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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 8 2586-2591
Copyright © 1997 by The Endocrine Society

Experimental Studies

Phe576 Plays an Important Role in the Secondary Structure and Intracellular Signaling of the Human Luteinizing Hormone/Chorionic Gonadotropin Receptor

Koichi Yano, Leonard D. Kohn, Motoyasu Saji, Akimasa Okuno and Gordon B. Cutler, Jr.

Developmental Endocrinology Branch, National Institute of Child Health and Human Development (K.Y., G.B.C.), and Cell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (L.D.K.), National Institutes of Health, Bethesda, Maryland 20892; Department of Surgery, Johns Hopkins University (M.S.), Baltimore, Maryland 21205; and Department of Pediatrics, Asahikawa Medical College (K.Y., A.O.), Asahikawa 078, Japan

Address all correspondence and requests for reprints to: Koichi Yano, Department of Pediatrics, Asahikawa Medical College, Nishikagura 4–5-3–11, Asahikawa 078, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent studies have identified multiple activating mutations in the sixth transmembrane domain of LH/chorionic gonadotropin receptor (LH/CGR) in patients with male-limited precocious puberty. Computer analysis suggested that these mutations had an effect on the secondary structure of the third cytoplasmic loop and sixth transmembrane domain, and that Phe576 was a critical conformational bridging residue between these regions that might be important for receptor activity. We made four amino acid substitutions of the Phe576 (F576I, F576G, F576Y, F576E) in the LH/CG receptor to analyze its functional role. Computer analysis of secondary structure predicted that the F576E mutant changed the secondary structure to a totally helical conformation in the region of the third intracellular and sixth transmembrane domain. In contrast, the F576G, F576I, and F576Y mutants were predicted to change the helical conformation in the region to an extended conformation. In expression studies, mutations of Phe576 produced functional changes in cAMP and inositol phosphate (IP) signaling, and human CG (hCG) binding. Mutations predicted to cause an extended conformation exhibited two functional patterns: first, constitutively activating in cAMP signaling without changes in IP signaling or hCG binding (F576I and F576G), and second, constitutively activating in cAMP signaling with decreased hCG-induced cAMP and IP signaling and with both higher affinity and lower capacity of hCG binding (F576Y). The mutation predicted to cause a totally helical conformation resulted in no cAMP response and a minimal IP response to hCG stimulation, with negligible hCG binding (F576E). These data suggest that the common change induced by the F576I, F576G, and F576Y mutations to an extended conformation on the third cytoplasmic loop and sixth transmembrane domain of the LH/CGR results in increased Gs coupling and activation of adenylyl cyclase. The F576Y mutation appears to have an additional effect, beyond a modification in receptor conformation, that leads to higher affinity and lower capacity of hCG binding, as well as altered Gq coupling and phospholipase C activation. The F576E mutation has a distinct and different impact on receptor conformation, which leads to negligible hCG binding and minimal function; however, the F576E mutation may provide a clue to understanding the receptor mutations that result in loss of function and pseudohermaphroditism. We conclude that Phe576 plays an important role in the human LH/CGR with respect to receptor conformation, Gs coupling, and cAMP signaling consistent with predictions from mutations associated with male-limited precocious puberty.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RECENT studies have identified activating or inactivating mutations of LH/chorionic gonadotropin receptors (LH/CGR) causing, respectively, male-limited precocious puberty or male pseudohermaphroditism. Thus, after discovery of the Asp578 to Gly mutation (D578G) in the sixth transmembrane region of the LH/CGR gene in patients with familial and sporadic male-limited precocious puberty (1, 2, 3, 5), seven other mutations in the fifth and sixth transmembrane regions, two mutations in the third intracellular loop, and one mutation in the second transmembrane region of the LH/CGR have been found in patients with male-limited precocious puberty (3, 4, 5, 6, 7, 8, 9, 10, 11), all of which caused constitutively elevated cAMP levels in transfected cells in vitro (2, 4, 5, 6, 7, 8, 10, 11). These results suggested that the Leydig cell activation and precocious puberty were caused by activating mutations of the LH/CGR.

