rFSHR) (9), but it cannot enhance the morphine-induced internalization of the µ-opioid receptor (5).

Recent studies from this laboratory have shown that the rFSHR becomes phosphorylated upon agonist activation (10, 11, 12). While the GRK-catalyzed phosphorylation of the majority of GPCRs occurs in the C-terminal tail, the rFSHR belongs to a small subfamily of GPCRs that are exclusively phosphorylated in the intracellular loops. Like the {alpha}2A-adrenergic (13) and the m2 muscarinic (14, 15) receptors, the agonist-induced phosphorylation of the rFSHR occurs in the third intracellular loop (11, 12). The rFSHR is unusual, however, in that agonist activation also promotes the phosphorylation of sites present in the first intracellular loop (12).

The possible involvement of rFSHR phosphorylation in agonist-induced uncoupling and internalization has been studied using a number of phosphorylation-impaired rFSHR mutants. Our studies have shown that the mutation of S/T residues present in the third intracellular loop of the rFSHR impairs agonist-induced phosphorylation and uncoupling without affecting agonist-induced internalization, whereas the mutation of S/T residues present in the first intracellular loop of the rFSHR impairs agonist-induced phosphorylation, uncoupling, and internalization (12). Two signaling-impairing mutations of the rFSHR that preserve the phosphorylation sites have also been shown to completely prevent agonist-induced phosphorylation and to impair internalization (9). Overexpression of arrestin-3, however, rescues the internalization of both signaling-impaired mutants, even in the absence of receptor phosphorylation (9). Thus, it appears that the agonist-induced phosphorylation of the rFSHR is important, but not essential, for agonist-induced internalization.

In spite of our increased understanding of the location and function of the residues that are phosphorylated in the rFSHR, there is a paucity of information about the identity of the kinases that mediate the agonist-induced phosphorylation. The possible involvement of the cAMP-dependent protein kinases has been excluded by the following two findings: 1) the hFSH-induced phosphorylation of the rFSHR proceeds normally in a transfected cell line that does not respond with an increase in cAMP accumulation (because of overexpression of cAMP phosphodiesterase); and 2) the addition of cAMP analogs or the stimulation of endogenous cAMP synthesis with PGE2 does not result in the phosphorylation of the rFSHR expressed in transfected cells (10). In contrast, protein kinase C seems to be partially responsible for the agonist-induced phosphorylation. This conclusion is supported by the following findings. First, the binding of hFSH to the rFSHR expressed in transfected cells stimulates not only the cAMP pathway, but also the inositol phosphate/diacylglycerol-signaling pathway (10, 11). Second, the pharmacological activation of C kinase with phorbol 12-myristate-13-acetate (PMA) enhances the phosphorylation of the rFSHR (10, 11, 12). Third, the down-regulation of C kinase by chronic treatment of transfected cells with PMA inhibits the hFSH-induced phosphorylation by approximately 30% (10). Thus, while the agonist-induced phosphorylation of the rFSHR may indeed be partially mediated by protein kinase C, one needs to invoke the participation of at least one other kinase. This suggestion is also supported by the finding that two inactivating mutations of the rFSHR that prevent agonist-induced phosphorylation do not affect PMA-induced phosphorylation (9).

With these findings in mind, the experiments presented here were designed to identify the GRKs that are involved in the agonist-induced phosphorylation of the rFSHR and to assess their involvement in agonist-induced internalization.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of the GRKs Present in 293 and MSC-1 Cells
As an initial approach to understanding the impact of GRKs on the phosphorylation and functions of the rFSHR, we examined the expression of the different members of the GRK family in 293 and MSC-1 cells. We chose 293 cells because all previous phosphorylation experiments with the rFSHR have been done using this cell type (10, 11, 12) and because these cells are a good host for cotransfection strategies designed to examine the impact of the different GRKs on the phosphorylation and functions of the rFSHR (see below). MSC-1 cells were also chosen because they represent a clonal cell line derived from mouse Sertoli cells that displays many of the differentiated functions of normal Sertoli cells (16, 17). Unlike normal Sertoli cells, however, the MSC-1 cells do not express the endogenous FSHR (16, 17).

We did not attempt to detect GRK1 because the expression of this kinase, also known as rhodopsin kinase, is restricted to the retina and the pineal (3, 18). The expression of the other members of the GRK family (GRK2-GRK6) was ascertained using Western blots with specific GRK antibodies. The results presented in Fig. 1Go show that we can readily detect GRK2 and GRK6 in 293 and MSC-1 cells. The data presented in Fig. 1Go show that MSC-1 cells express the {gamma}- and/or {delta}-isoforms of GRK4 as well. Although we do not have standards for the {gamma}- and {delta}-isoforms of GRK4, we conclude that one or both of these isoforms are present, as opposed to the {alpha}- and ß-isoforms for the following reasons. First, the antibody used in the Western blots shown in Fig. 1Go (I-20 from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) recognizes an epitope not present in the {alpha}- and ß-isoforms. Second, another antibody that recognizes only the {gamma}- and {delta}-isoforms of GRK4 (K-20 from Santa Cruz Biotechnology, Inc.) failed to give a signal in Western blots of MSC-1 cells (data not shown). Third, the apparent size of the GRK4 isoform detected in Western blots of MSC-1 cells (see Fig. 1Go) is closer to the predicted sizes for the {gamma}- and {delta}-isoforms of GRK4 (57–61 kDa) than to the predicted sizes for the {alpha}- and ß-isoforms (63–67 kDa) (19, 20). Antibodies directed against GRK3 or GRK5 failed to give a signal in Western blots from 293 or MSC-1 cells but readily detected the recombinant proteins used as controls (data not shown).



View larger version (52K):
[in this window]
[in a new window]
 
Figure 1. Western Blots of Different GRKs in Extracts of 293 and MSC-1 Cells

Cell lysates were prepared, and the indicated amounts of lysate protein were resolved on SDS gels and electrophoretically transferred to PVDF membranes as described in Materials and Methods. GRK2 (panel A) was detected using the 3A10 monoclonal antibody, GRK4-{delta}/{gamma} (panel B) and GRK6 (panel C) were detected using the GKR4(I-20) and GRK6(C-20) polyclonal antibodies from Santa Cruz Biotechnology, Inc.. Proteins were ultimately visualized using the ECL system of detection as described in Materials and Methods. The migration and size of mol wt markers are indicated next to each blot.

 
These results are in general agreement with the finding that GRK2 and GRK6 are ubiquitously expressed, whereas GRK4 is expressed mostly in several testicular cell types (3, 18, 19, 20). The expression of GRK2 in 293 cells has been previously documented (21, 22). In contrast to the results presented above, one of these publications also reported the presence of GRK5 in 293 cells (21).

GRK2, GRK4{alpha}, and GRK6 Can Phosphorylate the rFSHR
Since MSC-1 cells express GRK2, GRK4{delta}/{gamma}, and GRK6, we next attempted to determine whether these three kinases can phosphorylate the rFSHR. These experiments were done by measuring basal and agonist-stimulated phosphorylation of the rFSHR in 293 cells cotransfected with the rFSHR and the different GRKS.

The results presented in Fig. 2AGo display representative experiments documenting that transient transfection of 293 cells with optimal amounts of the appropriate plasmids results in increased expression of GRK2, GRK4{alpha}2, or GRK6. Note that Western blots of cells transfected with the GRK2 or GRK4{alpha} expression vectors not only reveal proteins of the appropriate size (i.e. 80 and 67 kDa, respectively), but they also reveal proteins of slightly higher molecular weights. Although the identity of these bands is not known, a similar phenomenon has been described by others upon transfection of GRK2 (23). These investigators suggested that the high molecular weight product detected in GRK2-transfected cells may represent improperly processed GRK2 (23).



