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Endocrinology Vol. 145, No. 10 4480-4488
Copyright © 2004 by The Endocrine Society

Serine Residues 338 and 339 in the Carboxyl-Terminal Tail of the Type II Gonadotropin-Releasing Hormone Receptor Are Critical for ß-Arrestin-Independent Internalization

Katharina Ronacher, Nosisa Matsiliza, Nonhlanhla Nkwanyana, Adam J. Pawson, Tasneem Adam, Colleen A. Flanagan, Robert P. Millar and Arieh A. Katz

Medical Research Council Research Group for Receptor Biology (K.R., N.M., N.N., T.A., C.A.F., A.A.K.), Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, and Department of Medicine (C.A.F., N.M.), University of Cape Town and Groote Schuur Hospital, Observatory 7925, Cape Town, South Africa; and Human Reproductive Sciences Unit (A.J.P., R.P.M.), Medical Research Council, Edinburgh EH16 4SB, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Dr. Arieh Katz, Medical Research Council Research Group for Receptor Biology, Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory 7925, South Africa. E-mail: katz{at}curie.uct.ac.za.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cloned mammalian type II GnRH receptors have a carboxyl-terminal tail in contrast to the mammalian type I GnRH receptors, which uniquely lack a carboxyl-terminal tail. Because this domain mediates internalization of many serpentine receptors, the internalization pathway of the marmoset monkey type II GnRH receptor and the functional role of the carboxyl-terminal tail in internalization was studied. The internalization pathway of the type II GnRH receptor was investigated in COS-1 cells by coexpressing G protein-coupled receptor kinases (GRKs), dynamin-1, and ß-arrestins. Internalization of the receptor requires GRKs and dynamin but does not require ß-arrestin. The type II GnRH receptor can also internalize via ß-arrestin in the presence of exogenous ß-arrestins, suggesting that the receptor can use two distinct internalization pathways. Receptor internalization appears to occur via clathrin-coated pits and caveolae because disruption of either structure inhibits internalization. Progressive truncations of the carboxyl-terminal tail identified a region containing serine residues 338 and 339 as critical for receptor internalization. Substitution of these serine residues with alanine residues inhibited internalization, whereas substitutions with glutamic acid residues rescued internalization. Furthermore, a dominant-negative GRK2 did not inhibit internalization of receptors having these serine substitutions, although it inhibited internalization of the wild-type receptor. These results together identify serine residues 338 and 339 in the carboxyl-terminal tail as critical for internalization of the type II GnRH receptor and suggest that these residues undergo phosphorylation by GRKs. However, neither of these residues, nor the carboxyl-terminal tail, is required for ß-arrestin-dependent internalization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MAMMALIAN TYPE I GnRH receptors (GnRH-RI) are unique among G protein-coupled receptors (GPCRs) because they lack a cytoplasmic carboxyl-terminal tail (1). A second form of GnRH receptor, designated type II GnRH receptor (GnRH-RII), has been cloned from several primate species including Marmoset monkey (2), African green monkey and Rhesus monkey (3). Unlike the mammalian type I GnRH receptors, the mammalian type II GnRH receptors possess a carboxyl-terminal tail.

In general, the carboxyl-terminal domains of GPCRs have an important role in regulating receptor activity (4, 5). The list of functions assigned to the carboxyl-terminal tail is growing and includes desensitization of G protein-mediated signaling, recruitment of G protein-independent signaling modules, receptor internalization, recycling, and degradation (4, 5). The carboxyl-terminal tail mediates these functions via interactions with a spectrum of protein partners. However, the precise role of many of these proteins and the structural elements underlying these interactions are poorly understood. More is known about the role of the carboxyl-terminal tail in receptor desensitization and internalization. For many GPCRs, as exemplified by the ß2-adrenergic receptor, the phosphorylation of certain serine and/or threonine residues in the carboxyl-terminal tail by G protein-coupled receptor kinases (GRKs) and subsequent binding of ß-arrestin leads to rapid desensitization and internalization of an agonist-stimulated receptor (4, 5). ß-Arrestins bind with higher affinity to the phosphorylated receptor and desensitize the receptor by sterically uncoupling the receptor from its cognate heterotrimeric G protein (6). In addition, ß-arrestin, through its interaction with the clathrin heavy chain (7) and the clathrin adaptor protein AP2 complex (8), target the desensitized GPCRs to clathrin-coated pits. Subsequently the GTPase, dynamin, catalyzes the budding and internalization of the clathrin-coated vesicles from the plasma membrane (9). However, studies done with dominant-negative ß-arrestin and dynamin mutants demonstrated that certain receptors use alternative internalization pathways, which vary with the cell type (4, 5). For example, vasoactive intestinal peptide 1 (VIP1) and endothelin type B receptors internalization is ß-arrestin and clathrin independent but dynamin dependent. These receptors apparently internalize through dynamin-catalyzed budding of caveolae (10, 11). The existence of additional endocytic routes has become evident because a few GPCRs, including the bradykinin type 2 and M2 muscarinic receptors, do not require ß-arrestin or dynamin for their internalization (10, 12).

