(Received for publication, September 25, 1995; and in revised form, December 15, 1995)
From the
The functional importance of cysteine residues in the extracellular domain and the extracellular loops (EL1 and EL2) to hormone binding of the rat luteinizing hormone receptor (LHR) was investigated. For this purpose, cysteines in the seven-transmembrane holoreceptor (Form A) and its hormone-binding splice variant (Form B) were replaced by serine residues, and mutant receptors were expressed in COS1 and/or insect cells. Within the extracellular domain, individual replacement of all four cysteines from Exon 1 abolished hormone binding activity, and replacement of Cys-109 and Cys-134 from exons 5 and 6 caused a 75% decrease in both cell surface and total cellular solubilized LHR hormone binding activity. Mutations of Cys-257 and -258 (Exon 9), Cys-321 and -331, and Cys-417 and -492 of EL1 and EL2, respectively (Exon 11), showed no surface hormone binding activity on intact cells, but exhibited wild type levels of total hormone binding activity when recovered from detergent-solubilized cellular extracts. This finding indicated that expression of high affinity LHR binding activity at the cell surface is independent of the acquisition of the high affinity binding conformation. Other cysteine residues, including Cys-282 (exon 10), and Cys-314 (exon 11) were not essential for hormone binding activity or plasma membrane insertion. This study demonstrates that the functional hormone binding domain utilizes all cysteines N-terminal to exon 7 and localizes the binding site to this N-terminal region of the extracellular domain.
The luteinizing hormone receptor (LHR) ()is a
seven-transmembrane (1) G protein-coupled receptor (GPCR) with
a high affinity hormone binding site on the N-terminal extracellular
domain (Fig. 1)(2, 3) . This hormone binding
domain is composed of two Cys-rich regions in exons 1 and 9, generally
conserved in the glycoprotein hormone receptor family (FSHR and TSHR),
and bordering a leucine-rich domain that is repeated from exons 2 to
8(4) . A third Cys-rich region (Cys-314, -321, and -331) is
located outside of the hormone binding domain although still in the
extracellular region and is also conserved in the FSHR (5) and
TSHR(6) . The LHR contains only three cysteines within the
extracellular domain that are not conserved in the FSHR and TSHR, at
Cys-109, Cys-134, and Cys-282 (Fig. 1). These cysteines are
within domains that have been implicated to be of importance to LH/hCG
binding in chimeric studies of the glycoprotein hormones(7) .
Outside of the N-terminal extracellular domain, two LHR cysteines on
EL-1 and EL-2 are conserved in most of the GPCR and are of structural
importance to the adrenergic/rhodopsin family(8, 9) .
Amino acid homology within the transmembrane and cytoplasmic domains of
the GPCR have led investigators to propose the existence of a common
mode of signal transfer and G coupling that may involve the formation
or disruption of disulfide bonds(10, 11) .
Figure 1: The rat LH receptor. The amino acid sequence of the rat LH receptor(1) . Amino acids corresponding to the cleavable signal peptide are within hexagons, and those of the mature polypeptide are within circles. The rat LHR Form B sequence diverges from holoreceptor at 294 (I) and continues with LLHGALPATHCLS peptide tail(2) . Cysteines are indicated by shaded circles, and those we have mutated are designated by amino acid number. Exon divisions are represented by vertical lines and exons are numbered 1-11.
To study the importance of cysteines and disulfide bonding to LHR hormone binding, the indicated cysteines (SH) (Fig. 1) in the rat holoreceptor were mutated to the amino acid serine (OH) and expressed in the mammalian COS1 cell. The substitution of Cys to Ser prevents the formation of a putative disulfide bond, without significantly affecting the charge characteristics of the protein(12) . The isolated hormone binding domain was studied in both the COS1 cell and the insect Sf9 cell with the soluble splice variant Form B that contains the high affinity hormone binding domain without the transmembrane, extracellular loops, or cytoplasmic domain(2) . This constitutes amino acids 1-294 of the holoreceptor and a unique 22-amino acid tail that contains one additional cysteine that may functionally substitute for a Cys in the holoreceptor. Our results demonstrate that only the four Cys within exon 1 are essential for hormone binding activity and that the two unique Cys at 109 and 134 are of importance, although not essential to hormone binding. The extracellular Cys (257, 258, 321, and 331) and EL1 417 and EL2 492 that are conserved in all of the glycoprotein hormone receptors are required for membrane insertion, but they do not contribute to hormone binding activity.