Of interest, however, among the cases of testicular resistance to LH caused by inactivating mutations of the LH/CGR gene reported recently (12, 13, 14), several have mutations in the region of the sixth or seventh transmembrane domains (12, 13). In one study, genomic DNAs from patients with male pseudohermaphroditism and Leydig cell hypoplasia were found to have a homozygous missense mutation, Ala593 to Pro, in the sixth transmembrane region of the LH/CGR (12). In another report (13), a boy with micropenis had a homozygous mutation of Ser616 to Tyr in the seventh transmembrane domain of the LH/CGR. In expression studies, these mutations demonstrated no increase in cAMP production with human CG (hCG) stimulation.

Using the Garnier program (15), we analyzed the secondary structure of receptors with activating mutations in the sixth transmembrane domain, where most mutations were discovered. The results suggested that Phe576 was a critical bridging residue important for receptor activity and conformation in the sixth transmembrane domain and third intracellular loop. All mutants but one induced changes in the computer-predicted secondary structure in the region between residues 571 and 583 (6), which changed the predicted structure at Phe576 from a helical array to an extended array. We therefore hypothesized that Phe576 might play an important role in the LH/CGR with respect to cAMP signaling and receptor conformation, although no mutations at Phe576 have been found in patients with male-limited precocious puberty. We hoped simultaneously to gain insight into the relationship between Gs and Gq coupling and receptor conformation, because some but not all mutations associated with increased Gs coupling and male-limited precocious puberty also exhibited altered hormone-induced Gq coupling and inositol phosphate formation. We also hoped that studies of Phe576 might provide clues about the role of receptor conformation in loss of Gs coupling in male pseudo-hermaphroditism.

To test this prediction and evaluate this question, we made four amino acid substitutions at residue Phe576 and studied cAMP production, inositol phosphate (IP) production, and hCG binding using cells transfected with each mutant LH/CGR complementary DNA (cDNA).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mutagenesis

Four amino acid substitutions of the Phe576 in LH/CGR cDNA were made by recombinant PCR (16), i.e. Phe to Gly (F576G), Phe to Glu (F576E), Phe to Ile (F576I), and Phe to Tyr (F576Y). In brief, two PCR products that overlap the mutated sequence were created, both of which contained each mutation introduced as part of the PCR primers. The second PCR step used the overlapped PCR products as template; primers contained human LH/CGR DNA sequence 5' or 3' to the mutated region plus a BstXI (5'-end) and a HpaI site(3'-end). The second-step PCR products containing each mutation were digested with BstXI and HpaI and ligated to wild-type human LH/CGR cDNA (2) in pSG5, which had been similarly digested to remove the normal hLH/CGR sequence in this interval. Amplified DNAs were purified by CsCl gradient centrifugation (17) and sequenced (18) to ensure that each DNA had the predicted mutation but no other changes.

Transfection and assays

Wild-type LH/CGR cDNA was the same preparation described previously (2). COS-7 cells were transfected with 25 µg plasmid mutant or wild-type cDNA by electroporation (5, 6, 11, 19, 20); cells transfected with pSG5 alone were negative controls. The same batch of transfected cells was plated in DMEM with 10% FCS in 6-well plates (5 x 105 cells/well) for binding assays or in 24-well plates (1 x 105 cells/well) for cAMP and IP assays (5, 6, 11, 19, 20). The medium was inositol free in the latter assays and supplemented with 2 mCi/L myo-[2-N-3H]inositol (SA, 22.9 Ci/mmol; DuPont-New England Nuclear, Billerica, MA). All assays were initiated simultaneously, 48 h after transfection and after washing the cells with assay buffer (HBSS containing 0.5% BSA and 20 mmol/L HEPES, pH 7.4) (5, 6, 11, 19, 20).