For many GPCRs, agonist-induced phosphorylation of the carboxyl-terminal tail is crucial for receptor internalization because truncations of the carboxyl-terminal tail or mutations of serine and threonine phosphorylation sites decrease internalization (13, 14, 15, 16, 17, 18). However, there are examples where truncation of the carboxyl-terminal tail of GPCRs has no effect on internalization or even enhances internalization (4, 5, 19, 20). The carboxyl-terminal tail is not the sole domain required for internalization and for several receptors, including the M1, M2, and M3 muscarinic receptors, it was found that phosphorylation of residues in intracellular loop (ICL) 3 is important for internalization, whereas for the FSH receptor, residues in ICL1 and ICL3 are important (4, 5, 21).

Consistent with the requirement of the carboxyl-terminal tail for enhanced receptor desensitization and internalization, mammalian type I GnRH receptors, which uniquely lack a carboxyl-terminal tail, do not display rapid, agonist-induced desensitization and internalization (1, 22). In contrast, nonmammalian type I GnRH receptors, which possess a carboxyl-terminal tail, undergo rapid desensitization and internalization (22, 23, 24). Truncation of the carboxyl-terminal tail of the chicken type I GnRH receptor results in an internalization pattern similar to that of the tail-less human receptor (22). In addition, the mammalian type I GnRH receptor internalization is ß-arrestin independent even in the presence of exogenous ß-arrestins, whereas internalization of the tailed nonmammalian receptors is ß-arrestin dependent (24, 25, 26). The importance of the tail in recruiting ß-arrestin and in enhancing receptor internalization was also demonstrated by addition of the carboxyl-terminal tail of the TRH receptor to the rat GnRH receptor (27). However, in the same set of experiments addition of the catfish GnRH receptor carboxyl-terminal tail did not confer ß-arrestin dependency (27). The role of dynamin in the internalization of the type I GnRH receptors was also examined. Comparative studies demonstrated that the internalization of the nonmammalian GnRH receptors is dynamin dependent (27), whereas contradictory results were obtained for the role of dynamin in the internalization of the mammalian GnRH receptors (23, 27). Very recently, the internalization of a bullfrog GnRH receptor equivalent to the GnRH-RII was reported (28). This receptor has a carboxyl-terminal tail, and in human embryonic kidney 293 cells the internalization of the receptor is dynamin dependent, but ß-arrestin independent.

Internalization of the mammalian type II GnRH receptor has not been studied. In this study, we have investigated the internalization pathway of a mammalian type II GnRH receptor in COS-1 cells and characterized the role of the carboxyl-terminal tail in internalization of the receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of mutant type II GnRH receptor constructs
Mutations were introduced into the marmoset monkey GnRH-RII using a PCR-based method with primers containing the indicated mutations. For truncated mutants, the codons for the amino acids Thr 372, Ser 366, Gln 357, Ser 344, Ser 335, and Gly 326 were replaced by stop codons resulting in the truncated type II GnRH receptors T372stop, S366stop, Q357stop, S344stop, S335stop, and G326stop. The substitution of serine residues 338 and 339 with alanine or glutamic acid residues was done by PCR of the carboxyl terminus with primers that contain these substitutions and subsequently replacing the carboxyl-terminal tail of the wild-type (WT) receptor with a carboxyl-terminal tail having the S338,339A or S338,339E mutations via the ApaI restriction site. The mutated PCR products were subcloned into the mammalian expression vector pcDNA 3.1+ (Invitrogen Life Technologies, Carlsbad, CA) and the mutations were confirmed by DNA sequencing (Epicenter Technologies, Madison, WI). Plasmid DNA for transient transfections was prepared using the Nucleobond PC 500 kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instructions.

Cell culture, DNAs, and transient transfection
COS-1 cells were cultured in DMEM (Invitrogen Life Technologies) containing 10% fetal calf serum, 2 mg/ml streptomycin sulfate, and 4000 U/ml sodium benzylpenicillin and were kept in a 10% CO2 incubator at 37 C. For inositol phosphates (IPs) and internalization assays, COS-1 cells were plated out on poly-D-lysine-coated 12-well plates (2 x 105 cells/well) and were transiently transfected using a modified DEAE-Dextran method (29).

WT ß-arrestins, dynamin 1, and GRKs and their dominant-negative forms [ß-arrestin 1 (V53D) and (319–418), ß-arrestin 2 (V54D), dynamin1 (K44A), and GRK2 (K220R)] were provided by Dr. M. G. Caron (Duke University Medical Center, Durham, NC). The dominant-negative mutant of caveolin-1 [cav-1({Delta}1–81)] was provided by Dr. J. Eggermont (Catholic University, Leuven, Belgium).

