Insertional Mutagenesis of the Arginine Cage Domain of the Gonadotropin-Releasing Hormone Receptor

Smiljka Kitanovic, Tony Yuen, Colleen A. Flanagan1, Barbara J. Ebersole and Stuart C. Sealfon

Fishberg Research Center for Neurobiology (S.K., T.Y., S.C.S.) and Department of Neurology (C.A.F., B.J.E., S.C.S.) Mount Sinai School of Medicine New York, New York 10029


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pattern of side-chain conservation at the cytoplasmic side of the third transmembrane domain of rhodopsin family G protein-coupled receptors, Asp/Glu-Arg-Tyr/X-X-X-Ile/Val, defines a structural "arginine cage" domain. Previous computational and mutagenesis studies of the GnRH receptor indicated an important contribution of local interactions to the function of this domain. We have investigated the functional importance of the intrahelical position and orientation of the arginine cage using insertional mutagenesis. Introduction of a single Ala proximal to the conserved Asp-Arg of this domain caused loss of detectable ligand binding. Inserting a second Ala, however, restored high-affinity agonist binding. Further insertion of three or four Ala residues at this site generated receptors that bound agonist with an affinity 3- to 10-fold higher than that of the wild-type receptor. Loss of detectable coupling to inositol phosphate turnover in all these mutant receptors confirms that the structure required in this region for efficient signaling is highly constrained. In contrast, the recovery of agonist binding with the progressive insertion of two to four Ala residues indicates that specific orientations of this segment can stabilize high-affinity receptor conformations that are uncoupled from signal transduction.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GnRH receptor (GnRHR) belongs to the large family of G-protein coupled receptors (GPCRs). Members of this protein group differ in their activators (neurotransmitter, hormone, light), as well as in their downstream signaling pathways. However, all rhodopsin-like GPCRs possess highly conserved residues that are important for contributing to normal receptor structure and function. Because one function shared by all GPCRs is the transition between active and inactive receptor states, it is likely that many of the highly conserved receptor side chains contribute to the structural mechanisms that mediate this transition.

The highly conserved Asp/Glu-Arg-Tyr/X motif in helix 3 of rhodopsin-like GPCRs is a hallmark of this large receptor family. Studies of this motif in various rhodopsin-like GPCRs support the hypothesis that conserved side chains contribute to receptor activation. Mutation of the absolutely conserved Arg 3.50 in many receptors either diminished or abolished signaling (see Materials and Methods for consensus numbering scheme) in many receptors (1, 2, 3, 4, 5). An Arg3.50Ala substitution in the oxytocin receptor was constitutively active (6). Similarly varying effects on the function of different GPCRs have been reported with mutations of Asp 3.49. In several receptors, mutations of Asp 3.49 lead to increased affinity for agonists, while receptor activation is either enhanced (constitutively active receptors) (7), unaffected (8), or undetectable (9, 10). These differing effects on activation suggest that this domain may contribute to the stabilization of both active and inactive conformers of these receptors.

We have previously studied the structure and function of this motif (Asp-Arg-Ser) in the GnRHR using molecular modeling and site-directed mutagenesis. Our study identified this segment as a component of a larger, highly conserved "arginine cage domain," Asp/Glu-Arg-Tyr/X-X-X-Ile/Val (1). Computational modeling shows that a clash between the side chains of Arg 3.50 and Ile 3.54 restricts the orientations of the conserved Arg 3.50 side chain during a repositioning that occurs with receptor activation (1). The steric constraint, provided by the ß-branched character of the Ile 3.54 side chain, supports efficient receptor activation (1). Such an interaction is feasible, providing that the branched residue at position 3.54 is located one helical turn below Arg 3.50 (1). The recent crystal structure of rhodopsin in the inactive conformation confirms the presence of these specific side-chain interactions among conserved residues that we had identified in the GnRHR (11). The study of the arginine cage domain demonstrated the importance of the relative positioning of conserved side chains for normal receptor function.

