Mutations of the Conserved DRS Motif in the Second Intracellular Loop of the Gonadotropin-Releasing Hormone Receptor Affect Expression, Activation, and Internalization

Krishan K. Arora, Zhengyi Cheng and Kevin J. Catt

Endocrinology and Reproduction Research Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GnRH receptor is an unusual member of the G protein-coupled receptor (GPCR) superfamily with several unique features. One of these is a variant of the conserved DRY motif that is located at the junction of the third transmembrane domain and the second intracellular (2i) loop of most GPCRs. In the GnRH receptor, the Tyr residue of the conserved triplet is replaced by Ser, giving a DRS sequence. The aspartate and arginine residues of the triplet are highly conserved in almost all GPCRs. The functional importance of these residues was evaluated in wild type and mutant GnRH receptors expressed in COS-7 cells. Mutants in which Asp138 was replaced by Asn or Glu were poorly expressed, but showed significantly increased internalization and exhibited augmented inositol phosphate generation to maximal agonist stimulation compared with the wild type receptor. In contrast, receptors in which Arg139 was substituted with Gln, Ala, or Ser showed reduced internalization, and the GnRH-induced inositol phosphate response for the Arg139Gln mutant was significantly impaired in proportion to its low expression level. Replacing Ser140 with Ala affected neither internalization nor signal transduction. The role of the polar amino acids at the C terminus of the 2i loop was evaluated in two additional mutants (Ser151Ala, Ser153Ala, and Ser151Ala, Ser153Ala, Lys154Gln, Glu156Gln). Both of these mutants exhibited agonist-induced inositol phosphate responses similar to that of the wild type receptor, but showed increased receptor internalization. This mutational analysis indicates that the conserved Asp and Arg residues in the DRY/S triplet make important contributions to the structural integrity of the receptor and influence receptor expression, agonist-induced activation, and internalization.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hypothalamic decapeptide, GnRH, acts via its specific high-affinity receptors in the anterior pituitary gland to regulate the synthesis and secretion of FSH and LH and thus plays a pivotal role in reproduction (1, 2). The cloning of cDNAs for the GnRH receptors of several species, including mouse (3, 4), rat (5, 6, 7), sheep (8, 9), cow (10), and human (11, 12), has shown that the receptor exhibits more than 85% amino acid identity among species. The hydropathy analysis of the GnRH receptor-coding region reveals the presence of seven putative transmembrane domains (TM I-VII), indicating a similar topology to those proposed for the other members of the G protein-coupled receptor (GPCR) superfamily (13). However, the GnRH receptor has several unique features, including the absence of a cytoplasmic carboxyl-terminal tail, replacement of Tyr by Ser in the highly conserved DRY sequence located at the junction of TM III and the 2i loop, and the presence of a long and highly basic first intracellular loop. Another interesting feature is that the highly conserved Asp in TM II and Asn in TM VII of most GPCRs are reciprocally exchanged in the GnRH receptor (13, 14).

Mutagenesis and chimeric studies have suggested that the intracellular regions of the GPCRs, in particular the second and third intracellular (2i and 3i) loops and sometimes the cytoplasmic tail, interact with G proteins and mediate signal transduction (15, 16, 17, 18, 19). Sequence alignment of various members of the GPCR superfamily shows that the acidic (Asp) and basic (Arg) residues of the DRY triplet are highly conserved (15, 16). Whereas the Arg residue in the triplet is invariant, in a few instances the Asp and Tyr residues are conservatively substituted with other amino acids (15, 20). Based on their conservation, it has been proposed that these residues have important functions in ligand binding and/or G protein interaction and activation. In studies on the structure/function relationships of the GnRH receptor, we examined the roles of the conserved acidic residue Asp138, the invariant basic residue Arg139, and the unique Ser140 residue (which is Tyr in most other GPCRs) in agonist-induced signal transduction and receptor internalization. Few studies have explored the roles of specific amino acids in the carboxyl-terminal portion of the 2i loop of the GPCRs in signaling and internalization, and no consensus sequences have been identified. We therefore evaluated the importance of several polar residues (Ser151, Ser153, Lys154 and Glu156) in this region in these cellular processes (see Fig. 1Go) by first making multiple replacements (carboxyl-terminal double and quadruple mutations, referred to as c-DM and c-QM, respectively), to be followed by single substitutions if effects were found. These conserved and polar residues in the GnRH receptor were replaced with other amino acids by site-directed mutagenesis, and the expressed receptors were analyzed for ligand binding, GnRH-stimulated inositol phosphate production, and agonist-induced internalization of the receptor-hormone complex.



