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Endocrinology Vol. 140, No. 10 4558-4563
Copyright © 1999 by The Endocrine Society

ARTICLES

Identification of Ionizable Amino Acid Residues on the Extracellular Domain of the Lutropin Receptor Involved in Ligand Binding1

Neil Bhowmick2, Prema Narayan and David Puett

Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229

Address all correspondence and requests for reprints to: Dr. David Puett, Department of Biochemistry and Molecular Biology, Life Sciences Building, Green Street, University of Georgia, Athens, Georgia 30602-7229. E-mail: puett{at}bchiris.bmb.uga.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The LH receptor (LHR) is a G protein-coupled receptor characterized by a relatively large N-terminal extracellular domain responsible for high affinity ligand binding. Based on a model proposed for a major portion of the extracellular domain that contains a number of leucine-rich repeats, nine ionizable amino acid residues (Glu57, Glu80, Lys158, Glu181, Lys183, Glu184, Glu188, Lys190, and Asp206) were selected for charge reversal mutagenesis based on their locations in the proposed model and their potential to serve as ligand contact sites. Mutant LHR complementary DNAs were transiently transfected into COS-7 cells, and the expressed receptors were characterized by Western blot analysis, competitive ligand (hCG) binding, and ligand-mediated cAMP production. The most interesting mutants were K158E, K183E, E184K, and D206K, which were present on the plasma membrane fraction, as judged by Western blots, but were incapable of binding hCG and, of course, were deficient in hCG-mediated cAMP production. Other replacements at these positions, K158R,Q,G; K183R, Q,G; E184N; and D206E,Q, led to cell surface binding and signaling. The mutants E57K, E189K, and K190E behaved similarly to wild-type LHR; E80K was trapped intracellularly, but bound ligand in solubilized cells; and E181K was not expressed or was rapidly degraded. These results, based on 18 point mutants of LHR, indicate that Lys158, Lys183, Glu184, and Asp206 are involved, either directly or indirectly, in gonadotropin binding and support the general nature of the proposed model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GLYCOPROTEIN hormone receptor family is unique within the G protein-coupled receptor superfamily by virtue of the relatively large N-terminal extracellular domain, accounting for about half the molecular size of the receptor, which is responsible for high affinity ligand binding (1). The two heterodimeric gonadotropins, LH and hCG, share a common {alpha}-subunit and have ß-subunits sufficiently similar that they act through the same heptahelical LH receptor (LHR) (2) that, via G protein activation, results in the enhancement of adenylyl cyclase activity and, in some cells, phospholipase Cß activity as well (3).

The crystal structure of hydrogen fluoride-treated hCG (4, 5) has provided a rational basis for efforts designed to elucidate the nature of the hormone-receptor contact interface. Unfortunately, there is no structure available for the LHR extracellular domain (ECD), although several models have been proposed based on the crystal structure of ribonuclease inhibitor (6) using the imperfect leucine-rich repeat (LRR) motif encoded by exons 2–10 of the LHR gene (7, 8, 9, 10, 11). The various models differ somewhat based on the size and number of LRRs chosen, but all suggest a horseshoe-shaped structure similar to that of ribonuclease inhibitor. Our model of amino acid residues 27–235 of rat LHR, based on nine LRRs (Fig. 1Go) like that reported by Kajava et al. (8), indicated that the electrostatic surface potential of the inner cusp, in addition to hydrophobic side-chains, might be important in directing ligand binding, a suggestion supported by site-directed mutagenesis of LHR within exon 6 (11).



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  		Figure 1. Ribbon diagram of working model and amino acid sequence of the ECD of rat LHR. Left, The ribbon diagram of amino acid residues 27–235 of the LHR ECD was adapted from Ref. 11, and residues 57–202 are represented by a solid ribbon. The amino acid residues that were studied by site-directed mutagenesis are indicated, and multiple replacements were made at those residues shown within boxes. Right, The amino acid sequence of the region represented by the parallel ß sheet motif in the model is shown. Only seven residues of each of the nine LRR units are shown, and the shaded region corresponds to the residues modeled in the solid ribbon. Ionizable residues that are unique to LHR and those homologous, i.e. conservation of charge, with other glycoprotein hormone receptors are also indicated.