Receptor internalization assay
For receptor internalization, a high-affinity agonist [His5,D-Tyr6]-GnRH radioiodinated by the chloramine-T method (30) was used. The internalization assays were based on an acid-wash method described previously (22). Briefly, transfected cells were washed once with ice-cold 10 mM HEPES buffered DMEM (pH 7.2) (HEPES/DMEM) and incubated with 2 x 105 cpm/well 125I-[His5,D-Tyr6] GnRH in 0.5 ml HEPES/DMEM for 5 h at 4 C. Cells were rapidly warmed to 37 C in a water bath for different periods of time, and internalization was stopped by washing the cells twice with ice-cold PBS. Surface-bound radioligand was removed by 1 ml ice-cold acid solution [50 mM acetic acid, 150 mM NaCl (pH 2.8)] and counted in a {gamma}-counter. Cells were lysed with 1 ml 1 M NaOH and acid-resistant (internalized) ligand was counted. Internalized radioligand was expressed as a percent of total cell-associated radioligand for each time point.

Pretreatment with monodansylcadavarine (MDC, Sigma-Aldrich Corp., St. Louis, MO), sucrose (Sigma-Aldrich Corp.) and filipin (Sigma-Aldrich Corp) was performed at 37 C for 30 min before receptor internalization assays.

Total IPs assay
Total IPs assays were performed as previously described (29). Briefly, 24 h after transfection COS-1 cells were incubated overnight with 1 µCi/well of myo-[2-3H]inositol (Amersham, Arlington Heights, UK) in 0.5 ml Medium 199 (Invitrogen Life Technologies). After another 24 h, cells were stimulated with various concentrations of GnRH II in 1 ml buffer [140 mM NaCl, 20 mM HEPES, 4 mM KCl, 8 mM glucose, 1 mM MgCl2, 1 mM CaCl2, 1 mg/ml BSA (pH 7.4)] containing 10 mM LiCl for 1 h at 37 C. Finally the medium was taken off, 1 ml of 10 mM formic acid was added per well, and the cells were incubated at 4 C for 30 min. Total IPs were extracted from the formic acid on DOWEX-1 ion exchange columns, and the radioactivity was determined by liquid scintillation counting.

Statistical analysis and data presentation
The figures show the mean ± SEM of combined results of at least two or three independent experiments performed in duplicates. Data were analyzed by one-way ANOVA, followed by Bonferroni’s multiple comparison test using PRISM graphing software (GraphPad, San Diego, CA). Data were considered significantly different from each other at P < 0.05. EC50 values were estimated by nonlinear regression using PRISM graphing software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Internalization of the GnRH-RII in COS-1 cells is ß-arrestin independent, but requires GRK and dynamin
To characterize the internalization pathway of the GnRH-RII, we coexpressed in COS-1 cells, the receptor with dominant-negative mutants and WT forms of GRK2, GRK3, ß-arrestin 1 and 2, and dynamin1 and examined internalization of the receptor. Expression of a dominant-negative mutant GRK2(K220R), together with the GnRH-RII, reduced the rate and extent of receptor internalization (reduced internalization was observed at all time points, P < 0.05, Fig. 1AGo), suggesting that GRK2 is required for the internalization of the GnRH-RII in COS-1 cells. Consistent with this result, coexpression with WT GRK2 or GRK3 enhanced the rate and extent of the receptor internalization (enhanced internalization in the presence of GRK2 and GRK 3 was observed at all time points; P < 0.05). Because GRK-mediated phosphorylation typically precedes ß-arrestin binding, the roles of ß-arrestin 1 and 2 in GnRH-RII internalization were examined. Coexpression of dominant-negative mutants of ß-arrestin 1 (V53D) and (319–418) as well as of ß-arrestin 2 (V54D) did not influence internalization of the GnRH-RII (internalization data at all time points was similar; P > 0.05; Fig. 1Go, B and C), indicating that neither of the arrestins is required for internalization of this receptor in COS-1 cells. However, coexpression of WT ß-arrestin 1 or 2 increased the rate and extent of GnRH-RII internalization. (Enhanced internalization in the presence of WT ß-arrestin 1 or 2 was observed at all time points; P < 0.05; Fig. 1Go, B and C). This enhanced internalization of GnRH-RII by WT ß-arrestins was blocked by coexpression of the dominant-negative mutants of ß-arrestins (data not shown), confirming that the transfected dominant-negative forms of ß-arrestins were expressed. These results indicate that GnRH-RII internalization in COS-1 cells is ß-arrestin independent; however, the receptor can also internalize via a ß-arrestin-dependent pathway in the presence of exogenous ß-arrestins.



