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J. Biol. Chem., Vol. 277, Issue 2, 879-882, January 11, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/2/879    most recent C100402200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jablonka-Shariff, A. Articles by Boime, I. Search for Related Content PubMed PubMed Citation Articles by Jablonka-Shariff, A. Articles by Boime, I. Social Bookmarking                      What's this?

ACCELERATED PUBLICATION
Evolution of Lutropin to Chorionic Gonadotropin Generates a Specific Routing Signal for Apical Release in Vivo^*

Albina Jablonka-Shariff, Vicenta Garcia-Campayo, and Irving Boime^§

From the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, July 17, 2001, and in revised form, November 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

One of the fundamental differences among mammals is the mechanism of maintaining the corpus luteum of pregnancy. Placentation in primates is associated with the production of the glycoprotein hormone chorionic gonadotropin (CG), which is secreted into the maternal serum and stimulates progesterone synthesis from the corpus luteum, which is essential for early development of the embryo. CG together with the pituitary hormones lutropin (LH), follitropin, and thyrotropin constitute the family of glycoprotein hormones comprised of a common  subunit and a hormone-specific  subunit. The LH and CG subunits share 85% amino acid sequence identity, and functionally LH and CG are interchangeable. CG evolved by a recent gene duplication event from the LH locus, and despite the close relationship between them, their modes of secretion are quite different. CG release from the placenta is apically directed, whereas LH is released from the basal side of the cell, and the determinant(s) for this redirected trafficking are unknown. Here, using the polarized Madin-Darby canine kidney (MDCK) cell line, we provide evidence for the molecular basis of the different secretory patterns of LH and CG in vivo. The apical targeting of CG is programmed by a carboxyl-terminal sequence, which encodes a novel sorting signal. It is also apparent that the presence of the O-linked oligosaccharides in the CTP sequence contributes to this apical routing. The CTP, which is absent in LH, redirects CG to the maternal serum and permits the unique arrangement for primate placentation. Our data also show that the MDCK cells can distinguish the different secretory pathways for the gonadotropins and will be a valuable model for elucidating the determinants associated with the unique sorting of these functionally related hormones.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

The evolution of primates is associated with a fundamental change in the morphogenesis of the placenta (1, 2). Placentation in primates is coupled to the synthesis of glycoprotein hormone chorionic gonadotropin (CG),¹ which is secreted into the maternal serum and stimulates progesterone synthesis from the corpus luteum, thereby allowing early implantation and development of the embryo (3, 4). CG and its pituitary counterpart, lutropin (LH), comprise a family of heterodimeric glycoprotein hormones, including follitropin and thyrotropin, that share a common  subunit, but differ in their hormone-specific  subunits (5). Although the  subunits determine biological specificity of each hormone, there is significant structural similarity between them. This is most evident for the LH and CG subunits, which share 85% amino acid identity in the first 114 amino acids (6). A major difference between the two subunits is the presence in the CG subunit of a 31-amino acid carboxyl-terminal extension (CTP) compared with a shorter 7-amino acid stretch in LH (see "Results").

