Biomedical subjects

D L Segaloff

Publications and source records attributed to D L Segaloff.

At least 37 records · Page 2Linked to original sources

PubMed1995

Intracellular retention of mutant gonadotropin receptors results in loss of hormone binding activity of the follitropin receptor but not of the lutropin/choriogonadotropin receptor.

It has recently been reported that Asp 397 of the rat lutropin/ choriogonadotropin receptor (rLHR) may be involved in transducing the signal from hormone binding to the stimulation of cAMP production. We examined the analogous region in the rat follitropin receptor (rFSHR) by substituting the Asp at position 404 (D404) of the rFSHR with either Glu (D404E), Ala (D404A), or Lys (D404K). Both in intact 293 cells and in detergent-solubilized extracts of 293 cells transiently transfected with the rFSHR constructs, only the wild type rFSHR exhibited detectable binding activity. Although the D404-substituted rFSHR mutants were visible on Western blots, in contrast to the wild type rFSHR which is present on Western blots as both mature and immature forms, only a single band comigrating with immature receptor was observed for the mutants. Furthermore, these mutants were sensitive to endoglycosidase H (Endo H), thus indicating that the mutant receptor proteins were retained intracellularly in the endoplasmic reticulum. To test whether the lack of binding of the D404-substituted rFSHR mutants was due to a perturbation of a binding site or to the intracellular retention of the mutants, a truncated rFSHR(t637) mutant, containing a cytoplasmic truncation that should not directly affect FSH binding, was examined. As with the D404-substitution mutants, rFSHR(t637) was stably expressed but sensitive to Endo H. Significantly, detergent-soluble extracts of cells expressing rFSHR(t637) were unable to bind FSH. From these results, we conclude that substitution of D404 of the rFSHR prevents hormone binding as a result of the intracellular retention of the mutants in the endoplasmic reticulum presumably in an incompletely folded state, as opposed to disruption of a hormone-binding site at D404. Comparable rLHR substitution (D397K) and truncation (t616) mutants were constructed and used to transfect 293 cells. For both rLHR(D397K) and rLHR(t616), human CG (hCG) binding to intact cells was not detectable, but high affinity hCG binding was observed in detergent-soluble extracts of the cells. Therefore, the rLHR differs from the rFSHR in that mutants of the rLHR that are retained in the endoplasmic reticulum have already been folded correctly and can bind hCG with high affinity as long as a hormone-binding site has not been perturbed by the mutation. In contrast, mutants of the rFSHR that are retained in the endoplasmic reticulum have not yet folded into a conformation that can bind hormone. This suggests a difference in the temporal pattern of folding between the two structurally related gonadotropin receptors. Our studies also demonstrate how mutagenesis studies of the rFSHR must be interpreted with caution, as FSHR mutants that are expressed but are retained intracellularly will most likely not be able to bind FSH even when a hormone-binding site has not been altered.

Animals↗

PubMed1995

Identification of the sites of N-linked glycosylation on the follicle-stimulating hormone (FSH) receptor and assessment of their role in FSH receptor function.

The FSH receptor (FSHR) contains a large extracellular domain in which exist three potential sites for N-linked glycosylation. A truncated form of the FSHR representing only the extracellular domain was created and expressed in mammalian cells. We show that this truncated receptor is glycosylated, through the carbohydrates are not as fully processed as those of the full-length receptor. This truncated receptor, which remains intracellular, binds FSH with an affinity comparable to that of the full-length FSHR. Therefore, although other regions of the FSHR may contribute to hormone binding, the extracellular domain alone can confer high affinity binding. The above results suggest that N-linked FSHR carbohydrates may, in some way, be required for FSH binding. Therefore, further experiments, done in the context of the full-length receptor, were performed to determine the actual sites of glycosylation in the FSHR as well as to elucidate their role in the functions of the FSHR. Site-directed mutagenesis was done to individually or collectively disrupt the potential sites for N-linked glycosylation. Western blot analyses of the wild type vs. mutant receptors demonstrate that, of the three potential sites for N-linked glycosylation, Asn's 174 and 276 are actually glycosylated. Binding assays demonstrate that these two N-linked FSHR carbohydrates are redundant in function since carbohydrate at either Asn174 or Asn276 allows the receptor to be expressed on the cell surface and to bind FSH with normal affinity. However, FSH binding activity is not observed with nonglycosylated mutant receptors where both sites have been collectively disrupted. Similarly, when cells expressing the wild type FSHR were treated with tunicamycin to prevent N-linked glycosylation, the resulting nonglycosylated FSHR was not able to bind FSH. In contrast, normal high affinity binding of FSH was maintained when N-linked carbohydrates were enzymatically removed from wild type receptors. Our results demonstrate that while N-linked carbohydrates on the FSH receptor are not required directly for the binding of hormone, a carbohydrate at either Asn174 or Asn276 is required for the efficient folding of the nascent receptor protein into a conformation that allows high affinity binding of hormone.