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  		FIG. 1. Effects of overexpression of WT and dominant-negative forms of ß-arrestins, GRKs, and dynamin on the internalization of GnRH-RII. COS-1 cells were grown in 12-well plates and transfected with 1 µg of GnRH-RII cDNA and 1 µg of vector or 1 µg of the indicated DNA constructs. For internalization experiments, cells were incubated with 125I-[His5,D-Tyr6]-GnRH, and the assay was performed at the indicated time points as described in Materials and Methods. Internalized radioligand was expressed as a percent of total cell-associated radioligand at each time point. A, Internalization of GnRH-RII in the presence of GRK2, GRK3 and dominant-negative GRK 2 (K220R). B, Internalization of GnRH-RII in the presence of ß-arrestin 1 and dominant-negative ß-arrestin 1 constructs (V53D and 319–418). C, Internalization of GnRH-RII in the presence of ß-arrestin 2 and a dominant-negative ß-arrestin 2 (V54D). D, Internalization of GnRH-RII in the presence of dynamin1 and the dominant-negative dynamin 1 (K44A). Data shown are mean ± SEM of two of three independent and similar experiments each performed in duplicate.

 
To test whether the internalization of the GnRH-RII is dynamin dependent, the internalization in the presence of WT and a dominant-negative mutant of dynamin1 (K44A) was measured. Coexpression of WT dynamin1 had no effect on internalization of the GnRH-RII (Fig. 1DGo), whereas coexpression of dominant-negative dynamin1 reduced the internalization of the receptor with highest inhibition in the first 30 min of internalization (internalization at 15 min was reduced by 38% ± 10%; P < 0.01). This inhibition indicates that dynamin is involved in the internalization pathway of the GnRH-RII in COS-1 cells.

Taken together, these results demonstrate that, in COS-1 cells, internalization of the GnRH-RII is ß-arrestin independent but requires GRK and dynamin. However, in the presence of overexpressed ß-arrestins, the GnRH-RII can also internalize in a ß-arrestin-dependent manner.

Internalization of the GnRH-RII in COS-1 cells requires intact caveolae and clathrin-coated pits
The finding that internalization of the receptor in COS cells is ß-arrestin independent but requires dynamin suggests that the receptor internalizes via caveolae. To test this possibility, we measured internalization of the GnRH-RII in the presence of disrupters of clathrin-coated pits or disrupters of caveolae. Disruption of clathrin-coated pit formation was done by pretreating cells expressing GnRH-RII with either 0.45 M sucrose or 400 µM MDC, which are known to disrupt clathrin-coated pit formation (31, 32). The disruption of caveolae formation was done by pretreating cells with 5 µg/ml filipin or coexpressing a dominant-negative mutant of caveolin-1 [cav-1({Delta}1–81)] because both have been shown to disrupt the formation of caveolae lipid rafts (33, 34). All four treatments were effective in inhibiting the internalization of the GnRH-RII (Fig. 2Go). Internalization of the receptor after 60 min in untreated cells was 46.1%; however, in the presence of the inhibitors internalization measured was less than 16% (P < 0.001). This marked inhibition of 65–90% in internalization of the receptor by disrupters of either clathrin-coated pits or caveolae function suggests that internalization of the GnRH-RII in COS-1 cells proceeds via clathrin-coated pits and caveolae.



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  		FIG. 2. Effect of disrupters of caveolae formation and clathrin-coated pit formation on internalization of the GnRH-RII. The effect of filipin, sucrose, and MDC on the internalization of GnRH-RII was measured in COS-1 cells grown in 12-well plates and transfected with 1 µg GnRH-RII cDNA and 1 µg vector. The effect of the dominant-negative caveolin ({Delta}1–81) was measured in cells transfected with 0.2 µg of GnRH-RII cDNA and 1.8 µg of dominant-negative caveolin ({Delta}1–81) cDNA. Pretreatment with filipin (5 µg/ml), sucrose (0.45 M), or MDC (400 µM) was done for 30 min before the addition of 125I-[His5,D-Tyr6] GnRH and the internalization assay was performed as described in Materials and Methods. Internalized radioligand was expressed as a percent of total cell-associated radioligand at 60 min at 37 C after the addition of radioligand. Control is the internalization of the GnRH-RII without treatments. Data shown are mean ± SEM of two of three independent and similar experiments each performed in duplicate. ***, Significant difference (P < 0.001) compared with the control value.