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Figure 1. Schematic Model of the GnRH Receptor Showing the Residues Mutated in This Study

The putative structure of the GnRH receptor, with cylinders representing transmembrane regions I–VII, is shown. The amino acid sequence of the 2i loop of the GnRH receptor is indicated. The consensus DRY sequence that is present in most of the GPCRs (15 ) is unique in the GnRH receptor, where Tyr is replaced by Ser. i and e Indicate intracellular and extracellular loops, respectively. The residues of the GnRH receptor that were mutated in the present study shown in bold are Asp138, Arg139, Ser140, Ser151, Ser153, Lys154, and Glu156.

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of 2i Loop Mutant GnRH Receptors in COS-7 Cells
Five substitution mutations were created in the 2i loop of the mouse GnRH receptor, as shown in Fig. 1Go. The conserved Asp residue at position 138 was changed to a neutral amino acid (Asn) to eliminate its negative charge, and to one that preserves the negative charge (Glu) but has a longer side chain. Replacement of Asp by Tyr or His was also performed, but these mutant receptors showed no radioligand binding, presumably due to lack of expression. The invariant Arg residue at position 139 was substituted with uncharged amino acids (Gln, Ala, and Ser), and with Lys to retain the same positive charge but with a longer side chain. The Lys139 receptor was poorly expressed and thus could not be further analyzed. Northern blot analysis of GnRH receptor mRNA in transfected COS-7 cells expressing mutant receptors revealed no reduction in transcript levels or molecular size as compared with the wild type receptor (data not shown), suggesting that the differences in cell-surface expression of the Asp138 and Arg139 mutant receptors do not reflect changes at the pretranslational level. The unique Ser140 was changed to Ala. The nonconserved polar amino acids in the carboxyl terminus of the 2i loop, namely Ser151, Ser153, Lys154, and Glu156, were replaced with Ala, Ala, Gln, and Gln, respectively.

[125I]GnRH-Ag binding was measured in intact COS-7 cells transfected with mutant or wild type GnRH receptors to determine the expression level and the functional integrity of these receptors at the plasma membrane. As indicated in Table 1Go, the wild type and all of the detectably expressed mutant receptors bound the radioligand with high affinity, and Scatchard analysis of the binding data yielded linear plots, reflecting a single class of GnRH-binding sites. Most of the modified receptors displayed similar dissociation constants, and the Asp138 mutants had slightly increased binding affinity. The expression levels of the DRS receptor mutants showed more significant variations. Although alanine replacement of Ser140 had no major effect on receptor expression, mutation of Asp138 and Arg139 in the DRS triplet reduced expression to approximately one-tenth and one-third to one-half of that of the wild type receptor, respectively (Table 1Go). However, receptors bearing double and quadruple mutations at the C-terminal end of the 2i loop (c-DM and c-QM) were expressed at almost the same level as the wild type receptor and displayed similar agonist-binding affinity.


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Table 1. Binding Characteristics of Wild Type and Mutant GnRH Receptors Expressed in COS-7 Cells

 
Effect of 2i Loop Mutations on GnRH-Mediated Inositol Phosphate Signaling
To determine the ability of the mutant receptors to couple to phospholipase C via Gq/G11 proteins, we measured the inositol phosphate response of transfected COS-7 cells stimulated with a maximal dose of GnRH in the presence of 10 mM LiCl. As reported previously (20), under these experimental conditions the major accumulated products of phosphoinositide hydrolysis in GnRH receptor-transfected COS-7 cells are inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3). Because the plasma membrane-binding sites of cells expressing the mutant GnRH receptors showed significant variations (Table 1Go), the correlation between cell-surface binding sites and the maximal inositol phosphate response was determined after COS-7 cells were transfected with increasing amounts of the wild type receptor cDNA. Despite the wide range of receptor expression in these cells, there was a near-linear relationship between the measured receptor sites and the inositol phosphate responses to GnRH stimulation (Fig. 2Go). Such linearity between cell-surface receptors and inositol phosphate responses has been also observed in COS-7 cells transfected with angiotensin II receptors (21). This finding indicates that valid comparisons between cells expressing mutant GnRH receptors can be made by normalizing their inositol phosphate responses to the number of plasma membrane-binding sites (Fig. 3BGo).