Amidohydrolases↗

PubMed1995

A role for increased lutropin/choriogonadotropin receptor (LHR) gene transcription in the follitropin-stimulated induction of the LHR in granulosa cells.

Follitropin (FSH) has been shown in previous studies to stimulate the induction of the LH/CG receptor (LHR) and LHR mRNA in the granulosa cells of diethylstilbesterol-primed immature rats. The present studies were undertaken to identify the mechanisms underlying the hormone-dependent induction of the LHR in rat granulosa cells. The effect of FSH on LHR mRNA stability was determined by measuring the decay of LHR mRNA after removal of FSH under conditions where transcription was inhibited. Under these conditions, readdition of FSH had little effect on mRNA stability. However, inhibitors of transcription themselves were found to have a marked effect on stabilizing the LHR mRNA, thus potentially masking an effect of FSH. These results suggest that there is a labile destabilizing factor that constitutively degrades LHR mRNA. At present, it cannot be ascertained whether FSH has any effect on this destabilizing factor. Transcriptional activity of the LHR gene was examined using nuclear run-on assays. It was found that 1) in the absence of FSH, LHR-binding activity and mRNA levels were negligible, but the LHR gene was transcriptionally active in granulosa cells of immature rats; 2) incubations of granulosa cells with FSH or 8-bromo-cAMP significantly increased endogenous LHR gene transcription (approximately 10-fold) under conditions where increases in LHR mRNA were observed; 3) the continuous presence of FSH or 8-bromo-cAMP was required to maintain elevated levels of LHR gene transcription and LHR mRNA; and 4) exogenous estradiol alone had no effect on transcription of the LHR gene although it was able to synergistically enhance FSH-induced LHR expression. These experiments suggest that while the effects of estradiol on LHR induction do not appear to be mediated by an increase in LHR gene transcription, the effects of FSH (or cAMP) on LHR induction are clearly mediated, at least in part, by significant increases in the rate of LHR gene transcription.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

Hormone-binding properties and glycosylation pattern of a recombinant form of the extracellular domain of the luteinizing hormone/chorionic gonadotropin receptor expressed in mammalian cells.

The present studies were undertaken to characterize the properties of a recombinant truncated rat LH/CG (rLH/CG) receptor representing the extracellular domain (rLHR-t338) expressed in mammalian cells. Using detergent-soluble extracts of stably transfected cells, we show that the binding of ovine (o) LH to the extracellular domain is different from the binding of oLH to the full-length receptor. Thus, whereas the full-length receptor from either rat corpora lutea or stably transfected 293 cells binds oLH with two apparent affinities, rLHR-t338 binds oLH with only one affinity, which is intermediate between the high and low affinities of the full-length receptors. However, rLHR-t338 as well as the full-length receptor binds hCG and hLH with similar high affinities and by a model assuming the presence of only one binding site. These data are consistent with the possibility that the carboxyl-terminal half of the LH/CG receptor may contribute (either directly or indirectly) to a second low affinity hormone-binding site. The truncated receptor retains normal binding specificity, as it does not bind hFSH, demonstrating that the extracellular domain alone contains all of the necessary elements for conferring specificity among the gonadotropin hormones. Our studies also show that rLHR-t338 is glycosylated and migrates as two proteins of 48 and 44 kilodaltons (kDa) on sodium dodecyl sulfate gels (where the 44-kDa species is probably a partially degraded form of the 48-kDa protein). However, the N-linked oligosaccharides of the truncated receptor do not appear to be fully processed, as they remain in a high mannose form. rLHR-t338 is not secreted from cells, but remains trapped intracellularly. It can be detected, though, in detergent-soluble extracts of cells or in homogenates. Upon centrifugation of homogenates, it was found that about 90% of the recoverable truncated receptor can be obtained in the supernatant fraction. Thus, mammalian cells could provide a source of a water-soluble extracellular domain of the LH/CG receptor.

