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BOR - Papers in Press, published online ahead of print October 29, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.022699
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BIOLOGY OF REPRODUCTION 70, 649–655 (2004)
DOI: 10.1095/biolreprod.103.022699
© 2004 by the Society for the Study of Reproduction, Inc.

Pituitary

Luteinizing Hormone and Follicle-Stimulating Hormone Exhibit Different Secretion Patterns from Cultured Madin-Darby Canine Kidney Cells

Albina Jablonka-Shariff, and Irving Boime1

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LH, FSH, and chorionic gonadotropin (CG) are comprised of a common {alpha} subunit and a hormone-specific ß subunit. Using Madin-Darby canine kidney (MDCK) epithelial cells to examine the polarized secretion of human CG/LH, we previously reported that CG and LH were detected in the apical and basolateral compartments, respectively, and the carboxyl terminal end of the CGß subunit contains a strong apical signal. Here we show that the carboxyl seven amino acids in the LHß subunit contribute to the basolateral secretion of LH, and an LH chimera bearing the CGß apical signal is redirected from the basolateral to the apical compartments. Because LH and FSH are synthesized in the same cell, we also compared the secretion polarity of LH with FSH. MDCK cells expressing the FSH dimer displayed an almost equal distribution of protein into the apical and basolateral compartments. Given that the LHß and CGß carboxy terminal sequences, which differ from that in the FSHß subunit, occupy a pivotal role in their polarized behavior, the results support the hypothesis that pituitary exit of LH and FSH occur via different secretion pathways, and are released spatially from the pituitary via different circulatory routes.

follicle-stimulating hormone, human chorionic gonadotropin, luteinizing hormone, pituitary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human luteinizing hormone (LH) and follicle-stimulating hormone (FSH) of the pituitary and the placental hormone human chorionic gonadotropin (CG) are members of the glycoprotein family, which also includes thyrotropin. These hormones are noncovalently associated heterodimers consisting of a common {alpha} subunit and a hormone-specific ß subunit [1].

When comparing biosynthesis of LH and CG, two important differences emerge: their pathways of secretion and their sites of release. LH is packaged into dense core secretion granules [2, 3], subject to regulated release by secretagogues [4], and secreted directly into the blood vessels via the basal laminal region of gonadotroph cells [5–7]. By contrast, FSH is released constitutively into the blood stream and its secretion is tightly coupled to synthesis [8–10]. CG is released constitutively from placenta but is apically directed through the villous to the uterine luminal compartment created by the implanted placenta [11–14].

The CGß subunit evolved by a gene duplication event from the LHß locus [15] and as a result, both ß subunits share 85% amino acid identity and functionally, LH and CG bind to a common gonadal receptor [16, 17]. A major difference between the two ß subunits is their carboxyl terminal region. The CGß subunit contains a 31-amino acid hydrophilic carboxy-terminal extension (CTP), which bears four O-glycosyl recognition sites [18–21]. The LHß subunit possesses a hydrophobic heptapeptide that terminates at residue 121 [17, 22]. Thus, the apparent major evolutionary change from LHß- to the CGß subunit was the appearance of the CTP. Although the overall crystal structure of the FSHß subunit [23] is similar to the CGß subunit [24], the amino acid sequence of the latter is quite distinct from the other ß subunits.

One of the unique features of most epithelial cells is that they exhibit polarized secretion of proteins by two different routes: apical and basolateral. The apical side faces luminal or exterior milieu and the basolateral surface is directed toward the blood supply [25, 26]. The secretion polarity of numerous proteins have been examined using polarized Madin-Darby canine kidney (MDCK) epithelial cell line [27–30]. Using this model, we demonstrated that CG is secreted apically from MDCK cells and this release is programmed by the CTP sequence [31]. By contrast, LH is secreted to the basolateral compartment in these cells [31]. Thus, the CTP, which is absent in the LHß subunit, directs CG to the maternal serum and permits the unique endocrine status of primate placentation.

FSH, like LH, is synthesized in the gonadotrophs and, although their release overlaps at the preovulatory surge [32], LH and FSH secretion diverges under a variety of physiological conditions [2, 10, 33, 34]. It has been reported that LH-containing secretory granules redistribute subcellularly and become polarized to the side of the gonadotroph nearest to the vascular sinusoid during the preovulatory surge [5, 6, 35], whereas FSH is apparently more dispersed in vesicles throughout the cytoplasm [6].