View larger version (34K):
[in this window]
[in a new window]
 
Figure 2. Effect of GRK Cotransfection on Agonist-Promoted Phosphorylation of the rFSHR in 293 Cells

293 cells were transiently cotransfected with an expression vector for the rFSHR (10 µg/100-mm dish) plus an empty expression vector GRK2, GRK4{alpha}, or GRK6 (10 µg/100-mm dish) as indicated. A, Cell lysates were prepared, and the indicated amounts of lysate protein were resolved on SDS gels and electrophoretically transferred to PVDF membranes. GRK2 was detected using the 9–7 polyclonal antibody; GRK4{alpha} and GRK6 were detected using the GRK4(K-20) and GRK6(C-20) polyclonal antibodies from Santa Cruz Biotechnology, Inc.. Proteins were ultimately visualized using the ECL system of detection as described in Materials and Methods. Only the relevant portions of the blots of a representative experiment are shown. B, The transiently transfected cells were metabolically labeled with 32Pi for 3 h and then incubated with or without 100 ng/ml hFSH for 60 min. Lysates were then prepared and equal amounts of rFSHR were immunoprecipitated as described in Materials and Methods. Autoradiograms of the dried gels were obtained using Kodak BioMax MS film and intensifying screens (Eastman Kodak Co.), scanned using a Molecular Imaging System (Bio-Rad Laboratories, Inc.) and captured in a digital format for presentation. Only the relevant portions of the autoradiograms of a representative experiment are shown.

 
The results presented in Fig. 2BGo display representative experiments in which the basal and agonist-induced phosphorylation of the rFSHR was assessed in 293 cells cotransfected with each of the three GRKs. The quantitation of several experiments is presented in Fig. 3Go. Since basal rFSHR phosphorylation (i.e. that detected in cells incubated with buffer only) varied by 10%, at most, in any of the cotransfection strategies (see Fig. 2BGo), the quantitation presented in Fig. 3Go and all subsequent figures is expressed as fold over basal.



View larger version (19K):
[in this window]
[in a new window]
 
Figure 3. Quantitation of the Effects of GRK Cotransfection on the Agonist-Induced Phosphorylation of the rFSHR

293 cells were transiently cotransfected with an expression vector for the rFSHR (10 µg/100-mm dish) plus an empty expression vector, GRK2, GRK4{alpha}, or GRK6 (10 µg/100-mm dish) as indicated. The transiently transfected cells were metabolically labeled with 32Pi for 3 h and then incubated with buffer or 100 ng/ml hFSH for 60 min. Lysates were then prepared, and equal amounts of rFSHR were immunoprecipitated as described in Materials and Methods. Autoradiograms of the dried gels were obtained using Kodak BioMax MS film and intensifying screens (Eastman Kodak Co.). After scanning and quantitation by densitometry, the hFSH-induced phosphorylation of the rFSHR in each group of cells was expressed as fold over their respective basal (see Materials and Methods for details). Each bar represents the mean ± SEM of three to six independent transfections. The asterisk indicates significant difference (P < 0.05) from cells transfected with the rFSHR and the empty vector and stimulated with FSH.

 
These results clearly show that, when expressed in 293 cells, the three GRKs present in MSC-1 cells are capable of enhancing the phosphorylation of the agonist-activated rFSHR. The experiments shown in Figs. 2Go and 3Go were done using optimal amounts of GRKs, but the enhancement in phosphorylation was only approximately 2-fold. The magnitude of this effect, however, is similar to that previously reported when the ß2-adrenergic receptor is cotransfected with these kinases in 293 cells. The finding that the different GRKs do not enhance basal rFSHR phosphorylation also highlights a known property of the GRKs, namely that they prefer the agonist-occupied receptor as a substrate (3, 18).

Kinase-Deficient Mutants of GRK2 or GRK6 Inhibit the hFSH-Stimulated Phosphorylation of the rFSHR by Kinases Endogenous to 293 Cells
While the experiments presented above show that GRK2, GRK4, and GRK6 can phosphorylate the agonist-stimulated rFSHR, overexpression studies cannot be used to discern the identity of the endogenous kinases that phosphorylate the agonist-occupied rFSHR (see Discussion). In an attempt to address this question we assessed agonist-induced receptor phosphorylation after transfection of 293 cells with the rFSHR and kinase-deficient mutants of GRK2 or GRK6. We did not attempt to use a kinase-deficient mutant of GRK4 because this kinase is not detectable in 293 cells (cf. Fig. 1Go).

Kinase-deficient mutants of many protein kinases have been prepared by mutation of a lysine residue that is universally conserved in their catalytic domain and is directly involved in the phosphotransfer reaction (25, 26). Such constructs can be readily used as dominant-negative mutants to inhibit endogenous kinases (23, 25, 26).

C20-GRK2-K220M, a previously described isoprenylated version of a kinase-deficient mutant of GRK2, has been shown to inhibit the agonist-induced phosphorylation of the ß2-adrenergic receptor expressed in 293 cells. The K220M mutation of this construct impairs its kinase activity, and the isoprenylation signal targets the kinase-deficient enzyme to the plasma membrane. The constitutive association of the C20-GRK2-K220M mutant with the plasma membrane is believed to enhance the ability of this construct to inhibit endogenous GRK2, which is a cytosolic enzyme that is translocated to the membrane by Gß/{gamma} as part of the agonist-induced activation of GPCRs (3, 18). GRK6-(K215M,K216M) is a new construct harboring a mutation in the catalytic domain3 of GRK6 that is expected to impair its kinase activity (25, 26). Like the wild-type GRK6, however, the GRK6-(K215M,K216M) construct should be constitutively associated with the plasma membrane by a covalently attached palmitate group (3, 18).

The ability of C20-GRK2-K220M and GRK6-(K215M,K216M) to act as specific dominant-negative mutants of GRK2 and GRK6, respectively, was tested in 293 cells cotransfected with the rFSHR, a suboptimal amount of the wild-type kinases (1 µg/100-mm dish), and an excess (9 µg/100-mm dish) of the dominant- negative mutants. As shown in Fig. 4Go, these transfection conditions result in increased expression of both the wild-type and the kinase-deficient mutants. As expected from the amounts of plasmid used, the expression of the transfected GRK6-(K215M,K216M) was higher than that of the transfected GRK6. Surprisingly, however, the expression of the transfected GRK2 was higher than that of the transfected C20-GRK2-K220M, in spite of the fact that we used 9 times more C20-GRK2-K220M than GRK2 in the transfections. This is perhaps due to the nature of the protein encoded by C20-GRK2-K220M, which, in addition to harboring a single-point K-to-M mutation in the catalytic site, has a C-terminal mutation that allows for isoprenylation of the mutant, whereas the wild-type GRK2 is not isoprenylated (see above).



View larger version (37K):
[in this window]
[in a new window]
 
Figure 4. Expression of GRK2, C20-GRK2-K220M, GRK6, and GRK6-(K215M,K216M) in Transfected 293 Cells

293 cells were transiently transfected with an empty vector or with expression vectors for GRK2, C20-GRK2-K220M, GRK6, or GRK6-(K215M,K216M) as indicated. Cell lysates were prepared, and 50 µg of lysate protein were resolved on SDS gels and electrophoretically transferred to PVDF membranes. GRK2 and C20-GRK2-K220M were detected using the GRK2(C-15) polyclonal antibody from Santa Cruz Biotechnology, Inc., and GRK6 and GRK6-(K215M,K216M) were detected using the GRK6(C-20) polyclonal antibody from Santa Cruz Biotechnology, Inc.. Proteins were ultimately visualized using the ECL system of detection as described in Materials and Methods. Only the relevant portions of the blots of a representative experiment are shown.