Animals↗

PubMed1993

Disruption of potential sites for N-linked glycosylation does not impair hormone binding to the lutropin/choriogonadotropin receptor if Asn-173 is left intact.

The rat lutropin/choriogonadotropin receptor (rLHR) is a G protein-coupled receptor, the large extracellular domain of which binds human choriogonadotropin (hCG) with high affinity. Within the extracellular domain are six potential sites for N-linked glycosylation. Although several studies have attempted to determine if N-linked carbohydrates are necessary for hormone binding, the results have been in apparent disagreement. In this study we have used site-directed mutagenesis to singly and collectively alter the consensus sequences for N-linked glycosylation in the rLHR. In particular, we examined the binding activity in both intact cells as well as detergent-solubilized extracts so that the effects on trafficking to the plasma membrane could be determined. In addition, we independently examined the effects of substituting a particular Asn versus Thr or Ser residue within a given glycosylation consensus sequence. Our data suggest that substitution of Asn-173 with Gln results in both a decreased ability of the receptor to be expressed on the plasma membrane as well as a vastly decreased binding affinity of the receptor for hCG. However, if the consensus sequence for N-linked glycosylation at Asn-173 is altered by substitution of Thr-175 with Ala (instead of Asn-173 to Gln), the resulting receptor binds hCG with high affinity although it is still impaired in its ability to be expressed at the plasma membrane. Furthermore, if all consensus sequences for N-linked glycosylation are mutated collectively while maintaining Asn-173 (by substituting Thr-175 with Ala instead of Asn-173 to Gln), the resulting deglycosylated receptor, although not expressed on the plasma membrane, binds hCG with high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Asparagine↗

PubMed1993

The lutropin/choriogonadotropin receptor ... 4 years later.

A comparison of this review with the review on the LH/CG receptor that we published in this journal 4 yr ago (1) clearly shows that the field has advanced tremendously in this short period of time. Many of the questions that were unanswered then have now been conclusively answered. On the other hand, as is always the case, the new knowledge generated has also resulted in many new questions that are yet to be answered. Hopefully it is clear from this review that the knowledge and experimental tools generated during the last 4 yr have given us new ammunition to address such important issues as the elucidation of the structural determinants of the LH/CG receptor that are involved in the different functions of this receptor, as well as on the molecular bases of receptor activation, inactivation, and regulation. In fact, since we finished writing this review in September of 1992, we are certain that some of the questions left unanswered here will already have an answer in 1993 when this review is published.

Amino Acid Sequence↗

PubMed1993

Positive charges in a putative amphiphilic helix in the carboxyl-terminal region of the third intracellular loop of the luteinizing hormone/chorionic gonadotropin receptor are not required for hormone-stimulated cAMP production but are necessary for expression of the receptor at the plasma membrane.

The LH/CG receptor (LHR) is a member of the family of G protein-coupled receptors and activates Gs when stimulated by LH or CG. Studies from other G protein-coupled receptors have implicated the carboxyl-terminal region of the third intracellular loop as being involved in the activation of G proteins. It has been suggested that the potential ability of this region to form an amphiphilic helix, with positively charged residues aligned to one face, may be important for this biological activity. To test whether the positively charged lysine residues, and thus an amphiphilic helix, in the carboxyl terminal region of the rat LHR (rLHR) are indeed important in the activation of Gs by the rLHR, a mutant rLHR was constructed in which lysines 541, 544, and 557 were simultaneously substituted with alanines. Clonal 293 cells expressing comparable numbers of cell-surface recombinant wild type rLHR or rLHR(K541,544,547A) were generated. Cells expressing the mutant receptor-bound human CG (hCG) with the same high affinity as those expressing the wild type rLHR. Since the numbers of receptors and binding affinities between the two cell lines were comparable, any changes in basal or hCG stimulated cAMP production could readily be interpreted as an alteration in the mutant receptor's ability to activate Gs. It was found, however, that basal cAMP production, the concentration of hCG required to elicit half-maximal cAMP production, and the maximal levels of cAMP produced in response to hCG were all unchanged in cells expressing rLHR(K541,544,547A).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Truncation of the cytoplasmic tail of the lutropin/choriogonadotropin receptor prevents agonist-induced uncoupling.

