0013-7227/91/1291-0133$03.00/0 Endocrinology Copyright ^ 1991 by The Endocrine Society
Vol. 129, No. 1 Printed in U.S.A.
Multiple Luteinizing Hormone/Chorionic Gonadotropin Receptor Messenger Ribonucleic Acid Transcripts* HAIYUN WANG, MARIO ASCOLI, AND DEBORAH L. SEGALOFF Department of Physiology and Biophysics (H. W., D.L.S.) and Department of Pharmacology (M.A.), The University of Iowa, Iowa City, Iowa 52242
ABSTRACT. 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. (Endocrinology 129: 133-138, 1991)
T
(341 amino acids with six potential sites for N-linked glycosylation), it was speculated that this domain might be responsible for binding the large glycoprotein hormones. Indeed, it has recently been shown that when cells are transfected with a cDNA encoding a truncated LH/CG receptor representing only the extracellular domain they produce a protein capable of binding hCG with the same high affinity as the full-length receptor (6). Interestingly, when one examines Northern blots of gonadal cells which express LH/CG receptors multiple LH/CG receptor messenger RNA (mRNA) transcripts are observed (7-10). In both the rat ovary and in MA-10 Leydig tumor cells (a clonal line of murine Leydig tumor cells that express functional LH/CG receptors, see Ref. 11 for a review) it has been shown that when levels of LH/CG receptor mRNA are altered, all the transcripts appear to be coordinately regulated (7-10). Since it is not known whether these different transcripts have any biological significance, we undertook the present study as an initial step towards better characterizing the LH/ CG receptor mRNA species.
HE LH/CG receptor is a cell surface receptor, present on specialized cells of the ovaries and testes, which activates adenylyl cyclase upon binding either LH or human CG (hCG). From biochemical studies on the receptor, it was determined that it is a single glycoprotein with a molecular mass, on sodium dodecyl sulfate (SDS)polyacrylamide gels, of 93 kilodaltons (kDa) (see Ref. 1 for a review). Recently, the cDNA for the LH/CG receptor has been cloned and expressed in mammalian cells (2). Analyses of the deduced amino acid sequence of the cDNA suggests that this receptor is composed of a large N-terminal extracellular domain attached to a region which is homologous to other G protein-coupled receptors. This C-terminal half of the receptor is therefore thought to similarly span the plasma membrane seven times, as has been shown for rhodopsin and the 0adrenergic receptor (3, 4). Using site-specific antibodies to the rat luteal LH/CG receptor it has been shown that the N-terminal region is indeed located extracellularly and that the C-terminal portion is located cytoplasmically (5). Given that the extracellular orientation of the N-terminal half of the LH/CG receptor and its large size
Materials and Methods
Received February 26, 1991. Address correspondence and requests for reprints to: Dr. Deborah L. Segaloff, Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242. * These studies were supported by NIH Grants HD-22196 (to D.L.S.), CA-40629 (to M.A.), the University of Iowa Diabetes and Endocrinology Research Center Grant AM-25295, and funds from the Roy J. Carver Charitable Trust (to M.A.).
Cells
The origin and handling of the MA-10 cells have been described (12). They were maintained in Waymouth MB752/1 modified to contain 1.1 g/liter NaHCO3, 20 mM HEPES, 50 gentamicin, and 15% horse serum, pH 7.4) in a humidi133
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
LH/CG RECEPTOR mRNA TRANSCRIPTS
134
Endo«1991 Vol 129 • No 1
fied atmosphere containing 2.5% CO2. Human embryonic kidney 293 cells (ATTC CRL 1573) were maintained in Dulbecco's modified Eagle's medium containing 50 ng/rci\ gentamicin and 10% heat-inactivated horse serum in a humidified atmosphere containing 5% CO2. 293 cells were transiently transfected with pCLHR, an expression vector encoding for the full-length rat luteal LH/CG receptor (2), following the procedure of Chen and Okayama (13). 293 cells transfected with this expression vector have previously been shown to bind hCG and respond with increased cAMP accumulation (2).
nucleotides encode for the first through fourth membranespanning domains. This probe was 32P-labeled by random priming. After hybridization, the blot was washed four times in 2x SSC (saline sodium citrate) and 0.1% SDS at room temperature (15 min/washing) and then two times in O.lx SSC and 0.1% SDS at 60 C (30 min/washing). The resulting blots were exposed to x-ray film at -70 C with intensifying screens. All samples shown in a given figure were analyzed in the same gel. Sizes of mRNA transcripts were determined using RNA markers (Boehringer Mannheim Biochemicals, Indianapolis, IN).
Chemical cross-linking of hCG to cells
Hormones and supplies
Culture dishes containing the MA-10 cells or transfected 293 cells were placed on ice for 30 min and washed two times with 2-ml portions of cold Medium A (Waymouth MB752/1 without NaHCO3, containing 20 mM HEPES, 1 mg/ml albumin, pH 7.4). The dishes were then incubated at 4 C for 4.5 h in 1 ml Medium A containing 125I-hCG alone or together with 50 IU unlabeled hCG (to correct for nonspecific binding). At the end of this incubation the dishes were scraped, rinsed with 1 ml cold Medium B (Hanks' balanced salt solution without NaHCO3, containing 20 mM HEPES, pH 7.4) and the contents transferred to a plastic tube on ice. The cells were collected by centrifugation (1300 x g, 15 min, 4 C) and washed twice with 2-ml portions of cold Medium B. The cell pellets were resuspended in 180 ix\ cold Medium B. The receptor-bound hormone was then cross-linked to the cells using disuccinimidyl suberate, the noncross-linked receptor-bound hormone was released by treatment of the cells with isotonic glycine buffer at pH 3 (14, 15), and the samples were prepared for SDS-polyacrylamide gel electrophoresis exactly as described previously (16). The crosslinked products were resolved on a 7.5% SDS-polyacrylamide gel in the presence of reducing agents.
Highly purified hCG (CR-125) was kindly provided by the National Hormone and Pituitary Agency of the NIDDK (NIH). The intact hormone was iodinated as described previously (21), which results in greater than 90% of the radioactivity being incorporated into the a-subunit of the hormone (22). Crude hCG (used for nonspecific binding determinations) was obtained from Sigma (St. Louis, MO). Disuccinimidyl suberate was obtained from Pierce (Rockford, IL).