The CG subunit is specific to primates and evolved by a gene duplication event from the LH locus (7); functionally the two are interchangeable. Similar to other mammals, primates express LH during pregnancy, raising the question as to why primates require CG. When comparing the biosynthesis of LH and CG, two important differences emerge: the pathways of secretion and polarity of their release. LH is packaged into storage granules (8), subject to regulated exocytosis by secretagogues (9), and released via the basolateral surface of pituitary gonadotrope cells (10, 11). In contrast, CG is secreted by an apical route through the villous and directly into the intervillous space created by the implanted placenta (1, 12-14). The molecular basis by which the intracellular trafficking of CG is directed to exit the chorionic villi into the maternal lumen is unknown. To examine whether the unique circulation profiles exhibited by LH and CG in vivo are reflected in differences in polarized secretion, and to identify potential targeting sequences, we co-transfected the common  subunit and LH/CG genes into Madin-Darby canine kidney (MDCK) cells. This cell line is an excellent model to study polarized secretion of endogenous and exogenous proteins in culture (15, 16). The plasma membrane of these epithelial cells is divided into apical and basolateral domains by tight junctions (17). The apical side faces the luminal or exterior milieu, which is covered by microvilli and is involved in absorptive or secretory processes, whereas the basolateral side faces the serosal environment (16, 17). Here we examined the polarized secretion of LH and CG in transfected MDCK cells and show that apical targeting of CG is programmed by the unique CTP, a novel sorting signal that is generated by a single frameshift mutation in the ancestral LH gene.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Polyclonal antisera directed against the  and CG subunit were prepared in this laboratory. Purified hCG (CR-127; 14,900 IU/mg) was provided by Dr. A. F. Parlow (National Hormone and Pituitary Program, National Institutes of Health, Torrance, CA).

Cell Culture-- MDCK cells (strain II) were a gift of Dr. Sharon Milgram (University of North Carolina). These cells were grown in DMEM/F-12 medium (for no more than 10-15 passages) supplemented with 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin and containing 5% FBS (v/v) at 37 °C in a humidified 5% CO₂ incubator (18). The cells (3.5 × 10⁵ cells/ml) were seeded on 12-mm or 24-mm Transwell filters (0.4 µm pore size, Corning-Costar, Cambridge, MA) allowing separate access of media to the apical and basolateral faces of the membrane. Filter-grown cells were cultured for 3-4 days with changes of medium daily.

To measure the integrity of epithelial monolayer and formation of tight junctions (as an indicator of achieving polarization), electrical resistance was measured across the cell monolayer using the Millicell Electrical Resistance System (Millipore Corp., Bedford, MA) (18).

Transfection and Clone Isolation-- Transfection was performed using the DNA-calcium-phosphate precipitation method as described previously (19). Cells grown on plastic dishes were stably transfected with pM²HA (20, 21) containing either the , LH, CG, or CG114 subunit genes.

Metabolic Labeling-- MDCK cells were labeled overnight with 25 µCi/ml [³⁵S]cysteine in DMEM/F-12 medium lacking cysteine and supplemented with 5% dialyzed fetal bovine serum, L-glutamine, penicillin, and streptomycin (20). Labeling medium was added to the apical and basolateral compartments, and media from them were collected into separate tubes and were immunoprecipitated with antisera directed against  and CG subunits using Pansorbin. The reduced (with 10 mM -mercaptoethanol) and heated (4 min) proteins were resolved as on 15% SDS-polyacrylamide gels described previously (19).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

We first examined the secretion patterns of the LH and CG dimers from transfected MDCK cells grown in Transwells and labeled overnight with [³⁵S]cysteine. The media were immunoprecipitated with CG antiserum, which reacts equally well with either the LH or CG subunits; co-precipitation of the  subunit is indicative of heterodimer formation. LH dimer released from the cells was primarily associated with the basolateral side, indicating that it is secreted in a polarized manner (Fig. 1A, lanes 1 and 2). The basolateral to apical ratio for LH was 3:1 (76.2 ± 1.6% and 23.8 ± 1.6%, respectively: n = 6; p < 0.05) (Fig. 1B). Control experiments demonstrated that when the integrity of the monolayer was disrupted with a cell scraper, determined by reduction in electrical resistance, LH dimer traversed the filter and distributed equally to both compartments (Fig. 1A, lanes 3 and 4). The preferential basolateral routing of LH is consistent with its known distribution and release from the pituitary (10, 11). LH-containing secretory granules redistribute subcellularly and become polarized to the side of the gonadotrope nearest to the vascular sinusoid during the preovulatory stage, and release of the granule content occurs in the basal region of the cell (10, 11).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Secretion of LH dimer. A, cells expressing the LH dimer were grown on Transwell filters and, after reaching confluence and forming a tight monolayer, the cells were metabolically labeled overnight with [35S]cysteine. The media collected separately from apical (Ap, lane 1) and basolateral (BL, lane 2) compartments were immunoprecipitated with antiserum directed against the CG subunit, which reacts equally with either the LH or CG subunits. Precipitation of the  subunit with the CG antiserum indicates dimer formation. The proteins were analyzed by SDS-polyacrylamide gels and autoradiography. Lanes 3 and 4 represent the control experiment and correspond to media collected from Transwell under the same conditions except that the monolayer was disrupted with a cell scraper, resulting in a low resistance (below 150 ohms/cm2). B, the relative percent secretion of LH into the apical (stippled bars) and basolateral (solid bars) compartments was quantitated by densitometry from autoradiographs. The total combined secretion of protein into the medium of apical and basolateral compartments was taken as 100%. The apical and basolateral ratio for LH was determined from the percent of protein present in each compartment. Results are shown as the mean ± S.E. (n = 6). The asterisk indicates significant difference, p < 0.05.