An agonist-induced change in the functional properties of a constant number of receptors seems to be a ubiquitous phenomenon involved in the regulation of cell surface receptors. Although the mechanisms responsible for this phenomenon (called uncoupling or desensitization) have been studied in detail using beta 2-adrenergic receptors it is unclear if the models derived from these studies are applicable to other members of the family of G protein-coupled receptors. Since it has been shown previously that truncation of the C-terminal cytoplasmic tail of the beta 2-adrenergic receptor results in a delay in the onset of agonist-induced uncoupling (Bouvier, M., Hausdorff, W.P., De Blasi, A., O'Dowd, B.F., Kobilka, B.K., Caron , M.G., and Lefkowitz, R.J. (1988) Nature 333, 370-373), we now present experiments designed to test the effects of a similar truncation of the lutropin/choriogonadotropin (LH/CG) receptor on its functional properties. The results presented herein show that (i) clonal lines of human embryonic kidney cells stably transfected with cDNAs encoding for the wild-type (rLHR-wt) or a mutant receptor truncated at amino acid residue 631 (rLHR-t631) express functional LH/CG receptors as judged by their ability to bind hCG and to respond to it with increased cAMP accumulation; (ii) a preincubation of the cells expressing rLHR-wt with hCG leads to a reduction in the ability of hCG to activate adenylylcyclase; and (iii) this reduction is severely blunted in cells expressing rLHR-t631. These results demonstrate that the C-terminal cytoplasmic tail of the LH/CG receptor is necessary for agonist-induced uncoupling.

Animals↗

PubMed1992

Evidence that the FSH receptor itself is not a calcium channel.

FSH has been shown to stimulate the uptake of calcium in cultured rat Sertoli cells, resulting in an increase in cytosolic calcium concentrations. One possibility which has been put forth is that the FSH receptor per se may act as a calcium channel. This is all the more tantalizing given the proposed structure of this receptor which, like all other G protein-coupled receptors, is thought to have the putative transmembrane helices forming a bundle-like structure in the plasma membrane. To test whether the FSH receptor could function as a calcium channel, we performed whole-cell voltage clamp experiments on 293 and 293F(wt1) cells, which are a clonal line of 293 cells expressing high levels of rat FSH receptors. The 293 cells, which do not express FSH receptors, were found to lack any detectable inward calcium currents, and therefore, serve as an excellent model for transfecting with potential calcium conducting FSH receptors. When the 293F(wt1) cells were then tested, no inward calcium currents could be detected in either control or FSH-stimulated cells. These results suggest that the FSH receptor itself is not a calcium channel and, therefore, FSH must be stimulating endogenous calcium channels in rat Sertoli cell plasma membranes.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1992

The 5'-flanking region of the rat luteinizing hormone/chorionic gonadotropin receptor gene confers Leydig cell expression and negative regulation of gene transcription by 3',5'-cyclic adenosine monophosphate.

The LH/CG receptor is a G protein-coupled receptor present on gonadal cells whose levels are modulated by a number of hormones, growth factors, and second messenger analogs. With the recently cloned cDNA for the LH/CG receptor, it has been shown that changes in the levels of the cognate mRNA are involved, at least in part, in the observed changes in receptor density. In order to study the transcriptional regulation of the LH/CG receptor we have isolated a 2-kilobase region of the 5'-flanking region of the rat LH/CG receptor gene and subcloned nucleotide -1 (relative to the translational initiation codon) to -1370 into a luciferase reporter plasmid. We show here that this region of the LH/CG receptor gene is able to enhance luciferase activity in MA-10 cells, a line of Leydig tumor cells that normally express LH/CG receptors, as opposed to human kidney 293 cells, which do not. Furthermore, the addition of 8-bromo-cAMP to MA-10 cells, under conditions known to decrease LH/CG receptor numbers and receptor mRNA levels, decreases the relative luciferase activity to about 26% of control. This decrease in reporter gene activity is severely blunted in a subclone of MA-10 cells with a cAMP-resistant phenotype. Our studies show, for the first time, that sequence(s) present with 1370 base pairs of the translational start site of the rat LH/CG receptor gene are sufficient for conferring expression of this gene in Leydig cells and for the negative modulation of LH/CG receptor gene transcription by high concentrations of cAMP.