We have now investigated the functional effects of rotating and displacing the arginine cage domain in the GnRHR using an insertional mutagenesis strategy. Ala residues have been sequentially inserted to displace and rotate the distal segment of the helix, and the effects of this alteration on receptor function have been investigated.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Agonist Binding to Ala Insertional Mutants
GnRH and [D-Ala6, Pro9-NHEt]-GnRH (GnRH-A) binding to the wild-type (WT) mouse GnRHR and Ala insertional mutants was characterized in transfected COS-1 cells. Introduction of one Ala just proximal to the Asp-Arg-Ser in the GnRHR (mutant ADRS) eliminated detectable agonist binding in both the whole-cell (Fig. 1Go and Table 1Go) and membrane binding assays (data not shown). However, the loss of binding was restored by insertion of additional alanines in mutants AADRS, AAADRS, and AAAADRS (Fig. 1Go and Table 1Go). The affinities of GnRH for the AADRS and WT receptors were comparable. In contrast, the AAADRS and AAAADRS mutants exhibited increased affinity for GnRH and GnRH-A. The changes in affinity for these constructs were greater when assayed at 4 C than at 22 C (Table 1Go; see Discussion).



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Figure 1. Binding to WT and Mutant Receptors

Representative whole-cell binding experiment with COS-1 cells transfected WT GnRHR ({Delta}) and ADRS (O), AADRS ({diamond}), AAADRS ({blacktriangleup}), and AAAADRS ({bullet}) insertional mutant receptors. Competition binding of GnRH was performed at 4 C. Data are represented as mean ± SEM. Nonspecific binding, determined with 1 µM GnRH, ranged from 233 to 340 cpm per well for these constructs. Most error bars are smaller than the markers used for each binding curve. Inset shows mutant binding curves at increased scale.

 

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Table 1. Agonist Binding and Activation of WT and Mutant Mouse GnRHRs

 
Expression of Epitope-Tagged WT and ADRS Receptors in COS-1 Cells
To determine whether the ADRS mutant, which had no detectable agonist binding, was expressed in the membranes of transiently transfected COS-1 cells, an epitope tag attached to a C-terminal tail segment was incorporated into ADRS and WT mouse GnRHRs to generate the mutants t-ADRS and t-WT, respectively (see Materials and Methods). Whole-cell binding and inositol phosphate (IP) assays confirmed that the t-ADRS has a phenotype similar to the untagged ADRS mutant, with no detectable binding or activation of the IP signaling pathway (Tables 1Go and 2Go). To determine whether the t-ADRS mutant was synthesized, immunoblot analysis was performed using both glycosylated and deglycosylated membranes. The t-ADRS mutant was present at lower intensity than the WT receptor. The deglycosylated mutant appeared to be full length, and the mutant had a similar pattern of glycosylation to that of the WT receptor (Fig. 2Go).


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Table 2. Binding and Activation of Carboxyl-Terminal Tailed Receptors

 


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Figure 2. Immunoblot of GnRHR Constructs

COS-1 cells were transfected with epitope-tagged WT and mutant GnRHR constructs. Cell membranes were solubilized, electrophoresed, and transferred to nitrocellulose. Epitope-tagged receptors were detected with an antibody generated against tetrahistidine. The four samples on the right were treated with N-glycosidase F. Equal amounts of protein (40 µg) were added to each lane.

 
Agonist-Stimulated Second Messenger IP Production of Alanine Insertional Mutants
None of the mutants containing Ala insertions preceding Asp-Arg-Ser mediated detectable IP production after GnRH stimulation (Fig. 3Go and Table 1Go). Basal levels of IP accumulation in cells expressing the mutant and WT receptors were comparable (see Fig. 3Go). Basal IP accumulation was also equivalent after transfection of WT receptor and vector alone (data not shown). Thus, neither the WT receptor nor any of the mutants possesses detectable constitutive activity.



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Figure 3. GnRH-Stimulated IP Accumulation

COS-1 cells were transfected with WT GnRHR ({Delta}) and ADRS (O), AADRS ({diamond}), AAADRS ({blacktriangleup}), AAAADRS ({bullet}), and DRSAAA ({square}) mutant receptors. Data are the mean ± SEM of a representative experiment performed in triplicate. For these constructs, basal levels of IP production (cpm per well), are as follows: WT, 1,148; ADRS, 980; AADRS, 804; AAADRS, 964; AAAADRS, 1,052; and DRSAAA, 978.