 
The ability of the kinase-deficient mutants of GRK2 and GRK6 to act as specific dominant-negative mutants of GRK2 and GRK6 was tested in the experiments shown in Fig. 5Go. In these experiments, 293 cells were cotransfected with the rFSHR and GRK2 or GRK6 and with C20-GRK2-K220M or GRK6-(K215M,K216M) to determine whether they could specifically inhibit the enhanced agonist-induced phosphorylation of the rFSHR induced by the expression of GRK2 or GRK6. These experiments were done using the same amounts of GRKs and dominant-negative GRKs used for the experiments summarized in Fig. 4Go. The results presented in Fig. 5Go show that transfection of suboptimal amounts of GRK2 or GRK6 resulted in approximately a 1.5-fold increase in the agonist-induced phosphorylation of the transfected rFSHR (compare the white bars with the dashed lines shown across each panel). Importantly, the data presented in Fig. 5Go also show that C20-GRK2-K220M inhibited the enhanced rFSHR phosphorylation induced by the transfected GRK2 (compare the gray and white bars in the top panel of Fig. 5Go) but did not inhibit the enhanced rFSHR phosphorylation induced by the transfected GRK6 (compare the gray and white bars in the bottom panel of Fig. 5Go). The data presented in the top panel of Fig. 5Go also show that under these conditions C20-GRK2-K220M inhibited the agonist-induced phosphorylation of the rFSHR promoted by GRK2 overexpression to a level below that detected in cells that express only the endogenous kinases (compare the gray bar with the dashed line shown in the top panel of Fig. 5Go). Conversely, GRK6-(K215M,K216M) inhibited the enhanced rFSHR phosphorylation induced by the transfected GRK6 (compare the black and white bars in the bottom panel of Fig. 5Go) but did not inhibit the enhanced rFSHR phosphorylation induced by the transfected GRK2 (compare the black and white bars in the top panel of Fig. 5Go). The data presented in the bottom panel of Fig. 5Go also show that under these conditions GRK6-(K215M,K216M) inhibited the agonist-induced phosphorylation of the rFSHR promoted by GRK6 overexpression to a level similar to that detected in cells that express only the endogenous kinases (compare the gray bar with the dashed line shown in the bottom panel of Fig. 5Go).



View larger version (33K):
[in this window]
[in a new window]
 
Figure 5. Effects of Kinase-Negative Mutants of GRKs on the GRK-Enhanced Phosphorylation of the rFSHR

Top panel, Three 100-mm dishes of 293 cells were transiently transfected with three different transfection mixtures. Two plasmids, an expression vector for the rFSHR (10 µg) and an expression vector for GRK2 (1 µg), were present in all three transfection mixtures. One mixture also contained 9 µg of the empty vector (white bar), a second mixture also contained 9 µg of C20-GRK2-K220M (gray bar), and a third mixture also contained 9 µg of GRK6-(K215M,K216M) (black bar). Bottom panel, Three 100-mm dishes of 293 cells were transiently transfected with three different plasmid mixtures. Two plasmids, an expression vector for the rFSHR (10 µg) and an expression vector for GRK6 (1 µg), were present in all three transfection mixtures. One mixture also contained 9 µg of the empty vector (white bar), a second mixture also contained 9 µg of C20-GRK2-K220M (gray bar), and a third mixture also contained 9 µg of GRK6-(K215M,K216M) (black bar). The transiently transfected cells were metabolically labeled with 32Pi for 3 h and then incubated with buffer or 100 ng/ml hFSH for 60 min. Lysates were then prepared, and equal amounts of rFSHR were immunoprecipitated as described in Materials and Methods. Autoradiograms of the dried gels were obtained using Kodak BioMax MS film and intensifying screens (Eastman Kodak Co.). After scanning and quantitation by densitometry, the hFSH-induced phosphorylation of the rFSHR in each group of cells was expressed as fold over their respective basal. Each bar represents the mean ± SEM of three independent experiments. The dashed line shown in each panel represents the extent of hFSH-induced phosphorylation (also expressed as fold-over basal) detected in 293 cells transiently transfected with the rFSHR expression vector but no additional kinases (i.e. 100-mm dishes transfected with 10 µg of the rFSHR expression vector and 10 g of empty vector). *, Significantly different (P < 0.05) from cells transfected with the rFSHR and GRK2 (top panel) or GRK6 (bottom panel).

 
In performing these experiments we were constrained by the amounts of plasmids that can be used in each transfection without affecting cell viability, and by the relatively small magnitude of the increased agonist-induced phosphorylation observed when maximally effective amounts of GRKs are transfected (cf. Fig. 3Go). This was a particular problem for GRK6-(K215M,K216M), which acted as a dominant-negative mutant of the transfected wild-type GRK6 only when the expression of the kinase-deficient mutant was higher than that of the wild-type plasmid (cf. Fig. 4BGo). In contrast, C20-GRK2-K220M acted as a dominant-negative mutant of the transfected wild-type GRK2 even when the expression of the dominant-negative mutant was lower than that of the wild-type plasmid (cf. Fig. 4AGo).

Since the rFSHR also becomes phosphorylated when transfected 293 cells are stimulated with PMA (9, 10, 11, 12), we conducted an additional test of the specificity of the kinase-deficient GRKs by testing their effects on the PMA-induced phosphorylation of the rFSHR. The results presented in Fig. 6Go show that transfection of 293 cells with C20-GRK2-K220M or GRK6-(K215M,K216M) (see expression data shown in Fig. 4Go) did not inhibit the PMA-induced phosphorylation of the cotransfected rFSHR.



View larger version (16K):
[in this window]
[in a new window]
 
Figure 6. Effects of Kinase-Negative Mutants of GRKs on the PMA-Induced Phosphorylation of the rFSHR

293 cells were transiently cotransfected with an expression vector for the rFSHR (10 µg/100-mm dish) plus an empty expression vector, C20-GRK2-K220M, or GRK6-(K215M,K216M) (at 10 µg/100-mm dish) as indicated. The transiently transfected cells were metabolically labeled with 32Pi for 3 h and then incubated with buffer or 200 nM PMA for 15 min. Lysates were then prepared and equal amounts of rFSHR were immunoprecipitated as described in Materials and Methods. Autoradiograms of the dried gels were obtained using Kodak BioMax MS film and intensifying screens (Eastman Kodak Co.). After scanning and quantitation by densitometry, the PMA-induced phosphorylation of the rFSHR in each group of cells was expressed as fold over their respective basal (i.e. cotransfected and 32Pi-prelabeled cells incubated with buffer only). Each bar represents the mean ± SEM of three independent transfections. None of the values shown are statistically different from the control (i.e. cells transfected with the rFSHR and empty vector and stimulated with PMA).

 
The data presented in Fig. 7Go show that C20-GRK2-K220M or GRK6-(K215M,K216M) was equally effective in inhibiting the agonist-induced phosphorylation of the rFSHR catalyzed by endogenous kinases. Approximately 50% inhibition of phosphorylation was detected when maximally effective concentrations of plasmids (cf. Fig. 4Go for relative expression of the transfected dominant-negative kinases to that of the endogenous kinases) were used. This degree of inhibition of phosphorylation of the transfected rFSHR catalyzed by endogenous kinases is somewhat higher than that reported using cotransfections of the C20-GRK2-K220M construct and the ß2-adrenergic receptor in 293 cells (23).