Animals↗

PubMed1992

Effects of truncations of the cytoplasmic tail of the luteinizing hormone/chorionic gonadotropin receptor on receptor-mediated hormone internalization.

The LH/CG receptor is a member of the family of G protein-coupled receptors and consists of a large N-terminal extracellular domain (which is responsible for binding hormone) attached to a region that spans the plasma membrane seven times, ending with an intracellularly located C-terminus. Binding of LH or human CG (hCG) to the LH/CG receptor causes a stimulation of adenylyl cyclase, presumably via activation of Gs. The binding of hormone also leads to its subsequent internalization by receptor-mediated endocytosis. In order to investigate the role of the cytoplasmic tail of this receptor in these events, we prepared a series of mutants in which progressively larger portions of the cytoplasmic tail were deleted. Deletion of 58 amino acids from the C-terminus, in which only 11 cytoplasmic residues remain, resulted in a receptor that was not expressed on the plasma membrane. Receptors rat LHR (rLHR)-t653 and rLHR-t631, in which 21 or 43 amino acids were removed, respectively, were properly expressed. These results suggest that a region(s) between residues 616 and 631 of the rLH/CG receptor are required for proper insertion and/or targeting of the receptor into the plasma membrane. Cells expressing rLHR-t653 or rLHR-t631 bound hCG with the same high affinity as cells expressing the full-length receptor, and basal levels of cAMP were the same among the cells. However, cells expressing the truncated receptors responded to hCG with approximately 2-fold greater levels of maximal cAMP accumulation than cells expressing the full-length receptor. Deletion of up to 43 amino acids from the C-terminus of the rLH/CG receptor had no deleterious effect on hCG internalization. In fact, mutants lacking 21 and 43 amino acids exhibited progressively faster rates of hCG internalization as compared to the full-length receptor. Once internalized, hCG was also degraded at a faster rate in cells expressing the truncated LH/CG receptors. Since hCG-stimulated cAMP stimulation and hCG internalization are retained by rLHR-t631, it can be concluded that the residues, not necessarily the same, required for these functions reside within the 26 amino acids of the cytoplasmic tail closest to the seventh transmembrane helix and/or residues within the intracellular loops. Our data show, however, that both hCG-stimulated cAMP production and hCG internalization are enhanced by the removal of the distal portion of the cytoplasmic tail.

Amino Acid Sequence↗

PubMed1992

The gonadotrophin receptors: insights from the cloning of their cDNAs.

The cloning and expression of the cDNA's for the gonadotrophin hormone receptors has confirmed that these receptors are each composed of a single polypeptide which can both bind hormone and activate adenylyl cyclase when occupied with agonist. Although some studies by others have suggested that the LH/CG receptor and the FSH receptor are composed of multiple subunits (Ascoli and Segaloff 1989; Reichert and Dattatreyamurty 1989; Shin and Ji 1985a, b, and c; Smith et al. 1985; Smith et al. 1986), biochemical studies on the LH/CG receptor have shown that it is composed of a single polypeptide with a molecular weight of 93,000 when analyzed on sodium dodecyl sulfate gels in the presence or absence of disulfide reducing agents (Ascoli and Segaloff 1989). As the LH/CG receptor has been shown to be readily proteolyzed into smaller-sized fragments (Ascoli and Segaloff 1986; Ascoli and Segaloff 1989), it is reasonable to postulate that the FSH receptor may be similarly susceptible to proteolysis, and that this may account for the discrepant reports on its structure. Clearly, the molecular cloning and functional expression of the cDNA's for the rat ovarian LH/CG receptor (McFarland et al. 1989) and the rat testicular receptor (Sprengel et al. 1990) demonstrate conclusively that the gonadotrophin receptors are indeed single polypeptides. As shown schematically in Fig. 5.12, the gonadotrophin hormone receptors (together with the thyrotrophin receptor, see Frazier et al. 1990; Libert et al. 1989; Nagayami et al. 1989; Parmentier et al. 1989) define a unique subclass of G protein-coupled receptors. Thus, in addition to the seven membrane-spanning domains which appear to be the hallmark of G protein-coupled receptors, the gonadotrophin receptors (and thyrotrophin receptor) also contain large extracellular domains. These extracellular domains are composed of a repeating leucine-rich motif which is found in a number of widely-diverse proteins, collectively termed leucine-rich glycoproteins. We have shown that the extracellular domain of the LH/CG receptor is entirely responsible for binding hormone with high affinity. This is in contrast to the rhodopsin-like G protein-coupled receptors which have relatively small amino-terminal extracellular domains and in which the ligands interact directly with amino acids within the transmembrane helices. As with other G protein-coupled receptors, however, it is thought that the gonadotrophin receptors interact with and activate Gs through residues located within the cytoplasmic loops and the amino-terminal region of the cytoplasmic tail.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Lutropin/choriogonadotropin down-regulates its receptor by both receptor-mediated endocytosis and a cAMP-dependent reduction in receptor mRNA.

