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Endocrinology Vol. 138, No. 4 1627-1633
Copyright © 1997 by The Endocrine Society

ARTICLES

A Functional Determinant in Human Luteinizing Hormone and Chorionic Gonadotropin: Differential Effect of Mutations about ß-GLN-541

Shuang-Bao Hu, Leslie Johnson, Patrick C. Roche and Henry T. Keutmann

Endocrine Unit and Reproductive Endocrine Sciences Center (S.-B.H., L.J., H.T.K.), Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and the Department of Laboratory Medicine and Pathology (P.C.R.) Mayo Medical School, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Henry T. Keutmann, M.D., Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: Keutmann{at}helix.mgh.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is evidence that the conserved glutamine at residue 54 in the ß-subunit of human LH and and CG (hCG) is important for biological activity. Mutation to Arg in LH has been reported to impair receptor binding, leading to a documented case of hypogonadism, whereas in hCG the mutation has been shown to result in defective subunit association. Functional distinctions between LH and hCG have been described, but the significance of peptide-chain differences between the two has not been investigated systematically. We therefore compared the role of Gln-54 and its neighboring residues in both hormones, through replacement by amino acids with contrasting properties using site-directed mutagenesis. The mutant subunits were coexpressed with {alpha}-subunit in mammalian (Chinese hamster ovary) cells and the secreted hormones assayed for heterodimer formation, receptor binding, and steroidogenesis in murine Leydig cell tumor (MA-10) cells. Basic (Arg, Lys) substitution for Gln-54 in either hormone markedly impaired subunit association (<20% of wild-type) and the heterodimers that were formed were inactive (<5% of wild-type) in both assays. Arg-substituted hCG was also inactive in an adenylate cyclase assay using HEK-293 cells expressing rat LH/hCG receptor. After acidic (Glu) or neutral (Ala) substitution, heterodimer formation was less impaired (50–60% of wild-type), but effects on receptor interaction differed between the two hormones. The LH mutants still lacked binding activity, whereas the hCG products were fully active. The importance of residue 54 for receptor interaction appears to be sharply localized because mutation at adjacent positions (Pro-53 and Val-55) did not impair the activity of either hormone. Diminished heterodimer formation by Ile-53 mutation in LH (but not hCG), together with the similar effects of basic mutations at 54, imply long-distance effects as these residues are remote from {alpha} in the crystal structure. Our findings indicate that position 54 in LH and hCG is a determinant for both subunit association and receptor interaction. The differing responses between LH and hCG to certain mutations suggest that structural characteristics of the peptide chains may confer functional differences despite their close sequence homology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN gonadotropins LH, CG, and FSH belong to the glycoprotein family of heterodimeric hormones that also includes TSH (1, 2, 3). The crystal structure of human CG (hCG) (4, 5) has revealed the extensive regions of contact between the common ({alpha}) and hormone-specific (ß) subunits, and holohormone surface sites likely to be available for receptor binding. The functional significance of these structural regions remains to be confirmed through continued analysis of structure-activity relations. To this end, naturally occurring defects associated with disease are particularly valuable because of their clinical as well as biochemical implications.

A prominent example has been provided by the characterization of mutant LH from a patient with hypogonadism, in which a point mutation encodes arginine in place of glutamine at position 54 in the ß-subunit (6, 7, 8). After expression from mammalian cells transfected with the mutant DNA sequence, the hormone product appeared immunologically intact but virtually devoid of LH/hCG receptor-binding activity (8). The importance of a Gln residue at this sequence position is emphasized by its conservation among all gonadotropins. Subsequent mutagenesis studies in hCG (9) affirmed the deleterious effect of a basic (Lys) substitution at ß54 but attributed the loss in activity to defective association with {alpha} subunit; the small amount of heterodimer that was formed appeared to retain at least partial steroidogenic activity.

A differential effect of mutation at this clearly important residue might result from structural differences between LH and hCG. The two hormones are closely related and exert similar actions through a common receptor. Despite this apparent structural similarity, certain functional differences between the two hormones have long been recognized. LH characteristically has a lower receptor-binding affinity and steroidogenic potency (10, 11), attributed principally to faster rates of dissociation (12, 13) and internalization (14). LH also shows increased sensitivity to changes in ionic environment (15, 16), and a shorter metabolic half-life (17, 18). Synthetic peptides representing analogous binding sequences in the respective hormones also differ in their binding affinities (19).