Results Figure 1 shows a comparison of LH/CG receptor mRNA transcripts in MA-10 cells (lane A), in mammalian 293 human kidney cells transiently transfected with A I It I C I I)
Preparation of RNA and Northern blots Cytoplasmic RNA was prepared from cultured cells (MA-10 cells and transiently transfected 293 cells) exactly as described previously (17). The RNA obtained from the transfected 293 cells was further treated with DNAse I to digest any of the LH/ CG receptor cDNA expression plasmid that might be cytoplasmically located. Total RNA from ovaries of pseudopregnant rats (18), Sertoli cells from adult rats (19), ovaries or testes of adult rats, or testes of adult mice were prepared by extraction with LiCl2 (20). These preparations of cytoplasmic or total RNA were further purified using oligo-(dT) cellulose following the instructions supplied by the manufacturer. For Northern analyses the RNA samples were denatured and electrophoresed in a 1% formaldehyde-agarose gel and then transferred to a nylon (Biodyne, ICN) membrane. The blots were prehybridized for 6 h at 42 C and then hybridized overnight at 42 C with a nick-translated 32P-labeled pGEM-4Z vector (Promega, Madison, WI) containing nucleotides 1-622 of the rat luteal LH/CG receptor cDNA (2). This portion of the LH/CG receptor cDNA encodes for the initial two-thirds of the extracellular domain of the receptor. In some experiments a probe corresponding to the transmembrane domain of the receptor was used. This was a cDNA produced by the polymerase chain reaction that corresponded to nucleotides 1102-1574 of the rat luteal LH/CG receptor cDNA (2). These
kl)
kh
FIG. 1. LH/CG receptor mRNAs in different LH/CG receptor-bearing cells/tissues. Poly (A)+-mRNA was prepared and analyzed by Northern analysis using a cDNA probe corresponding to the extracellular domain of the rat luteal LH/CG receptor. The lanes correspond to (A) 30 ng from mouse MA-10 cells; (B) 25 fig from human kidney 293 cells transiently transfected with an expression vector encoding the rat luteal LH/CG receptor; (C) 25 ng from rat Sertoli cells; and (D) 1.25 fig from ovaries from pseudopregnant rats. The autoradiogram shown was developed for 5 days at -70 C. The amounts of RNA loaded onto the gel were varied to optimize visualization of the transcripts in the different samples. Therefore, a quantitative comparison between the samples cannot be made.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
LH/CG RECEPTOR mRNA TRANSCRIPTS
a cDNA encoding for the full-length rat luteal LH/CG receptor (2) (lane B), in rat Sertoli cells (lane C), and in rat luteal tissue (lane D). These different cells/tissues were chosen because the MA-10 cells, transfected 293 cells, and luteal tissue express cell surface LH/CG receptors. Whereas Sertoli cells do not express LH/CG receptors, they do express FSH receptors, which is a closely related protein (23). This Northern, and all other Northerns shown unless noted otherwise, was probed using a partial cDNA (nucleotides 1-622) of the rat luteal LH/ CO receptor (2). This partial cDNA encodes for approximately the first two thirds of the extracellular domain of the LH/CG receptor. It is readily apparent from Fig. 1 that in cells/tissues expressing LH/CG receptors the numbers and sizes of LH/CG receptor mRNA transcripts vary among the different sources of RNA. The pattern of LH/CG receptor mRNAs in both the MA-10 cells and rat luteal tissue is quite complex (Fig. 1). In MA-10 cells there are six discernable LH/CG receptor transcripts of 1.2, 1.6, 1.9, 2.6, 4.3, and 7.7 kilobases (kb) in length. The most abundant of these is the 1.2 kb species. In contrast, the most abundant LH/ CG receptor mRNA in rat ovaries is 6.7 kb in size. In addition to this transcipt, there are less abundant LH/ CG receptor mRNA species of 4.3, 2.6, and 1.2 kb in size. In Northern blots of rat gonadal tissues we do not consistently observe the 1.2 kb transcript, probably because of its low abundance. For example, it cannot be clearly discerned in Fig. 1; however, it has been shown in previously published Northern blots of rat ovarian tissues (2, 7). In comparing the MA-10 cells and rat luteal tissue, therefore, there are three LH/CG receptor mRNA transcripts in common, these being the 1.2, 2.6, and 4.3 kb species. Although the relative abundance of the 1.2 kb transcript is quite different between these two sources of LH/CG receptor mRNA, the 2.6 and 4.3 kb species are both minor species in these two sources. Figure 1 also shows that no LH/CG receptor mRNA transcripts can be detected under these conditions in RNA prepared from FSH receptor-bearing rat Sertoli cells. It should be noted, however, that at much longer exposure, a faint band at 1.7 kb can be discerned. The multiple LH/CG receptor mRNAs observed in MA-10 cells and rat gonadal tissue may arise from a number of different phenomenon including: 1) different sites of polyadenylation, 2) different lengths of polyadenylation, and/or 3) alternate or incorrect splicing of the LH/CG receptor gene. To eliminate contributions of the latter possibility, we examined the LH/CG receptor mRNAs present in 293 cells transfected with an expression vector encoding for the full-length rat luteal LH/ CG receptor cDNA. It has previously been shown that 293 cells transfected with this plasmid express cell surface 125I-hCG binding activity (2). The prominent LH/
135
CG receptor mRNA identified in these cells (see lane B of Fig. 1) is 3.1 kb long, which is not observed in either the MA-10 cells or rat luteal tissue (compare lanes A, B, and D of Fig. 1). Furthermore, the transfected 293 cells also appear to express other less abundant and less well resolved LH/CG receptor mRNAs both larger and smaller than the 3.1 kb species. The most apparent of these is a species slightly smaller than the 3.1 kb transcript. Therefore, in the absence of transcription of the endogenous LH/CG receptor gene, and thus in the absence of any potential alternate splicing of this gene, there is still some differential processing of the LH/CG receptor mRNA in the 293 cells. These data suggest that even if alternate or incorrect gene splicing was contributing to the different sizes of LH/CG receptor mRNA transcripts in gonadal cells, other factors such a differences in polyadenylation are also likely to be involved. Given the different patterns of LH/CG receptor mRNA expression in MA-10 cells, transfected 293 cells, and rat luteal tissue, it was important to determine whether these cells/tissues expressed the same size cell surface LH/CG receptor. We have previously shown that rat luteal tissue and MA-10 cells both express a 93 kDa LH/CG receptor (18, 24). In order to characterize the LH/CG receptor expressed in the transfected 293 cells, the following experiment was performed. Intact 12'I-hCG (iodinated in the a-subunit) was allowed to bind to the transfected cells, the cells were washed, and the receptorbound hormone was cross-linked. After solubilization, the iodinated cross-linked products were resolved by SDS-polyacrylamide gel electrophoresis. The results of this experiment, and one performed in parallel with MA10 cells, are shown in Fig. 2. It is apparent that the same cross-linked products are generated in the transfected 293 cells and in the MA-10 cells. Thus, radioactivity is present at the tracking dye and at positions corresponding to 53 and 107 kDa proteins. From previous experiments cross-linking 125I-hCG to MA-10 cells, we determined that the radioactivity present at the tracking dye corresponds to the free a-subunit of the hormone, the 53 kDa band corresponds to the a- and /3-subunit of hCG cross-linked to the cell surface LH/CG receptor, and the 107 kDa band corresponds to the a-subunit of hCG crosslinked to the cell surface LH/CG receptor (16). Since the LH/CG receptor of MA-10 cells was shown to be 93 kDa in size (on SDS polyacrylamide gels) (24), we conclude that the LH/CG receptor expressed in the transfected 293 cells is also processed to 93 kDa. Since the size of the rat luteal LH/CG receptor has also been estimated to be 93 kDa (18), we conclude that despite the varying patterns of LH/CG receptor mRNA expression in MA10 cells, transfected 293 cells, and rat luteal tissue {cf. Fig. 1), the same size cell surface receptor is expressed in each.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
LH/CG RECEPTOR mRNA TRANSCRIPTS
136 293 Cells
MA-10 Cells
A |
Endo • 1991 Vol 129 • No 1
B | C
I)
-6.7
107 —
-4.3 kDa
kl)
53—
-2.6 -1.9 -1.6 Tracking dye — -1.2 Unlabeled hCG
—
+
—
+
FIG. 2. Cross-linking of hCG to the LH/CG receptor expressed on transfected 293 cells and MA-10 cells. 293 cells transiently transfected with an expression vector encoding the rat luteal LH/CG receptor and MA-10 cells were incubated with 125I-hCG at 100 ng/ml and 40 ng/ml, respectively, in the absence or the presence of 50 IU unlabeled hCG as indicated. After washing, the cells were cross-linked with disuccinimidyl suberate and analyzed on SDS gels as described in Materials and Methods.