 
We have previously demonstrated the importance of Ile 3.54 in restricting the movement of the Arg 3.50 side chain and in contributing to efficient receptor activation (1). The insertional mutagenesis alters the position of both Asp-Arg-Ser and distal residues, including Ile 3.54. To discriminate the contribution of displacement of Asp-Arg-Ser from that of the distal residues to the phenotype of the AAADRS mutant, we studied the effect of displacing only the distal residues by inserting three alanines distal to Asp-Arg-Ser. The DRSAAA mutant had an affinity for GnRH comparable to that of the WT receptor (Table 1Go). Unlike the AAADRS mutant, the DRSAAA receptor mediated agonist-stimulated IP production, albeit at a reduced level in comparison with the WT receptor (Fig. 3Go and Table 1Go). The coupling of the DRSAAA mutant was similar to that of the Ile3.54->Ala mutant previously reported (1).

Effects of GTP Analogs on Agonist Binding
Lack of IP stimulation may be due to the failure of mutant receptors to interact with Gq/11. It is also possible that high-affinity agonist binding of these receptors reflects their interaction with another type of G protein. To study the potential of insertional mutants to interact with G proteins other than Gq/11, the effect of guanine nucleotides on agonist binding was assessed. For these studies, the epitope-tagged WT and AAADRS mutant receptors containing additional C-terminal sequence were used because this modification was previously found to enhance receptor expression (12) and was therefore expected to improve the signal-to-noise ratio in these assays. The increase in tagged WT and AAADRS GnRHR protein expression was reflected in elevated maximum binding (Bmax) values for both constructs (Table 2Go). However, the functions of receptors t-WT and t-AAADRS were unchanged, as assessed by agonist binding affinity and coupling, which were comparable with the untagged WT and AAADRS constructs, respectively (Table 2Go). Because the modification did not alter receptor function, these constructs were used to study the effects of GTP analogs on agonist binding.

In COS-1 membrane preparations, increasing concentrations of GTP analogs [5'-guanylylimidodiphosphate (GppNHp) and guanosine-5'-O-(3-thiotriphosphate (GTP{gamma}S)] decreased the specific binding of [125I] GnRH-A to t-WT GnRHR (Fig. 4Go, A and B). This is consistent with a productive interaction between the receptor and cytosolic G proteins: the stimulated G protein dissociates from the receptor, decreases receptor affinity for agonist, and leads to the loss of detectable high-affinity agonist binding sites, as previously reported for the bovine pituitary GnRHR at 37 C (17). In contrast, under the same conditions, the specific binding of [125I] GnRH-A to t-AAADRS was not affected by either GppNHp (Fig. 4AGo) or GTP{gamma}S (Fig. 4BGo). In control experiments, 5'-adenylylimidodiphosphate (AppNHp) (Fig. 4CGo) and 1 µM adenosine-5'-O-(3-thiothriphosphate) (ATP{gamma}S) (data not shown) had no effect on the binding of radiolabeled agonist to either t-WT or t-AAADRS GnRHRs. These data indicate that the effects of GTP analogs on the WT receptor were specifically mediated through G proteins and that binding of agonist to the alanine insertional mutant is not affected by guanine nucleotides.