View larger version (14K):
[in this window]
[in a new window]
 
Figure 7. Inhibition of the Phosphorylation of the rFSHR by Endogenous Kinases Present in 293 Cells

293 cells were transiently cotransfected with an expression vector for the rFSHR (10 µg/100-mm dish) plus an empty expression vector, C20-GRK2-K220M, G{alpha}t, or GRK6-(K215M,K216M), each at 10 µg/100-mm dish as indicated. The transiently transfected cells were metabolically labeled with 32Pi for 3 h and then incubated with or without 100 ng/ml hFSH for 60 min. Lysates were then prepared and equal amounts of rFSHR were immunoprecipitated as described in Materials and Methods. Autoradiograms of the dried gels were obtained using Kodak BioMax MS film and intensifying screens (Eastman Kodak Co.). After scanning and quantitation by densitometry, the hFSH-induced phosphorylation of the rFSHR in each group of cells was expressed as fold over their respective basal (i.e. cotransfected and 32Pi-prelabeled cells incubated with buffer only). All experiments were done using cells transfected with maximally effective amounts of plasmids. Each bar represents the mean ± SEM of three to six independent transfections. *, Significantly different from cells transfected with the rFSHR and the empty vector (P < 0.05).

 
While additional inhibitors for GRK6 are not available, one additional co-transfection strategy, overexpression of G{alpha}t, was used as an independent attempt to inhibit endogenous GRK2. This construct acts as Gß/{gamma} scavenger (29, 30) and should inhibit GRK2-catalyzed phosphorylation by preventing the agonist-induced translocation of GRK2 to the plasma membrane, an event that is mediated by Gß/{gamma} (3, 18). This construct is not expected to inhibit GRK6 because GRK6 is already associated with the plasma membrane by a covalently attached palmitate group. As shown in Fig. 7Go, G{alpha}t inhibited the hFSH-induced phosphorylation of the rFSHR catalyzed by endogenous kinases by approximately 70%.

These results indicate that the two GRKs that are endogenously expressed in 293 cells participate in the hFSH-induced phosphorylation of the transfected rFSHR.

Functional Correlates of GRK-Mediated Phosphorylation of the rFSHR
Since a binary complex formed by some phosphorylated GPCRs and arrestins serve as common molecular intermediates in the functional uncoupling of the receptors from their effector systems and the internalization of the agonist-receptor complex (see Introduction), we tested the effects of different GRKs, as well as arrestin-3 and dominant-negative mutants, on the receptor-mediated internalization of FSH.

The data presented in Fig. 8Go show that cotransfection of the rFSHR with GRK2, GRK4{alpha}, or GRK6 result in a 2- to 3-fold increase in the receptor-mediated internalization of hFSH. These data also show that cotransfection with arrestin-3 enhanced internalization 2- to 3-fold and that combinations of the GRKs and arrestin-3 enhanced internalization 3- to 4-fold. Thus, the effects of coexpression of arrestin-3 with GRK2, GRK4{alpha}, or GRK6 were more pronounced than those of arrestin-3 alone or GRKs alone, but were certainly not additive.



View larger version (17K):
[in this window]
[in a new window]
 
Figure 8. Effects of GRKs and Arrestin-3 Cotransfection on the rFSHR-Mediated Internalization of [125I]hFSH

Transient cotransfections of rFSHR plus an empty vector GRK2, GRK4{alpha}, GRK6, and arrestin-3 were performed as indicated. After an overnight transfection with the indicated plasmids, the cells were washed, trypsinized, and distributed into six-well plates. Twenty four hours later the cells were incubated with 40 ng/ml [125I]hFSH for 12 min at 37 C, and the internalization index was measured as described in Materials and Methods. All experiments were done using cells transfected with maximally effective amounts of plasmids. The bars represent the average ± SEM of results obtained in four to eight independent transfections. *, Significantly different (P < 0.05) from cells transfected with the empty vector without arrestin-3 (i.e. compare white bars of GKR2, GRK4{alpha}, or GRK6 with white bar of empty vector). **, Significantly different (P < 0.05) from paired transfection conditions without arrestin-3 (i.e. compare black and white bars of each pair).

 
Since the role (if any) of endogenous GRKs on internalization is best determined using dominant-negative mutants (see Discussion), we tested the two inhibitors of GRK2 (C20-GRK2-K220M and G{alpha}t) and the dominant-negative mutant of GRK6 (GRK6-(K215M,K216M) on the rFSHR-mediated internalization of hFSH. These results (Table 1Go) show that GRK6-(K215M,K216M) had little or no effect on internalization, while C20-GRK2-K220M and G{alpha}t inhibited internalization to approximately 65% of control. The divergent effects of the inhibitors of GRK2 and GRK6 on internalization stands in contrast to the finding that all of them inhibited rFSHR phosphorylation catalyzed by endogenous kinases to 30–50% of control (cf. Fig. 7Go).


View this table:
[in this window]
[in a new window]
 
Table 1. Effects of Inhibitors of GRKs, Nonvisual Arrestins, and Dynamin on the rFSHR-Mediated Internalization of [125I]hFSH

 
The experiments presented in Table 1Go with the inhibitors of GRK2 and GRK6 were done by transfecting cells with an amount of plasmid that had been previously determined to yield optimal inhibition of phosphorylation (i.e. 10 µg/100-mm dish). Ultimately, however, the optimal degree of inhibition of internalization obtained with the inhibitors of GRK2 and GRK6 (Table 1Go) was relatively poor when compared with a dominant-negative mutant of the non-visual arrestins or a dominant-negative mutant of dynamin. Thus, as shown in Table 1Go, ß-arrestin-(319–418), a dominant-negative mutant of arrestin-mediated internalization (31) and dynamin-K44A, a dominant-negative mutant of dynamin-mediated internalization (32, 33), inhibited the internalization of the rFSHR to approximately 30% of control (Table 1Go).

Since the inhibitors of GRK2 and GRK6 have a similar effect on phosphorylation but display a differential effect on internalization, these results suggest that the GRK2-catalyzed phosphorylation of the rFSHR is more important for internalization than the GRK6-catalyzed phosphorylation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The experiments presented herein were designed to identify members of the GRK family that participate in the agonist-induced phosphorylation of the rFSHR and to assess the impact of GRK-mediated phosphorylation on the agonist-induced internalization of this receptor.

We concentrated our efforts on characterizing the involvement of GRK2 and GRK6 on the phosphorylation and internalization of the rFSHR because these two kinases are present in MSC-1 cells (a Sertoli-like cell line) and in 293 cells, the cells used for transfections (Fig. 1Go). Moreover, experiments in which GRK2 or GRK6 were cotransfected with the rFSHR in 293 cells indicate that both of these GRKs are capable of phosphorylating the agonist-occupied rFSHR (Figs. 2Go and 3Go). The majority of cotransfection experiments have revealed no specificity in the ability of GRKs to phosphorylate a given GPCR (reviewed in Refs. 3, 18). On the other hand, there is one report showing that cotransfection of the {alpha}2C10-adrenergic receptor with GRK5 or GRK6 does not result in increased agonist-induced receptor phosphorylation, while cotransfection with GRK1 or GRK2 does. This report is particularly relevant because, like the rFSHR (9, 12), the {alpha}2C10-adrenergic receptor is phosphorylated in an acidic S/T cluster present in the third intracellular loop (13). In spite of the similarity in the phosphorylation sites of the {alpha}2C10-adrenergic and the rFSHR receptors, however, our overexpression studies with GRK2 and GRK6 (Figs. 2Go and 3Go) did not reveal the kind of GRK specificity documented with the {alpha}2C10-adrenergic receptor.