All experiments described above were performed at least 3 times in triplicate.
Transfection of the LHR holoreceptor Form A into the COS1
cell yields a high affinity hCG hormone binding receptor that is
translocated to the cell surface. Scatchard analysis of the surface
wild-type holoreceptor reveals that the dissociation constant K of the LHR holoreceptor expressed in COS1 cell
is similar to that of the rat ovarian LH
receptor(2, 20) . Similar hormone binding affinities
were also obtained with expression of the Form B splice variant in COS1
or insect Sf9 cells(17) . Individual mutation of each of the
cysteines in exon 1 to Ser of the holoreceptor expressed from the COS1
cell (Cys 8, 12, 14, 22) or the Form B splice variant expressed in the
insect cell (Cys 8, 12, 14, 22) resulted in total loss of hormone
binding activity (Table 1, Fig. 2and 3). Receptor
activity was not recovered in the detergent solubilized fraction of the
COS1 cell (Fig. 2), indicating that loss of activity was not due
to entrapment within the cells. To confirm that Cys-8
Ser,
Cys-12
Ser, Cys-14
Ser, and Cys-22
Ser was
expressed, the concentration of receptors was determined by specific
radioimmunoassay. Mutant receptor levels of Cys-8
Ser and Cys-22
Ser were within 60% that of wild-type, and Cys-12
Ser and
Cys-14
Ser levels were not significantly different from
wild-type (Table 2).
Figure 2:
Percent wild-type I-hCG
binding activity of designated CysLHRSer holoreceptor mutant proteins
expressed from COS1 cells. *, no significant difference from basic
vector without insert.
Substitution of the unique cysteines from exon 5 and 6 (Cys-109 and Cys-134) caused a 75% decrease in hormone binding activity on the surface of the intact cell that could not be recovered in detergent-solubilized extracts ( Fig. 2and Fig. 4). Expression levels of these mutant LHRs were not significantly different from wild-type (Table 2, Fig. 4), indicating that substitution of Cys-109 and -134 specifically affects hormone binding but not receptor expression. In addition, these cysteines do not appear to play a role in membrane insertion since the proportion of active mutant to wild-type LHR was the same for surface and solubilized receptors (approximately 25%). Ser-134 LHR exhibited a marked reduction in maximal hormonal activation of cAMP in comparison with the wild-type. This reduction of 80 ± 15% was consistent with the 75% decrease in hormone binding activity, and similarly substitution of Cys-109 reduced maximal hormonal stimulation (Table 2).
Figure 4:
Displacement curves of I-hCG
binding to wild-type and mutant Cys-109 and -134 mutant LHR
holoreceptor expressed in COS1 cells. Binding to intact cells (Surface) (left) and to detergent extracts (total) (center). Right, dose-related displacement of
I-LHR(36-51) peptide binding to peptide
(36-51) antibody by unlabeled LHR (36-51) peptide (standard
curve) (20) and by designated LHR wild-type and mutant
receptors. The extracts of samples displayed parallel displacement to
the standard curve. No displacement was observed by extracts from cells
transfected with vector in the absence of LHR
insert.
Individual substitution of the two consecutive
cysteines within exon 9 (Cys-257 and -258) (Fig. 1), which are
conserved in all of the glycoprotein hormone receptors, resulted in
total loss of surface binding activity (Fig. 2). However,
70-100% of the wild-type hormone binding activity was recovered
following detergent solubilization. (Table 1, Fig. 2). To
determine whether Cys-257 functionally replaced Cys-258 in the Cys-258
Ser mutant, the double mutant Cys-257
Ser/Cys-258
Ser was expressed from COS1 cells and characterized for hormone binding
activity. Detergent extracts from cells expressing the double mutant
also displayed 90% of the wild-type hormone binding activity. The
binding affinities (K
) of these mutant receptors
were identical to that of wild-type (Table 1). Thus, it does not
appear that Cys-257 or -258 are important for hCG hormone binding.
Substitution of Cys-282 Ser (Fig. 1), which is not
conserved in the FSHR or TSHR, resulted in a mutant receptor with
impaired but not total loss of surface hormone binding activity (Fig. 2; Table 1and Table 2). Cys-282
Ser
exhibited a 50% reduction in surface receptor hormone binding activity,
with a binding affinity that was similar to wild-type (Table 1).