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Figure 2. Correlation between GnRH Receptor Expression Level and Inositol Phosphate Responses to GnRH Stimulation

COS-7 cells subcultured in 24-well plates were transfected with increasing amounts of wild type GnRH receptor cDNA (0.03–2.0 µg) using lipofectamine (7 µg/well). The total amount of plasmid DNA per well was kept constant at 2.0 µg by the addition of pcDNAI/Amp plasmid DNA. For inositol phosphate measurements, the cells were labeled for 24 h with [3H]inositol and stimulated with 100 nM GnRH in the presence of 10 mM LiCl. The extracellular GnRH receptor-binding sites were measured by analyzing [125I]GnRH-Ag displacement curves as described in Methods. The combined InsP2 and InsP3 responses are shown as means of duplicates from a representative experiment, with similar results from three independent experiments. Values shown were obtained using 0.015, 0.031, 0.062, 0.125, 0.25, and 0.50 µg DNA transfected per well.

 


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Figure 3. Effects of Site-Directed Mutations in the 2i Loop of GnRH Receptor on GnRH-Induced Inositol Phosphate Responses

COS-7 cells transiently expressing wild type (WT) or mutant GnRH receptors were labeled with [3H]inositol for 24 h, then preincubated in the presence of 10 mM LiCl for 30 min followed by 15 min of stimulation with 100 nM GnRH. Inositol phosphates were extracted and separated by anion exchange chromatography as described in Methods. Panel A shows the combined radioactivity (cpm) of the InsP2 and InsP3 fractions after incubation with (+) or without (-) GnRH. The data shown are means ± SE from three or more independent experiments, each performed in duplicate. Panel B shows the combined InsP2 and InsP3 responses normalized to the number of [125I]GnRH-Ag binding sites. These data were calculated after subtracting the respective basal levels in nonstimulated cells and are expressed as percent of the wild type receptor response, which was 14,270 ± 600 cpm/pmol binding sites (n = 3). For values shown in panel B, the SEs were less than 10% of the mean.

 
The GnRH-induced inositol phosphate responses mediated by each of the mutant GnRH receptors were measured after maximal agonist stimulation with 100 nM GnRH. Except for the D138E and R139Q mutants, the inositol phosphate responses of cells expressing the mutant GnRH receptors were similar to those of the wild type receptor (Fig. 3AGo). Normalization of the data based on receptor number showed that the impaired responses of cells transfected with the D138E (or D138N) receptor were attributable to their lower expression level, and that these mutants, in fact, activated phospholipase C more effectively than the wild type receptor (Fig. 3BGo). In contrast, maximal inositol phosphate signaling by the R139Q receptor was found to be significantly impaired (by about 50%) even when the data were normalized for the reduced number of binding sites (Fig. 3BGo). Cells transfected with the R139S and R139A receptors also exhibited significantly reduced (55–65% of the wild type) inositol phosphate production after normalization for receptor number (not shown). The InsP2/InsP3 responses mediated by the S140A, c-DM, and c-QM mutant receptors were similar to that of the wild type receptor (Fig. 3Go, A and B).

Effect of Guanosine Thiotriphosphate (GTP{gamma}S) on [125I]GnRH Agonist Binding to Wild Type and Mutant GnRH Receptors
The ability of the Asp138 and Arg139 mutant receptors to interact with G proteins was further evaluated by measuring the effect of GTP{gamma}S on [125I]GnRH agonist binding to COS-7 cell membranes expressing wild type, D138N, and R139Q mutant receptors. As shown in Fig. 4Go, treatment with GTP{gamma}S reduced agonist binding to the wild type receptor by about 55%. This reduction in agonist binding was due to a decrease in the affinity of the receptor for GnRH and reflects the normal coupling of the activated receptor to G protein(s). The inhibitory effect of the GTP analog on agonist binding to the D138N receptor was essentially the same as for the wild type receptor (Fig. 4Go). However, GTP{gamma}S had relatively little effect on agonist binding to the R139Q receptor (Fig. 4Go), consistent with the impaired ability of this mutant to mediate inositol phosphate production in response to GnRH stimulation (Fig. 3BGo).