It is generally accepted that while GRK overexpression studies provide useful information (see above), they cannot be used to discern the role of endogenous GRKs on the phosphorylation of a given GPCR because overexpression of GRKs may force a reaction that does not normally occur at the ratio of GRK/receptor that prevails in the cell. A better way to address this question is to use overexpression of dominant-negative mutants as specific inhibitors of a given GRK. Thus, the possible involvement of endogenous GRK2 or GRK6 on the agonist-induced phosphorylation of the rFSHR expressed in 293 cells was probed by overexpression of G{alpha}t, as well as kinase-negative mutants of GRK2 and GRK6. Overexpression of G{alpha}t, a Gß/{gamma} scavenger (30), is expected to specifically inhibit GRK2 by preventing the Gß/{gamma}-promoted translocation of this kinase to the membrane (3, 18, 29). It should not affect the activity of GRK6, however, because this kinase is constitutively associated with the plasma membrane via a covalently attached palmitate (3, 18). Specificity studies performed against the transfected wild-type kinases show that overexpression of a C20-GRK2-K220M inhibited the cotransfected GRK2 but not the cotransfected GRK6, and overexpression of GRK6-(K215M,K216M) inhibited the cotransfected GRK6 but not the cotransfected GRK2 (Fig. 5Go). In addition, overexpression of C20-GRK2-K220M or GRK6-(K215M,K216M) had no effect on the PMA-induced phosphorylation of the transfected rFSHR (Fig. 6Go).

Together with the studies discussed above, the ability of C20-GRK2-K220M, G{alpha}t, and GRK6-(K215M,K216M) to inhibit the agonist-induced phosphorylation of the rFSHR catalyzed by kinases endogenous to 293 cells (Fig. 7Go) show that GRK2 and GRK6 participate in the phosphorylation of the rFSHR. When considered together with previous data from this laboratory, these results allow us to conclude that three different kinases, protein kinase C, GRK2, and GRK6, participate in the agonist-induced phosphorylation of the rFSHR. Although it is difficult to ascertain the relative importance of each of these three kinases, the most effective inhibitor of phosphorylation was G{alpha}t, an inhibitor of GRK2 (cf. Fig. 6Go), and the least effective inhibitor of agonist-induced phosphorylation of the rFSHR was the down-regulation of protein kinase C (10). The dominant-negative mutants of GRK2 and GRK6 were intermediate, but equally effective (cf. Fig. 7Go). it is also important to note that while C20-GRK2-K220M and G{alpha}t are both inhibitors of GRK2, G{alpha}t was more effective than C20-GRK2-K220M in inhibiting the agonist-induced phosphorylation of the rFHSR catalyzed by endogenous kinases (Fig. 7Go). The reasons for this difference were not explored, but since these plasmids encode for different proteins, it is possible that G{alpha}t is expressed at higher levels than C20-GRK2-K220M leading to a more efficient inhibition of GRK2. By virtue of its ability to scavenge Gß/{gamma}, G{alpha}t should also be an inhibitor of any kinase that depends on Gß/{gamma} for activity or for translocation to the plasma membrane. There is only one other kinase, GRK3, that is known to be dependent on Gß/{gamma} for membrane translocation, however, and this kinase was not detectable in 293 cells by Western blots (see Results). Cotransfection studies with mixtures of the dominant-negative mutants have not yet been performed, but addition of a protein kinase C inhibitor (Bisindolylmaleimide) to cells overexpressing C20-GRK2-K220M failed to enhance the effects of the dominant-negative kinase on rFSHR phosphorylation (data not shown).

To our knowledge this is the first report on the involvement of GRKs in the phosphorylation of the glycoprotein hormone receptors. While previous studies from other laboratories have examined the functional impact of GRK overexpression or ablation on some functions of the glycoprotein hormone receptors, none of them have examined receptor phosphorylation. Thus, it has been reported that GRK2 and GRK5 may be responsible for the agonist-induced desensitization of the TSH receptor in a rat thyroid cell line (35, 36). Likewise, cotransfection of GRK2 or the four isoforms of GRK4 have been previously reported to enhance the agonist-induced uncoupling of the lutropin/choriogonadotropin receptor (19).

In the studies presented here we also attempted to correlate rFSHR phosphorylation with the rFSHR-mediated internalization of FSH because the agonist-induced internalization of some GPCRs is facilitated by GRK-catalyzed phosphorylation (see Introduction). Overexpression of GRK2, GRK4{alpha}, or GRK6 is similarly effective in enhancing hFSH-stimulated rFSHR phosphorylation (Figs. 2Go and 3Go) and the internalization of [125I]hFSH (Fig. 8Go). Moreover, arrestin-3 alone is capable of enhancing the internalization of [125I]hFSH, and the effects of GRKs on internalization are enhanced by arrestin-3 (Fig. 8Go). Thus, it is clear that the enhanced phosphorylation elicited by GRK overexpression or the overexpression of arrestin-3 result in an increase in receptor-mediated agonist internalization (also see Ref. 9).

While the overexpression studies establish a link between phosphorylation and internalization, the results obtained with the kinase-deficient mutants and with G{alpha}t indicate that the GRK2-mediated rFSHR phosphorylation is important for internalization, but the GRK6-mediated rFSHR phosphorylation is not (Table 1Go). The idea that the rFSHR phosphorylation mediated by different GRKs may have a different functional impact merits further consideration in view of the finding that the agonist-induced phosphorylation of the rFSHR occurs in two distinct domains, the first and third intracellular loops. Moreover, the phosphorylation of these two domains may not be functionally equivalent, as documented by the finding that mutation of the phosphorylation sites present in the third intracellular loop of the rFSHR inhibits uncoupling but not internalization, while mutation of the phosphorylation sites present in the first intracellular loop of the rFSHR inhibits uncoupling as well as internalization (12). Thus, there are now two lines of evidence, mutation of individual phosphorylation domains of the rFSHR (12) and inhibition of different GRKs (this paper) that suggest different roles for different phosphorylation events in the rFSHR. It will now be of interest to determine whether the two domains phosphorylated upon agonist activation of the rFSHR are preferentially phosphorylated by GRK2 or GRK6. If we can also establish an experimental paradigm to examine agonist-induced uncoupling in transiently transfected cells, we should also be able to determine whether the dominant-negative mutants of GRK2 or GRK6 have a differential effect on the agonist-induced uncoupling of the rFSHR.

The idea that phosphorylation of different domains, or even different residues, has a different impact on the functional properties of GPCRs is also supported by recent studies from our laboratory and others. Thus, individual mutations of the four serine residues that become phosphorylated in the LH/CG receptor in response to agonist stimulation show that two of these residues are involved in uncoupling and internalization, the third residue is involved in internalization but not uncoupling, and the fourth residue is not involved in either (37). Similar conclusions can be made from individual mutation of the two phosphorylation clusters present in the third intracellular loop of the m2 muscarinic receptor. In this receptor the phosphorylation of only one of the two clusters is necessary for uncoupling, but the phosphorylation of both clusters is necessary for internalization (15).

Finally, the data presented here also show that a dominant-negative mutant of the nonvisual arrestins is a more effective inhibitor of internalization than the two inhibitors of GRK2 (Table 1Go). The dominant-negative mutant of the nonvisual arrestins is, in fact, as effective in inhibiting internalization as a dominant-negative mutant of dynamin (Table 1Go). This finding suggests that the binding of nonvisual arrestin to the rFSHR is more important than phosphorylation in the internalization of the agonist-rFSHR complex. This conclusion is in agreement with previous results from this laboratory showing that the loss of agonist-induced phosphorylation of two signaling-deficient mutants of the rFSHR can be rescued by overexpression of GRK2, but this enhanced phosphorylation rescues the slow rate of internalization of the agonist-rFSHR complex in only one of the two mutants. Overexpression of arrestin-3, however, rescues the internalization of both mutants (12).

In summary, the results presented here show that GRK2 and GRK6 participate in the agonist-induced phosphorylation of the rFSHR and suggest that only the GRK2-induced phosphorylation facilitates the internalization of the agonist-rFSHR complex. When considered with previous data from this laboratory (12), our results also suggest that even when the phosphorylation of the rFSHR is impaired by inhibiting GRK2 (this paper) or by preventing agonist-induced activation (12), the agonist-rFSHR complex can still be internalized by a pathway that depends on a nonvisual arrestin.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Cells
The cloning of a cDNA containing the full-length coding region plus portions of the 5'- and 3'-untranslated regions of the wild-type rFSHR have been previously described. This cDNA was subcloned into the eukaryotic expression vector pcDNAI/Neo (Invitrogen, San Diego, CA) and used for transfections.