Purified hCG (CR-127; 14,900 IU/mg) used in cAMP, IP, or binding assays was kindly provided by the National Hormone and Pituitary Program (NIDDK, Center for Population Research, of the National Institute of Child Health and Human Development, and the Agricultural Research Service of the United States Department of Agriculture). For binding studies, it was iodinated (SA, 44 µCi/µg) using lactoperoxidase (5, 6, 11, 19, 20). [125I]hCG binding was measured after 8 h at 22 C in incubations containing 1 mL assay buffer, 4 x 104 cpm [125I]hCG, and 0–10-7 mol/L unlabeled hCG. Specific binding was calculated by subtracting values obtained in cells transfected with the pSG5 control plasmid and incubated with the same concentrations of radiolabeled and unlabeled hCG. Nonspecific binding was less than 10% and was the same as [125I]hCG binding in the presence of 10-7 mol/L unlabeled hCG (5, 6, 11, 19, 20).

Total cAMP and IP levels were measured (5, 6, 11, 19, 20) in the same wells. Incubations were performed for 2 h at 37 C in 0.2 mL assay buffer containing 10 mmol/L LiCl and 0.5 mmol/L isobutylmethylxanthine alone or with 10-11 to 10-7 mol/L hCG. After adding 1.0 mL 5% perchloric acid, samples were centrifuged to remove protein debris, neutralized with KOH, and centrifuged to remove insoluble salts. Total cAMP was measured in aliquots of the supernatant with a RIA kit (New England Nuclear-DuPont); IP formation was determined in aliquots of the same supernatant using Dowex AG1-X8 columns (5, 6, 11, 19, 20). Data were expressed as the fold increase above the negative control value, i.e. cells transfected with pSG5 and exposed to buffer alone.

The cAMP and IP assays were performed in triplicate; binding assays were performed in duplicate. Values in each well were corrected either for cell protein, measured using a Bio-Rad protein assay kit (Bio-Rad Labs., Richmond, CA) and a BSA standard, or for total tritiated inositol incorporated (5, 6, 11, 19, 20). The program LIGAND (21) was used to calculate dissociation constant (Kd) values (5, 6, 11, 19, 20).

Statistic analyses

Values are presented as the mean ± SE from multiple experiments. Statistical differences were calculated using the two-tailed Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Basal and hCG-induced cAMP Levels in Cells Transfected with Mutant LH/CGRs

In the absence of hCG, cells transfected with the F576I or the F576G mutants exhibited 3.2 ± 0.4-fold or 2.7 ± 0.2-fold higher basal cAMP levels, respectively, than cells transfected with wild-type receptor or with vector alone (Fig. 1Go). hCG could still induce a concentration-dependent increase in cAMP levels similar to the levels in cells with wild-type receptor. Maximal (10-7 mol/L) hCG-stimulated cAMP levels in cells transfected with the F576I mutant, F576G mutant, and the wild-type hLH/CGR were 10.0 ± 0.6-fold, 10.8 ± 0.7-fold, and 10.0 ± 0.7-fold higher than the control value, respectively. These values did not differ from each other statistically.



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  		Figure 1. Basal and hCG-induced cAMP levels in COS-7 cells transfected with F576I, F576G, F576Y, F576E mutant, and wild-type hLH/CGR cDNA. In absence of hCG, cells transfected with F576I or F576G mutants exhibited 3.2-fold or 2.7-fold higher basal cAMP levels, respectively, than cells transfected with wild-type receptor or with vector alone. Cells transfected with F576Y cDNA exhibited 2.1-fold higher basal cAMP levels. F576E mutant did not alter basal cAMP levels and did not exhibit a statistically significant increase in cAMP levels when exposed to hCG.