 
A region in the carboxyl-terminal tail of the GnRH-RII is required for rapid internalization
In contrast to the mammalian type I GnRH receptors, the cloned mammalian type II GnRH receptors have a carboxyl-terminal tail. To identify structural determinants in the carboxyl-terminal tail of the GnRH-RII that mediate rapid internalization, a series of receptor mutants with progressive truncations of the carboxyl terminus was tested. Receptors were truncated by substitution of a stop codon for the following amino acids: T372, S366, Q357, S344, S335, and G326 (Fig. 3Go). All truncated receptors were functional in signaling as determined by GnRH II-induced IP production (Table 1Go). The EC50 values of all the truncated mutant receptors (up to S335) were similar to the EC50 value of the WT receptor. Interestingly, a receptor lacking the entire carboxyl-terminal tail (G326stop) was found to be active. However, the G326stop mutant showed only 50% of maximum IP produced by the WT receptor. This mutant also showed a 92.5% reduction in ligand binding (data not shown), suggesting that the decreased level of receptor expression is responsible for the reduced maximum IP. Due to the very low ligand binding, it was not possible to measure internalization of this mutant.



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  		FIG. 3. Schematic representation of the carboxyl-terminal tail of WT and mutant GnRH-RII. Amino acids indicated in gray circles were mutated to stop codons to obtain carboxyl-terminal truncation mutants of the receptor. Serine residues 338 and 339, indicated in black circles, were mutated to alanine or glutamic acid residues.

 

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  		TABLE 1. Summary of IPmax and EC50 for WT and mutant type II GnRH receptors stimulated with GnRH II

 
Internalization assays showed that receptor mutants truncated by up to 37 amino acids (up to S344stop) internalized to the same extent as the WT receptor (internalization of receptor mutants was similar to WT receptor at all time points; P > 0.05; Fig. 4Go). However, deletion of a further nine amino acids resulted in a receptor (S335stop), which exhibited a 75% reduction in internalization compared with the WT receptor in the first 30 min of internalization (P < 0.001). These findings indicate that the region between Gly 343 and Ser 335 in the carboxyl-terminal tail of the GnRH-RII is important for rapid internalization. This region contains two serine residues (Ser 338 and Ser 339) and in view of the important role of phosphorylation of serines and threonines in the carboxyl-terminal tail in internalization of many GPCRs, it is likely that these serine residues undergo phosphorylation that leads to rapid internalization of the type II GnRH receptor.



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  		FIG. 4. Internalization of WT and carboxyl-terminal truncation mutants of the GnRH-RII. COS-1 cells in 12-well plates were transfected with 2 µg of GnRH-RII or 2 µg plasmid DNA encoding the indicated truncated mutants. Cells were incubated with 125I-[His5,D-Tyr6] GnRH and internalization assay was performed at the indicated time points as described in Materials and Methods. Internalized radioligand was expressed as a percent of total cell-associated radioligand at each time point. Data shown are mean ± SEM of two of three independent and similar experiments each performed in duplicate.

 
Serine 338 and Serine 339 in the carboxyl-terminal tail are required for rapid internalization of GnRH-RII because they are putative phosphorylation sites for G protein-coupled receptor kinases
To verify whether the putative serine phosphorylation sites (S338 and S339) play a role in rapid internalization, these serine residues were mutated to alanine residues that do not undergo phosphorylation. The double mutant receptor S338,339A showed an internalization profile that is identical with the profile of the truncated S335stop mutant (internalization data at all time points was similar; P > 0.05; Fig. 5Go). This indicates that serine 338 and serine 339 in the carboxyl-terminal tail of the GnRH-RII are critical for rapid internalization of the receptor. In light of the finding that GRK activity is required for internalization of the GnRH-RII (Fig. 1AGo), it is likely that these serine residues undergo phosphorylation by GRK after ligand binding, and this phosphorylation is required for rapid internalization of the type II GnRH receptor.



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  		FIG. 5. Internalization of the GnRH-RII and S335stop and S338,339A receptor mutants. Internalization assays were performed on the indicated expression constructs transfected into COS-1 cells (2 µg DNA/well). Internalized radioligand was expressed as a percent of total cell-associated radioligand at the indicated time points and was determined as described in Materials and Methods. Data shown are mean ± SEM of two of three independent and similar experiments each performed in duplicate.

 
To test whether phosphorylation of serine residues 338 and 339 enhances GnRH-RII internalization, these residues were substituted by glutamic acid, which mimics the negative charge produced by phosphorylation. Compared with the markedly impaired internalization of the receptor having alanine residues, the receptor having glutamic acid residues at these positions had markedly enhanced internalization (P < 0.001; Fig. 6Go), which shows that the negative charge of these residues rescued internalization significantly. Furthermore, although the dominant-negative mutant of GRK2 (K220R) can inhibit WT GnRH-RII receptor internalization (P < 0.001; Fig. 6Go), it did not inhibit internalization of the mutant receptor lacking these putative phosphorylation sites (S338,339A) or having glutamic acid residues that mimic phosphorylated serines (S338,339E), (P > 0.05). Taken together, these results strongly suggest that serine residues 338 and 339 of the GnRH-RII undergo phosphorylation by GRKs and that this phosphorylation is required for internalization.