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Figure 4. Effect of Nucleotide on Agonist Binding

Specific binding of agonist [125I] GnRH-A to t-WT GnRHR (dark bars) and mutant t-AAADRS (light bars) in membranes at 37 C in the presence of increasing concentrations of GppNHp (A), GTP{gamma}S (B), and AppNHp (C). In these representative experiments, data obtained from triplicate points (mean ± SEM) are presented relative to the specific binding of agonist in the absence of nucleotides. The cpm values obtained in triplicate ± SEM in this experiment in the absence of nucleotide were for the GppNHp study, t-WT: 3,208 ± 68, 288 ± 50; M: 1,962 ± 24, 330 ± 15; for the GTP{gamma}S study, t-WT: 4,446±55, 239 ± 18; M: 1,454 ± 24, 331 ± 30; for the AppNHp study, t-WT: 3,738 ± 72, 351 ± 16; M: 1,805 ± 30, 284 ± 16 with the total and nonspecific values separated by commas and M representing the t-AAADRS mutant. Each experiment was replicated at least three times.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The functional role of the distal segment of helix 3 in the GnRHR has been studied using insertional mutagenesis, an approach previously used for studying the muscarinic receptor (13). The periodic nature of side chain conservation in the arginine cage domain (1) and the results of spin-labeling studies performed on solubilized rhodopsin (14) support an {alpha}-helical secondary structure of the arginine cage domain, Asp/Glu-Arg-Tyr/X-X-X-Ile/Val. Assuming an {alpha}-helical structure of this domain, in each of the mutant receptors the inserted alanines would be expected to progressively rotate the side chains of subsequent residues of the domain relative to other transmembrane domains (TMDs), as well as to displace them toward the cytosol (Fig. 5Go).



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Figure 5. Proposed Rotation and Extension of Distal Helix 3 with Insertions

The cytoplasmic end of the helix 3 backbone is shown in yellow. Most side chains are omitted for clarity. The Ile3.46 side chain (gray) is included to provide a point of reference. The Ala insertions are shown in green. Asp3.49 and Arg3.50 are red and blue, respectively. A, WT. B, ADRS mutant. C, AADRS mutant. D, AAADRS mutant. E, AAAADRS mutant. With each additional Ala insertion, the position of Arg 3.50 shifts by 100°.

 
Inserting one to four Ala residues proximal to the Asp-Arg-Ser motif generated the mutants ADRS, AADRS, AAADRS, and AAAADRS. None of these mutants were found to elicit IP second messenger production, either constitutively or in response to agonist stimulation (Fig. 3Go and Table 1Go). These experiments cannot exclude the possibility that the absence of detectable GnRH-stimulated IP production in these mutants resulted from the lower levels of receptor expression. However, we believe this interpretation is unlikely. The mutants with detectable binding express at 10–45% the level of the WT receptor. We have previously reported that GnRHR mutants that are expressed at levels as low as 5% that of the WT mediate IP production (12). The results from the guanine nucleotide radioligand binding experiments also support the lack of G protein coupling of the insertional mutant tested.

Mutant ADRS, having a single alanine insertion, showed no detectable agonist binding in either COS-1 whole-cell (Fig. 1Go and Table 1Go) or membrane preparations (data not shown). The protein detected on Western blot analysis is not necessarily plasma membrane protein and could represent endoplasmic reticulum membrane protein. Nonetheless, the absence of high-affinity binding to ADRS most likely did not result from a loss of protein synthesis or stability as indicated by expression of glycosylated receptor. Progressive rotation of the Asp-Arg-Ser motif by further insertion of two to four alanines in mutants AADRS, AAADRS, and AAAADRS partially restored the high affinity binding of GnRH, which was undetectable in the ADRS mutant.