Here we examine the secretion behavior of FSH and LH from MDCK cells. The data show that FSH displays no secretion polarity. This contrasts with LH, which exhibits a basolateral preference. We also show that the carboxy terminal region of the LHß subunit contributes to its basolateral signaling. Moreover, while fusion of the CTP to the LHß subunit targets LH secretion to the apical side of the MDCK cells, addition of CTP to the FSHß subunit did not alter polarity. These results support the hypothesis that pituitary exit of LH and FSH occurs via different secretion pathways. Moreover, in the case of LH/CG, the carboxy terminal region is an evolutionarily functional determinant, responsible for the apical polarity of CG secretion in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies

Restriction enzymes were purchased from New England BioLabs, Inc. (Beverly, MA) and Promega Corp. (Madison, WI). Oligonucleotides used for PCR amplification and sequencing were prepared by Washington University Sequencing Facility (St. Louis, MO). Dulbecco Modified Eagle Medium/Ham F-12 medium (DMEM/F-12), Dulbecco PBS (with and without calcium and magnesium), L-cysteine, and normal rabbit serum were purchased from Sigma Chemicals (St. Louis, MO). Fetal bovine serum (FBS) was obtained from Harlan (Indianapolis, IN), FBS dialyzed against 150 mM NaCL (DFBS) and neomycin analogue G418 were purchased from Gibco (Invitrogen, San Diego, CA). Pansorbin was obtained from Calbiochem (San Diego, CA). L-glutamine, trypsin, penicillin, and streptomycin were prepared by Washington University Center Basic Research (St. Louis, MO). [35S]Cysteine (>1000 Ci/mmol) was purchased from DuPont-New England Nuclear Corp. (Boston, MA). Polyclonal antisera directed against human {alpha} subunit and human CGß subunit (which cross-reacts with the free and dimer forms of the LHß subunit) were prepared in this laboratory. Recombinant FSH and FSHß specific (FSH 4B) monoclonal antibody were from Organon (Oss, The Netherlands). Polyclonal antiserum directed against human FSHß subunit and purified human CG (CR-121; 14 900 IU/mg) was kindly provided by Dr. A.F. Parlow (National Hormone and Pituitary Program, Torrance, CA).

Cell Culture

MDCK cells (strain II), a gift of Dr. Sharon Milgram (University of North Carolina, Chapel Hill, NC), were grown in DMEM/F-12 (for no more than 10–15 passages) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and containing 5% FBS (vol/vol) at 37°C in a humidified 5% CO2 incubator. The cells were cultured on plastic dishes or, to obtain polarized monolayer, on 24-mm (3.5 x 105 cells/ml) Transwell filters (0.4 µm pore size; Corning-Costar, Cambridge, MA) allowing separate access of media to the apical and basolateral membrane. Filter-grown cells were cultured for 4–5 days with changes of medium daily.

To assess the integrity of epithelial monolayer and formation of tight junctions (as an indicator of achieving polarization), transepithelial electrical resistance was measured using the Millicell ERS (Millipore Corp., Bedford, MA) [36]. All experiments were performed when electrical resistance was >300 ohms/cm2 [31].

Transfection and Clone Isolation

Transfection was performed using the DNA-calcium-phosphate precipitation method as previously described [31, 37, 38]. MDCK were stably transfected with pM2HA vector containing the {alpha}, FSHß, LHß, LHß114, LHß114-CTP subunit genes or the LHß114-bearing deglycosylated (dg) CTP, which lacks the O-linked oligosaccharides (LHß114-dgCTP) (Fig. 1). The mutant LHß114 is a truncated LHß lacking the seven amino acids (115–121) at the carboxyl terminus [38]. The construction of {alpha}, LHß, LHß114, LHß114-CTP expression plasmids were previously described [37, 38] (Fig. 1). The LHß114-CTP contains the first 114 amino acids of LHß and the entire CTP sequence of CGß subunit [20]. The construction of the FSHß-CTP chimera was previously described [39].



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  		FIG. 1. Structure of wild-type CGß/LHß/FSHß subunits and LHß and FSHß mutants. The CGß, LHß, and FSHß subunits are designated by the closed, open, and grey boxes, respectively. The number of the amino acid residues defines the amino and carboxyl termini and the CTP sequence of CGß (amino acid residues 115–145; cross hatched box) with four O-linked oligosaccharides. LHß114 lacks the carboxy terminal region (residues 115–121). The chimeras LHß114-CTP and FSHß-CTP contain the first 114 amino acids of LHß and 111 amino acids of FSHß, respectively, and the CTP. The LHß114-dgCTP contains the first 114 amino acids of LHß, and the CTP, which lacks the O-linked oligosaccharides