In comparing the different LH/CG receptor mRNAs expressed in MA-10 cells us. rat gonadal tissues, it must be kept in mind that there are three differences between the sources of cells/tissues. Thus the former are from mouse, are testicular in origin, and are tumorigenic; while the latter are from rat, are ovarian in origin, and are nonmalignant. To determine which of these factors contributes to the differences in LH/CG receptor mRNAs present, the experiment shown in Fig. 3 was performed. Lanes A and B compare the LH/CG receptor mRNAs in ovaries from pseudopregnant rats and in testes from adult rats, respectively. There is a 1.7 kb transcript present in the rat testes which is not as visible in rat ovaries. With that exception, the sizes of LH/CG receptor transcripts in the two rat gonadal tissues are the same. Note, however, that as shown previously (2), the abundance of LH/CG receptor mRNA in the ovaries of pseudopregnant rats is far greater than that in rat testes. In examining the LH/CG receptor mRNAs in mouse testes (lane C), it is readily apparent that the number and sizes of transcripts are different from those of rat testes (compare lanes B and C) but the sizes of transcripts present in mouse testes are the same as those observed in MA-10 cells (compare lanes C and D). Thus, the primary reason for the difference in LH/CG receptor mRNAs between rat gonadal tissues and MA-10 cells is simply due to the difference in species. The greater abundance of LH/CG receptor mRNA in MA-10 cells as compared to mouse testes is due to the small percentage of Leydig cells present in testes. The only apparent
FIG. 3. LH/CG receptor mRNAs in mouse and rat gonadal tissues. Poly(A)+-mRNA was prepared and analyzed by Northern analysis using a cDNA probe corresponding to the extracellular domain of the rat luteal LH/CG receptor. The lanes correspond to (A) 25 ng adult (nonpregnant) rat ovary; (B) 35 ng adult rat testes; (C) 50 ng from adult mouse testes; and (D) 25 ng from mouse MA-10 cells. Although all of the lanes are from the same blot, lane A was developed for 3 days, whereas lanes B-D were from an autoradiogram developed for 5 days. The amounts of RNA loaded onto the gel and the times of exposure of the autoradiograms were varied to optimize visualization of the transcripts in the different samples. Therefore, a quantitative comparison between the samples cannot be made.
difference between MA-10 cells and mouse testes is the relatively greater proportion of the 1.2 kb message in MA-10 cells. As the open reading frame of the rat luteal LH/CG receptor is 2.1 kb, one must question the identity of those LH/CG receptor mRNA transcripts that are smaller than 2.1 kb. In order to address this question, we reprobed blots of rat ovarian mRNA and MA-10 cell mRNA with a cDNA probe corresponding to the transmembrane region (specifically the first through fourth membrane-spanning domains) of the receptor. These were compared with hybridizations that had been done using a complementary DNA (cDNA) encoding for amino acids in the initial two-thirds of the extracellular domain. As shown in Fig. 4, the 1.2 kb LH/CG receptor mRNA that is so clearly detected in MA-10 cells using a probe corresponding to the extracellular region of the receptor is not detected by a probe corresponding to the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
LH/CG RECEPTOR mRNA TRANSCRIPTS Km (>\;irk's
MA-10 Cells
6.7-
•U -I -2.6
kh
-1.9 -1.6 -i.:
FlG. 4. Differential hybridization of LH/CG receptor mRNAs with different cDNA probes. Poly(A)+-mRNA was prepared from rat ovaries and MA-10 cells and analyzed by Northern analysis (25 ^/g/lane from rat ovaries and 30 ^g/lane from MA-10 cells). Each lane was probed with a cDNA probe corresponding to the extracellular (EC) domain or the transmembrane (TM) domain of the rat luteal LH/CG receptor. The autoradiogram shown was developed for 5 days at —70 C.
transmembrane portion of the receptor. Due to the low abundance of the 1.7 kb transcript in MA-10 cells, it is not possible to determine if there is also a differential detection of this LH/CG receptor mRNA. For the same reasons, it is not possible to detect a difference in the 1.2 kb band in the different blots of rat ovarian mRNA. It can be seen, however, that all the LH/CG receptor mRNAs equal to or greater than 2.1 kb can be detected by both probes. The results of this experiment suggest that the 1.2 kb LH/CG receptor mRNA encodes for a truncated form of the receptor which lacks the transmembrane and cytoplasmic domains.