The work reported here examines the effects of selected mutations at residue ß54 and its neighbors in LH and hCG on three processes essential to gonadotropin action: subunit association, receptor binding, and steroidogenesis. Besides probing the mechanisms underlying hypogonadism as a result of LH mutation, our investigations provide a systematic comparison of structure-activity relations involving the peptide chains of the two hormones in a manner not previously undertaken.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Cell culture media, horse serum, geneticin (G418), and HEPES buffers were purchased from Life Technology (Gaithersburg, MD) and FBS and antibiotics (penicillin/streptomy-cin/Fungizone) from Bio-Whittaker (Walkersville, MD). Restriction enzymes and DNA purification reagents were purchased from Promega (Madison, WI), and the DNA sequencing reagents from USB (Cleveland, OH). pM2-hCG{alpha} plasmid for stable transfection was kindly provided by Dr. I. Boime (Washington University, St. Louis, MO), and the human LH-ß minigene by Dr. J. Fiddes through the NIH (Bethesda, MD). The pGEM plasmid containing hCG-ß cDNA was a gift of Dr. O. Lockridge (Eppley Institute, Omaha, NE). Murine Leydig tumor (MA-10) cells were obtained courtesy of Dr. Mario Ascoli (University of Iowa, Iowa City, IA). Reagents and tracer for progesterone assay were purchased from ICN (Horsham, PA).

Plasmid construction and site-directed mutagenesis
The LHß minigene was trimmed at the 5'-end with BstEII, blunt ended, and a BamH1 linker attached. The BamH1-EcoRI fragment of the minigene was then inserted into the expression vector pcDNA1 (Invitrogen, San Diego CA). The hCG-ß cDNA was removed from the pGEM vector by Bam-H1 and ligated into the unique Bam-HI sites in the pcDNA1 vector. Oligonucleotide-directed mutagenesis was performed according to Kunkel et al. (20). The mutant plasmids were confirmed by DNA sequencing according the instruction of the supplier (United States Biochemicals).

Transfection, expression, and processing of media
Wild-type and mutant ß-subunit DNA was isolated using the Wizard (Promega) DNA purification system. Chinese hamster ovary (CHO) cells were stably transfected with pM2-hCG{alpha} using calcium phosphate (21), selected with geneticin, and maintained until use in {alpha}-MEM containing 10% heat-inactivated FBS and antibiotics. Cells were cotransfected transiently with wild-type or mutant ß-subunit DNA using the diethylaminoethyl-dextran procedure as described by Seed and Aruffo (22). Four to 6 days after transfection, the media were harvested; although subject to variation in incubation time and amounts of DNA incorporated, recoveries of wild-type dimer averaged 35–40 ng/ml for LH and 15–25 ng/ml for hCG. Media were concentrated about 10 times using Centriprep tubes (Amicon, Beverley MA) for radioreceptor and steroidogenic assays. Holohormone formation was also evaluated on a larger scale using the expression product from CHO cells stably transfected with pM2{alpha}/LHß as described by Weiss et al. (8). Stably-transfected CHO cells were also used in coexpressing the pM2{alpha} and pCDNA1-hCGß vectors for adenylate cyclase assay of mutant and wild-type hCG.

Gonadotropin RIAs
Dimer-specific RIAs of the harvested expression media were used to establish hormone doses for the receptor binding and steroidogenic assays and, in combination with ß-specific assays, to determine the relative content of dimer vs. total (free and associated) ß as a measurement of subunit association. hCG concentrations (ng/ml medium) were assayed using ß-hCG and dimer Tandem hCG kits purchased from Hybritech (San Diego, CA); the Tandem kit has been employed in several previous studies of mutant hCG (9, 23, 24). LH concentrations were determined in the Assay Core of the MGH Reproductive Endocrine Sciences Center, using a dimer-specific two-site ELISA procedure (25) and a ß-specific RIA (7). These assays provided media concentrations in mIU (2nd IRP HMG and 2nd IS-WHO 71/233, respectively), which were converted to mass units (ng/ml) after calibration of the respective standards against purified dimer LH and ß-subunit quantitated by amino acid analysis. Recognition of mutant and native LH by these antibodies had been established in earlier investigations (7, 8) of circulating natural and recombinant ß-54 mutant LH.

Binding assays
MA-10 cells (26) were maintained in Waymouth’s medium supplemented with 15% heat inactivated horse serum, 500 µg/ml gentamicin, and 20 mM HEPES. Three days after seeding in 24-well plates, the cells were washed once in binding buffer (Waymouth’s medium with 1 mg/ml BSA). The concentrated media containing wild-type or mutant recombinant hormones were serially diluted in the binding buffer and incubated with the cells in presence of 125I-hCG overnight (14–16 h) at 20 C with constant agitation (9, 23). After washing the cells twice with binding buffer, 5 nM NaOH was added and the harvested cells counted on a gamma counter. Control incubations demonstrated that neither the mutant nor wild-type hormones were degraded by the cultured cells, and cellular uptake of labeled hormone was insufficient to adversely affect the binding response profiles.