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  		FIG. 6. Internalization of the GnRH-RII and S338,339E and S338,339A receptor mutants in the absence and presence of dominant-negative GRK 2. COS-1 cells were transfected with 1 µg of the receptor constructs and 1 µg of vector or GRK 2 (K220R). Internalized radioligand was expressed as a percent of total cell-associated radioligand and determined after 5 and 15 min at 37 C after addition of ligand as described in Materials and Methods. Data shown are mean ± SEM of three independent experiments each performed in duplicate. ***, Significant difference (P < 0.001).

 
Serine residues 338 and 339 are not required for ß-arrestin-dependent internalization
The putative phosphorylation sites, serines 338 and 339 of the carboxyl-terminal tail have been shown to be crucial for internalization of the GnRH-RII in COS-1 cells. However, the internalization pathway of the receptor in these cells is ß-arrestin independent (Fig. 1Go, B and C). As GnRH-RII can also use a ß-arrestin-dependent pathway in the presence of overexpressed ß-arrestins, we sought to test whether these serine residues are required for the ß-arrestin-dependent internalization. Coexpression of ß-arrestin 1 with mutant S338,339A or the truncated mutant S335stop increased the rate and extent of internalization of both mutant receptors (enhanced internalization of the S338,339A and the S335stop mutant receptors in the presence of WT ß-arrestin 1 was observed at all time points; P < 0.05; Fig. 7Go, A and B). Similar results were obtained with coexpression of ß-arrestin 2 (not shown). In contrast, as a control, ß-arrestin did not augment internalization of the human type I GnRH receptor (not shown), which is in agreement with previous reports showing that mammalian type I GnRH receptors do not use ß-arrestin for internalization (27). These observations indicate that neither the putative phosphorylation sites, Ser 338 and Ser 339 of the GnRH-RII, nor the carboxyl-terminal tail is required for interaction with ß-arrestin.



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  		FIG. 7. Effect of coexpression of ß-arrestin 1 on the internalization of GnRH-RII and receptor mutants. COS-1 cells were grown in 12-well plates and transfected with 1 µg of receptor constructs and 1 µg of vector or 1 µg of the ß-arrestin 1 construct. Internalization of a mutant lacking putative phosphorylation sites (S338,339A) (A) and a carboxyl-terminal truncated mutant (S335stop) (B) was measured in presence and absence of ß-arrestin 1. Internalized radioligand was expressed as a percent of total cell-associated radioligand at the indicated time points and was determined as described in Materials and Methods. Data shown are mean ± SEM of two of three independent and similar experiments each performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ligand-induced internalization of the mammalian type II GnRH receptor and functional role of the carboxyl-terminal tail have not been previously characterized. This study demonstrates that a mammalian type II GnRH receptor internalizes in COS-1 cells more rapidly and to a greater extent than mammalian type I GnRH receptors, which lack a carboxyl-terminal tail. Approximately 55% of type II GnRH receptors internalize after 90 min of agonist stimulation in comparison to 25% of the tail-less human GnRH receptors (22). The internalization of the type II GnRH receptor in COS-1 cells is ß-arrestin independent, but GRK and dynamin dependent. Although the archetypal internalization pathway involves GRK phosphorylation and subsequent binding of ß-arrestin to the activated and phosphorylated receptor, the internalization of the GnRH-RII in COS-1 cells requires GRK phosphorylation but does not require ß-arrestin, because dominant-negative ß-arrestins 1 and 2 failed to inhibit the rate and extent of receptor internalization. However, in contrast to what has been previously described for the mammalian GnRH-RI (26), overexpression of ß-arrestins 1 and 2 enhanced internalization of the GnRH-RII, demonstrating that the GnRH-RII can also undergo internalization in a ß-arrestin-dependent manner. The finding that the internalization of GnRH-RII in COS-1 cells is ß-arrestin independent can be explained by the low levels of ß-arrestins in these cells (35). Nevertheless, the TRH, platelet-activating factor and AT1 angiotensin receptors have been reported to internalize in a ß-arrestin-dependent manner in COS cells, demonstrating the existence of a ß-arrestin-dependent internalization pathway in these cells (26, 36, 37). One likely explanation for this apparent discrepancy in the ß-arrestin dependency is that the GnRH-RII may have a low affinity for ß-arrestins and at low levels of ß-arrestin, as in COS cells, the receptor uses an alternative internalization pathway. Because this receptor is widely expressed (2), it is likely that ß-arrestins play a role in internalization of the GnRH-RII in at least some of the cell types that express the receptor. Dynamin functions in ß-arrestin-dependent and -independent internalization pathways because dynamin is required for the fission of nascent clathrin-coated vesicles, as well as nascent caveolae vesicles (10, 11). We therefore examined the role of dynamin by measuring internalization of the GnRH-RII in the presence of a dominant-negative dynamin 1. Our results show that the dominant-negative dynamin 1 inhibited internalization of GnRH-RII and provides evidence that dynamin is required for internalization of the receptor in COS cells. The dominant-negative dynamin 1 only partially inhibited internalization of the receptor. One possible explanation for this is that the major dynamin in COS cells is dynamin 2 (36). However, dominant-negative forms of dynamin 1 and 2 are reported to be equally effective in inhibiting dynamin 2 in COS cells (36). Nevertheless, the possibility that a subpopulation of the GnRH-RII internalizes in COS cells via a dynamin-independent pathway cannot be excluded.