However, total detergent solubilization of this mutant yielded total
receptor binding concentrations that were similar to wild-type ( Table 1and Table 2). Hormonal stimulation caused
dose-related increases in cAMP levels in cells transfected with this
mutant, and maximal levels were 51 ± 2% that of wild-type. This
correlated with the 50% reduction in surface receptor (Table 2).
Thus, Cys-282, which is unique to the LHR, is not important for either
hCG hormone binding or signal transfer, but it does contribute to
surface membrane expression.
Cys-314, -321, and -331; Cys-417; and
Cys-492 are not contained in the Form B hormone binding domain,
although these are present in the extracellular N-terminal, and EC
loops 1 and 2, respectively, of the holoreceptor (Fig. 1).
Substitution of Cys-314 Ser did not affect either hormone
binding affinity or membrane insertion (Table 2, Fig. 2),
or cAMP levels (not shown). Thus Ser-314 is functionally equivalent to
Cys-314, and Cys-314 does not have to be paired in a disulfide bond in
the holoreceptor for binding activity. Substitution of Cys-321,
Cys-331, Cys-417, and Cys-492 by Ser resulted in mutant receptors that
exhibited no surface binding activities. However, binding activity of
these mutant LHRs were recovered in detergent-solubilized extracts ( Table 1and II; Fig. 2).
The importance of disulfide bonds to hormone binding activity was investigated in ligand blots using the Form B splice variant expressed in insect cells and the affinity-purified Form A holoreceptor isolated from ovarian membranes. Treatment of the 38-kDa Form B receptor with reducing agent prior to electrophoretic separation, and transfer to nitrocellulose, did not reduce the ability of the electroblotted receptor to bind labeled hCG (Fig. 5, LHRB, reducing and nonreducing). These results, and those reported recently(17) , indicate that the initial oxidation state does not affect the ability of the Form B LHR to renature during the electroblotting transfer to nitrocellulose membrane.
Figure 5:
Western and ligand blots of Form A
(holoreceptor) and B (splice variant) wild-type LHR under reducing and
nonreducing conditions. Left, LHR in sample buffer was either
reduced with -mercaptoethanol (R) or not reduced (NR) prior to SDS-PAGE as described under ``Experimental
Procedures.'' Samples were subsequently electroblotted to
nitrocellulose and probed with the LHR peptide (36-51) antibody. Right, ligand blot probed with
I-hCG.
In contrast, prior incubation of the Form A holoreceptor with reductant severely impaired the ability of the receptor to bind hormone after electrophoresis in ligand blots (Fig. 5, LHRA, reducing versus nonreducing). Thus, the addition of the transmembrane/cytoplasmic domains presumably has a negative effect on renaturation of the reduced LHR on nitrocellulose (see ``Discussion''). A minor aggregate (>200 kDa), more pronounced under nonreducing conditions, exhibits no detectable ligand binding (Fig. 5, left, LHRA, reducing and nonreducing versus right, LHRA, reducing and nonreducing).
The ability of the reduced, denatured wild-type Form B
LHR to recover its active configuration and bind hormone in ligand
blots raises the question of whether the exon 1 cysteines are essential
for renaturation of the mature receptor. Renaturation of the reduced
Form B receptor on ligand blots may require the reformation of
essential disulfide bonds, which would presumably be prevented in the
exon 1 mutant LHRs. The Cys Ser conservative substitution
primarily impacts on loss of disulfide formation in the mutant
protein(11) , but it may also result in the loss of essential
free cysteines in the exon 1 mutants. Ligand blotting was performed
with each of the exon 1 Cys mutant LHRs (Cys-8
Ser, Cys-12
Ser, Cys-14
Ser, Cys-22
Ser) to resolve two
questions. First, whether ligand binding of the reduced wild-type Form
B receptor on nitrocellulose was due to renaturation that involved exon
1 cysteines, and perhaps the reformation of disulfide bonds during
electroblotting, or the hormone binds to a surface that is exposed on
nitrocellulose without the aid of disulfide loops. Second, does the
loss of hormone binding activity by the Cys/Ser substitutions in exon 1
result from the loss of required free or disulfide bonded cysteines on
the mature receptor or impairment of initial processing of the nascent
LHR in the endoplasmic reticulum.