Discussion Previous studies have shown that in both rat gonadal tissues and in mouse MA-10 Leydig tumor cells there are numerous LH/CG receptor mRNA transcripts (7-10). Furthermore, the numbers and sizes of the transcripts differ between these two sources of mRNA. We show here that this difference is entirely due to a difference in species. Thus, mouse testes contain the same LH/CG receptor mRNAs as MA-10 cells. Neither MA-10 cells nor rat luteal tissue, however, have the same size LH/ CO receptor mRNA as that in human kidney 293 cells transfected with an expression vector containing the rat luteal LH/CG receptor cDNA. Nonetheless all three of these different cell types express a 93 kDa cell surface LH/CG receptor. These data suggest that each of these cell types contain a LH/CG receptor mRNA containing the same coding sequence, but differing lengths of 5'and/or 3'-untranslated regions. These might arise from simple differences in the sites and/or lengths of polyadenylation or they may reflect more substantial altera-
137
tions in the length or composition of the untranslated regions. Further evidence that a given mRNA may be differentially processed comes from the results of Northern blots of 293 cells transfected with the LH/CG receptor cDNA. As untransfected or mock-transfected cells do not express any detectable 12r>I-hCG binding activity (2), all LH/CG receptor mRNA must be derived from trancription of the exogenously added cDNA rather than the endogenous gene. Although these cells express one predominant LH/CG receptor mRNA, there are also other much less abundant mRNAs (cf. Fig. 1). The different LH/CG receptor mRNAs in this case could not arise from alternate splicing of the gene. An additional mechanism accounting for the different LH/CG receptor mRNAs in gonadal cells appears to be alternative or incorrect splicing of the LH/CG receptor gene. When the rat luteal and the porcine testicular LH/ CG receptor cDNAs were originally isolated, several variants were described (25, 26). These included cDNAs that encoded for receptors that were prematurely truncated and/or contained deletions. Recently, it has been reported that the human LH/CG receptor gene contains at least nine introns located within the region encoding the extracellular domain (27), which may help explain the diversity of the LH/CG receptor cDNAs. Thus, some of the different mRNAs observed in gonadal cells may also correspond to transcripts with different coding sequences arising from alternate splicing of the gene. Indeed, we show here that the 1.2 kb LH/CG receptor mRNA encodes for a truncated receptor corresponding to the extracellular domain. Of primary concern though is whether this or any other mRNA encoding for a structurally different form of the LH/CG receptor is actually translated and, if so, if the protein is functionally expressed (as opposed to being rapidly degraded). Given that the 1.2 kb mRNA encodes for a protein that contains a signal sequence (2) and no longer contains membranespanning domains, it would be predicted that this truncated receptor would be secreted from the cells. However, we found that when 293 cells are transfected with a cDNA encoding the extracellular domain of the rat luteal LH/CG receptor, the truncated receptor remains trapped intracellularly (6). Although it has been reported that COS-1 cells transfected with a cDNA encoding the extracellular domain of the human testicular LH/CG receptor secrete hCG binding activity into the medium, the levels reported were relatively low (27). In our own experiments with COS-7 cells we have found that although a small percentage of the total binding activity is secreted into the media, the majority remains trapped intracellularly (unpublished observations). In MA-10 cells the most abundant LH/CG receptor transcript is the 1.2 kb species which encodes for the extracellular domain. In these cells, however, no hCG binding activity
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
LH/CG RECEPTOR mRNA TRANSCRIPTS
138
is detected intracellularly (28). Neither were we able to detect hCG binding activity secreted into the medium (unpublished data). Thus, the expression of a 1.2 kb mRNA does not necessarily indicate that a functional hormone-binding protein is produced. These data do not, however, rule out the possibility that other gonadal cells might express such a protein (or other alternate receptor form). Further studies are underway to ascertain which of the various gonadal LH/CG receptor mRNAs encodes for the cell surface 93 kDa receptor and to determine whether there is any biological significance to other mRNA species.
Acknowledgment We wish to thank Kimberly Collison for expert technical assistance.
References 1. Ascoli M, Segaloff DL 1989 On the structure of the luteinizing hormone/chorionic gonadotropin receptor. Endocr Rev 10:27-44 2. McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science 245:494-499 3. Wang H, Lipfert L, Malbon CC, Bahouth S 1989 Site-directed anti-peptide antibodies define the topography of the /J-adrenergic receptor. J Biol Chem 264:14424-14431 4. Findlay JBC, Pappin DJC 1986 The opsin family of proteins. Biochem J 238:625-642 5. Rodriguez MC, Segaloff DL 1990 The orientation of the LH/CG receptor as revealed by site-specific antibodies. Endocrinology 127:674-681 6. Xie YB, Wang H, Segaloff DL 1990 Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity. J Biol Chem 265:21411-21414 7. Segaloff DL, Wang H, Richards JS 1990 Hormonal regulation of LH/CG receptor mRNA in rat ovarian cells during follicular development and luteinization. Mol Endocrinol 4:1856-1865 8. Wang H, Segaloff DL, Ascoli M 1991 Lutropin/choriogonadotropin down-regulates its receptor by both receptor-mediated endocytosis and a cAMP-dependent reduction in mRNA. J Biol Chem 266:780785 9. LaPolt PS, Oikawa M, Jia X-C, Dargan C, Hsueh AJW 1990 Gonadotropin induced up- and down-regulation of rat ovarian LHreceptor message levels during follicular growth, ovulation, and luteinization. Endocrinology 126:3277-3279 10. Hoffman Y, Peegel H, Sprock MJE, Zhang ZY, Menon KMJ 1991 Evidence that human chorionic gonadotropin/luteinizing hormone receptor down-regulation involves decreased levels of receptor messenger ribonucleic acid. Endocrinology 128:388-393
Endo • 1991 Voll29«Nol
11. Ascoli M 1985 Functions and regulation of cell surface receptors in cultured Leydig tumor cells. In: Conn PM (ed) The Receptors. Academic Press, Boca Raton, FL, pp 368-400 12. Ascoli M 1981 Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 108:88-95 13. Chen C, Okayama H 1987 High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745-2752 14. Ascoli M 1982 Internalization and degradation of receptor-bound human choriogonadotropin in Leydig tumor cells. Fate of the hormone subunits. J Biol Chem 257:13306-13311 15. Segaloff DL, Ascoli M 1981 Removal of the surface-bound human choriogonadotropin results in the cessation of hormonal responses in cultured Leydig tumor cells. J Biol Chem 256:11420-11423 16. Ascoli M, Segaloff DL 1986 Effects of collagenase on the structure of the lutropin/choriogonadotropin receptor. J Biol Chem 261:3807-3815 17. Wang H, Ascoli M 1990 Reduced gonadotropin responses in a novel clonal strain of Leydig tumor cells established by transfection of MA-10 cells with a mutant gene of the Type I regulatory subunit of the cAMP-dependent protein kinase. Mol Endocrinol 4:80-90 18. Rosemblit N, Ascoli M, Segaloff DL 1988 Characterization of an antiserum to the rat luteal luteinizing hormone/chorionic gonadotropin receptor. Endocrinology 123:2284-2290 19. Mather JP, Zhuang LZ, Perez-Infante V, Phillips DM 1982 Culture of testicular cells in hormone-supplemented serum-free medium. In: Bardin CW, Sherins RJ (eds) The Cell Biology of the Testis. New York Academy of Sciences, New York, pp 2284-2293 20. Auffray C, Fougeon F 1980 Purification of mouse immunoglobin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem 107:303-314 21. Ascoli M 1980 Degradation of the subunits of receptor-bound human choriogonadotropin by Leydig tumor cells. Biochem Biophys Acta 629:409-417 22. Morgan FJ, Kaye GI, Canfield RG 1974 Characterization of preparations of radioiodinated human chorionic gonadotropin. Isr J Med Sci 10:1263-1270 23. Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH 1990 The testicular receptor for follicle stimulating hormone: Structure and functional expression of cloned cDNA. Mol Endocrinol 4:525530 24. Kim I-C, Ascoli M, Segaloff DL 1987 Immunoprecipitation of the lutropin/choriogonadotropin receptor from biosynthetically labeled Leydig tumor cells: a 92-kDa glycoprotein. J Biol Chem 262:470-477 25. Segaloff DL, Sprengel R, Nikolics K, Ascoli M 1990 The structure of the lutropin/choriogonadotropin receptor. Rec Prog Hormone Res 46:261-303 26. Loosfelt H, Misrahi M, Atger M, Salesse R, Vu Hai-Luu Thi MT, Jolivet A, Guiochon-Mantel A, Sar S, Jallal B, Gamier J, Milgrom E 1989 Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science 245:525-528 27. Tsai-Morris CH, Buczko E, Wang W, Dufau ML 1990 Intronic nature of the rat luteinizing hormone receptor gene defines a soluble receptor subspecies with hormone binding activity. J Biol Chem 265:19385-19388 28. Ascoli M 1983 An improved method for the solubilization of stable gonadotropin receptors. Endocrinology 113:2129-2134
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
Vol. 129, No. 1 Printed in U.S.A.