The findings that the type II GnRH receptor internalization in COS cells is ß-arrestin independent, but requires dynamin, suggest that the internalization of GnRH-RII is apparently not via clathrin-coated vesicles, but via caveolae. Similar observations have been made for the VIP1 receptor and the endothelin type B receptor, and both receptors are thought to internalize via caveolae (10, 11). In contrast, the internalization of the rat type I GnRH receptor, which is also ß-arrestin independent, but dynamin dependent, was found to occur probably via a clathrin-dependent mechanism (26). Therefore, it is possible that ß-arrestin dependency is not obligatory for internalization via clathrin-coated vesicles. The inhibition of type II GnRH receptor internalization by disrupters of either caveolae or clathrin-coated pits function indicates that the receptor internalization requires caveolae and clathrin-coated pits. The marked inhibition of receptor internalization (65–90% inhibition at 60 min of internalization) by either inhibitor suggests that the receptor internalization uses caveolae and clathrin-coated pits in tandem and not that there are two subpopulations of type II receptors: the first internalizes via clathrin-coated pits, and the second via caveolae. A recent study has suggested that caveolae and clathrin-coated pits might be positioned in sequence or interlinked in the same internalization pathway (38). This pathway has been implicated in the internalization of several growth factor receptors (38, 39, 40, 41). These receptors may first redistribute to caveolae, and subsequently the receptors associate with clathrin-coated pits before they internalize. Very recently, such a mechanism has been suggested for the internalization of the chicken GnRH receptor (42). Furthermore, this internalization route could explain the ß-arrestin-independent but clathrin-coated pit-dependent internalization of the rat type I GnRH receptor (26). The identity of the adaptor molecules that can mimic ß-arrestin function in targeting receptors to clathrin-coated pits and in addition can target receptors to caveolae is not known. The recent findings that GRK2 can directly interact with clathrin via a clathrin box present in its carboxyl terminus and with caveolin via caveolin-binding motifs present in its pleckstrin homology domain and in its amino-terminal domain (43, 44) could suggest that GRK2 may act as an adaptor molecule for clathrin and/or caveolae-mediated endocytosis. Because GRK2 was found to be required for the internalization of type II GnRH receptor, it is possible that GRK2 acts as a kinase and as an adaptor molecule for the internalization of the receptor.

The findings that the mammalian GnRH-RII internalizes more rapidly and to a greater extent than the tail-less human GnRH-RI and that ß-arrestin can enhance internalization of the mammalian GnRH-RII and not the GnRH-RI were the impetus for examining the functional role of the GnRH-RII carboxyl-terminal tail. This was done by measuring internalization of receptor mutants with carboxyl-terminal truncations or substitutions of putative phosphorylation sites. The internalization assay measures internalized radiolabeled ligand as an index for receptor internalization and is calculated by determining the fraction of internalized ligand of total cell-associated ligand. Because this assay measures ratios of ligand-bound receptors, it is not affected by variations in receptor expression or affinity (as long as ligand binding is measurable). Examination of the role of the carboxyl terminus in the internalization of the GnRH-RII in COS cells identified two serine residues (S338,339) that when mutated to alanine residues, lead to a 75% reduction in internalization within the first 30 min of agonist stimulation compared with the WT receptor. In contrast, substituting these serine residues with glutamic acid residues that mimic a phosphorylated serine effectively restored internalization of the receptor. The enhanced internalization for a receptor carrying acidic substitutions over a receptor having alanine substitutions is a strong indication that serines 338 and 339 are putative phosphorylation sites that undergo phosphorylation that is required for efficient receptor internalization. Furthermore, the ability of the dominant-negative GRK2 to inhibit internalization of the intact type II GnRH receptor and not to inhibit internalization of receptor mutants lacking these serine residues (S338,339A and S338,339E) indicates that these residues (Ser 338 and 339) undergo phosphorylation by GRK before internalization. Together, these findings suggest that the internalization of type II GnRH receptor is mediated by GRK catalyzed phosphorylation of serine residues 338 and 339 in the carboxyl-terminal tail. Consistent with this notion, serines 338 and 339 are adjacent to acidic residues, which favor GRK2-mediated phosphorylation (45). Further support for the importance of these residues is the conservation of serine residues 338 and 339 among the human and other monkey species GnRH-RII receptors (3, 46). Interestingly, these residues are not conserved in the bullfrog type II receptor, which has a longer and different carboxyl terminus (47).