Steroidogenic assays
MA-10 cells were maintained as described for the section on binding assays. Cells were washed once with binding buffer before incubating with serial dilutions of mutant or wild-type media for 1 h at 37 C. The supernatant was harvested and either stored at -20 C, or immediately subjected to progesterone RIA (ICN, Horsham, PA). Hormone doses for all assays were adjusted to true heterodimer content by RIA.

Assay for adenylate cyclase activation
Assays for adenylate cyclase activation were carried out using monolayer cultures of mammalian cells stably transfected with the rat LH/hCG receptor. From luteinized rat ovaries (10), full-length rat receptor cDNA was produced by reverse transcription of oligo (dT)-primed mRNA. PCR-amplified cDNA was purified by gel electrophoresis and incorporated into the pBacPAK9 Baculovirus expression vector. A resulting clone, Oligo-31, encoded the full-length LH/hCG receptor as demonstrated by sequence analysis. This was subcloned into the pcDNA3 (Invitrogen) expression vector for liposome-mediated transfection into human embryonic kidney (HEK-293) cells. A stable cell line was selected and maintained in presence of geneticin under conditions described by Wang et al. (27). Scatchard analysis with 125I-hCG indicated 2.9 x 105 receptor sites/cell. For assay, Oligo 3l cells were cultured in 24 x l6-mm wells for 48 h. Wild-type and mutant expression products were incubated in serial dilution for 1 h at 37 C, and cAMP content determined by RIA (27).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Alternative forms of wild-type LHß
At the outset, we needed to establish the appropriate form of LH to use for expression. Although LHß cDNA is encoded for 122 residues followed by a stop codon (28), all reported preparations isolated from natural pituitary sources have terminated at residue 114 (1, 2, 3, 29). The additional, predominantly hydrophobic 7-residue (115–122) sequence appears to retard subunit assembly and secretion (30), but its effect on binding activity of secreted homone has not been tested. We expressed both LH (ß-114) and LH (ß-122) using separate plasmids with suitably placed stop codons. As shown in Fig. 1Go, the binding inhibition curves and affinities of the secreted holohormones were identical. Our subsequent studies used the shorter pituitary (ß-114) form of the subunit.



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  		Figure 1. Binding inhibition curves for LH with alternative forms of ß-subunit differing in length at the carboxyl-terminus. The truncated LHß-114 form, found in natural pituitary extracts, was used for mutagenesis in these studies.

 
Holohormone formation by Gln-54 mutants
We replaced Gln-54 in LHß and hCGß with the two basic residues (Arg, Lys), a neutral residue (Ala), and an acidic residue (Glu). Calculated as the recovery of dimer-specific ß relative to total ß in the expression media, holohormone formation by the wild-type expression products averaged 28 ± 6% for hCG and 19 ± 3% for LH. Subunit association by the mutant ß-subunits varied according to the substituted residue as summarized in Fig. 2Go. Relative to the wild-type (100%), mutation to Ala or Glu in either hormone reduced subunit association by less than half. Hence, heterodimer formation by Q54E mutant hCG was 57 ± 1.9% and human LH 60 ± 3.7% (based on four transfections), with similar proportions for the Q54A mutants. The holohormone recoveries were ample for analysis of receptor binding and steroidogenesis.



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  		Figure 2. Effect of mutation on heterodimer formation by expressed hCG (filled bars) and human LH (hatched bars). Ratio of heterodimer-ß to total ß for each mutant was determined by RIA and plotted relative to the wild-type (1.0); data for position 53 and 55 mutations are mean of duplicate transfections, whereas position 54 mutations are based on three or more transfections (see Results in text).

 
On the other hand, recoveries of mutant Q54R and Q54K heterodimer were markedly reduced, to 20% or less relative to wild-type (Fig. 2Go). Because precise RIA quantitation of dimer relative to total ß was difficult at these concentrations, we confirmed this finding by long-term expression of the Q54R LHß mutant in cultures of stably transfected CHO cells (8). Heterodimer formation was 18.6 ± 1.5% of wild-type, a recovery similar to the transiently-expressed Q54R peptide and significantly lower than the Q54E and Q54A mutants.

Receptor binding and activation by Arg- and Lys- substituted Gln-54 mutants
Despite the low amounts of heterodimer formed, we could test the transiently expressed Q54R and Q54K mutant hormones at doses up to 10-fold (for hCG) and 20-fold (for LH) above that required for 50% binding inhibition by the respective wild-type hormones. No inhibition of labeled hCG binding by either mutant was observed (Fig. 3Go; Table 1Go). Similarly, no steroidogenic activity was found at doses providing a significant response with the respective wild-type hormones.