In contrast to LH, CG was secreted preferentially into the apical compartment (Fig. 2A, lanes 1 and 2). Densitometric analysis of labeled bands demonstrated an apical to basolateral ratio of 2.7:1 (73 ± 1.5% versus 27 ± 1.5%, respectively: n = 6; p < 0.05) (Fig. 2B). These data reflect differences in the secretion behavior of CG compared with its pituitary homologue LH. The apical secretion of CG is consistent with its in vivo release, since CG enters the maternal blood lakes of the uterus via an apical route through the villous (1, 12-14).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Secretion of CG and CG114 dimers. A, MDCK cells expressing wild type CG or the truncated carboxyl-terminal mutant CG114 dimer were metabolically labeled overnight with [35S]cysteine. The media from the apical (Ap) and basolateral (BL) compartments were immunoprecipitated with CG antiserum and analyzed as described for Fig. 1. The migration of CG114 species bearing two (N2) or one (N1) asparagine-linked oligosaccharides and the  subunit are indicated by arrows. N2 indicates the presence of both of the CG Asn-linked oligosaccharides and N1 is a minor form bearing only one Asn-linked carbohydrate unit. CG114 co-migrates with the  subunit on SDS gels. That the N2 form of CG114 and  subunit reflect heterodimer formation was confirmed by nondenatured Western blots, which demonstrated a molecular mass of 35 kDa, corresponding to the intact dimer (data not shown). B, the relative percent secretion of CG and CG114 dimers was quantitated by densitometry. Results are shown as the mean ± S.E. (n = 6-8). Asterisks indicate significant difference, p < 0.05.

To examine the possibility of nonspecific transfer of CG and LH across the MDCK cell monolayer, conditioned media containing ³⁵S-labeled CG or LH from the MDCK cell-line was added to nontransfected MDCK cells grown on Transwell filters. After overnight incubation lysates and media from the apical and basolateral compartments were immunoprecipitated. No transfer of protein from one compartment to the other was detected (data not shown), indicating direct and specific sorting of CG and LH into the apical and basolateral compartments, respectively. These data demonstrate that the MDCK model recapitulates the secretion polarity for pituitary LH and placental CG as seen in vivo.

Numerous studies have shown that the uncombined  subunit is primarily secreted constitutively (22, 23). To assess the specificity of the MDCK model, cells expressing only the  subunit were examined (Fig. 3). In this case the secretion pattern was random, i.e. the  subunit was observed equally in both compartments (Fig. 3A, lanes 1 and 2). Presumably, the differential sorting exhibited by LH and CG is due to the  subunit, since the  subunit is the same for both hormones. To test this prediction, and to address the question if dimer formation is a prerequisite for sorting, MDCK cells expressing either the LH or CG subunits were grown on Transwells (Fig. 3, lanes 3-6). The  subunits exhibited the same polarity as the corresponding dimers, indicating that the sorting signals are encoded in the hormone-specific  subunit, and heterodimer formation is not required for the secretion polarity.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Secretion of , LH, and CG subunits. A, cells expressing subunits grown on Transwell filters were metabolically labeled overnight with [35S]cysteine. Media from the apical (Ap) and basolateral (BL) compartments were immunoprecipitated with  or CG antiserum. B, the relative percent secretion of , LH, and CG subunits was quantitated by densitometry as described for Fig. 1. Results are shown as the mean ± S.E. (n = 5). Asterisks indicate significant differences, p < 0.05.