Although serine residues 338,339 in the carboxyl terminus are required for internalization of the GnRH-RII in COS cells, these residues are not required for recruitment of ß-arrestin because the mutant receptor lacking these putative phosphorylation sites (S338,339A) internalizes in a ß-arrestin-dependent manner. Similarly, a carboxyl-terminal truncation receptor mutant having a carboxyl-terminal tail of only nine residues and lacking any carboxyl-terminal tail putative phosphorylation sites (S335stop) internalizes weakly in COS cells but can also internalizes in a ß-arrestin-dependent manner. These findings demonstrate that domains of GnRH-RII required for ß-arrestin-independent internalization are distinct from domains required for ß-arrestin-dependent internalization. The finding that GnRH-RII does not require its carboxyl terminus for ß-arrestin-dependent internalization, whereas the tail-less type I GnRH receptor is unable to use ß-arrestin for internalization demonstrates the complex nature of receptor-ß-arrestin interaction. An obvious question is which region in GnRH-RII interacts with ß-arrestin. ICL3 of several receptors was found to bind ß-arrestin, and this binding was suggested to require basic residues located mainly in the amino terminus of ICL3 (48). Amino acid comparison between ICL3 of mammalian type I and type II GnRH receptors shows that only type II receptors have basic residues in the amino terminus of ICL3, and it is possible that these residues specify the GnRH-RII-ß-arrestin interaction.

The complexity of the ß-arrestin-receptor interaction is further exemplified by the diverse effects of casein kinase II (CKII) phosphorylation sites in the carboxyl-terminal tail on ß-arrestin-GPCR interactions. A previous report demonstrated that the addition of carboxyl-terminal tails to the tail-less mammalian type I GnRH receptor will confer ß-arrestin dependency if CKII phosphorylation sites are present and undergo phosphorylation (49). In contrast, we find that although both serine residues 338 and 339 are located in a consensus sequence for CKII phosphorylation, these serine residues are not required for ß-arrestin dependency. This contrasting role for CKII consensus sites in ß-arrestin dependency suggests that phosphorylation sites are not contact sites with ß-arrestin but rather function in stabilizing the ß-arrestin binding surface of the receptor that, possibly involves multiple domains of the receptor. Further support for the notion that receptor ß-arrestin interaction can be phosphorylation independent derives from studies done on the LH/choriogonadotropin receptor showing that ß-arrestin can interact with the receptor in a phosphorylation-independent manner (50, 51). However, receptor activation is required, suggesting that a conformational change of the receptor is critical for ß-arrestin interaction.

In summary, this study demonstrates that serine residues 338 and 339 in the carboxyl-terminal tail of a mammalian type II GnRH receptor are critical for the internalization of the receptor in COS-1 cells because they are putative GRK phosphorylation sites. The receptor internalization in COS cells requires GRK and dynamin but is ß-arrestin independent and proceeds via clathrin-coated pits and caveolae, which are apparently positioned in tandem. Finally, in contrast to the mammalian type I GnRH receptor that uniquely lacks a carboxyl-terminal tail, the type II GnRH receptor can also internalize in a ß-arrestin-dependent pathway; however, this dependency does not require serine residues 338 and 339 or the carboxyl-terminal tail.


   Acknowledgments
 
The authors thank M. G. Caron (Duke University Medical Center, Durham, NC), J. Eggermont (Catholic University, Leuven, Belgium), S. Maudsley (Human Reproductive Sciences Unit, Medical Research Council, Edinburgh, Scotland, UK) and Anna Aragay (Faculty of Medicine, Bergen University, Bergen, Norway) for providing plasmids encoding WT and/or dominant-negative mutants of GRK2 and 3, ß-arrestin1, ß-arrestin 2, dynamin 1, and [cav-1({Delta}1–81)].


   Footnotes
 
This work was supported by The Wellcome Trust, UK (to A.A.K.), the Medical Research Councils of South Africa and the United Kingdom (to A.A.K. and R.P.M.), and by the Austrian Ministry of Science (to K.R.).

Abbreviations: CKII, Casein kinase II; GnRH-RI, type I GnRH receptor; GnRH-RII, type II GnRH receptor; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; IP, inositol phosphates; ICL, intracellular loop; MDC, monodansylcadavarine; VIP1, vasoactive intestinal peptide 1; WT, wild-type.

Received January 22, 2004.

Accepted for publication June 8, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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