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  		Figure 3. Effect of ß-subunit position 54 mutations on receptor binding (left panels) and steroidogenesis (right panels) by LH and hCG in murine Leydig tumor (MA-10) cells. Binding curves represent inhibition of 125I-labeled hCG by increasing doses of CHO cell media, calibrated for hormone content by dimer-specific RIA.

 

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  		Table 1. Receptor binding by mutant gonadotropins

 
Mutant Q54R-hCG from stably transfected cells was likewise inactive in the competitive binding assay. As an independent assessment of bioactivity, this mutant was tested for adenylate cyclase activation in cultured HEK-293 cells incorporating recombinant rat LH/hCG receptor. The cAMP response to wild-type expressed hormone was comparable with purified hCG. No increase above basal cAMP levels was observed with Q54R-hCG at concentrations up to 50 ng/ml, a dose that elicited a 25-fold elevation of cAMP with the wild-type (Fig. 4Go).



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  		Figure 4. Adenylate cyclase response to wild-type and Q54R mutant hCG from stable transfection, assayed in HEK-293 cells expressing recombinant rat LH/hCG receptor. Basal response in presence of media alone was 30 pmol/106 cells. Half-maximal response (EC50) to purified hCG in this system is 2 x 10-10 M.

 
Binding and activation by Ala- and Glu-substituted Gln-54 mutants
Responses to the Q54A and Q54E mutants differed between the two hormones. Both hCG mutants inhibited [125I]-hCG binding, with concomitantly productive progesterone responses (Fig. 3Go). The corresponding LH mutants bound to receptor with markedly lower affinity than their hCG counterparts, partial binding inhibition appearing at a concentration 20-fold higher than that required for wild-type LH (Fig. 3Go; Table 1Go). Unlike the basic substitutions, however, binding of these mutants did result in a progesterone response, about 10-fold weaker than wild-type (Fig. 3Go).

Mutations at adjacent sequence positions (53 and 55)
After mutation of the proline at 53 to several contrasting residues (Arg, Glu, Ala, or Ile), both LH and hCG retained activity in the RRA, with commensurate steroidogenic responses (Fig. 5Go). An exception was P53I-LH, which formed very little heterodimer (Fig. 2Go), sufficient for only the lowest dose levels that suggested at least partial binding activity. At residue 55, Valine was replaced by a basic (Arg) and aromatic (Phe) residue with no adverse effect on subunit association by either hormone (Fig. 2Go). For hCG, binding and steroidogenic response curves were comparable with the wild-type (Fig. 6Go and Table 1Go). Although not evaluated in full-range dose-response assays, the LH heterodimers showed complete binding and steroidogenic activity at doses eliciting similar responses using the wild-type. This tolerance to mutation appears consistent with the wide sequence variation at this position among species and hormones.



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  		Figure 5. Effect of ß-subunit position 53 mutations in LH and hCG on receptor binding (left panels) and steroidogenesis (right panels) in MA-10 cells. Assay conditions were as described for Fig. 3Go and in the Materials and Methods section.

 


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  		Figure 6. Effect of hCGß position 55 mutations on receptor binding (left panel) and steroidogenesis (right panel) by MA-10 cells, as described in Fig. 3Go legend. Responses comparable to the wild-type were also observed after ß-55 mutation in LH (not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The foregoing results show that the nature of residue 54 in LH/hCGß influences both subunit association and receptor binding, in a manner dependent on the hormone as well as the specific residue used for replacement. In terms of heterodimer formation, LH and hCG are affected similarly by mutation. Basic substitutions markedly impaired subunit association, whereas neutral and acidic substitutions reduced association by 40–50%–an increment unlikely to be functionally apparent in vivo. For receptor interaction, LH appears less tolerant to mutation than hCG; all substitutions markedly diminished binding activity in LH in contrast to more selective decreases with hCG. Despite low secreted heterodimer concentrations, the Q54R and Q54K mutants of either hormone could be obtained in sufficient amounts to show that binding affinities were less than 5% of the respective wild types. The most striking differences between the two hormones occurred with substitutions other than basic residues. Q54A- and Q54E-hCG bound to receptor with high affinity, whereas these mutations significantly impaired LH binding as with the basic substitutions.

In both hormones, the basic mutants Q54R and Q54K were devoid of steroidogenic activity. On the other hand, the LH Q54A and Q54E mutants did retain partial efficacy, suggesting that signal transduction was intact or even enhanced in proportion to their very weak binding. These same mutations in hCG effectively promoted steriodogenesis, in keeping with their higher binding affinities, and we also found Q54E-hCG to be highly active in the adenylate cyclase assay (data not shown). An efficient steroidogenic response to Q54E-hCG was consistently observed by Huang and Puett (9). They also found evidence for at least partial steroidogenic activity in hCG after basic (Q54K) mutation; although the limited holohormone concentration permitted assays only at the low end of the progesterone response range, the response to a single 0.15 ng/ml dose approached that of the wild type (9). We could not detect activity at up to 0.5 ng/ml, but our narrower total progesterone response range may have obscured a minimal effect at these low doses. Our findings are supported, however, by the lack of activity of Q54R-hCG in the adenylate cyclase assay which covered a much wider dose and response range.