 
In the present work, we used our receptor model to investigate 9 ionizable amino acid residues, encoded by exons 3, 7, 8, and 9, 7 of which are located in the inner circumference of the LHR ECD model and may serve as contact points with hCG, analogous to the contacts noted in the ribonuclease-ribonuclease inhibitor complex (6), and 2 of which are located on the outer circumference. A total of 18 point mutants of LHR were prepared and characterized, and the results suggest that Lys158, Lys183, Glu184, and Asp206 are involved, either directly or indirectly, with hCG binding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction and expression of LHR mutants
The complementary DNA (cDNA) for rat LHR, provided by Dr. William Moyle (Robert Wood Johnson Medical Center, Piscataway, NJ), was subcloned into the mammalian expression vector, pcDNA3 (Invitrogen, San Diego, CA). Two PCR-based methods were used to generate the site-directed mutants in the LHR cDNA. The first, described by Li and Wilkinson (12), was employed for the mutagenesis of Glu57->Lys, Glu80->Lys, Lys158->Glu, Glu181->Lys, Lys183->Glu, Glu184->Lys, Glu189->Lys, Lys190->Glu, and Asp206->Lys. Briefly, 27- to 30-base oligonucleotide mutagenic primers and their reverse complementary oligonucleotides were used to amplify the LHR-pcDNA3 construct with Pfu polymerase (Stratagene, La Jolla, CA). Then, the parental DNA was digested with DpnI, and DH5{alpha} cells were transformed. The second method was used for the mutagenesis of Lys158 and Lys183 each to Gly, Gln, and Arg, of Glu184->Asn, and of Asp206->Glu and Gln. An oligonucleotide complementary to the T7 promoter sequence present in pcDNA3 and a degenerate mutagenic antisense oligonucleotide were used to amplify the 5'-end of the cDNA insert. The resulting PCR product and a primer to the SP6 promoter sequence in pcDNA3 were used to amplify the remaining portion of the cDNA insert. The mutant cDNAs were subsequently digested with appropriate restriction enzymes and subcloned into pcDNA3. The mutant clones were verified by dideoxy sequencing (13).

LHR and its mutant cDNAs were transiently transfected into COS-7 cells (grown in DMEM, 10% FBS, and antibiotics) using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) according to the recommended protocol. The cellular localization of LHR mutant expression was assessed 48 h posttransfection by Con A-assisted sucrose gradient centrifugation to achieve plasma membrane separation (14, 15, 16). Briefly, the plasma and intracellular membrane fractions collected from the sucrose gradient were washed twice in 0.15 M NaCl and 20 mM HEPES, pH 7.4, containing 0.5 mM N-ethylmaleimide, 0.2 mM phenylmethylsulfonylfluoride, and 0.5 mM EDTA and solubilized with 0.5% Nonidet-P40 in the same buffer for 20 min at 0 C. The solubilized fraction, separated by centrifugation, was deglycosylated with 0.6 U N-glycosidase F (Roche Molecular Biochemicals, Indianapolis, IN)/3 µg protein for 2 h at 37 C. The deglycosylated product was run on a 10% SDS-polyacrylamide gel under denaturing conditions and probed with rabbit anti-LHR antibody (Ab-114) raised against Escherichia coli-expressed LHR ECD (Bhowmick, N., N. Menon, J. Gray, A. Przybyla, D. Puett, and P. Narayan, unpublished results). Deglycosylation was necessary because Ab-114 does not recognize the fully glycosylated form of LHR.

Bioassay of LHR mutants
The functionality of the LHR mutants was assayed in transiently transfected COS-7 cells via competitive binding and cAMP assays. For binding studies, cells were incubated with 0–250 ng/ml hCG in the presence of 2 ng/ml [125I]hCG at room temperature for 18 h. Cells were then washed, and bound [125I]hCG was measured by {gamma}-counting. If necessary, cells were solubilized before binding using the protocol described previously (11). Ligand-mediated adenylyl cyclase stimulation was assessed in cells expressing wild-type (WT) and mutant LHRs 30 min after the addition of 100 ng/ml hCG, in the presence of 0.5 mM isobutylmethylxanthine at 37 C, by determining intracellular cAMP concentrations with an [125I]cAMP kit (NEN Life Science Products, Boston, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ionizable amino acid residues in the rat LHR ECD were selected for site-directed mutagenesis on the basis of their location in the concave region of our receptor model (11). Of the 12 ionizable side-chains in the proposed ß-sheet region (Fig. 1Go), three, Lys104, Glu132, and Asp135, were studied earlier (11), and 7 residues, Glu57, Glu80, Lys158, Glu181, Lys183, Glu184, and Asp206, were investigated herein, each being replaced with an oppositely charged side-chain. In addition, multiple replacements were created at positions 158, 183, 184, and 206 using primers containing 3- and 4-fold degeneracy by the PCR-based mutagenesis procedure. Also, 2 adjacent residues, Glu189 and Lys190, were investigated as negative controls based upon their proposed location outside the cusp-shaped section where no binding of ligand is predicted.