Inserts encoding the full-length bovine GRK2 (39) and human GRK6 (28) were subcloned into pcDNA1.1/Amp for expression studies. A mutant form of GRK6 predicted to be deficient in kinase activity (25, 26) was prepared using PCR strategies to mutate two adjacent lysine residues present in GRK6 to methionine. This mutant, designated GRK6-(K215M,K216M), was also subcloned into pcDNA1.1/Amp for expression studies. A full-length human GRK4{alpha} subcloned into pcDNA 1.1 (19) was generously donated by Dr. Robert Lefkowitz (Duke University, Durham, NC). An expression vector (pcDNA1.1) encoding for a prenylated, kinase-deficient mutant of GRK2 (designated C20-GRK2-K220M4, see Ref. 23) was generously donated by Dr. Marc Caron (Duke University). A plasmid encoding for a human influenza hemagglutinin-tagged dominant-negative mutant form of dynamin (i.e. dynamin K44A, see Ref. 32) was obtained from Dr. Sandra Schmid (Scripps Research Institute, La Jolla, CA) and subcloned into pcDNA3.1 (Invitrogen) for transfection. The expression vectors (both in pcDNA3.1) encoding for arrestin-3 and ß-arrestin(319–418) have been described (31). An expression vector for bovine transducin {alpha} (designated G{alpha}t) in pcDNA1 was purchased from the American Type Culture Collection (Manassas, VA).

Transient cotransfections of human embryonic kidney (293) cells were done using calcium phosphate as described by Chen and Okayama (40). Cells plated in 100-mm dishes were transfected when 70–80% confluent using a maximum of 20 µg of plasmid DNA (the total amount of plasmid transfected was kept constant by including the appropriate amounts of empty expression vector). After an overnight incubation the cells were washed, trypsinized, and replated in 100- mm dishes (1–2 x 107 cells per dish) for phosphorylation experiments or in 35-mm wells (5–10 x 105 cells per well) for binding and internalization experiments. All cells were used 24 h later.

Intact Cell Phosphorylation Assays
Transiently transfected cells (in 100-mm dishes) were metabolically labeled with 200–300 µCi/ml of 32Pi for 3 h at 37 C. The methods used to lyse the cells, to immunoprecipitate the rFSHR, and to resolve the immunoprecipitates on SDS gels have been described previously (10, 11, 41). Receptor phosphorylation was determined after incubation of the 32Pi-prelabeled cells at 37 C with buffer only for 15–60 min, 100 ng/ml hFSH for 60 min, or 200 nM PMA for 15 min (11). Although there were some variations on receptor expression among the different conditions tested, there were no consistent effects of arrestin or GRK overexpression (or their dominant-negative mutants) on FSHR expression. Regardless of the plasmids transfected, the amount of receptor used in all immunoprecipitation experiments was equalized based on measurements of [125I]hFSH binding performed in the same batch of cells used for the phosphorylation assays (see above). In 70 independent transfections, the binding of [125I]hFSH to transiently transfected cells (determined during a 1-h incubation of intact cells with 100 ng/ml [125I]hFSH) was found to be 3.2 ± 0.3 ng/106 cells.

After immunoprecipitation and electrophoresis, autoradiograms of the dried gels were obtained using Kodak BioMax MS film and intensifying screens (Eastman Kodak Co., Rochester, NY). The autoradiograms were scanned and quantitated using a molecular imaging system (Bio-Rad Laboratories, Inc., Hercules, CA). All images were captured in a digital format for presentation. Basal receptor phosphorylation (i.e. that detected in 32Pi-labeled cells incubated with buffer only, see above) varied by at most 10% among all the conditions tested, and thus all data presented are expressed as fold over basal.

Internalization Assays
Transiently transfected cells (in 35-mm wells) were preincubated in 1 ml of Waymouths MB752/1 containing 1 mg/ml BSA and 20 mM HEPES, pH 7.4 for 60 min at 37 C. Each well then received 40 ng/ml [125I]hFSH, and the incubation was continued for 12 min at 37 C. The cells were then washed twice with 2-ml aliquots of cold HBSS containing 1 mg/ml BSA. 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 2–4 min (42, 43). The acidic buffer was removed, and the cells were washed once more with another aliquot of the same buffer. The acid buffer washes were combined and counted, and the cells were solubilized with 100 µl of 0.5 N NaOH, collected with a cotton swab, and counted to determine the amount of internalized hormone. The results of these experiments are expressed as an internalization index (which is defined as the ratio of internalized [125I]hFSH/surface-bound [125I]hFSH, see Ref. 44) because plots of internalization index vs. time are linear for at least 30 min. Under the experimental conditions used here, such plots can be readily used to calculate rates of internalization.

Immunoblots
Cell were lysed in a solution containing 1% Triton X-100, 200 mM NaCl, 20 mM HEPES, 1 mM EDTA, pH 7.4, during a 30-min incubation at 4 C. The lysates were clarified by centrifugation (100,000 x g for 30 min), and the amount of protein present in the supernatants was measured using the DC protein assay from Bio-Rad Laboratories, Inc.. The lysates were resolved on SDS gels and electrophoretically transferred to polyvinylidenefluoride (PVDF) membranes as described elsewhere (41). After blocking (41), expression of the different proteins was determined by incubating the blots overnight with the indicated concentrations of the primary antibodies listed below. Horseradish peroxidase-labeled secondary antibodies were then used during a 1-h incubation at a final dilution of 1:5,000, and the proteins were finally visualized using the enhanced chemiluminescence (ECL) system of detection from Amersham Pharmacia Biotech (Arlington Heights, IL).

GRK2 and C20-GRK2-K220M were detected using a mouse monoclonal antibody (3A10, at a final dilution of 1:100) (45) a rabbit polyclonal antibody (AB9, at a final dilution of 1:3,000) (46), or a commercially available rabbit polyclonal antibody (C-15 from Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a final concentration of 0.5 µm/ml. GRK3 was detected using a rabbit polyclonal antibody (C-14, final concentration = 0.5 µg/ml) from Santa Cruz Biotechnology, Inc.. GRK4 was detected using rabbit polyclonal antibodies specific for the {alpha}- and ß-isoforms (K-20) or the {gamma}- and {delta}-isoforms (I-20) from Santa Cruz Biotechnology, Inc. at final concentrations of 0.5 µg/ml. GRK5 was detected using 0.5 µg/ml of a rabbit polyclonal antibody designated GRK5(C-20) from Santa Cruz Biotechnology, Inc.. GRK6 and GRK6-(K215M,K216M) were detected using 0.5 µg/ml of a rabbit polyclonal antibody designated GRK6(C-20) from Santa Cruz Biotechnology, Inc.. Arrestin-3 was detected with a mouse monoclonal antibody (F4C1, final dilution = 1:2,000) directed against an epitope common to all known arrestins (47). ß-Arrestin (319–418) was detected using a polyclonal antibody (KEE, final dilution = 1:2,000) raised against a C-terminal 16 residue peptide (48). The hemagglutinin-tagged dynamin was detected using the 12CA5 monoclonal antibody at a final concentration of 5 µg/ml (Boehringer Mannheim, Indianapolis, IN).

Other Methods
Statistical analysis (t test with two-sided P values) was performed using Instat (Graph Pad Software, San Diego, CA).