 
Cells transfected with F576Y cDNA exhibited 2.1 ± 0.2-fold higher basal cAMP levels; however, these cells exhibited a significantly lower hCG-induced increase in cAMP levels than did wild-type, F576I, or F576G receptors. Maximal (10-7 mol/L) hCG-stimulated cAMP level in cells transfected with the F576Y mutant was only 5.6 ± 0.3-fold higher than the control value.

The F576E mutant did not alter basal cAMP levels and did not exhibit a statistically significant increase in cAMP levels when exposed to hCG. Basal cAMP levels and the levels at 10-7 mol/L hCG were 1.5 ± 0.2-fold and 1.9 ± 0.5-fold higher than the control value, respectively.

Basal and hCG-induced IP Levels in Cells Transfected with Mutant LH/CGRs

There were no significant differences between basal IP levels of cells expressing any of the mutant receptors and basal IP levels of cells expressing the wild-type LH/CGR (Fig. 2Go).



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  		Figure 2. Basal and hCG-induced IP levels in COS-7 cells transfected with F576I, F576G, F576Y, F576E mutant, and wild-type hLH/CGR cDNA. Values were obtained from same experiments described in Fig. 1Go. There were no significant differences between basal IP levels of cells expressing any of mutant receptors and basal IP levels of cells expressing wild-type LH/CGR.

 
Cells transfected with the F576I mutant exhibited a similar hCG-induced increase in IP levels as cells transfected with wild-type receptor. Maxmal (10-7 mol/L) hCG-stimulated IP levels in cells transfected with the F576I mutant and the wild-type hLH/CGR were 2.29 ± 0.04-fold and 2.45 ± 0.01-fold higher than the control value, respectively. The maximal stimulated levels by hCG for the F576G mutant, 1.85 ± 0.05-fold, were slightly lower than the levels for wild-type receptor. Cells transfected with the F576E and the F576Y mutants showed minimal hCG-induced increases in IP levels at high hCG concentrations; i.e. 1.35 ± 0.02-fold and 1.27 ± 0.01-fold higher than the control value at 10-7 mol/L hCG, respectively. The increase in IP formation by cells transfected with either mutant and exposed to 10-7 mol/L hCG was, nevertheless, significant (P < 0.005).

hCG Binding in Cells Transfected with Mutant LH/CGRs

Binding of hCG to the F576I or the F576G mutants was similar to wild-type receptor. COS-7 cells transfected with the F576I, the F576G mutants, and wild-type receptor cDNAs specifically bound 9.1%, 8.8%, and 10.5% of added [125I]hCG, respectively (Fig. 3AGo). Kd values were 10.2 x 10-10 M, 9.2 x 10-10 M, and 9.2 x 10-10 M, respectively. Cells transfected with the F576Y mutant bound 4.9% of added [125I]hCG; the Kd value was 5.1 x 10-10 M. These cells exhibited a lower capacity as well as a higher affinity, as shown in Scatchard analysis (Fig. 3BGo). Cells transfected with F576E exhibited negligible hCG binding.



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  		Figure 3. Displacement of [125I]hCG by unlabeled hCG in COS-7 cells transfected with F576I, F576G, F576Y, F576E mutant, and wild-type hLH/CGR cDNA. A, Data were expressed as bound/total ratio for each receptor, as determined in LIGAND plot. A Scatchard plot of same data is presented in B. Binding of hCG to F576I or F576G mutants was similar to wild-type receptor. Cells transfected with F576Y exhibited a lower capacity as well as a higher affinity. Cells transfected with F576E exhibited negligible hCG binding.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study, we made four amino acid substitutions of the Phe576 in the human LH/CGRs and studied cAMP production, IP production, and hCG binding using cells transfected with each mutant LH/CGR DNA.