The expression of WT and the nine charge reversal mutant LHRs in COS-7 cells was initially determined by immunodetection with an anti-aglyco-LHR antibody. However, to distinguish cell surface expression from receptors trapped intracellularly, the plasma membrane and intracellular membrane fractions were separated by sucrose gradient centrifugation after Con A binding to intact cells at 0 C (14, 15, 16). Western blots of the two membrane fractions showed a major 58- to 60-kDa band (Fig. 2Go), corresponding to the 57-kDa protein detected on multiple Ala replacements of N-linked glycosylation sites of LHR (17). Seven of the nine mutant LHRs exhibited immunoreactive bands with the plasma membrane fraction, but there was no detectable E80K or E181K (Fig. 2AGo); with the exception of E181K, all other charge reversal mutants were detected within the intracellular membrane fractions. These results show that E80K is trapped intracellularly and that E181K is either not expressed or is rapidly degraded. (It is somewhat surprising to find WT and mutant LHRs in both plasma membrane and intracellular fractions; this observation is probably attributable to biosynthesis exceeding the trafficking capacity or the presence of immature precursors in the transiently transfected COS-7 cells.)



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  		Figure 2. Detection of LHR mutant proteins expression by Western blotting. N-Glycosidase F-digested membrane, detergent-solubilized extracts of transiently expressed WT, and mutant LHR cDNAs were run (2 µg protein) under reducing conditions on 10% SDS-gels, transferred to nitrocellulose, and probed with the Ab-114 antibody. After sucrose gradient centrifugation, the plasma membrane fraction at the 60–35% sucrose interface (A) and the intracellular membrane fraction isolated from the 0–35% sucrose interface (B) were washed and solubilized with Nonidet P-40-containing buffer before digestion.

 
These nine LHR charge reversal mutants as well as other replacements of Lys158, Lys183, Glu184, and Asp206 were analyzed by competitive binding (Table 1Go and Figs. 3Go and 4Go) and maximal hCG-mediated cAMP production (Table 1Go). Considering first the nine mutant LHRs analyzed by Western blots, there was minimal binding and cAMP production in the E80K and E181K mutants, consistent with the data presented in Fig. 2Go. The mutants, E57K, E189K, and K190E, which exhibited Kd values identical to that of WT LHR, expressed well on the cell surface and responded to hCG with cAMP production similar to that of WT LHR. Even the reduced apparent cell surface expression of E189K is consistent with Western blot analysis (Fig. 2Go).


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  		Table 1. Summary of the binding and ligand-mediated cAMP data for WT and mutant LHRs

 


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  		Figure 3. Competitive binding of hCG to three LHR charge reversal mutants at positions Glu57, Glu189, and Lys190. Transiently expressing COS-7 cells were incubated with 2 pg/ml [125I]hCG and competing amounts of unlabeled hCG, followed by washing, extraction, and {gamma}-counting. WT LHR binding is indicated by a dashed line. At least two independent experiments, performed in duplicate, were analyzed using the Prism computer program (GraphPad Software, Inc., San Diego, CA).

 


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  		Figure 4. Competitive binding of hCG to mutant LHRs with replacements at Lys158 (A), Lys183 (B), Glu184 (C), and Asp206 (D). The cDNAs of the LHR point mutants were transiently transfected in COS-7 cells, and competitive binding of hCG with [125I]hCG was performed in at least two independent experiments, each performed in duplicate. WT LHR binding is indicated by a dashed line; the same set of data is used in all four panels.

 
The results on the remaining four mutants, K158E, K183E, E184K, and D206K, are particularly interesting. In each case, despite the presence of immunoreactive protein on the cell surface and associated with intracellular membranes (Fig. 2Go), there was minimal binding to intact or solubilized cells and, consistent with this finding, minimal hCG-mediated cAMP production.