Hormones and Supplies
The rabbit antibody to the rFSHR (Anti-F) has been described. The National Hormone and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases kindly provided purified hFSH (AFP-5720D) and PMSG. [125I]hFSH was prepared as previously described (49). [32P]orthophosphate was obtained from DuPont-New England Nuclear (Boston, MA). Phosphate-free DMEM was purchased from ICN Biomedicals, Inc. (Irvine, CA). Nonidet P-40, Triton X-100, protease inhibitors, N,N',N''-triacetylchitotriose, protein A-agarose, and BSA were from Sigma Chemical Co. (St. Louis, MO). Okadaic acid and cypermethrin were purchased from Alexis Biochemicals (Woburn, MA). Wheat germ agglutinin agarose was from Vector Laboratories, Inc. (Burlingame, CA). Cell culture supplies and reagents were obtained from Corning, Inc. (Corning, NY) and Gibco BRL (Grand Island, NY), respectively. The enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech (Arlington Heights, IL), and the horseradish peroxidase-labeled secondary antibodies were from Bio-Rad Laboratories, Inc.). Human embryonic kidney cells (293) were obtained from the American Type Culture Collection (CRL-1573) and MSC-1 cells (16, 17) were generously donated by Dr. Michael Griswold (Washington State University, Pullman, WA). All other materials were obtained from commonly used suppliers.


    ACKNOWLEDGMENTS
 
We wish to thank Julie Jaquette, JoEllen Fabritz, and Lynn Harrison for performing some of the experiments presented here. We also thank Dr. Deborah L. Segaloff (University of Iowa, Iowa City, IA) for a critical reading of this manuscript. Lastly, we thank Dr. Marc Caron (Duke University, Durham, NC) for providing us with the C20-GRK2-K220M expression vector; Dr. Robert J. Lefkowitz (Duke University) for providing us with the GRK4{alpha} expression vectors, Dr. Sandra Schmidt (Scripps Research Institute) for the dynamin K44A plasmid, Dr. Allen Spiegel (NIH, Bethesda, MD) for the KEE antibody, Dr. Larry Donoso (Wills Eye Hospital, Philadelphia, PA) for the F4C1 antibody, and Dr. Mike Griswold (Washington State University, Pullman, WA) for MSC-1 cells.


    FOOTNOTES
 
Address requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2–512 BSB, The University of Iowa, Iowa City, Iowa 52242-1109. E-mail: mario-ascoli{at}uiowa.edu

This work was supported by NIH Grants HD-28962 to M.A and GM-47417 and GM-44944 to J.L.B. The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa were supported by NIH Grant DK-25295. J.L.B. is an Established Investigator of the American Heart Association. M. de F.M.L. was supported by a fellowship from the Fudaçao de Amparo A Pesquisa Do Estado de São Paulo, Brazil (FAPESP, 96/1454–8). K.N. was partially supported by a fellowship from the Lalor Foundation.

1 Present address: Laboratorio de Farmacologia, Instituto Butantan, Av. Dr. Vital, Brazil 1500, 05503–900, Sao Paulo, Brazil. Back

2 There are four splice variants of GRK4 (cf. Refs. 19 and 20) and only two of them, {delta} and {gamma}, were identified in MSC-1 cells (cf. Fig. 1Go). However, all expression experiments were done using GRK4{alpha} (the longest splice variant) because there appear to be no differences in the ability of these four variants to enhance the agonist-induced desensitization of the gonadotropin receptors (19 ). Back

3 Since GRK6 has two adjacent lysines (codons 215 and 216) in the conserved kinase catalytic domain (28 ), both of them were mutated to methionine. Back

4 This construct was initially named C20ßARK1-K220M (23 ). In order to be consistent with the more current nomenclature used in this paper, we renamed it C20-GRK2-K220M, because ßARK1 is now known as GRK2. Back