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Figure 4. Effects of GTP{gamma}S on [125I]GnRH-Ag Binding to Wild Type and Mutant GnRH Receptors

Binding to crude membranes prepared from transfected COS-7 cells expressing wild type, D138N, or R139Q receptors was performed in the absence or presence of increasing concentrations of GTP{gamma}S as described in Methods. Results are expressed as percent of the binding determined in the absence of GTP{gamma}S (Bo) and are shown as means of values obtained from two separate experiments, each performed in duplicate.

 
Effect of 2i Loop Mutations on GnRH Receptor Internalization
GnRH receptors expressed in COS-7 cells undergo ligand-induced internalization, similar to that of the native receptors in pituitary gonadotrophs and {alpha}T3–1 cells (22, 23). The effects of mutations on receptor internalization were evaluated by measuring the kinetics of [125I]GnRH-Ag uptake over a period of 60 min at 37 C in cells expressing wild type or mutant receptors. A direct comparison between the wild type and mutant receptors was made by plotting the percent of bound radioligand that was internalized with increasing time of incubation (see Fig. 5Go, A-D). Single replacements of Asp138 by Asn or Glu increased the rate of internalization (Fig. 5AGo), and the sequestration of radioligand at 60 min was at least 100% higher than that of the wild type receptor (Fig. 5EGo). The endocytotic rate constant, a cellular constant that defines the probability of an occupied receptor being internalized in 1 min at 37 C, was also calculated using these data. Values for D138N and D139E receptors were 200–250% higher than that of the wild type receptor. On the other hand, mutation of Arg139 to Gln, Ala, or Ser caused much slower internalization (Fig. 5BGo), and the amount of tracer sequestered after 60 min was only 47% of that of the wild type receptor (Fig. 5EGo). The endocytotic rate constants were 20% of that of the wild type receptor. The kinetics and rate constant for the internalization of S140A receptors were virtually identical to that of the wild type receptor (Fig. 5Go, C and E). The internalization kinetics of the c-DM and c-QM receptors were rapid compared with the wild type receptor (Fig. 5DGo), and the amounts sequestered at 60 min were 28% and 56% higher, respectively, than that of the wild type receptor (Fig. 5EGo). The endocytotic rate constants for the c-DM and c-QM receptors were 50–150% increased compared with the wild type receptor.



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Figure 5. Effects of Site-Directed Mutations in the 2i Loop on Internalization of GnRH Receptors

Wild type or mutant GnRH receptors were transiently expressed in COS-7 cells, and the internalization kinetics of [125I]GnRH-Ag/receptor complexes were measured at 37 C as described in Methods. Panels A through D show the time course of internalization of the radioligand by wild type (WT) and mutant receptors. Values are expressed as percent of total binding for each time point and are means ± SE from three or more independent experiments, each performed in triplicate. In panel E, data on internalization at 60 min for mutants (from panels A, B, C, or D), expressed as percent of the internalization of the wild type receptor, are shown as bar graphs. In this study, 27.1 ± 0.9% (n = 4) of the radioligand bound to the wild type receptors was internalized after 60 min.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The present mutational analysis of residues in the amino- and carboxyl-terminal portions of the 2i loop in the GnRH receptor, summarized in Table 2Go, has revealed that in addition to their structural role, these regions are functionally important determinants of receptor expression, signaling, and internalization. Our findings indicate that mutations of residues Asp138, Arg139, Ser151, Ser153, Lys154, and Glu156 alter agonist-induced internalization of the GnRH receptor. Most of these mutations, including replacement of Asp138 and the combined mutations (c-DM and c-QM), increased the endocytotic rate constants by 50–250%. On the other hand, substitution of Arg139 caused a significant reduction in the rate of receptor internalization (Fig. 5EGo). Agonist-induced signal transduction, measured as the stimulation of inositol phosphate production by GnRH, was largely unaffected by mutations that increased receptor endocytosis. Conversely, mutations that reduced internalization showed impaired signal transducing ability (Fig. 3Go). The latter findings are consistent with our recent report (20) that mutation of a highly conserved hydrophobic amino acid (Leu147) in the 2i loop significantly impaired both receptor-G protein coupling and receptor internalization.