Based on their extensive sequence identity, it was suggested that CG evolved from an ancestral LH gene (7). The sequences of the carboxyl ends of the LH and CG termini are shown in Scheme 1. (The shaded areas indicate identical sequence, and the asterisks show the O-linked serine acceptor site in the CG subunits.) A single base pair deletion at codon 114 in the ancestral gene lead to generation of an mRNA encoding the CG subunit composed of 145 amino acids compared with the 121 amino acids of the LH subunit, and thus the two proteins have different COOH-terminal sequences (7). The seven amino acids at the carboxyl terminus of LH are very hydrophobic; in contrast, the CG carboxyl end is hydrophilic containing several serine residues, of which four are O-glycosylated. Thus, the apparent major evolutionary change from LH to the CG subunit was the appearance of the CTP. CG serum levels rise rapidly in the first trimester of pregnancy and decline thereafter (4, 24). It is widely accepted that the main role for the CTP sequence is to enhance the circulatory half-life of CG during peak synthesis (21, 25, 26), maintaining a bolus of activity in early gestation to stimulate maternal progesterone production in the corpus luteum. However, our data implicate a novel role for this sequence as a routing signal. To examine whether the CTP is essential for polarity, a stop codon was introduced at residue 115 of the CG subunit (21), resulting in a truncated protein of 114 amino acids (CG114) (Fig. 2A, lanes 3 and 4). In contrast to wild type CG dimer, the dimer bearing the CG114 mutant (CG114) was secreted basolaterally (68.9% ± 1.7: n = 6; p < 0.05) (Fig. 2B). These results confirm that the carboxyl-terminal sequence of the CG subunit is a major routing signal for the apical secretion of CG dimer.

View larger version (27K): [in this window] [in a new window]   Scheme 1.   Sequences of the carboxyl ends of the LH and CG termini.

It has been reported that N- and O-linked oligosaccharides are associated with apical sorting signals for some glycoproteins. The O-glycosylated stalked region of the neurotrophin receptor has been implicated in its sorting to the apical membrane, since deletion of this region lead to a mistargeting of the mutant protein to the basolateral membrane in MDCK cells (27). Moreover, eliminating or reducing the number of O-glycans affects the polarity of intestinal brush border enzymes released from MDCK cells (28, 29). However, deleting the O-linked oligosaccharides from aminopeptidase N had no effect on its apical secretion pattern from MDCK cells (28). Because there are four O-linked oligosaccharides in the CTP sequence, it is unclear whether the targeting function of CTP is encoded in the protein sequence and/or the carbohydrate. To address whether the O-linked carbohydrate in the CTP contributes to the apical secretion of CG, we constructed a CG mutant (CG-Odg) (Fig. 4A) in which the four serine acceptor sites at residues 121, 127, 132, and 138 in the CTP were changed to alanine. (The Ser at position 130 was also mutated in this variant because we observed that some alternative O-glycosylation occurred at this site when the mutant was expressed in CHO cells.)² Synthesis of CG dimer bearing this mutation (Fig. 4A, lanes 1 and 2) or the uncombined CG mutant (Fig. 4A, lanes 3 and 4) displayed a basolateral preference (68.3% ± 2.8 and 79% ± 2.2, respectively; p < 0.05) (Fig. 4B). These results suggest that the apical sorting signaling of the CTP is mediated by the O-linked oligosaccharides.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Secretion of CG dimer bearing the CG subunit devoid of O-linked carbohydrate (CG-Odg) and uncombined mutated subunit (CG-Odg). A, MDCK cells expressing CG-Odg dimer and CG-Odg subunit were metabolically labeled overnight with [35S]cysteine. The media from the apical (Ap) and basolateral (BL) compartments were immunoprecipitated with CG antiserum and analyzed as described in the legend to Fig. 1. Dimerized O-deglycosylated CG subunit appears as two bands (presumably N1 and N2), which co-migrate with the  subunit (arrow). B, the relative percent secretion of CG-Odg dimer and CG-Odg subunit was quantitated by densitometry. Results are shown on the mean + S.E. (n = 5-7).