Received for publication December 8, 1998. Revision received March 10, 1999. Accepted for publication March 16, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Ferguson SSG, Zhang J, Barak LS, Caron MG 1997 Pleiotropic role for GRKs and b-arrestins in receptor regulation. News Physiol Sci 12:145–151[Abstract/Free Full Text]
  2. Lefkowitz RJ 1998 G protein-coupled receptors. III. New roles for receptor kinases and ß-arrestins in receptor signaling and desensitization. J Biol Chem 273:18677–18680[Free Full Text]
  3. Krupnick JG, Benovic JL 1998 The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annu Rev Pharmacol Toxicol 38:289–319[CrossRef][Medline]
  4. Whistler JL, von Zastrow M 1998 Morphine-activated opioid receptors elude desensitization by ß-arrestin. Proc Natl Acad Sci USA 95:9914–9919[Abstract/Free Full Text]
  5. Zhang J, Ferguson SSG, Barak LS, Bodduluri SR, Laporte SA, Law P-Y, Caron MG 1998 Role for G protein-coupled receptor kinase in agonist-specific regulation of µ-opioid receptor responsiveness. Proc Natl Acad Sci USA 95:7157–7162[Abstract/Free Full Text]
  6. Murray SR, Evans CJ, von Zastrow M 1998 Phosphorylation is not required for dynamin-dependent endocytosis of a truncated mutant opioid receptor. J Biol Chem 273:24987–24991[Abstract/Free Full Text]
  7. Malecz N, Bambino T, Bencsik M, Nissenson RA 1998 Identification of phosphorylation sites in the G protein-coupled receptor for parathyroid hormone. Receptor phosphorylation is not required for agonist-induced internalization. Mol Endocrinol 12:1846–1856[Abstract/Free Full Text]
  8. Ferguson SSG, Downey WE, Colaprieto A, Barak LB, Menard L, Caron MC 1996 Role of ß-arrestin in mediating agonist-promoted G protein-coupled receptor internalization. Science 271:363–365[Abstract]
  9. Nakamura K, Krupnick JG, Benovic JL, Ascoli M 1998 Signaling and phosphorylation-impaired mutants of the rat follitropin receptor reveal and activation- and phosphorylation-independent but arrestin-dependent pathway for internalization. J Biol Chem 273:24346–24354[Abstract/Free Full Text]
  10. Quintana J, Hipkin RW, Sánchez-Yagüe J, Ascoli M 1994 Follitropin (FSH) and a phorbol ester stimulate the phosphorylation of the FSH receptor in intact cells. J Biol Chem 269:8772–8779[Abstract/Free Full Text]
  11. Hipkin RW, Liu X, Ascoli M 1995 Truncation of the C-terminal tail of the follitropin (FSH) receptor does not impair the agonist- or phorbol ester-induced receptor phosphorylation and uncoupling. J Biol Chem 270:26683–26689[Abstract/Free Full Text]
  12. Nakamura K, Hipkin RW, Ascoli M 1998 The agonist-induced phosphorylation of the rat follitropin receptor (rFSHR) maps to the first and third intracellular loops. Mol Endocrinol 12:580–591[Abstract/Free Full Text]
  13. Eason MG, Moreira SP, Liggett SB 1995 Four consecutive serines in the third intracellular loop are the sites for b-adrenergic receptor kinase-mediated phosphorylation and desensitization of the a2A-adrenergic receptor. J Biol Chem 270:4681–4688[Abstract/Free Full Text]
  14. Pals-Rylaarsdam R, Xu Y, Witt-Enderby P, Benovic JL, Hosey M 1995 Desensitization and internalization of the m2 muscarinic acetylcholine receptor are directed by independent mechanisms. J Biol Chem 270:29004–29011[Abstract/Free Full Text]
  15. Pals-Rylaarsdam R, Hosey MM 1997 Two homologous phosphorylation domains differentially contribute to desensitization and internalization of the m2 muscarinic acetylcholine receptor. J Biol Chem 272:14152–14158[Abstract/Free Full Text]
  16. McGuinness MP, Linder CC, Morales CR, Heckert LL, Pikus J, Griswold MD 1994 Relationship of a mouse sertoli cell line (MSC-1) to normal Sertoli cells. Biol Reprod 51:116–124[Abstract]
  17. Peschon JJ, Behringer RR, Cate RL, Harwood KA, Idzerda RL, Brinster RL, Palmiter RD 1992 Directed expression of an oncogene to Sertoli cells in transgenic mice using Mullerian inhibiting substance regulatory sequences. Mol Endocrinol 6:1403–1411[Abstract]
  18. Pitcher JA, Freedman NJ, Lefkowitz RJ 1998 G protein-coupled receptor kinases. Annu Rev Biochem 67:653–692[CrossRef][Medline]
  19. Premont RT, Macrae AD, Stoffel RH, Chung N, Pitcher JA, Ambrose C, Inglese J, MacDonald ME, Lefkowitz RJ 1996 Characterization of the G protein-coupled receptor kinase GRK4. J Biol Chem 271:6403–6410[Abstract/Free Full Text]
  20. Sallese M, Mariggio S, Collodel G, Moretti E, Piomboni P, Baccetti B, De Blasi A 1997 G protein-coupled receptor kinase GRK4. J Biol Chem 272:10188–10195[Abstract/Free Full Text]
  21. Freedman NJ, Liggett SB, Drachman DE, Pei G, Caron MG, Lefkowitz RJ 1995 Phosphorylation and desensitization of the human ß1-adrenergic receptor. J Biol Chem 270:17953–17961[Abstract/Free Full Text]
  22. Menard L, Fergusson SSG, Zhang J, Lin F-T, Lefkowitz RJ, Caron MG, Barak LS 1997 Synergistic regulation of ß2-adrenergic receptor sequestration: intracellular complement of ß-adrenergic receptor kinase and b-arrestin determine kinetics of internalization. Mol Pharmacol 51:800–808[Abstract/Free Full Text]
  23. Ferguson SSG, Menard L, Barak LS, Koch WJ, Colapietro A-M, Caron MG 1995 Role of phosphorylation in agonist-promoted ß2-adrenergic receptor sequestration. J Biol Chem 270:24782–24789[Abstract/Free Full Text]
  24. Menard L, Ferguson SSG, Barak LS, Bertrand L, Premont RT, Colaprieto A-M, Lefkowitz RJ, Caron MG 1996 Members of the G protein-coupled receptor kinase family that phosphorylate the ß2-adrenergic receptor facilitate sequestration. Biochemistry 35:4155–4160[CrossRef][Medline]
  25. Hanks SK, Quinn AM, Hunter T 1988 The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241:42–52[Medline]
  26. Robinson MJ, Harkins PC, Zhang J, Baer R, Haycock JW, Cobb MH, Goldsmith EJ 1996 Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5[prime]-triphosphate. Biochemistry 35:5641–5646[CrossRef][Medline]
  27. Inglese J, Koch WJ, Caron MG, Lefkowitz RJ 1992 Isoprenylation in regulation of signal transduction by G-protein-coupled receptor kinases. Nature 359:147–150[CrossRef][Medline]
  28. Benovic JL, Gomez J 1993 Molecular cloning and expression of GRK6. J Biol Chem 268:19521–19527[Abstract/Free Full Text]
  29. Koch WJ, Hawes BE, Inglese J, Luttell LM, Lefkowitz RJ 1994 Cellular expression of the carboxyl termminus of a G protein-coupled receptor kinase attenuates Gß{gamma}-mediated signaling. J Biol Chem 269:6193–6197[Abstract/Free Full Text]
  30. Federman AD, Conklin BR, Schrader KA, Reed RR, Bourne HR 1992 Hormonal stimulation of adenylyl cyclase through Gi-protein ß{gamma} subunits. Nature 356:159–161[CrossRef][Medline]
  31. Krupnick JG, Santini F, Gagnon AW, Keen JH, Benovic JL 1997 Modulation of the arrestin-clathrin interaction in cells. J Biol Chem 272:32507–32512[Abstract/Free Full Text]
  32. Damke H, Baba T, Warnock DE, Schmid SL 1994 Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol 127:915–934[Abstract]
  33. Zhang J, Ferguson SSG, Barak LS, Menard L, Caron MG 1996 Dynamin and ß-arrestin reveal distinct mechanisms of G protein-coupled receptor internalization. J Biol Chem 271:18302–18305[Abstract/Free Full Text]
  34. Jewell-Motz EA, Liggett SB 1996 G protein-coupled receptor kinase specificity for phosphorylation and desensitization of {alpha}2-adrenergic receptor subtypes. J Biol Chem 271:18082–18087[Abstract/Free Full Text]
  35. Iacovelli L, Francchetti R, Masini M, De Blasi A 1996 GRK2 and ß-arrestin as negative regulators of thyrotropin receptor-stimulated response. Mol Endocrinol 10:1138–1146[Abstract]
  36. Nagayama Y, Tanaka K, Hara T, Namba H, Yamashita S, Taniyama K, Niwa M 1996 Involvement of G protein-coupled receptor kinase 5 in homologous desensitization of the thyrotropin receptor. J Biol Chem 271:10143–10148[Abstract/Free Full Text]
  37. Lazari MFM, Bertrand JE, Nakamura K, Liu X, Krupnick JG, Benovic JL, Ascoli M 1998 Mutation of individual serine residues in the C-terminal tail of the lutropin/choriogonadotropin (LH/CG) receptor reveal distinct structural requirements for agonist-induced uncoupling and agonist-induced internalization. J Biol Chem 273:18316–18324[Abstract/Free Full Text]
  38. Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH 1990 The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA. Mol Endocrinol 4:525–530[Abstract]
  39. Benovic JL, DeBlasi A, Stone CW, Caron MG, Lefkowitz RJ 1989 ß-adrenergic receptor kinase: primary structure delineates a multigene family. Science 246:235–240[Medline]
  40. Chen C, Okayama H 1987 High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752[Medline]
  41. Quintana J, Hipkin RW, Ascoli M 1993 A polyclonal antibody to a synthetic peptide derived from the rat FSH receptor reveals the recombinant receptor as a 74 kDa protein. Endocrinology 133:2098–2104[Abstract]
  42. Ascoli M 1982 Internalization and degradation of receptor-bound human choriogonadotropin in Leydig tumor cells. Fate of the hormone subunits. J Biol Chem 257:13306–13311[Abstract/Free Full Text]
  43. Fletcher PW, Reichert Jr LE 1984 Cellular processing of follicle-stimulating hormone by Sertoli cells in serum-free culture. Mol Cell Endocrinol 34:39–49[CrossRef][Medline]
  44. Wiley HS, Cunningham DD 1982 The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. J Biol Chem 257:4222–4229[Free Full Text]
  45. Loudon RP, Perussia B, Benovic JL 1996 Differentially regulated expression of the G protein-coupled receptor kinases, ßARK and GRK6, during myelomonocytic cell development in vitro. Blood 88:4547–4557[Abstract/Free Full Text]
  46. Garcia-Higuera I, Penela P, Murga C, Egea G, Bonay P, Benovic JL, Mayor JF 1994 Association of the regulatory ß-adrenergic receptor kinase with rat liver microsomal membranes. J Biol Chem 269:1348–1355[Abstract/Free Full Text]
  47. Donoso LA, Gregerson DS, Smith L, Robertson S, Knospe V, Vrabec T, Kalsow CM 1990 S-antigen: preparation and characterization of site-specific monoclonal antibodies. Curr Eye Res 9:343–355[Medline]
  48. Sterne-Marr R, Gurevich VV, Goldsmith P, Bodine RC, Sanders C, Donoso LA, Benovic JL 1993 Polypeptide variants of ß-arrestin and arrestin 3. J Biol Chem 268:15640–15648[Abstract/Free Full Text]
  49. Ascoli M, Puett D 1978 Gonadotropin binding and stimulation of steroidogenesis in Leydig tumor cells. Proc Natl Acad Sci USA 75:99–102[Abstract]