Function-restoring mutants are an important protein-engineering tool that can reveal the structural mechanisms contributing to specific receptor properties. This approach had been used previously in the GnRHR to elucidate how the interaction between two conserved residues in helices 2 and 7 contributes to certain receptor functions (15, 16). In the GnRHR, the single mutation of Asn2.50Asp eliminates binding, coupling, and receptor expression (16). Introducing a second mutation to the impaired construct, Asp7.49Asn in helix 7, restores receptor expression and allows detection of binding and coupling. In the present study, the most detrimental mutation, ADRS, disrupts receptor function without abolishing the expression of the receptor protein. However, in mutants AADRS, AAADRS, and AAAADRS, additional Ala insertions restore high-affinity binding of agonists. As the Asp-Arg-Ser motif of helix 3 is located in a region of {alpha}-helical secondary structure, each Ala insertion would be expected to rotate the distal segment of the helix, including the side chains of the Asp-Arg-Ser motif, by approximately 100o. Thus, recovery of binding in mutants with two to four alanines inserted suggests that a large range of Asp-Arg-Ser orientations are permissive for establishing a high-affinity agonist binding site. The mutants AAADRS and AAAADRS possess increased affinity for GnRH and GnRH-A relative to the WT receptor (Table 1Go). The increased affinity was specifically found with displacement of the Asp-Arg-Ser segment; the DRSAAA mutant, which displaces only the distal portion of the arginine cage motif, had an affinity comparable to the WT receptor. Contrary to mutants ADRS and AADRS, insertion of three or four alanines adjacent to the Asp-Arg-Ser motif rotates and "returns" the residues of this motif to the same face of the helix as they occupy in the WT receptor (Fig. 5Go). Thus, in the AAADRS and AAAADRS constructs, the residues of the Asp-Arg-Ser motif may contribute to the formation of intramolecular bonds that freeze the receptor in a high-affinity state, which is, however, not able to activate G proteins.

Because assay temperature can influence receptor-G protein interactions, which in turn regulate binding of agonists, we studied the effects of altering assay temperature on agonist binding (17). In addition to the standard binding assays performed at 4 C, ligand binding was also determined at 22 C. The AAADRS and AAAADRS receptors showed both an increase in agonist affinity relative to the WT receptor and a sensitivity of their affinity for agonists to assay temperature (Table 1Go). The affinity of these two mutants for GnRH and GnRH-A decreased approximately 2-fold with an increase in assay temperature from 4 to 22 C (Table 1Go). However, agonist affinity for the WT GnRHR remained unchanged when assessed at either 4 or 22 C. At 22 C, the Bmax values for the WT and all mutant receptors decreased with respect to those measured at 4 C (Table 1Go). The decrease in Bmax with increasing temperature has been reported previously for the bovine GnRHR (18). The increased affinity of two of the insertional mutants suggests that the family of conformations adopted by the AAADRS and AAAADRS receptors differ from those of the WT GnRHR.

The phenotypes of mutants AAADRS and AAAADRS are similar to phenotypes described for two arginine cage domain rhodopsin mutants. In one mutant, the conserved Glu-Arg-Tyr motif was altered into Arg-Glu-Tyr by an exchange of residues Glu 3.49 and Arg 3.50 (19). In the other, Arg 3.50 was mutated into Gly (20). With exposure to light, the Arg-Glu-Tyr mutant rhodopsin underwent transition into the activated state, metarhodopsin ll, but failed to stimulate second messenger production due to a lack of transducin binding (19). In the second mutant, activated receptor bound transducin but failed to stimulate GDP dissociation (20). Attempts to distinguish between the two mechanisms for loss of coupling of the GnRHR insertional mutants, loss of G protein binding and loss of G protein activation, through receptor-G protein coimmunoprecipitation experiments were inconclusive. We propose that the increased affinity GnRHR insertional mutants adopt a partial active-state receptor conformation, reflected by their increase in agonist affinity, but fail to stimulate second messenger due to either impaired G protein binding or activation.

The present data indicate that when the Asp-Arg-Ser segment is displaced, the receptor conformations that are assumed to form a high-affinity agonist binding site fail to transmit the signal to the G protein. This dissociation of high-affinity agonist complexing and signaling are consistent with the proposed role of the arginine cage domain in propagating signal transduction through the membrane. Molecular modeling suggests that the active-state partners for Arg 3.50 are the Asn 2.50 and Asp 7.49 pair, previously identified as forming a functional microdomain in GPCRs (12, 15, 16). In mutants AAADRS and AAAADRS, the displacement of the arginine cage apparently disrupts the formation of these critical interhelical bonds that facilitate receptor activation, while interhelical bonds that stabilize high-affinity agonist binding are preserved. Our studies support an emerging model of sequential side chain rearrangements that underlie signal transduction through the GnRHR.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Consensus Residue Numbering Scheme
A consensus residue numbering scheme was developed to allow comparison of corresponding residues among the helices of different rhodopsin-like GPCRs (21). Each helix contains a highly conserved side chain, which is used as a reference to identify the remaining residues of the helix. For example, in helix 3, the most highly conserved residue corresponds to Arg 139 of the Asp-Arg-Ser motif in GnRHR, which is designated as Arg3.50(139). The number "3" denotes helix 3 and "50" indicates the position of Arg 139. The residues on the C-terminal side of Arg 3.50 are labeled with increasing numbers, such as Ser3.51(140) in the GnRHR, while those on the N-terminal side have decreasing numbers, such as Asp3.49(138).