Impairment of function as result of a natural mutation within a gondatotropin molecule is relatively uncommon, in comparison to well-known instances such as familial precocious puberty or hyperthyroidism that involve defects in the receptor (for example, see Refs. 31–33). Besides the Q54R mutation in LH (8), an active, abundant ßLH variant (W8R + I15T) is associated with impaired immunoreactivity and enhanced steroidogenic activity but as yet uncertain clinical consequences (34). Primary amenorrhea and infertility resulted from a deletion mutant leading to a premature termination codon in FSHß (35). In TSH, a ß-subunit G37R mutation blocked subunit association in a hypothyroid subject (36) and, in a striking recent report (37), mutation of Cys-105 to Val impaired both subunit association and receptor binding in two hypothyroid kindreds. This represents a 2-fold effect of mutation similar to our findings with the Q54R mutant in LH.

Between these effects, deficient receptor binding appeared to predominate in the hypogonadal patient reported by Axelrod, Weiss, and colleagues (6, 7, 8). Although their reports of adequate or even elevated Q54R mutant heterodimer may reflect differences in immunoassay conditions, their findings were supported by serum gel filtration profiles that revealed primarily intact hormone (7). Conceivably, the intracellular environment in the gonadotrope in vivo is more favorable for subunit association than our cultured cells, although Matzuk et al. (30) reported close similarity in LH synthesis and assembly among a number of different cell types. It should also be noted that an observed effect on subunit association may involve either an outright defect in synthesis, folding, and/or assembly resulting in low holohormone content in the medium (30), or instability of the secreted holohormone that would prevent or curtail subsequent receptor occupancy (38). In fact, accelerated subunit dissociation during the binding process would be difficult to distinguish from a true structural incompatibility between hormone and receptor.

The importance of residue 54 appears to be sharply localized, as effects on receptor binding and activation could not be replicated by mutations at adjacent sites (Pro-53 or Val-55). As described further below, this does not rule out a conformational effect of Gln-54 mutation, but it is unlikely that this residue is a representative component of a larger linear determinant. An example of the latter would be the segment LVY (residues 37–39) in FSHß studied by Roth and Dias (38). A direct effect of residue 54 on bioactivity would not be surprising, as it resides in the solvent-exposed C-terminal half of the long intercysteine (38–>57) loop in the crystal structure of hCG (Fig. 7Go). Other loop mutations or internal enzymatic cleavages diminish activity (39, 40), and synthetic peptides representing this sequence from both LH and hCG bind to receptor and promote steroidogenesis (41).



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  		Figure 7. Molecular model based on crystal structure of hCG (Refs. 4, 5) showing location of Gln-54 and neighboring residues in the long loop (ßL2; residues 38–57) of the ß-subunit. Adjacent region of the {alpha}-subunit ({alpha}L1; residues 12–40) is shown, with side-chains removed for clarity. ßGln-54 is oriented across the loop and slightly downward into the plane of the paper. ßArg-43 is also cross-loop in orientation, angled slightly outward toward the reader.

 
The effects on subunit association are more unexpected, given the remote location of Gln-54 and its neighbors from any {alpha} contact surface in the crystal structure. This implies that the unfavorable substitutions can exert remote effects on subunit folding or conformation. The crystal structure shows the Gln-54 side-chain to be oriented back across the loop (Fig. 7Go), where it might contact or stabilize residues closer to {alpha} such as Thr-40, which has been implicated in subunit interaction (42). A hydrogen bond involving the Gln-54 side-chain and Met-41 is described in one of the published crystal structures (4). The Gln side-chain may thus maintain the shape of the loop as a whole, subject to distortion by inappropriate groups that introduce steric constraints or relocate cross-loop hydrogen bonds.

Eighteen sequence differences are found between the LHß-subunit and the corresponding core (1–114) region in hCG, any of which could influence a differential effect of mutation on either receptor interaction or heterodimer formation. Hence, besides the differences in receptor binding among the Gln-54 mutants, we also found heterodimer recovery to be markedly diminished after replacement of Pro-53 by Ile in LH, but not in hCG. Because nearby position 51 differs between LH (Pro) and hCG (Ala), it is possible that the bulky Ile sidechain cannot be accommodated in LH without long-range distortion of subunit contacts. Two other substitutions (positions 42 and 47) also occur within the long intercysteine (38–57) loop shared with Pro-53 and Gln-54. The three sequence differences are sufficient to induce a 5-fold difference in binding activity in the respective synthetic peptides (41), and far more sites for differential interactions are clearly possible in the whole hormone.