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Table 2. Summary of Functional Properties of Mutant GnRH Receptors

 
In contrast, the ligand binding, agonist-induced internalization, and signal transduction properties of the S140A receptor, with a mutation adjacent to Arg139, were indistinguishable from those of the wild type receptor, suggesting that the effects of the above mutations are not nonspecific. Neither the double nor quadruple mutations of nonconserved residues in the C-terminal region of the 2i loop had a major effect on signal transduction, indicating that this locus is not important for coupling of GnRH receptor to its cognate G protein(s). However, in the TSH (24) and angiotensin II (25) receptors, the carboxyl-terminal region of 2i loop was found to be important in mediating signal transduction.

Another interesting observation was the significantly reduced expression of receptors bearing Asp138 and Arg139 mutations (Table 1Go). The low radioligand binding capacity of cells expressing these mutant receptors could be due to decreased receptor expression or to deficient localization of the receptors at the cell surface. This possibility cannot be tested in the absence of a suitable permeant ligand or a highly specific GnRH receptor antibody. However, the agonist-binding affinity of the Asp138 mutants was increased rather than decreased and was largely unchanged for the Arg139Gln receptors, indicating that these changes did not alter the integrity of the receptor. These results also suggest that the impairment of signal generation by the Arg139 mutant receptor was not caused by reduction of binding affinity.

There is increasing evidence for the concept that positively charged amino acids, located near the boundaries of transmembrane domains, are important determinants of the topology of membrane-spanning proteins (26, 27, 28). Because the Asp138 and Arg139 residues are located at the boundary of the third transmembrane domain and the 2i loop, it is probable that mutation of either residue disturbs the charge balance in this region. This in turn could disrupt interactions and destabilize helix formation and thereby exert a deleterious effect on receptor expression. The acidic and basic residues in the DRY triplet are conserved in almost all GPCRs and are located in a region that terminates as an {alpha}-helical structure that forms an extension of the third transmembrane domain (29). The positively charged Arg residue may participate in ionic interactions with water, G proteins, and/or charged lipid head groups. The negatively charged Asp residue could likewise interact with G protein(s) during receptor activation.

The highly conserved Arg residue in the DRY triplet has been shown to participate in G protein coupling. In a recent study (30), a point mutation of this residue in the m1 muscarinic receptor abolished its ability to mediate inositol phosphate production and binding of a labeled GTP analog. Mutation of the corresponding Arg residue in the m2 muscarinic receptor resulted in partial loss of receptor-G protein coupling in terms of inhibiting cAMP formation. Likewise, mutation of the corresponding Arg residue in the N-formyl peptide receptor impaired ligand binding and its ability to mobilize calcium (31). In the present study, we also observed a significant reduction in the ability of Arg139 mutants to mediate inositol phosphate formation during GnRH stimulation. The invariant Arg residue in the GnRH receptor has been suggested to be part of a conserved structural motif (I/LxxDRY/SxxI/V) in which the flanking ß-branched amino acid residues provide a hydrophobic cage that restricts its rotamer positions, thus enabling it to achieve the most favorable orientation(s) required for efficient G protein coupling (32). In regard to the structural basis of coupling specificity, this is possibly determined by cooperation between multiple regions of the receptor (15, 16, 17, 18, 19, 33).

In contrast to the above findings, no effects of Asp138 mutations on GnRH receptor signaling were observed. Previous studies have suggested an important role for the corresponding aspartate residue in the function of the {alpha}2A-adrenergic (34, 35), ß2-adrenergic (36), and m1 muscarinic (37) receptors. In these reports, substitution of aspartate by asparagine had no influence on high-affinity agonist binding, but significantly reduced the ability of the mutant receptors to couple to their respective G protein/effector systems. The effects of mutation of the corresponding Glu residue in rhodopsin have likewise been attributed to impaired interaction with its G protein, transducin (38). Studies on the TSH (24) and angiotensin II (25) receptors showed that multiple mutations in the DRY region resulted in complete abolition of G protein coupling. On the other hand, mutation of the corresponding single acidic residue (Glu to Asp or Asn) in the LH/CG receptor did not affect either hormone binding or signal transduction (39). The present finding that mutation of Asp138 to Asn or Glu does not impair GnRH receptor function is in general agreement with the data reported for the LH/CG receptor and argues against the generality of an essential role of this residue in signal transduction.