Multiple Luteinizing Hormone/Chorionic Gonadotropin Receptor Messenger Ribonucleic Acid Transcripts* HAIYUN WANG, MARIO ASCOLI, AND DEBORAH L. SEGALOFF Department of Physiology and Biophysics (H. W., D.L.S.) and Department of Pharmacology (M.A.), The University of Iowa, Iowa City, Iowa 52242
ABSTRACT. 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. (Endocrinology 129: 133-138, 1991)
T
(341 amino acids with six potential sites for N-linked glycosylation), it was speculated that this domain might be responsible for binding the large glycoprotein hormones. Indeed, it has recently been shown that when cells are transfected with a cDNA encoding a truncated LH/CG receptor representing only the extracellular domain they produce a protein capable of binding hCG with the same high affinity as the full-length receptor (6). Interestingly, when one examines Northern blots of gonadal cells which express LH/CG receptors multiple LH/CG receptor messenger RNA (mRNA) transcripts are observed (7-10). In both the rat ovary and in MA-10 Leydig tumor cells (a clonal line of murine Leydig tumor cells that express functional LH/CG receptors, see Ref. 11 for a review) it has been shown that when levels of LH/CG receptor mRNA are altered, all the transcripts appear to be coordinately regulated (7-10). Since it is not known whether these different transcripts have any biological significance, we undertook the present study as an initial step towards better characterizing the LH/ CG receptor mRNA species.
HE LH/CG receptor is a cell surface receptor, present on specialized cells of the ovaries and testes, which activates adenylyl cyclase upon binding either LH or human CG (hCG). From biochemical studies on the receptor, it was determined that it is a single glycoprotein with a molecular mass, on sodium dodecyl sulfate (SDS)polyacrylamide gels, of 93 kilodaltons (kDa) (see Ref. 1 for a review). Recently, the cDNA for the LH/CG receptor has been cloned and expressed in mammalian cells (2). Analyses of the deduced amino acid sequence of the cDNA suggests that this receptor is composed of a large N-terminal extracellular domain attached to a region which is homologous to other G protein-coupled receptors. This C-terminal half of the receptor is therefore thought to similarly span the plasma membrane seven times, as has been shown for rhodopsin and the 0adrenergic receptor (3, 4). Using site-specific antibodies to the rat luteal LH/CG receptor it has been shown that the N-terminal region is indeed located extracellularly and that the C-terminal portion is located cytoplasmically (5). Given that the extracellular orientation of the N-terminal half of the LH/CG receptor and its large size
Materials and Methods
Received February 26, 1991. Address correspondence and requests for reprints to: Dr. Deborah L. Segaloff, Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242. * These studies were supported by NIH Grants HD-22196 (to D.L.S.), CA-40629 (to M.A.), the University of Iowa Diabetes and Endocrinology Research Center Grant AM-25295, and funds from the Roy J. Carver Charitable Trust (to M.A.).
Cells
The origin and handling of the MA-10 cells have been described (12). They were maintained in Waymouth MB752/1 modified to contain 1.1 g/liter NaHCO3, 20 mM HEPES, 50 gentamicin, and 15% horse serum, pH 7.4) in a humidi133
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
LH/CG RECEPTOR mRNA TRANSCRIPTS
134
Endo«1991 Vol 129 • No 1
fied atmosphere containing 2.5% CO2. Human embryonic kidney 293 cells (ATTC CRL 1573) were maintained in Dulbecco's modified Eagle's medium containing 50 ng/rci\ gentamicin and 10% heat-inactivated horse serum in a humidified atmosphere containing 5% CO2. 293 cells were transiently transfected with pCLHR, an expression vector encoding for the full-length rat luteal LH/CG receptor (2), following the procedure of Chen and Okayama (13). 293 cells transfected with this expression vector have previously been shown to bind hCG and respond with increased cAMP accumulation (2).
nucleotides encode for the first through fourth membranespanning domains. This probe was 32P-labeled by random priming. After hybridization, the blot was washed four times in 2x SSC (saline sodium citrate) and 0.1% SDS at room temperature (15 min/washing) and then two times in O.lx SSC and 0.1% SDS at 60 C (30 min/washing). The resulting blots were exposed to x-ray film at -70 C with intensifying screens. All samples shown in a given figure were analyzed in the same gel. Sizes of mRNA transcripts were determined using RNA markers (Boehringer Mannheim Biochemicals, Indianapolis, IN).
Chemical cross-linking of hCG to cells
Hormones and supplies
Culture dishes containing the MA-10 cells or transfected 293 cells were placed on ice for 30 min and washed two times with 2-ml portions of cold Medium A (Waymouth MB752/1 without NaHCO3, containing 20 mM HEPES, 1 mg/ml albumin, pH 7.4). The dishes were then incubated at 4 C for 4.5 h in 1 ml Medium A containing 125I-hCG alone or together with 50 IU unlabeled hCG (to correct for nonspecific binding). At the end of this incubation the dishes were scraped, rinsed with 1 ml cold Medium B (Hanks' balanced salt solution without NaHCO3, containing 20 mM HEPES, pH 7.4) and the contents transferred to a plastic tube on ice. The cells were collected by centrifugation (1300 x g, 15 min, 4 C) and washed twice with 2-ml portions of cold Medium B. The cell pellets were resuspended in 180 ix\ cold Medium B. The receptor-bound hormone was then cross-linked to the cells using disuccinimidyl suberate, the noncross-linked receptor-bound hormone was released by treatment of the cells with isotonic glycine buffer at pH 3 (14, 15), and the samples were prepared for SDS-polyacrylamide gel electrophoresis exactly as described previously (16). The crosslinked products were resolved on a 7.5% SDS-polyacrylamide gel in the presence of reducing agents.
Highly purified hCG (CR-125) was kindly provided by the National Hormone and Pituitary Agency of the NIDDK (NIH). The intact hormone was iodinated as described previously (21), which results in greater than 90% of the radioactivity being incorporated into the a-subunit of the hormone (22). Crude hCG (used for nonspecific binding determinations) was obtained from Sigma (St. Louis, MO). Disuccinimidyl suberate was obtained from Pierce (Rockford, IL).