Using MA-10 Leydig tumor cells as a model system we have examined the possibility that the lutropin/choriogonadotropin (LH/CG)-induced down-regulation of the LH/CG receptor is accompanied by changes in LH/CG receptor mRNA. We show that LH or CG are indeed capable of reducing the levels of LH/CG receptor mRNA, but that the time course and magnitude of the reduction in receptor mRNA are such that this phenomenon cannot account entirely for the down-regulation of the receptor. In fact, we estimate that LH/CG can reduce the number of LH/CG receptors by at least 80% with little or no change in the levels of LH/CG receptor mRNA. These data are consistent with our previous hypothesis that the LH/CG-induced down-regulation of the LH/CG receptor is primarily due to an increase in the rate of degradation of the receptor that occurs as a result of the receptor-mediated endocytosis of LH/CG. Our studies also show that the LH/CG-induced down-regulation of the LH/CG receptor mRNA is mediated by cAMP. Thus, addition of 8-bromo-cAMP to MA-10 cells leads to a similar reduction in the levels of LH/CG receptor and receptor mRNA; while deglycosylated human CG, a hormone derivative that binds to the LH/CG receptor but has a reduced ability to stimulate cAMP synthesis, does not reduce the levels of LH/CG receptor mRNA. Last, human CG or 8-bromo-cAMP are unable to reduce LH/Cg receptor mRNA in a mutant MA-10 cell line that express a cAMP-resistant phenotype.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Epidermal growth factor and phorbol esters reduce the levels of the cognate mRNA for the LH/CG receptor.

The LH/CG receptor of MA-10 Leydig tumor cells can be down-regulated by LH/CG, mouse epidermal growth factor (mEGF), cAMP analogues and phorbol esters. In a recent publication we showed that the LH/CG-induced down regulation of the LH/CG receptor is primarily due to an increase in the rate of degradation of the receptor, and occurs with little or no change in the levels of LH/CG receptor mRNA. Although relatively unimportant for the LH/CG-induced down-regulation of the LH/CG receptor, we also found that LH/CG can in fact elicit a reduction in LH/CG receptor mRNA and that this reduction is mediated by cAMP. These findings raised the possibility that the ability of other compounds to down-regulate the LH/CG receptor may be accompanied by decreases in LH/CG receptor mRNA. In an attempt to answer this question, we examined the possibility that the mEGF- or phorbol ester-induced down regulation of the LH/CG receptor is due to changes in LH/CG receptor mRNA. The experiments presented herein show that this is indeed the case.

Animals↗

PubMed1991

Multiple luteinizing hormone/chorionic gonadotropin receptor messenger ribonucleic acid transcripts.

It has previously been shown that multiple messenger RNA (mRNA) species can be identified in gonadal tissues by probes specific for the LH/CG receptor. Here we show that the sizes and relative abundancies of gonadal LH/CG receptor transcripts are quite variable between such closely related species as rat and mouse. These patterns of LH/CG receptor mRNAs are yet different from that observed in human embryonic kidney 293 cells that have been transfected with a cDNA encoding for the rat luteal LH/CG receptor. In spite of the diversity in the number and sizes of LH/CG receptor mRNA transcripts, however, our data also show that the size of the cell surface receptor expressed in these three cells/tissues is identical. We further show that the most abundant LH/CG receptor mRNA present in MA-10 cells, a clonal strain of cultured Leydig tumor cells, is a 1.2 kilobase transcript which encodes for a truncated version of the LH/CG receptor corresponding to the extracellular hormone-binding domain. It does not appear, however, that this transcript is translated into a functional protein.