Several techniques, including mutagenesis, synthetic peptides, and antibodies, have been employed in the past to probe for signaling function(s) in the intracellular loops of various GPCRs. Although no general consensus has been reached concerning the location and nature of the intracellular determinants of receptor-G protein activation, one or more of several intracellular regions have been implicated in various GPCRs (15, 16, 19, 33). For example, studies on ß1-adrenergic, muscarinic, and rhodopsin receptors have shown that the amino- and carboxyl-terminal regions of the 3i loop, and often the amino-terminal region of the cytoplasmic tail, are important for coupling to G proteins (19, 33, 40). In the TSH receptor, the 1i loop and the carboxyl-terminal regions of the 2i and 3i loops are involved in signal transduction (24). These results suggest that 1) GPCRs contain multisite, noncontiguous intracellular determinants of agonist-induced receptor signaling; and 2) the presence and location of the regions involved in G protein coupling vary among individual receptors. It has been postulated that regions of the carboxyl-terminal tail, and the 2i and 3i loops adjacent to the transmembrane domains, may form amphipathic {alpha}-helices. These, together with charged residues in the 2i and 3i loops, i.e. DRY (or DRS in the GnRH receptor), may interact cooperatively to permit efficient binding to G proteins and their subsequent activation (15).

Our findings on receptor internalization indicate that single-point mutations of Asp138 to Asn or Glu increase receptor endocytosis by 100%, whereas mutation of the adjacent Arg139 residue to Gln, Ala, or Ser reduce receptor endocytosis by almost 50%. The critical importance of Ser/Thr-rich sequences in the 3i loop or carboxyl-terminal tail of TRH (41), gastrin-releasing peptide (GRP) (42), muscarinic (43), and yeast {alpha}-factor receptors (44), in maintaining efficient internalization has been demonstrated. Also, in ß2-adrenergic (45) and GnRH (46) receptors, the aromatic amino acid in the NPXXY sequence (or its variant) in the seventh transmembrane domain was shown to be important in receptor internalization. The present results suggest that the Asp and Arg residues in the Asp-Arg-Ser triad are directly or indirectly involved in GnRH receptor internalization. Because the GnRH receptor has no Ser/Thr-rich regions in any of its intracellular loops and lacks a cytoplasmic tail, other residues or as yet unidentified motifs must be involved in its internalization. Recently, a Ser-Thr-Leu sequence in the carboxyl-terminal region of the angiotensin II receptor has been shown to be essential for agonist-induced endocytosis (47). In receptors for PTH and PTH-related protein, which lack the conserved NPXXY sequence, both positive and negative regulatory elements for internalization are present in the carboxyl-terminal tail (48). The nature of the regions/residues that are involved in the internalization of the GnRH receptor will be investigated in future studies to provide more information about the structural basis for its internalization. The impaired internalization of the Arg139 mutant receptors suggests that more than one region of the GnRH receptor may be involved in internalization and/or that more than one internalization pathway exists.