Results Figure 1 shows a comparison of LH/CG receptor mRNA transcripts in MA-10 cells (lane A), in mammalian 293 human kidney cells transiently transfected with A I It I C I I)
Preparation of RNA and Northern blots Cytoplasmic RNA was prepared from cultured cells (MA-10 cells and transiently transfected 293 cells) exactly as described previously (17). The RNA obtained from the transfected 293 cells was further treated with DNAse I to digest any of the LH/ CG receptor cDNA expression plasmid that might be cytoplasmically located. Total RNA from ovaries of pseudopregnant rats (18), Sertoli cells from adult rats (19), ovaries or testes of adult rats, or testes of adult mice were prepared by extraction with LiCl2 (20). These preparations of cytoplasmic or total RNA were further purified using oligo-(dT) cellulose following the instructions supplied by the manufacturer. For Northern analyses the RNA samples were denatured and electrophoresed in a 1% formaldehyde-agarose gel and then transferred to a nylon (Biodyne, ICN) membrane. The blots were prehybridized for 6 h at 42 C and then hybridized overnight at 42 C with a nick-translated 32P-labeled pGEM-4Z vector (Promega, Madison, WI) containing nucleotides 1-622 of the rat luteal LH/CG receptor cDNA (2). This portion of the LH/CG receptor cDNA encodes for the initial two-thirds of the extracellular domain of the receptor. In some experiments a probe corresponding to the transmembrane domain of the receptor was used. This was a cDNA produced by the polymerase chain reaction that corresponded to nucleotides 1102-1574 of the rat luteal LH/CG receptor cDNA (2). These
kl)
kh
FIG. 1. LH/CG receptor mRNAs in different LH/CG receptor-bearing cells/tissues. Poly (A)+-mRNA was prepared and analyzed by Northern analysis using a cDNA probe corresponding to the extracellular domain of the rat luteal LH/CG receptor. The lanes correspond to (A) 30 ng from mouse MA-10 cells; (B) 25 fig from human kidney 293 cells transiently transfected with an expression vector encoding the rat luteal LH/CG receptor; (C) 25 ng from rat Sertoli cells; and (D) 1.25 fig from ovaries from pseudopregnant rats. The autoradiogram shown was developed for 5 days at -70 C. The amounts of RNA loaded onto the gel were varied to optimize visualization of the transcripts in the different samples. Therefore, a quantitative comparison between the samples cannot be made.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
LH/CG RECEPTOR mRNA TRANSCRIPTS
a cDNA encoding for the full-length rat luteal LH/CG receptor (2) (lane B), in rat Sertoli cells (lane C), and in rat luteal tissue (lane D). These different cells/tissues were chosen because the MA-10 cells, transfected 293 cells, and luteal tissue express cell surface LH/CG receptors. Whereas Sertoli cells do not express LH/CG receptors, they do express FSH receptors, which is a closely related protein (23). This Northern, and all other Northerns shown unless noted otherwise, was probed using a partial cDNA (nucleotides 1-622) of the rat luteal LH/ CO receptor (2). This partial cDNA encodes for approximately the first two thirds of the extracellular domain of the LH/CG receptor. It is readily apparent from Fig. 1 that in cells/tissues expressing LH/CG receptors the numbers and sizes of LH/CG receptor mRNA transcripts vary among the different sources of RNA. The pattern of LH/CG receptor mRNAs in both the MA-10 cells and rat luteal tissue is quite complex (Fig. 1). In MA-10 cells there are six discernable LH/CG receptor transcripts of 1.2, 1.6, 1.9, 2.6, 4.3, and 7.7 kilobases (kb) in length. The most abundant of these is the 1.2 kb species. In contrast, the most abundant LH/ CG receptor mRNA in rat ovaries is 6.7 kb in size. In addition to this transcipt, there are less abundant LH/ CG receptor mRNA species of 4.3, 2.6, and 1.2 kb in size. In Northern blots of rat gonadal tissues we do not consistently observe the 1.2 kb transcript, probably because of its low abundance. For example, it cannot be clearly discerned in Fig. 1; however, it has been shown in previously published Northern blots of rat ovarian tissues (2, 7). In comparing the MA-10 cells and rat luteal tissue, therefore, there are three LH/CG receptor mRNA transcripts in common, these being the 1.2, 2.6, and 4.3 kb species. Although the relative abundance of the 1.2 kb transcript is quite different between these two sources of LH/CG receptor mRNA, the 2.6 and 4.3 kb species are both minor species in these two sources. Figure 1 also shows that no LH/CG receptor mRNA transcripts can be detected under these conditions in RNA prepared from FSH receptor-bearing rat Sertoli cells. It should be noted, however, that at much longer exposure, a faint band at 1.7 kb can be discerned. The multiple LH/CG receptor mRNAs observed in MA-10 cells and rat gonadal tissue may arise from a number of different phenomenon including: 1) different sites of polyadenylation, 2) different lengths of polyadenylation, and/or 3) alternate or incorrect splicing of the LH/CG receptor gene. To eliminate contributions of the latter possibility, we examined the LH/CG receptor mRNAs present in 293 cells transfected with an expression vector encoding for the full-length rat luteal LH/ CG receptor cDNA. It has previously been shown that 293 cells transfected with this plasmid express cell surface 125I-hCG binding activity (2). The prominent LH/
135
CG receptor mRNA identified in these cells (see lane B of Fig. 1) is 3.1 kb long, which is not observed in either the MA-10 cells or rat luteal tissue (compare lanes A, B, and D of Fig. 1). Furthermore, the transfected 293 cells also appear to express other less abundant and less well resolved LH/CG receptor mRNAs both larger and smaller than the 3.1 kb species. The most apparent of these is a species slightly smaller than the 3.1 kb transcript. Therefore, in the absence of transcription of the endogenous LH/CG receptor gene, and thus in the absence of any potential alternate splicing of this gene, there is still some differential processing of the LH/CG receptor mRNA in the 293 cells. These data suggest that even if alternate or incorrect gene splicing was contributing to the different sizes of LH/CG receptor mRNA transcripts in gonadal cells, other factors such a differences in polyadenylation are also likely to be involved. Given the different patterns of LH/CG receptor mRNA expression in MA-10 cells, transfected 293 cells, and rat luteal tissue, it was important to determine whether these cells/tissues expressed the same size cell surface LH/CG receptor. We have previously shown that rat luteal tissue and MA-10 cells both express a 93 kDa LH/CG receptor (18, 24). In order to characterize the LH/CG receptor expressed in the transfected 293 cells, the following experiment was performed. Intact 12'I-hCG (iodinated in the a-subunit) was allowed to bind to the transfected cells, the cells were washed, and the receptorbound hormone was cross-linked. After solubilization, the iodinated cross-linked products were resolved by SDS-polyacrylamide gel electrophoresis. The results of this experiment, and one performed in parallel with MA10 cells, are shown in Fig. 2. It is apparent that the same cross-linked products are generated in the transfected 293 cells and in the MA-10 cells. Thus, radioactivity is present at the tracking dye and at positions corresponding to 53 and 107 kDa proteins. From previous experiments cross-linking 125I-hCG to MA-10 cells, we determined that the radioactivity present at the tracking dye corresponds to the free a-subunit of the hormone, the 53 kDa band corresponds to the a- and /3-subunit of hCG cross-linked to the cell surface LH/CG receptor, and the 107 kDa band corresponds to the a-subunit of hCG crosslinked to the cell surface LH/CG receptor (16). Since the LH/CG receptor of MA-10 cells was shown to be 93 kDa in size (on SDS polyacrylamide gels) (24), we conclude that the LH/CG receptor expressed in the transfected 293 cells is also processed to 93 kDa. Since the size of the rat luteal LH/CG receptor has also been estimated to be 93 kDa (18), we conclude that despite the varying patterns of LH/CG receptor mRNA expression in MA10 cells, transfected 293 cells, and rat luteal tissue {cf. Fig. 1), the same size cell surface receptor is expressed in each.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
LH/CG RECEPTOR mRNA TRANSCRIPTS
136 293 Cells
MA-10 Cells
A |
Endo • 1991 Vol 129 • No 1
B | C
I)
-6.7
107 —
-4.3 kDa
kl)
53—
-2.6 -1.9 -1.6 Tracking dye — -1.2 Unlabeled hCG
—
+
—
+
FIG. 2. Cross-linking of hCG to the LH/CG receptor expressed on transfected 293 cells and MA-10 cells. 293 cells transiently transfected with an expression vector encoding the rat luteal LH/CG receptor and MA-10 cells were incubated with 125I-hCG at 100 ng/ml and 40 ng/ml, respectively, in the absence or the presence of 50 IU unlabeled hCG as indicated. After washing, the cells were cross-linked with disuccinimidyl suberate and analyzed on SDS gels as described in Materials and Methods.