Mutagenesis
The mouse GnRHR mutants, ADRS, AADRS, AAADRS, AAAADRS, and DRSAAA, were generated using PCR mutagenesis. In the ADRS mutant, one alanine residue was introduced proximal to the Asp-Arg-Ser motif, while in the AADRS, AAADRS, and AAAADRS mutants, two, three, and four alanines were inserted at the same site, respectively. A silent BglII restriction site was incorporated in each primer to facilitate positive identification of mutant receptors. The mutated DNA fragments were subcloned into ApaI and PflM 1 restriction sites of WT mouse GnRHR in the expression vector pcDNA/Amp 3 (Invitrogen, San Diego, CA), thus replacing the corresponding WT receptor sequence. Mutations were confirmed by BglII digestion and sequencing of mutant constructs.

Epitope Tagging of WT and Mutant ADRS and AAADRS GnRHRs
Using PCR and multifragment subcloning into the pcDNA/Amp 3 expression vector, the WT mouse GnRHR was modified by 1) insertion of the hemagglutinin (HA) tag (YPYDVPDYA) after the initial Met residue, and 2) addition of a carboxy-terminal domain derived from a putative type ll human GnRHR, followed by a hexa-histidine tag (12). The epitope-tagged WT mouse GnRHR was designated t-WT GnRHR. Epitope-tagged mutants t-ADRS and t-AAADRS were generated by subcloning the mutated fragments of ADRS and AAADRS into the ApaI and PflM 1 sites of t-WT GnRHR.

Transfection
COS-1 cells grown in DMEM with 10% FBS were seeded into 100-mm plates at a density of 3 million cells per plate. These cells were transfected with DNA plasmids encoding the WT and mutant mouse GnRHR receptors, using lipofectamine (Life Technologies, Inc., Gaithersburg, MD) as previously described (22).

Whole-Cell Agonist Binding Assay
COS-1 cells were assayed for ligand binding 2 days after transfection with plasmid DNA encoding mouse WT and mutant GnRHRs. Ligand [D-Ala6, Pro9-NHEt]-GnRH (GnRH-A, Bachem, Torrance, CA) radiolabeled with iodogen (Pierce Chemical Co., Rockford, IL) was used in the whole-cell competition binding assay with increasing concentrations of cold GnRH-A or GnRH (Bachem) in Ringer-HEPES buffer (pH 7.4) with 0.1% BSA, at 4 or 22 C as previously described (22). The equilibrium dissociation constant (Kd) and Bmax values for GnRH-A were estimated from homologous competition binding assays, using LIGAND software (23). Nonspecific binding was subtracted from total binding before data analysis. IC50 values for GnRH were obtained with Kaleidagraph software (specific binding = B0/(1+[GnRH]/IC50); where B0 is maximum [125I]GnRH-A bound; and IC50 is the concentration of GnRH that inhibited [125I ]GnRH-A binding by 50%), and subsequently converted into Ki (equilibrium dissociation constant for GnRH) values using the Cheng-Prusoff equation (24). Protein amounts were determined with the Bradford reagent (Sigma, St. Louis, MO).