Other replacements of Lys158 (Arg, Gln, Gly), Lys183 (Arg, Gln, Gly), Glu184 (Asn), and Asp206 (Glu, Gln) resulted in variable apparent expression levels, Kd values comparable to or slightly higher than those of WT LHR, and robust hCG-mediated cAMP responses. Lastly, several of the mutant LHRs that exhibited a low degree of [125I]hCG binding to intact cells, e.g. E80K and to some degree E189K, bound ligand at levels approaching those of WT LHR in solubilized cells; these results, particularly with E80K, are consistent with the Western blots (Fig. 2Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This work has identified four ionizable amino acid residues in the ECD of LHR, Lys158, Lys183, Glu184, and Asp206, predicted to lie within the concave region of our receptor model (11) where ligand binding is expected, that appear to be involved in high affinity binding of hCG based on charge reversal mutants. For each of these mutants, Western blot analysis established the presence of immunoreactive protein on the cell surface at about the same level as that of WT LHR, but specific binding of [125I]hCG to cells expressing each of the mutant LHRs was only 1–2% that of WT receptor; moreover, binding was not increased in solubilized cells. Another possible explanation of these results is that each mutation results in a conformationally altered form of LHR on the cell surface that is incompatible with ligand binding. This explanation, however, seems unlikely based on the following observations. Functional mutants at each of these four positions could be expressed on the cell surface even with dramatic replacements, such as K158G and K183G. Also, other charge reversal mutants in this immediate region of LHR, e.g. of Glu189 and Lys190, yielded functional receptors at the cell surface. Thus, the loss of ligand binding at positions Lys158, Lys183, Glu184, and Asp206 occurred only with charge reversal replacements, suggesting that each of these side-chains forms an ion pair or perhaps an ion-dipole interaction with a complementary site on hCG.

Of the nine ionizable amino acid residues investigated herein coupled with data on Lys104, Glu132, and Asp135 reported previously (11), we mapped 10 of the 12 ionizable residues within the inner cusp of our model and found that Glu132, Asp135, Lys158, Lys183, Glu184, and Asp206 are involved, either directly or indirectly, with ligand binding, whereas Glu57, Glu80, and Lys104 do not appear to be. The predicted electrostatic surface potential of this putative cusp region was published previously (11) and shown to exhibit, as expected, a predominant negative surface. Using the program GRASP (18), we also examined the electrostatic surface potential of several charge reversal LHR mutants that expressed at the cell surface but failed to significantly bind ligand in intact cells or cellular lysates, e.g. K158E, K183E, E184K, and D206K. Not surprisingly, the K158E and K183E mutants exhibited greatly increased surface negative electrostatic potential, whereas the E184K and D206K mutants were characterized by decreased negative electrostatic potential in the cusp region (data not shown).

Thus, the net electrostatic potential arising from the distribution of charges in the cusp region appears to greatly influence ligand binding. In this context, it is noteworthy that Szkudlinski et al. (19) found a significant increase in the binding and signaling potencies of TSH and the TSH receptor and of hCG binding to LHR in mutant hormones obtained by increasing the net positive charge on the N-terminal region of the {alpha}-subunit. For example, the quadruple mutant {alpha}, Q13K + E14K + P16K + Q20K, increased the potency of TSH binding and signaling nearly 40-fold and that of hCG binding and signaling severalfold.

Based on a limited number of ligand-receptor cocrystal structures in the literature, it has been observed that, in general, hydrophobic residues are the principal contributors to the free energy of binding; however, the contact epitopes and signaling events can often be attributed to a few ionizable side-chains (20, 21, 22, 23). We hypothesize that contact sites for hCG and LHR follow these guidelines as well. Also, it is possible that the ionizable side-chains contribute to the specificity of appropriate ligand binding to the glycoprotein hormone receptors. Of the six ionizable amino acid residues in LHR that we have identified as possible contact sites for hCG, only Lys183 is specific to LHR. The homologous positions are occupied by Ser in FSHR and Asn in TSHR (1); it is possible that the Lys183 side-chain pairs with negatively charged groups present on the ß-subunit of hCG and LH, but not on FSH or TSH, e.g. Asp105 or Asp111. Identical, e.g. Asp135, or conserved, e.g. Lys/Arg and Glu/Asp replacements, amino acid residues in the three receptors may indicate pairing with identical or conserved residues on either of the subunits of glycoprotein hormones.

Our earlier work emphasized the importance of negative charges at positions 132 and 135, proposed to be localized in LRR 5, of LHR (11). The present study has extended the area of the inner cusp of the receptor model to include three more LRRs, 6–8. In this localized region of the model, the net charge is, overall, negative; yet, the two positive charges arising from lysines 158 and 183 are, as we have shown, involved in high affinity ligand binding. To date, the various LHR ECD models (7, 8, 9, 10, 11) exhibit a similar overall shape, which is not surprising as these were all developed based on porcine ribonuclease inhibitor, a LRR protein with an {alpha}ß coiled fold (6). Alternately, it is possible that the ECD may adopt a conformation characterized by another coiled form (24). Our results cannot differentiate between these two possibilities.