In comparing the different LH/CG receptor mRNAs expressed in MA-10 cells us. rat gonadal tissues, it must be kept in mind that there are three differences between the sources of cells/tissues. Thus the former are from mouse, are testicular in origin, and are tumorigenic; while the latter are from rat, are ovarian in origin, and are nonmalignant. To determine which of these factors contributes to the differences in LH/CG receptor mRNAs present, the experiment shown in Fig. 3 was performed. Lanes A and B compare the LH/CG receptor mRNAs in ovaries from pseudopregnant rats and in testes from adult rats, respectively. There is a 1.7 kb transcript present in the rat testes which is not as visible in rat ovaries. With that exception, the sizes of LH/CG receptor transcripts in the two rat gonadal tissues are the same. Note, however, that as shown previously (2), the abundance of LH/CG receptor mRNA in the ovaries of pseudopregnant rats is far greater than that in rat testes. In examining the LH/CG receptor mRNAs in mouse testes (lane C), it is readily apparent that the number and sizes of transcripts are different from those of rat testes (compare lanes B and C) but the sizes of transcripts present in mouse testes are the same as those observed in MA-10 cells (compare lanes C and D). Thus, the primary reason for the difference in LH/CG receptor mRNAs between rat gonadal tissues and MA-10 cells is simply due to the difference in species. The greater abundance of LH/CG receptor mRNA in MA-10 cells as compared to mouse testes is due to the small percentage of Leydig cells present in testes. The only apparent
FIG. 3. LH/CG receptor mRNAs in mouse and rat gonadal tissues. Poly(A)+-mRNA was prepared and analyzed by Northern analysis using a cDNA probe corresponding to the extracellular domain of the rat luteal LH/CG receptor. The lanes correspond to (A) 25 ng adult (nonpregnant) rat ovary; (B) 35 ng adult rat testes; (C) 50 ng from adult mouse testes; and (D) 25 ng from mouse MA-10 cells. Although all of the lanes are from the same blot, lane A was developed for 3 days, whereas lanes B-D were from an autoradiogram developed for 5 days. The amounts of RNA loaded onto the gel and the times of exposure of the autoradiograms were varied to optimize visualization of the transcripts in the different samples. Therefore, a quantitative comparison between the samples cannot be made.
difference between MA-10 cells and mouse testes is the relatively greater proportion of the 1.2 kb message in MA-10 cells. As the open reading frame of the rat luteal LH/CG receptor is 2.1 kb, one must question the identity of those LH/CG receptor mRNA transcripts that are smaller than 2.1 kb. In order to address this question, we reprobed blots of rat ovarian mRNA and MA-10 cell mRNA with a cDNA probe corresponding to the transmembrane region (specifically the first through fourth membrane-spanning domains) of the receptor. These were compared with hybridizations that had been done using a complementary DNA (cDNA) encoding for amino acids in the initial two-thirds of the extracellular domain. As shown in Fig. 4, the 1.2 kb LH/CG receptor mRNA that is so clearly detected in MA-10 cells using a probe corresponding to the extracellular region of the receptor is not detected by a probe corresponding to the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
LH/CG RECEPTOR mRNA TRANSCRIPTS Km (>\;irk's
MA-10 Cells
6.7-
•U -I -2.6
kh
-1.9 -1.6 -i.:
FlG. 4. Differential hybridization of LH/CG receptor mRNAs with different cDNA probes. Poly(A)+-mRNA was prepared from rat ovaries and MA-10 cells and analyzed by Northern analysis (25 ^/g/lane from rat ovaries and 30 ^g/lane from MA-10 cells). Each lane was probed with a cDNA probe corresponding to the extracellular (EC) domain or the transmembrane (TM) domain of the rat luteal LH/CG receptor. The autoradiogram shown was developed for 5 days at —70 C.
transmembrane portion of the receptor. Due to the low abundance of the 1.7 kb transcript in MA-10 cells, it is not possible to determine if there is also a differential detection of this LH/CG receptor mRNA. For the same reasons, it is not possible to detect a difference in the 1.2 kb band in the different blots of rat ovarian mRNA. It can be seen, however, that all the LH/CG receptor mRNAs equal to or greater than 2.1 kb can be detected by both probes. The results of this experiment suggest that the 1.2 kb LH/CG receptor mRNA encodes for a truncated form of the receptor which lacks the transmembrane and cytoplasmic domains.
Discussion Previous studies have shown that in both rat gonadal tissues and in mouse MA-10 Leydig tumor cells there are numerous LH/CG receptor mRNA transcripts (7-10). Furthermore, the numbers and sizes of the transcripts differ between these two sources of mRNA. We show here that this difference is entirely due to a difference in species. Thus, mouse testes contain the same LH/CG receptor mRNAs as MA-10 cells. Neither MA-10 cells nor rat luteal tissue, however, have the same size LH/ CO receptor mRNA as that in human kidney 293 cells transfected with an expression vector containing the rat luteal LH/CG receptor cDNA. Nonetheless all three of these different cell types express a 93 kDa cell surface LH/CG receptor. These data suggest that each of these cell types contain a LH/CG receptor mRNA containing the same coding sequence, but differing lengths of 5'and/or 3'-untranslated regions. These might arise from simple differences in the sites and/or lengths of polyadenylation or they may reflect more substantial altera-
137
tions in the length or composition of the untranslated regions. Further evidence that a given mRNA may be differentially processed comes from the results of Northern blots of 293 cells transfected with the LH/CG receptor cDNA. As untransfected or mock-transfected cells do not express any detectable 12r>I-hCG binding activity (2), all LH/CG receptor mRNA must be derived from trancription of the exogenously added cDNA rather than the endogenous gene. Although these cells express one predominant LH/CG receptor mRNA, there are also other much less abundant mRNAs (cf. Fig. 1). The different LH/CG receptor mRNAs in this case could not arise from alternate splicing of the gene. An additional mechanism accounting for the different LH/CG receptor mRNAs in gonadal cells appears to be alternative or incorrect splicing of the LH/CG receptor gene. When the rat luteal and the porcine testicular LH/ CG receptor cDNAs were originally isolated, several variants were described (25, 26). These included cDNAs that encoded for receptors that were prematurely truncated and/or contained deletions. Recently, it has been reported that the human LH/CG receptor gene contains at least nine introns located within the region encoding the extracellular domain (27), which may help explain the diversity of the LH/CG receptor cDNAs. Thus, some of the different mRNAs observed in gonadal cells may also correspond to transcripts with different coding sequences arising from alternate splicing of the gene. Indeed, we show here that the 1.2 kb LH/CG receptor mRNA encodes for a truncated receptor corresponding to the extracellular domain. Of primary concern though is whether this or any other mRNA encoding for a structurally different form of the LH/CG receptor is actually translated and, if so, if the protein is functionally expressed (as opposed to being rapidly degraded). Given that the 1.2 kb mRNA encodes for a protein that contains a signal sequence (2) and no longer contains membranespanning domains, it would be predicted that this truncated receptor would be secreted from the cells. However, we found that when 293 cells are transfected with a cDNA encoding the extracellular domain of the rat luteal LH/CG receptor, the truncated receptor remains trapped intracellularly (6). Although it has been reported that COS-1 cells transfected with a cDNA encoding the extracellular domain of the human testicular LH/CG receptor secrete hCG binding activity into the medium, the levels reported were relatively low (27). In our own experiments with COS-7 cells we have found that although a small percentage of the total binding activity is secreted into the media, the majority remains trapped intracellularly (unpublished observations). In MA-10 cells the most abundant LH/CG receptor transcript is the 1.2 kb species which encodes for the extracellular domain. In these cells, however, no hCG binding activity
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
LH/CG RECEPTOR mRNA TRANSCRIPTS
138
is detected intracellularly (28). Neither were we able to detect hCG binding activity secreted into the medium (unpublished data). Thus, the expression of a 1.2 kb mRNA does not necessarily indicate that a functional hormone-binding protein is produced. These data do not, however, rule out the possibility that other gonadal cells might express such a protein (or other alternate receptor form). Further studies are underway to ascertain which of the various gonadal LH/CG receptor mRNAs encodes for the cell surface 93 kDa receptor and to determine whether there is any biological significance to other mRNA species.