A distinguishing structural feature between the LH and CG subunits is that the LH subunit contains one N-linked oligosaccharide, whereas CG contains two units. Thus, while the CTP is sufficient to route CG apically, we cannot exclude that the observed sorting is achieved in concert with the additional N-linked oligosaccharide on the CG subunit. There is evidence that N-glycans can act as apical signals for some secretory and integral membrane proteins (30), but their exact role is unclear (31), since many glycoproteins do not display glycan-dependent apical targeting (32). This indicates that the contribution of N-glycans to apical targeting is protein specific. It has been suggested that N-glycans do not function as sorting signals directly but rather play an accessory role necessary for the function of an apical determinant (31, 33).

There are several reports showing that motifs consisting of a tyrosine residue near at least one hydrophobic amino acid, or a motif bearing a leucine/leucine or a leucine/isoleucine pair are involved in basolateral sorting of membrane proteins (34-36). It is intriguing that the unique carboxyl-terminal heptapeptide in the LH subunit contains the dileucine motif.

Why only the CG subunit among the glycoprotein hormone family contains CTP is unclear. Expression of CG in the placenta suggests that this extension is a critical factor in the gestational role of CG. A major function of the placenta during pregnancy is the exchange of metabolic products between the fetal and maternal blood streams (1). Thus, the CTP directs CG apically from the fetally derived placenta into the maternal intervillus spaces; these are luminal compartments filled with maternal blood derived from pressure gradients generated in the spiral arteries. Our data reveal that LH, which is elaborated by the pituitary directly into a single vascular compartment, is distinct from the production of humoral agents in the feto-placental unit. The use of the MDCK model has permitted the identification of a novel targeting domain that reversed the polarity of two evolutionarily related heterodimers, which activate the identical receptor. The appearance of CTP represents not only an adaptive response to maintain a high level of circulating gonadotropin, but also a targeting signal redirecting a pituitary function to the placenta, thus ensuring a successful implantation and development of the primate embryo.

	ACKNOWLEDGEMENTS

We are grateful to Drs. Rajendra Kumar, Ross Cagan, David Ornitz, Mark Boothby, and Sharon Milgram for their helpful comments regarding the manuscript. We also thank Dr. Thomas Woolsey for his advice during the study, and Kristy Chamberlain for her excellent assistance in the preparation of the manuscript.

	FOOTNOTES

^* This work was supported by a grant from Organon.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of an National Research Service Award award from the National Institutes of Health.

^§ To whom correspondence should be addressed: Dept. of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid, St. Louis, MO 63110. Tel.: 314-362-2556; Fax: 314-361-3560; E-mail: iboime@pcg.wustl.edu.

Published, JBC Papers in Press, November 26, 2001, DOI 10.1074/jbc.C100402200

² V. Garcia-Campayo and I. Boime, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: CG, chorionic gonadotropin; LH, lutropin; MDCK, Madin-Darby canine kidney; DMEM, Dulbecco's modified Eagle's medium.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/2/879    most recent C100402200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jablonka-Shariff, A. Articles by Boime, I. Search for Related Content PubMed PubMed Citation Articles by Jablonka-Shariff, A. Articles by Boime, I. Social Bookmarking                      What's this?