In the absence of hCG, cells transfected with the F576I or the F576G mutants exhibited 3-fold higher basal cAMP levels than cells transfected with wild-type receptor or with vector alone (Fig. 1Go). hCG could still induce a concentration-dependent increase in cAMP levels similar to the levels in cells with wild-type receptor. Cells transfected with the F576Y mutant exhibited 2-fold higher basal cAMP levels; however, these cells exhibited a lower hCG-induced increase in cAMP levels. The F576E mutant did not alter basal cAMP values and did not respond to hCG.

In contrast to the increased basal cAMP levels, there were no significant differences between basal IP levels of cells expressing any of the mutant receptors and cells expressing the wild-type LH/CGR (Fig. 2Go). Cells transfected with F576I exhibited a similar hCG-induced increase in IP levels as cells transfected with wild-type receptor; the maximal stimulated levels by hCG for F576G were slightly lower than the levels for wild-type receptor. Cells transfected with F576E and F576Y showed minimal but significant hCG-induced increases in IP levels at high hCG concentration.

Binding of hCG to F576I or F576G was similar to wild-type receptor. COS-7 cells transfected with each of the three cDNAs specifically bound 9–10% of added [125 I]hCG (Fig. 3Go). Cells transfected with F576Y bound 5% of added [125I]hCG and exhibited higher affinity and lower capacity. Cells transfected with F576E exhibited negligible hCG binding.

We analyzed protein secondary structure of these mutants using a computer program (15) that predicted secondary structure (helical, extended, turn, or coil conformations). The F576G, F576I, and F576Y mutants are all predicted to change the helical conformation similarly to an extended conformation in the region of the sixth transmembrane domain (Fig. 4Go). In contrast, the F576E mutant changes the secondary structure to a totally helical conformation in the region.



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  		Figure 4. Protein secondary structure of mutant hLH/CGRs as calculated by method of Garnier (15). This program computes helical (x), turn (>), and extended (-) conformations in a protein sequence. Sequence studied represents transition region between third cytoplasmic loop and sixth transmembrane domain of human LH/CGR, as noted at top of figure. Numbers locate mutated residues.

 
In expression studies, mutations of Phe576 produced functional changes in cAMP and IP signaling, and hCG binding. Mutations predicted to cause an extended conformation exhibited two functional patterns: first, constitutively activated cAMP signaling without changes in IP signaling or hCG binding (F576I and F576G), and second, constitutively activated cAMP signaling with decreased hCG-induced cAMP and IP signaling, as well as higher affinity and lower capacity of hCG binding (F576Y). The mutation predicted to cause a totally helical conformation resulted in no cAMP response and minimal but significant IP response to hCG stimulation, with negligible hCG binding (F576E).

These data suggested that the common denominator of the F576I, F576G, and F576Y mutants is altered activation of adenylyl cyclase, i.e. the common change to an extended conformation in this region is linked to increased Gs coupling. The totally different impact on receptor conformation of the F576E mutant and its association with decreased rather than increased Gs coupling strengthens the possibility that the extended conformation induced by the other mutations in this region is important for increased Gs coupling.

Two additional points emerge from these studies. First, the F576Y mutant has effects that lead to higher binding affinity, lower capacity of hCG binding, and decreased Gq coupling and phospholipase C activation, in addition to its effects on Gs coupling, but these changes cannot be linked to the common conformational shift, because they are different from F576I and F576G. The impact of the tyrosine, as opposed to the phenylalanine, glycine, and isoleucine side chains, all of which are hydrophobic but devoid of the hydroxyl group, must be considered to result in these changes, in addition to altered conformation. We already reported similar changes in activating mutant LH/CGRs that were found in patients with male-limited precocious puberty (6, 11). The A572V mutant in the sixth transmembrane domain in the LH/CGR exhibited increased basal cAMP and IP levels with decreased hCG-induced IP levels (6). The M398T mutant in the second transmembrane domain in the LH/CGR exhibited increased basal cAMP levels, no increase in basal IP levels, and decreased hCG-induced IP levels (11). In addition, these two mutants exhibited higher affinity and lower capacity of hCG binding. Similar changes in affinity and capacity have been noted also in mutations altering G protein coupling of the TSH receptor and {alpha}1B adrenergic receptor (19, 22, 23, 24, 25). These changes in affinity and capacity have been postulated to reflect distant effects on the extracellular domain of the conformational changes induced by the mutations in the third cytoplasmic loop-sixth transmembrane region that cause the constitutively increased G protein coupling.