Acknowledgment We wish to thank Kimberly Collison for expert technical assistance.
References 1. Ascoli M, Segaloff DL 1989 On the structure of the luteinizing hormone/chorionic gonadotropin receptor. Endocr Rev 10:27-44 2. McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science 245:494-499 3. Wang H, Lipfert L, Malbon CC, Bahouth S 1989 Site-directed anti-peptide antibodies define the topography of the /J-adrenergic receptor. J Biol Chem 264:14424-14431 4. Findlay JBC, Pappin DJC 1986 The opsin family of proteins. Biochem J 238:625-642 5. Rodriguez MC, Segaloff DL 1990 The orientation of the LH/CG receptor as revealed by site-specific antibodies. Endocrinology 127:674-681 6. Xie YB, Wang H, Segaloff DL 1990 Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity. J Biol Chem 265:21411-21414 7. Segaloff DL, Wang H, Richards JS 1990 Hormonal regulation of LH/CG receptor mRNA in rat ovarian cells during follicular development and luteinization. Mol Endocrinol 4:1856-1865 8. Wang H, Segaloff DL, Ascoli M 1991 Lutropin/choriogonadotropin down-regulates its receptor by both receptor-mediated endocytosis and a cAMP-dependent reduction in mRNA. J Biol Chem 266:780785 9. LaPolt PS, Oikawa M, Jia X-C, Dargan C, Hsueh AJW 1990 Gonadotropin induced up- and down-regulation of rat ovarian LHreceptor message levels during follicular growth, ovulation, and luteinization. Endocrinology 126:3277-3279 10. Hoffman Y, Peegel H, Sprock MJE, Zhang ZY, Menon KMJ 1991 Evidence that human chorionic gonadotropin/luteinizing hormone receptor down-regulation involves decreased levels of receptor messenger ribonucleic acid. Endocrinology 128:388-393
Endo • 1991 Voll29«Nol
11. Ascoli M 1985 Functions and regulation of cell surface receptors in cultured Leydig tumor cells. In: Conn PM (ed) The Receptors. Academic Press, Boca Raton, FL, pp 368-400 12. Ascoli M 1981 Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 108:88-95 13. Chen C, Okayama H 1987 High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745-2752 14. Ascoli M 1982 Internalization and degradation of receptor-bound human choriogonadotropin in Leydig tumor cells. Fate of the hormone subunits. J Biol Chem 257:13306-13311 15. Segaloff DL, Ascoli M 1981 Removal of the surface-bound human choriogonadotropin results in the cessation of hormonal responses in cultured Leydig tumor cells. J Biol Chem 256:11420-11423 16. Ascoli M, Segaloff DL 1986 Effects of collagenase on the structure of the lutropin/choriogonadotropin receptor. J Biol Chem 261:3807-3815 17. Wang H, Ascoli M 1990 Reduced gonadotropin responses in a novel clonal strain of Leydig tumor cells established by transfection of MA-10 cells with a mutant gene of the Type I regulatory subunit of the cAMP-dependent protein kinase. Mol Endocrinol 4:80-90 18. Rosemblit N, Ascoli M, Segaloff DL 1988 Characterization of an antiserum to the rat luteal luteinizing hormone/chorionic gonadotropin receptor. Endocrinology 123:2284-2290 19. Mather JP, Zhuang LZ, Perez-Infante V, Phillips DM 1982 Culture of testicular cells in hormone-supplemented serum-free medium. In: Bardin CW, Sherins RJ (eds) The Cell Biology of the Testis. New York Academy of Sciences, New York, pp 2284-2293 20. Auffray C, Fougeon F 1980 Purification of mouse immunoglobin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem 107:303-314 21. Ascoli M 1980 Degradation of the subunits of receptor-bound human choriogonadotropin by Leydig tumor cells. Biochem Biophys Acta 629:409-417 22. Morgan FJ, Kaye GI, Canfield RG 1974 Characterization of preparations of radioiodinated human chorionic gonadotropin. Isr J Med Sci 10:1263-1270 23. Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH 1990 The testicular receptor for follicle stimulating hormone: Structure and functional expression of cloned cDNA. Mol Endocrinol 4:525530 24. Kim I-C, Ascoli M, Segaloff DL 1987 Immunoprecipitation of the lutropin/choriogonadotropin receptor from biosynthetically labeled Leydig tumor cells: a 92-kDa glycoprotein. J Biol Chem 262:470-477 25. Segaloff DL, Sprengel R, Nikolics K, Ascoli M 1990 The structure of the lutropin/choriogonadotropin receptor. Rec Prog Hormone Res 46:261-303 26. Loosfelt H, Misrahi M, Atger M, Salesse R, Vu Hai-Luu Thi MT, Jolivet A, Guiochon-Mantel A, Sar S, Jallal B, Gamier J, Milgrom E 1989 Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science 245:525-528 27. Tsai-Morris CH, Buczko E, Wang W, Dufau ML 1990 Intronic nature of the rat luteinizing hormone receptor gene defines a soluble receptor subspecies with hormone binding activity. J Biol Chem 265:19385-19388 28. Ascoli M 1983 An improved method for the solubilization of stable gonadotropin receptors. Endocrinology 113:2129-2134
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 20:02 For personal use only. No other uses without permission. . All rights reserved.