None of the exon 1 Form B LHR mutant receptors exhibited hormone binding activity when immobilized on nitrocellulose (Fig. 6), indicating that in contrast to the denatured, reduced wild-type Form B LHR, the exon 1 mutants are incapable of renaturation during electroblotting (Fig. 6). This indicates that Cys-8, -12, -14, and -22 are essential for renaturation of the mature LHR and that therefore these cysteines play an essential role beyond processing of the nascent receptor, perhaps as disulfide-bonded cysteines.
Figure 6:
Western and ligand blots of Form B LHR
wild-type and Exon 1 Cys mutants. Left, LHR in sample buffer
was reduced as in Fig. 5, resolved on SDS-PAGE, and transferred
to nitrocellulose. Blot was probed with the LHR peptide (36-51)
antibody. Right, after transfer to nitrocellulose, blot was
incubated with I-hCG.
Site-directed mutagenesis of the cysteines of the rat LHR, including residues that are unique to the LHR (Cys-109, -134, and -282), of importance to the rhodopsin/adrenergic family (Cys-417 and -492)(8, 9) , and those that are conserved in the extracellular domain of the glycoprotein hormone receptor family (Cys-8, -12, -14, and -22 of exon 1, Cys-257 and -258 of exon 9, and Cys-314, -321, and -331 of exon 11), reveal that only the extracellular N-terminal cysteines in exon 1 and Cys-109 and -134 are of importance for hormone binding. A second group of conserved cysteines within the extracellular domain (Cys-257, -258, -321, and -331) and the extracellular loops 1 (417) and 2 (492) are essential for surface expression but not for hormone binding (Table 2).
The importance of cysteines to hormone binding and signal transfer in the class 1 GPCR has been well documented(8, 9) , with the implication that disulfide bonds are important for structural integrity and signal relay. The LHR differs from the class 1 GPCR (adrenergic family) by an extended 300-amino acid N-terminal extracellular hormone binding domain that can be physically separated from the transmembrane/cytoplasmic domain without loss of hCG hormone binding affinity(2) . Putative disulfide bonds that connect the extracellular domain to the transmembrane/cytoplasmic domain are not required for hormone binding activity, and in this study we have found no cysteines including and following the exon 9 position 257/258 that are important for hormone binding.
The isolated high affinity hormone binding domain that is represented by the splice variant Form B and consists of exons 1 to 10 carries a unique carboxyl 22-amino acid tail with an extra cysteine (Fig. 1) that is not in the holoreceptor. This cysteine does not function as a replacement for one of the extracellular exon 11 cysteines (Cys-314, -321, -331, -417, or -492), since none of these exon 11 cysteines are important for hormone binding. However, the addition of the Form B cysteine (Cys-305) does cancel out a potential unpaired cysteine (Fig. 1) and may serve to reduce intermolecular aggregation that could result from the reactivity of an unpaired cysteine.
The four cysteines in exon 1 are each essential for hormone binding activity, and no other cysteines can replace their functions in the mutant LHRs. In the TSH receptor, mutation of the analogous first three cysteines of exon 1 did not affect TSH binding(21) , suggesting that these have a unique function in LH/hCG binding, perhaps forming disulfide bonds with the unique LHR cysteines 109 and 134, which are not conserved in the TSH receptor. Although the four cysteines of the TSHR exon 1 can substitute for the LHR exon 1 in chimeric receptor constructs, hCG binding was only achieved when the segment that includes Cys-109 and -134 of the LHR was also present in the construct (7) . However, putative disulfide bonds between cysteines in exon 1 and Cys-109 or Cys-134 should yield equivalent reductions in activity upon mutation of each half cystine, which was not the case in these studies ( Table 1and Table 2). Rather, a putative disulfide bond is suggested between Cys-109 and Cys-134 in the hormone binding domain since mutation of each of these resulted in equivalent reductions of binding activity.
There is some indication that the extracellular and transmembrane domains are linked by disulfide bonds in the TSH receptor, and LHR-specific interactions between the two domains have also been proposed to account for membrane insertion of the LH receptor(22) . Our experiments suggest that the LHR extracellular/transmembrane interactions may be in the form of disulfide bonds, similar to the TSHR. Cys-257, -258, -321, and -331 of the extracellular domain and the Cys-417 of EL1 and Cys-492 of EL2, all conserved in the glycoprotein hormone receptors LHR, TSHR, and FSHR, are required for membrane insertion but not hormone binding activity (Table 1). Individual mutation of each of these cysteines prevented surface expression, indicating that the disulfide configuration is highly specific and that other cysteines cannot functionally substitute. Most of these extracellular cysteines that are essential for insertion into the membrane may be important in signal transduction, since they are conserved in all members of the glycoprotein hormone receptor family. The acquisition of the correct disulfide configuration to initiate signal transfer may be a prerequisite for membrane insertion, although, as we have shown, acquisition of the high affinity binding conformation is not a prerequisite for membrane insertion.