Although substantial, the difference in carboxyl-terminal structure beyond residue 114 does not appear to play a role in the differential response of the hormones to mutation. The 31-residue, carbohydrate-rich (115–145) tail of hCG stabilizes the hormone in vivo (18), but it has little direct effect on target-cell activation: steroidogenic activity of hCG after C-terminal truncation or deletion is comparable to the wild-type (11, 18, 24). The precise C-terminus of the ß-subunit in circulating, bioactive LH remains uncertain; all reported structures of isolated pituitary LH terminate at residue 114 (1, 2, 3), whereas the nucleotide sequence predicts an extra seven residues, through residue 122 (28). This hydrophobic and potentially unstable sequence, if present even transiently, may be in part responsible for the slow rate of assembly observed for LH compared with hCG (30). Nevertheless, our results show that once secreted as the heterodimer, the ß-122 and truncated ß-114 forms of LH are comparable in binding activity.

This investigation demonstrates that the ß-subunit primary sequence as well as previously recognized differences in carbohydrate structure can influence the biological properties of LH relative to hCG. In another recent study, point mutagenesis of cysteine residues in the common {alpha}-subunit was used to show that the (7–31) and (59–87) disulfide linkages are critical for assembly of LH heterodimer, but not hCG or FSH (43). Differential effects among gonadotropins after mutations in the midregion and the C-terminus of the {alpha}-subunit have also been documented (44, 45, 46). These and other distinctions would not be predicted, nor can their structural bases be deduced, from scrutiny of the crystal structure alone. While useful as a framework for suggesting mutants or analogs, it is increasingly apparent that the crystal structure cannot reliably predict the functional role of a given residue or region, especially if one attempts to simply build residues from other hormones into the hCG structure. This emphasizes the continued need for structure-activity studies to complement the crystal structure in mapping critical functional regions in all of the glycoprotein hormones.


   Acknowledgments
 
We acknowledge with gratitude Dr. Tom Gardella (Endocrine Unit, Massachusetts General Hospital) for his advice and guidance in preparation of the mutant plasmids, Kathleen Kitzmann (Department of Laboratory Medicine and Pathology, Mayo Medical School) for her expert technical assistance in performing the adenylate cyclase assays and preparing the Oligo-31 cell cultures, Dr. Sven Hyberts (Department of Biochemistry and Molecular Pharmacology, Harvard Medical School) for his help in preparation of the molecular model, and Karen Minyard (Endocrine Unit, Massachusetts General Hospital) for her patient work with preparation of the manuscript.