Mutations in the carboxyl end of the third intracellular loop, which is adjacent to the sixth transmembrane region of the {alpha}1B-adrenergic receptor, constitutively activate the receptor, resulting in G protein coupling in the absence of agonist (24). All 19 amino acid substitutions at Ala293 confer constitutive activity. This set of mutated receptors exhibits a graded range of elevated biological activities. The fact that all possible mutations at this particular site result in increased activity suggests that this region may function to constrain the G protein coupling of the receptor, a constraint that is normally relieved by agonist occupancy. Our results of the LH/CGR study was different from the results of the {alpha}1B-adrenergic receptor study. LH/CGR mutations at Phe576 in the sixth transmembrane domain exhibited three different functional patterns depending on their predicted change in the secondary structure. Two patterns were constitutively activating in cAMP signaling with or without changes in IP signaling or hCG binding. The other pattern was no cAMP response and a minimal IP response to hCG stimulation with negligible hCG binding. There must be different mechanisms of constitutive activation even in similar regions of the different G protein-coupled receptors.

Second, the fact that there is minimal but significant Gq coupling by the F576E mutant, despite negligible hCG binding and Gs coupling, suggests that at least some receptor has been incorporated into the bilayer, and that it is not a null mutation that results in a failure of the receptor to enter the bilayer; nevertheless, this cannot be excluded at this time. Recently, inactivating mutations of the LH/CGR have been reported in patients with male pseudohermaphroditism (12, 13, 14). Of these, one, A593P, is in the C-terminal portion of the sixth transmembrane domain (12). Interestingly, this mutation does not alter conformation of the Phe576 region (Fig. 5Go, dark bar 1), but rather has a profound impact on the conformation of the C-terminal portion of the sixth transmembrane domain (Fig. 5Go, dark bar 2). This mutation results in a receptor with a reduced maximal hormone binding but normal affinity. This result is consistent with the present studies, because there is no change in the Phe576 region to an extended conformation that would increase Gs coupling. Nevertheless, this observation raises the possibility that mutations in the C-terminal portion of the sixth transmembrane domain that similarly break up the helical conformation in the region between residues 588–594 will decrease Gs coupling. Of interest, a mutation in the seventh transmembrane domain that results in decreased receptor activity and micropenis, S616Y (13, 14), has no effect on the conformation of the sixth transmembrane domain, suggesting diverse mechanisms through which mutations produce loss of activity.



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  		Figure 5. Protein secondary structure of two mutant hLH/CGRs associated with male pseudohermaphroditism, A593P and S616Y, as calculated by method of Garnier (15). This program computes helical (x), coil (*), turn (>), and extended (-) conformations in a protein sequence. Sequence studied represents region between third cytoplasmic loop and seventh transmembrane domain of human LH/CGR, as noted at top of figure. Dark lines below wild-type receptor and A593P mutant denote regions associated with increased Gs and decreased Gs coupling.

 
In summary, the analysis of secondary structure of the human LH/CGR in the present study suggests an association between an extended conformation in the third intracellular loop and sixth transmembrane domain and increased Gs coupling. Because many seven-transmembrane receptors couple to Gs, these observations may be applicable to other G protein coupled receptors. Last, we can conclude that Phe576 plays an important role in the LH/CGR with respect to receptor conformation and cAMP signaling.

Received January 24, 1997.

Revised April 23, 1997.

Accepted May 1, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Hum. Reprod., November 1, 1999; 14(11): 2844 - 2850.
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