In summary, this analysis of the functional significance of the residues in the Asp-Arg-Ser triplet located in the N-terminal region of the 2i loop in the GnRH receptor has provided evidence for the importance of the aspartate and arginine residues in GnRH receptor signaling and internalization. Replacement of conserved Asp138 with either Asn or Glu reduced expression levels, but the mutant receptors showed increased agonist-induced internalization and activated phospholipase C more effectively than the wild type receptor. The invariant Arg139 residue appears to play an important role both in receptor-G protein coupling and in agonist-induced internalization of the receptor. The highly conserved nature of the Asp and Arg residues in this region in almost all members of the GPCR superfamily suggests that these residues are of general importance in receptor function.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
The cDNA encoding the mouse GnRH receptor was isolated by expression cloning in pCDM8 as previously reported (4). The expression vector pcDNAI/Amp was obtained from Invitrogen (San Diego, CA). GnRH native and its agonist, des-Gly10-[D-Ala6]GnRH N-ethylamide (GnRH-Ag), were obtained from Peninsula Laboratories Inc. (Belmont, CA). Lipofectamine and OPTI-MEM media were purchased from Life Technologies, Inc. (Gaithersburg, MD); cell culture-related products were obtained from Biofluids (Rockville, MD); restriction and DNA-modifying enzymes were from New England BioLabs (Beverly, MA); and Sequenase II was purchased from US Biochemical Corp (Cleveland, OH). Oligonucleotide primers for site-directed mutagenesis were synthesized in a Beckman Oligo 1000 DNA Synthesizer (Beckman Instruments Inc., Fullerton, CA). The Muta-Gene phagemid in vitro mutagenesis kit (Version 2) was obtained from Bio-Rad (Hercules, CA) and was used according to the manufacturer’s instructions. AG-1-X8 resin (100–200 mesh formate form) and the Poly-Prep Chromatography Columns for anion exchange chromatography were also obtained from Bio-Rad. All other reagents were of HPLC or analytical grade quality. myo-[3H]inositol (80–100 Ci/mmol) was from Amersham Corp (Arlington Heights, IL). 125I-des-Gly10-[D-Ala6]GnRH N-ethylamide (125I-GnRH-Ag) was prepared by Hazleton Laboratories (Vienna, VA).

Methods
Construction of Wild Type and Mutant GnRH Receptors
The 1.22-kb GnRH receptor cDNA subcloned into pcDNAI/Amp at the XbaI site (20) was used as a template for creating site-directed mutations according to the method of Kunkel et al (49) using a Muta-Gene phagemid in vitro mutagenesis kit. The sequence of the 25 mer mutagenic primer for Asp138 was 5'-GATTAGCCTGGAG/GAACGCTCCCTGGCC-3'; at the underlined bases, codon GAC for Asp was replaced with either GAG (for Glu) or GAA (for Asn). For Arg139, the 28 mer mutagenic primer was: 5' GATTAGCCTGGACCAG/GCC/AGC TCCCTGGCCATC-3'; at the underlined bases the codon CGC for Arg was altered to CAG for Gln, GCC for Ala, or AGC for Ser. These mutations were performed using separate primers. For Ser140, the 20 mer mutagenic primer was: 5'-CCTGGACCGCGCCCTGGCCA-3'; at the underlined bases the codon TCC for Ser was altered to GCC for Ala. For the double mutation (Ser151, Ser153) at the carboxyl-terminal (c-DM), the 49 mer mutagenic primer was: 5'-CCCCTTGCTGTACAAGCCAACGCCAAGCTTGAACAGTCTATGATCAGC-C-3'; at the underlined bases the codons AGC for Ser were altered to GCC for Ala. For the quadruple mutation (Ser151, Ser153, Lys154, Glu156) at the carboxyl terminus (c-QM), the 49 mer mutagenic primer was: 5'-CCCCTTGCTGTACAA-GCCAACGCCCAGCTTCAACAGTCTATGATCAGCC-3'; at the underlined bases the codons AGC for Ser were altered to GCC for Ala, the codon AAG for Lys was replaced with CAG for Gln, and the codon GAA for Glu was changed to CAA for Gln. Mutations were confirmed by the dideoxy sequencing method of Sanger et al. (50) using Sequenase II.

Transient Transfection in COS-7 Cells
Wild type and mutant GnRH receptors were transiently expressed in COS-7 cells. To measure inositol phosphate responses and internalization kinetics, or [125I]GnRH-Ag binding to intact cells, the cells were seeded in 24-well plates (Costar, Cambridge, MA) at a density of 4 x 104 cells per well and cultured in DMEM supplemented with 10% heat-inactivated FBS containing 100 U/ml of penicillin and 100 µg/ml streptomycin (Pen-Strep) at 37 C in an atmosphere consisting of 5% CO2-95% humidified air. At 60–70% confluence, the cells were transfected in 0.5 ml of serum-free OPTI-MEM I medium with 1 µg of wild type or mutant plasmid DNA and 6–8 µg lipofectamine per well. For membrane-binding studies, 1 x 106 cells were cultured in 100-mm petri dishes for 3 days. Transfections were performed for 6 h using 5 ml OPTI-MEM I containing 10 µg plasmid DNA and 16 µg/ml lipofectamine. Six hours later, the medium was replaced with fresh medium and cultures were maintained for 48 h before use in ligand binding, membrane preparation, and functional assays.