   Footnotes
 
1 This work was supported by Grant HD-12851 and Center Grants P30-HD28138 and P30-HD09140 from the NIH. Back

Received September 16, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pierce JG, Parsons TF 1981 Glycoprotein hormones: structure and function. Annu Rev Biochem 50:465–495[CrossRef][Medline]
  2. Gordon WL, Ward DN 1985 Structural aspects of luteinizing hormone actions. In: Ascoli M (ed) Luteinizing Hormone Receptors and Action. CRC Press, Paris, pp 173–198
  3. Ryan RJ, Keutmann HT, Charlesworth MC, McCormick DJ, Milius RP, Calvo FO, Vutyavanich T 1987 Structure and function of the glycoprotein hormones. Recent Prog Horm Res 43:383–429
  4. Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, Morgan FJ, Isaacs NW 1994 Crystal structure of human chorionic gonadotropin. Nature 369:455–461[CrossRef][Medline]
  5. Wu H, Lustbader JW, Liu Y, Canfield RE, Hendrickson WA 1994 Structure of human chorionic gonadotropin at 2.6A resolution from MAD analysis of the selenomethionyl protein. Structure 2:545–558[Medline]
  6. Axelrod L, Neer RM, Kliman B 1979 Hypogonadism in a male with immunologically active, biologically inactive luteinizing hormone: an exception to a venerable rule. J Clin Endocrinol Metab 48:279–287[Medline]
  7. Beitins IZ, Axelrod L, Ostrea T, Little R, Badger TM 1981 Hypogonadism in a male with an immunologically active, biologically inactive luteinizing hormone: characterization of the abnormal hormone. J Clin Endocrinol Metab 52:1143–1149[Medline]
  8. Weiss J, Axelrod L, Whitcomb RW, Harris PE, Crowley WF, Jameson JL 1992 Hypogonadism caused by a single amino acid substitution in the beta-subunit of luteinizing hormone. New Engl J Med 326:179–183[Medline]
  9. Huang J, Puett D 1994 On the role of the invariant glutamine at position 54 in the human choriogonadotropin ß-subunit. Mol Cell Biochem 136:183–186[CrossRef][Medline]
  10. Lee CY, Ryan RJ 1973 Interaction of ovarian receptors with human luteinizing hormone and human chorionic gonadotropin. Biochemistry 12:4609–4619[CrossRef][Medline]
  11. El-Deiry S, Chen T-M, Puett D 1991 Comparison of steroidogenic potencies of homologous and heterologous gonadotropins in rat and mouse Leydig cells. Mol Cell Endocrinol 76:105–113[CrossRef][Medline]
  12. Segaloff DL, Puett D, Ascoli M 1981 The dynamics of the steroidogenic response of perifused Leydig tumor cells to human chorionic gonadotropin, ovine luteinizing hormone, cholera toxin, and adenosine 3'5'-cyclic monophosphate. Endocrinology 108:632–638[Abstract]
  13. Strickland TW, Puett D 1981 Contribution of subunits to the function of luteinizing hormone/human chorionic gonadotropin recombinants. Endocrinology 109:1933–1942[Abstract]
  14. Niswender G, Schwall R, Fitz T, Farin C, Sawyer H 1985 Regulation of luteal function in domestic ruminants: new concepts. Recent Prog Horm Res 41:101–151
  15. Buettner K, Ascoli M 1984 Na+ modulates the affinity of the lutropin/choriogonadotropin receptor. J Biol Chem 259:15078–15084[Abstract/Free Full Text]
  16. Ascoli M, Segaloff DL 1987 On the fates of receptor-bound ovine luteinizing hormone and human chorionic gonadotropin in cultured Leydig tumor cells. Definition of similar rates of internalization. Endocrinology 120:1161–1172[Abstract]
  17. Wehmann RE, Nisula BC 1979 Metabolic clearance rates of the subunits of human chorionic gonadotropin in man. J Clin Endocrinol Metab:753–759
  18. Matzuk MM, Hsueh AJW, LaPolt P, Tsafriri A, Keene JL, Boime I 1990 The biological role of the carboxyl-terminal extension of human chorionic gonadotropin ß-subunit. Endocrinology 126:376–383[Abstract]
  19. Keutmann HT 1994 Receptor binding regions of hLH and hCGß subunit: structural and functional properties. In: Lustbader JW, Puett D, Ruddon RW (eds) Glycoprotein Hormones: Structure, Function and Clinical Implications. Serono Symposia, USA, Springer-Verlag, New York, pp 103–117
  20. Kunkel TA 1985 Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492[Abstract/Free Full Text]
  21. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning: a Laboratory Manual, 2nd edition, Vol 3, Cold Spring Harbor Press, Plainview, NY, pp 16.30–16.55
  22. Seed B, Aruffo A 1987 Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc Natl Acad Sci USA 84:3365–3369[Abstract/Free Full Text]
  23. Chen F, Puett D 1991 Delineation via site-directed mutagenesis of the carboxyl-terminal region of human choriogonadotropin beta required for subunit assembly and biological activity. J Biol Chem 266:6904–6908[Abstract/Free Full Text]
  24. Huang J, Chen F, Puett D 1993 Amino/carboxyl-terminal deletion mutants of human choriogonadotropin ß. J Biol Chem 268:9311–9315[Abstract/Free Full Text]
  25. Taylor AE, Khoury RH, Crowley WF 1994 A comparison of 13 different immunometric assay kits for gonadotropins: implications for clinical investigation. J Clin Endocrinol Metab 79:240–247[Abstract]
  26. Ascoli M 1981 Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 108:88–95[Abstract]