Animals↗

PubMed1990

Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity.

The lutropin-choriogonadotropin (LH/CG) receptor is a cell surface receptor comprised of two domains of roughly equivalent size. The amino-terminal half of the receptor is relatively hydrophilic and is located extracellularly, whereas the carboxyl-terminal half of the receptor shares amino acid homology with other receptors that couple to G proteins and is similarly thought to span the plasma membrane seven times, ending with a relatively short carboxyl-terminal tail. In order to test the role of the extracellular domain in binding hormone, we constructed a mutated rat luteal LH/CG receptor cDNA (termed pCLHR-D2), which encodes for only the extracellular domain, and used it to transiently transfect human kidney 293 cells. Here we report that the expressed extracellular domain of the LH/CG receptor is capable of binding human CG with a high affinity, comparable with that of the full-length receptor. Thus, not only is the extracellular domain of the glycoprotein hormone receptors involved in binding hormone, but it alone is capable of conferring high affinity binding. Unexpectedly, it was also found that this truncated receptor is not secreted into the culture media but remains trapped within the cells.

Amino Acid Sequence↗

PubMed1990

Structure of the lutropin/choriogonadotropin receptor.

In summary, the LH/CG receptor is a single polypeptide which contains a large hydrophilic domain that is situated extracellularly, attached to a region that spans the plasma membrane seven times, the carboxy-terminal region being intracellular. This topology was predicted by the amino acid sequence and has been confirmed by our immunofluorescence studies. The extracellular domain, which is related to a family of leucine-rich glycoproteins, is presumably involved in binding the large glycoprotein hormones hCG and LH. The carboxy-terminal half of the receptor, which is related to the family of rhodopsinlike receptors, is (by analogy with these receptors) presumably involved in the coupling of the receptor to the G protein. Our transfection studies confirm that this single polypeptide is capable of binding hormone and activating adenylyl cyclase. Therefore, not only is the structure of the LH/CG receptor unique compared to other cell surface receptors characterized to date, but also its structure suggests that the mechanism of the translation of hormone binding to G protein coupling in this receptor is different from other G protein-coupled receptors whose ligands are much smaller and intercalcate among the transmembrane helices. We predict that, due to the homology among the glycoprotein hormones, the structures of the FSH and TSH receptors share extensive amino acid and structural homology with the LH/CG receptor. Last, our newly acquired knowledge about the structure of the LH/CG receptor, and the development of a cDNA and antibodies for this receptor, should enable more detailed studies on the function and regulation of the LH/CG receptor, not previously possible.

Amino Acid Sequence↗

PubMed1990

The orientation of the lutropin/choriogonadotropin receptor in rat luteal cells as revealed by site-specific antibodies.

Polyclonal antibodies were raised against peptides corresponding to specific regions of the rat luteal lutropin/choriogonadotropin (LH/CG) receptor. One of these (anti-LHR02) was directed against a 13 amino acid sequence that occurs within the large N-terminal hydrophilic domain, and another (anti-LHR06) was directed against the 14 most C-terminal amino acids of the receptor (as deduced from the open reading frame of the cDNA). These antisera, which we show to be highly specific for the LH/CG receptor, were used in indirect immunofluorescence experiments with primary cultures of rat luteal cells to determine the orientation of this receptor within the plasma membrane. Anti-LHR02 was shown to specifically bind to intact cells. In contrast, anti-LHR06 did not bind to intact cells. When the cells were first permeabilized with detergent and then incubated with this antibody, however, an intense and specific immunofluorescence was observed. These studies clearly show that the orientation of the LH/CG receptor in the plasma membrane is such that the N-terminal domain is located extracellularly, while the C terminus is located intracellularly. Thus, we have experimentally confirmed that the orientation of this receptor as predicted from the amino acid sequence is indeed correct.

Amino Acid Sequence↗