Thomas et al. (25) reported that high affinity ligand binding by the LHR ECD occurs with the amino acid sequence encoded by exons 1–6, and Zhang et al. (26) found that all Cys residues in this region were necessary for ligand binding. In contrast to the report by Thomas et al. (25), Hong et al. (27) found a significant increase in the Kd when the amino acid sequence corresponding to exon 10 was deleted from the ECD, little change with progressive removal of the sequences encoded by exons 7–9, and another increase in Kd when the region encoded by exon 5 was removed; indeed, binding was detected in the N-terminal region of the receptor encoded by only exons 1–2. Our findings herein and reported previously (11) indicate that certain ionizable amino acid residues encoded by exons 6–8 contribute to ligand binding in the full-length receptor.

Other than this region of LHR and the Cys residues located in the sequence encoded by exons 1–6 (26), little is known about other amino acid residues that affect ligand binding or signaling. A recent study of a patient with familial gestational hyperthyroidism revealed that she was heterozygous for a K183R mutation in her TSHR (28). Lys183 in TSHR is in a homologous position to Lys158 in LHR: TLKLY (residues 181–185 in TSHR and 156–160 in LHR, the rat and human receptors having identical sequences in this region). Of considerable interest is the observation that the K183R mutation in TSHR, although having no significant effect on TSH-mediated signaling, greatly increases the sensitivity of TSHR signaling to hCG. Our results show that the K158R mutation in LHR has no profound effect on hCG binding and signaling, whereas the charge reversal mutant, K158E, in LHR appears to express at the cell surface, but exhibits negligible ligand binding. With several mutants (K158Q, K158G, K183G, and D206E), reduced expression resulted in maximal cAMP levels comparable to those of WT LHR, suggesting that a limited number of occupied receptors is sufficient for effective signaling.

Based on individual replacements with Lys, we have shown that two adjacent acidic amino acid residues in the ECD near transmembrane helix 1, Glu332 and Asp333, are involved in ligand-mediated signaling, but not binding (29). Confirming these in vitro mutagenesis studies, a report recently appeared establishing that a naturally occurring mutation, leading to the same replacement of Glu332 by Lys, resulted in primary amenorrhea in a 46,XX individual and pseudohermaphroditism in her two 46,XY siblings (30). Another report of a 46,XY individual presenting with incomplete male pseudohermaphroditism localized a mutation in the LHR gene that resulted in a replacement of Cys133 by Arg (31), consistent with the mutagenesis studies of Dufau and co-workers (26).

In summary, the results presented herein show that four ionizable amino acid residues in the ECD of rat LHR, Lys158, encoded by exon 7, Lys183 and Glu184, encoded by exon 8, and Asp206, encoded by exon 9, are involved, either directly or indirectly, in high affinity hCG binding to the full-length receptor.


   Footnotes
 
1 This work was supported by NIH Research Grant DK-33973. Back

2 Present address: Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232. Back

Received May 3, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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The Differential Binding Affinities of the Luteinizing Hormone (LH)/Choriogonadotropin Receptor for LH and Choriogonadotropin Are Dictated by Different Extracellular Domain Residues
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S. Neumann, M. Claus, and R. Paschke
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G. B. Fralish, B. Dattilo, and D. Puett
Structural Analysis of Yoked Chorionic Gonadotropin-Luteinizing Hormone Receptor Ectodomain Complexes by Circular Dichroic Spectroscopy
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H.F. Vischer and J. Bogerd
Cloning and Functional Characterization of a Gonadal Luteinizing Hormone Receptor Complementary DNA from the African Catfish (Clarias gariepinus)
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M. Ascoli, F. Fanelli, and D. L. Segaloff
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G. Smits, C. Govaerts, I. Nubourgh, L. Pardo, G. Vassart, and S. Costagliola
Lysine 183 and Glutamic Acid 157 of the TSH Receptor: Two Interacting Residues with a Key Role in Determining Specificity toward TSH and Human CG
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M. W. Szkudlinski, V. Fremont, C. Ronin, and B. D. Weintraub
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Hormone Interactions to Leu-rich Repeats in the Gonadotropin Receptors. I. ANALYSIS OF LEU-RICH REPEATS OF HUMAN LUTEINIZING HORMONE/CHORIONIC GONADOTROPIN RECEPTOR AND FOLLICLE-STIMULATING HORMONE RECEPTOR
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H. Zeng, T. Phang, Y. S. Song, I. Ji, and T. H. Ji
The Role of the Hinge Region of the Luteinizing Hormone Receptor in Hormone Interaction and Signal Generation
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A. Schmidt, R. MacColl, B. Lindau-Shepard, D. R. Buckler, and J. A. Dias
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S. D. Mahale, J. Cavanagh, A. Schmidt, R. MacColl, and J. A. Dias
Autologous Biological Response Modification of the Gonadotropin Receptor
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