  27. Wang Z, Wang H, Ascoli M 1993 Mutation of a highly conserved acidic residue present in the second intracellular loop of G-protein coupled receptors does not impair hormone binding or signal transduction of the luteinizing hormone/chorionic gonadotropin receptor. Mol Endocrinol 7:85–92[Abstract]
  28. Talmadge K, Vamvakopoulos NC, Fiddes JC 1984 Evolution of the genes for the ß subunits of human chorionic gonadotropin and luteinizing hormone. Nature 307:37–40[CrossRef][Medline]
  29. Ryan RJ, Charlesworth MC, McCormick DJ, Millius RP, Keutmann HT 1988 The glycoprotein hormones: recent studies of structure-function relationships. FASEB J 2:2661–2669[Abstract]
  30. Matzuk MM, Spangler MM, Camel M, Suganuma N, Boime I 1989 Mutagenesis and chimeric genes define determinants in the ß subunits of human chorionic gonadotropin and lutropin for secretion and assembly. J Cell Biol 109:1429–1438[Abstract/Free Full Text]
  31. Parma J, Duprez L, van Sande J, Cochaux P, Gervy C, Mockel J, Dumont J, Vassart G 1993 Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature 365:649–651[CrossRef][Medline]
  32. Shenker A, Laue L, Kosugi S, Merendino JJJ, Minegishi T, Cutler GBJ 1993 A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365:652–654[CrossRef][Medline]
  33. Latronico AC, Anasti J, Arnhold IJP, Mendonca BB, Domenice A, Albano MC, Zachman K, Wajchenberg BL, Tsigos C 1995 A novel mutation of the luteinizing hormone receptor gene causing male gonadotropin-independent precocious puberty. J Clin Endocrinol Metab 80:2490–2494[Abstract]
  34. Suganuma N, Furui K, Kikkawa F, Tomoda Y, Furuhashi M 1996 Effects of the mutations (Trp9->Arg and Ile15->Thr) in human luteinizing hormone (LH) ß-subunit on LH bioactivity in vitro and in vivo. Endocrinology 137:831–838[Abstract]
  35. Matthews CH, Borgato S, Peck-Peccoz P, Adams M, Tone Y, Gambino G, Casagrande S, Tedeschini G, Benedetti A, Chatterjee VKK 1993 Primary amenorrhoea and infertility due to a mutation in the ß-subunit of follicle-stimulating hormone. Nature Genet 5:83–85[CrossRef][Medline]
  36. Hayashizaki Y, Hiroaka Y, Endo Y, Matsubara K 1989 Thyroid-stimulating hormone (TSH) deficiency caused by a single base substitution in the CAGYC region of the beta subunit. EMBO J 8:2291–2296[Medline]
  37. Medieros-Neto G, Herodotou DT, Rajan S, Kommareddi S, de Lacerda L, Sandrini R, Boguszewski MCS, Hollenberg AM, Radovick S, Wondisford FE 1996 A circulating, biologically inactive thyrotropin caused by a mutation in the beta subunit gene. J Clin Invest 97:1250–1256[Medline]
  38. Roth KE, Dias JA 1995 Scanning-alanine mutagenesis of long loop resides 33–53 in follicle stimulating hormone beta subunit. Mol Cell Endocrinol 109:143–149[CrossRef][Medline]
  39. Bousfield GR, Ward DN 1988 Selective proteolysis of ovine lutropin or its beta subunit by endoproteinase Arg-C. J Biol Chem 263:12602–12607[Abstract/Free Full Text]
  40. Cole LA, Kardana A, Andrade-Gordon P, Gawinowicz MA, Morris JC, Bergert ER, O’Connor J, Birken S 1991 The heterogeneity of human chorionic gonadotropin (hCG). III. The occurrence and biological and immunological activities of nicked hCG. Endocrinology 129:1559–1567[Abstract]
  41. Keutmann HT, Charlesworth MC, Mason KA, Ostrea T, Johnson L, Ryan RJ 1987 A receptor-binding region in human choriogonadotropin/lutropin ß subunit. Proc Natl Acad Sci USA 84:2038–2041[Abstract/Free Full Text]
  42. Xia H, Puett D 1994 Identification of conserved amino acid residues in the ß-subunit of human choriogonadotropin important in holoprotein formation. J Biol Chem 269:17944–17953[Abstract/Free Full Text]
  43. Furuhashi M, Suzuki S, Suganuma N 1996 Disulfide bonds 7–31 and 59–87 of the {alpha}-subunit play a different role in assembly of human chorionic gonadotropin and lutropin. Endocrinology 137:4196–4200[Abstract]
  44. Yoo J, Zeng H, Ji I, Murdoch WJ, Ji TH 1993 COOH-terminal amino acids of the {alpha} subunit play common and different roles in human choriogonadotropin and follitropin. J Biol Chem 268:13034–13042[Abstract/Free Full Text]
  45. Grossmann M, Szkudlinski MW, Dias JA, Xia H, Wong R, Puett D, Weintraub BD 1996 Site-directed mutagenesis of amino acids 33–44 of the common {alpha}-subunit reveals different structural requirements for heterodimer expression among the glycpoprotein hormones and suggests that cyclic adenosine 3',5' monophosphate production and growth promotion are potentially dissociable functions of human thyrotropin. Mol Endocrinol 10:769–779[Abstract]
  46. Szkudlinski MW, Grossman M, Weintraub BD 1996 Structure-function studies of human TSH. Trends Endocrinol Metab 7:277–286[Medline]



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J. Pantel, P. Robert, F. Troalen, M. Kujas, D. Bellet, and J.-M. Bidart
Characterization of Human Lutropin Carboxyl- Terminus Isoforms
Endocrinology, February 1, 1998; 139(2): 527 - 533.
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