Preparation of Membranes for Western Blotting and Agonist Binding in the Presence of GTP Analogs
Two days after transfection, COS-1 cells from two 100-mm plates were each rinsed at room temperature with PBS and subsequently detached with 3 ml of ice-cold buffer A (50 mM Tris-HCl, 1 mM EGTA, 5 µg/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride, pH 7.4). Cells remaining on the plates were collected with an additional 2 ml of buffer and added to the tube. After centrifugation for 10 min at 500 x g, the cell pellet was resuspended in buffer A and homogenized by 10 strokes in a Dounce homogenizer. The suspension was transferred to a microfuge tube and centrifuged at 500 x g for 10 min. Without disturbing the pellet, the supernatant was collected and centrifuged at 17,000 x g for 30 min. The resulting membrane pellet was resuspended in 5 ml of buffer A with 0.1% BSA for use in membrane binding assays. For Western blotting experiments, membranes were solubilized in 0.5 ml of buffer A containing CHAPS ([(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, final concentration 1 mM) at 4 C for 30 min with gentle rocking, and then centrifuged at 17,000 x g to remove the insoluble material. Samples to be deglycosylated were treated with N-glycosidase F (0.2 U per 20 µl of supernatant, Roche Molecular Biochemicals, Indianapolis, IN) for 30 min at 37 C, before electrophoresis.

Agonist Binding to GnRHR Constructs in the Presence of GTP Analogs
Cell membranes (5 µg protein) were incubated for 20 min at 37 C with 200,000 cpm of [125I] GnRH-A, 5 mM MgCl2, and increasing concentrations of GTP/ATP analogs (GTP{gamma}S, GppNHp, ATP{gamma}S, AppNHp; Sigma) in a final volume of 0.4 ml of buffer A with 0.1% BSA. This assay was performed at 37 C to facilitate guanine nucleotide exchange and to allow correlation with the signal transduction assay that is performed at the same temperature. Nonspecific binding was determined by adding 1 µM GnRH-A. The reaction was stopped by rapid filtration and washing with 3 x 5 ml of ice-cold buffer B (50 mM Tris-HCl, 1 mM EDTA, and 0.1% BSA, pH 7.4) in a cell harvester. Radioactivity bound to GF/C filters (Brandel Inc., Gaithersburg, MD) was counted in a {gamma}-counter.

IP Assay
The IP assay was performed at 37 C, 3 days after transfection of COS-1 cells with WT and mutant GnRHRs (22). For each concentration of GnRH, IP production was converted to fold-stimulation of basal IP, then analyzed and plotted using Kaleidagraph software (Synergy Software, Reading, PA) and the equation E = Emax/(1 + EC50/[GnRH]), where E represents the measured IP production, Emax represents the maximum IP production, and EC50 is the concentration of agonist that elicits the half-maximal response.

Immunoblot Analysis
Solubilized COS-1 membranes (10 µl, 5 µg protein) were mixed with 2x Laemli buffer and incubated for 30 min at 37 C. Samples were electrophoresed on 12% SDS-PAGE gels, and proteins were transferred to nitrocellulose membranes using a semidry blotting apparatus. After blocking for 1 h in 5% milk/Tris-buffered saline (TBS) at room temperature, the nitrocellulose was incubated with Tetra-His antibody (QIAGEN, Valencia, CA; dilution 1:1000) in 5% milk/TBS overnight at 4 C. On the next day, the blot was washed in TBS and incubated with horseradish peroxidase-conjugated antimouse IgG (Amersham Pharmacia Biotech, Arlington Heights, IL; dilution 1:1000) in 5% milk/TBS for 1 h at room temperature, and afterward washed in TBS. The enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech) was used to visualize the epitope-tagged WT, ADRS, and AAADRS receptor proteins adsorbed to the blot.


    ACKNOWLEDGMENTS
 
We thank Dr. Daqun Zhang and Professor Harel Weinstein for providing the computational graphic shown in Fig. 5Go. The technical assistance of Vladimir Rodic is gratefully appreciated.


    FOOTNOTES
 
Address requests for reprints to: Stuart C. Sealfon M.D., Neurology Box 1137, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029. E-mail: sealfs01{at}doc.mssm.edu

1 Current address: Department of Medical Biochemistry, University of Cape Town Medical School, Observatory, 7925, South Africa. Back

This work was supported by NIH Grant DK-46943.

Received for publication May 25, 2000. Revision received December 5, 2000. Accepted for publication December 8, 2000.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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