Agonist Binding to Transfected Cells
The binding affinity and sites of the mutant receptors were determined in transfected COS-7 cells incubated with 2 nM [125I]GnRH-Ag in binding medium (M199 containing 25 mM HEPES and 0.1% BSA), in the absence or presence of increasing concentrations of unlabeled peptide for 4 h at 4 C. The cells were rapidly washed twice with ice-cold PBS (pH 7.4) and solubilized in 0.2 M NaOH-1% SDS solution, and the cell-associated radioactivity was measured by {gamma}-spectrometry. All time studies were performed in duplicate on at least three occasions, and displacement curves were analyzed by the LIGAND program (obtained from Dr. Peter J. Munson, National Institutes of Health, Bethesda, MD) using a one-site model (51).

Internalization Assays
Transfected COS-7 cells were washed once with binding medium before the addition of 2 nM 125I-labeled GnRH agonist. Nonspecific binding was determined in the presence of 1000-fold excess of the unlabeled GnRH agonist. After incubation at 37 C for the indicated times, the cells were washed twice with ice-cold PBS (pH 7.4) and incubated with 1 ml of 50 mM acetic acid-150 mM NaCl (pH 2.8) for 12 min to remove surface-bound tracer. The acid-released radioactivity was collected to determine the receptor-bound radioactivity, and the internalized (acid-resistant) radioactivity was quantitated after solubilizing the cells in NaOH-SDS solution. Radioactivities were measured by {gamma}-spectrometry, and the internalized radioligand at each timepoint was expressed as a percent of the total (acid-resistant + acid-released) binding. The endocytotic rate constant (52) was calculated using algorithms obtained from Dr. H. Steven Wiley (University of Utah Medical Center, Salt Lake City, UT). For these calculations, correction values of 4% and 10% were used for surface to intracellular, and intracellular to surface spillover, respectively. Values for endocytotic rate constant for the wild type and various mutant receptors were: wild type, 0.010; D138E/N, 0.031–0.038; R139Q/S/A, 0.002; c-DM, 0.015; c-QM, 0.025.

Radioligand Binding to COS-7 Cell Membranes
Transfected cells were washed twice with ice-cold 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA and scraped into 1 ml of the same medium. Cells were lysed by freezing and thawing, and crude membranes were prepared by centrifuging the samples at 16,000 x g. The pellets were washed in the same medium, and the protein content was measured by the BCA method (Pierce Chemical Co., Rockford, IL). Radioligand-binding assays were conducted using 20- to 25-µg membranes in the presence or absence of GTP{gamma}S (20). The bound radioactivity was separated by rapid filtration followed by three washes with ice-cold PBS (pH 7.4) and measured by {gamma}-spectrometry.

Inositol Phosphate Production
COS-7 cells were labeled 24 h after transfection by incubation in inositol-free DMEM medium containing 20 µCi/ml [3H]inositol as described previously (20). After 24 h of labeling, cells were washed with inositol-free M199 medium and preincubated in the same medium containing 10 mM LiCl for 30 min at 37 C, then stimulated with 100 nM GnRH for 15 min. Incubations were terminated by the addition of ice-cold perchloric acid [5% (vol/vol) final concentration]. The inositol phosphates were extracted as described previously (53) and separated by anion exchange chromatography. Briefly, after neutralization, the samples were applied to Poly-Prep columns containing Dowex AG 1-X-8 resin. The columns were washed four times with water (3 ml/wash) and twice with 0.2 M ammonium formate in 0.1 M formic acid (3 ml/wash) to remove inositol and inositol monophosphate, respectively. Then, InsP2 + InsP3 fractions were eluted from the columns by washing twice with 1 M ammonium formate in 0.1 M formic acid (3 ml/wash), and their radioactivities were measured by liquid scintillation ß-spectrometry. The mean inositol phosphate formation of cells expressing mutant receptors was expressed as a percentage of that mediated by the wild type receptor in the same experiment.


    FOOTNOTES
 
Address requests for reprints to: Kevin J. Catt, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Building 49, Room 6A36, National Institutes of Health, Bethesda, Maryland 20892.

Received for publication October 25, 1996. Revision received April 17, 1997. Accepted for publication May 6, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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