In studies with bovine opsin, which does not contain an extended extracellular domain, cysteines from extracellular loop 1 and 2 (Cys-110 and -187) were required for membrane insertion(23) , similar to our observation with Cys-417 and -492 mutant LHRs. Cys-621 and -622 from the cytoplasmic domain have been reported to be important for membrane insertion(24) . However, another report attributes the decrease in surface Cys mutant receptors to an increase in the rate of hormone/receptor internalization(25) . In addition, non-Cys mutations in the transmembrane/cytoplasmic module such as E441Q can also impair membrane insertion(26) . Taken together, these studies indicate a wide range of extracellular and transmembrane/cytoplasmic amino acids that are required for membrane insertion of the LHR, suggesting that the total integrity of the exon 7-11 domain is a prerequisite for translocation to the plasma membrane.
Previous studies with tunicamycin show that folding of the nascent Form B LHR receptor that lacks the transmembrane/cytoplasmic domain, to an active configuration is dependent on the presence of N-linked carbohydrates and not on the initial oxidation state of the receptor(17) . We have established that the LHR Form B receptor is only capable of spontaneous renaturation during ligand blotting, following SDS denaturation and boiling in the presence or absence of reductant, as long as the receptor carries the proximal N-acetylglucosamine on Asn-173 and Asn-152 (17) and, in this study, the exon 1 cysteines (Fig. 6).
Although renaturation of the Form B splice variant did not depend on the initial oxidation state of the receptor, hormone binding of the Form A holoreceptor was greatly reduced when the receptor was treated with reductant prior to electrophoresis and ligand blotting (Fig. 5). The transmembrane domain appears to interfere with renaturation of the extracellular hormone binding domain only when the LHR is in the reduced, free cysteine state prior to electrophoresis/blotting. Fig. 6suggests that renaturation of the isolated Form B hormone binding domain involves the reformation of exon 1 disulfide bonds, since the primary impact of the conservative substitution of Ser for Cys is in the loss of the ability to form disulfide bonds(12) . In the Form A holoreceptor, reactive cysteines from the transmembrane or cytoplasmic domain, exposed only under reducing conditions, may interfere with these putative disulfide interactions in the extracellular hormone binding domain, impairing ligand binding.
The observation that all but two of the cysteines characterized in this study that were determined to be functional for hormone binding or membrane insertion are conserved indicates that a common disulfide configuration may exist for the glycoprotein receptor family, designed for the general function of membrane insertion. The body of evidence that has accumulated on LHR hormone binding suggests that the extracellular hormone binding domain may be a minimal secondary structural element such as an amphipathic helix, since measurable hCG hormone binding has been surprisingly achieved with small receptor fragments and transmembrane helical domains(27) . With this in mind, the ``spontaneous renaturation'' of the hormone binding domain that occurs in ligand blots, even under the extraordinary denaturing conditions that were used for deglycosylation(17) , may instead indicate that extensive renaturation of the LHR does not have to occur for hormone binding and that a minimal Form B receptor domain, exposed on the nitrocellulose membrane, may be sufficient for binding.
In summary, the extracellular cysteines in exon 1 (Cys-8, -12, -14, and -22) and exons 5 (Cys-109) and 6 (Cys-134) are important for hormone binding activity; those in exon 1 being essential for activity. The relatively conservative substitution of serine for cysteine suggests that the loss of binding activity may be attributed to the loss of potential disulfide bonds. Cys-257, -258, -282, -321, -331, -417, and -492 were important to various degrees for membrane insertion. Radioimmunoassay studies indicate that while substitution of many of the LHR cysteines resulted in retention of the mutant proteins in the nonsurface membrane fraction, no changes in steady-state concentrations of the receptor were observed. These single substitution experiments demonstrate that specific cysteines in exons 1-6, perhaps through disulfide interaction, are essential/important for the active receptor configuration.