The LHß114-dgCTP (Fig. 1) was constructed in several steps. First, we generated a polymerase chain reaction (PCR) fragment (PCR I) comprising LHß exon 1, exon 2, and a newly created SalI site in the intron of the LHß gene by using LHß114-CTP-{alpha} single chain as a template [40]. The resulting fragment was ligated into the SalI site of pM2HA. A second PCR (PCR II) was performed using FSHß-CTP-LHß114-CTP-{alpha} single chain as a template [41] to construct a fragment containing part of the intron bearing the SalI site and exon 3 devoid of the coding sequence encoding carboxyl-terminal heptapeptide (-SalI-LHß114 exon3-). A parallel reaction (PCR III) containing the gene encoding the O-deglycosylated CGß mutant (CGß-dgCTP) [42] generated a PCR fragment comprising the coding sequence of deglycosylated CTP sequence and SalI site (dgCTP-SalI). The overlapping PCR (PCR IV) was performed using PCR II and PCR III products generating a fragment containing -SalI-LHß114 exon 3-dgCTP-SalI. This product was ligated into pM2-HA-LHß exon 1, exon 2-SalI. The final product, LHß114-dgCTP, was sequenced to ensure no errors occurred during the PCR reactions.

Clones expressing individual subunits and dimers were selected approximately 12 days later with G418 (0.25 mg/ml) [37, 38]. Single colonies were isolated and maintained in culture medium containing 0.125 mg/ml G418. Several clones expressing the dimers FSH (N = 4), LH (N = 4), LH114 (N = 3), LH114-CTP (N = 10), LH114-dgCTP (N = 7), FSH-CTP (N = 6), or their corresponding subunits (N = 3–10 clones for each subunit) grown on culture dishes, were selected by metabolic labeling and immunoprecipitation of media using {alpha}-, CGß-, or FSHß-specific antisera (see below).

To determine the basolateral and apical distribution, cells were grown on Transwell filters, and after achieving confluence and forming a tight monolayer, the cells were metabolically labeled with 25 µCi/ml [35S]cysteine in DMEM/F-12 medium lacking cysteine and supplemented with 5% DFBS, L-glutamine, penicillin, and streptomycin [20, 31, 38]. Following overnight incubation, the media were immunoprecipitated with subunit-specific antiserum following Pansorbin treatment. Precipitation of the {alpha} subunit with CGß/FSHß antiserum indicates dimer formation. The reduced and boiled (4 min) proteins were analyzed by 15% SDS-PAGE gels [31, 37].

Expression of Data and Statistical Analysis

Labeled proteins from autoradiography were quantified by densitometry. Equal exposure times for the autoradiograms were used when comparing the results of protein secretion into the apical and basolateral compartments. The total combined secretion of protein into the medium of apical and basolateral compartments was taken as 100%. The apical and basolateral ratio for all dimers and subunits was determined from the relative percent of protein present in each compartment. For each subunit and dimer, several clones were analyzed. Each experiment was repeated at least three times and the results are expressed as mean (%) ± SEM; P < 0.05 was considered as significantly different.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Secretion of LH114 Dimer and the LHß114 Subunit

We previously demonstrated that LH is released from the basolateral side of MDCK cells while CG is secreted apically [31]. Although the LHß and CGß subunits share greater than 85% amino acid identity [1], a major sequence divergence occurs at their carboxyl termini: CGß subunit terminates with a 31-amino acid hydrophilic carboxyl terminal extension containing four O-linked glycosyl units [18–21] compared with a seven-amino acid hydrophobic stretch that terminates the LHß carboxyl terminus [17, 22] (Fig. 1). Given that the apical route of CG from MDCK cells is primarily due to the CTP sequence [31], we suspected that the LH basolateral preference is related, at least in part, to the heptapeptide on the LHß subunit. To examine this point, MDCK cells were cotransfected with the {alpha} subunit and either the wild-type LHß subunit or a truncated LHß mutant lacking the last seven codons corresponding to amino acids 115–121 (LHß114) (Figs. 1 and 2). In addition, cells were transfected in the absence of the {alpha} subunit. The cells were metabolically labeled and immunoprecipitated with CGß antiserum (Fig. 2A). Deletion of the seven amino acids significantly reduced (P < 0.05) the basolateral fraction of the LH114 dimer (Fig. 2, A and B) compared with wild-type LH dimer (n = 3 experiments and N = 4 clones) and LHß subunit (n = 5 experiments and N = 3 clones; Fig. 2, C and D); only 61% ± 1.4% of total secreted LH114 dimer (n = 9 experiments and N = 3 clones) was released basolaterally whereas 39% ± 1.4% was detected in the apical medium (Fig. 2B). When the uncombined LHß114 subunit was examined (n = 8 experiments and N = 4 clones), the pattern of secretion (Fig. 2B) resembled that seen for the corresponding dimer. These data indicate that the heptapeptide of LHß contributes to the basolateral secretion of LH dimer.



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  		FIG. 2. Secretion of LH114 dimer and the LHß114 subunit. A) MDCK cells expressing the truncated carboxyl-terminal mutants LH114 dimer (lanes 1 and 2) or LHß114 subunit (lanes 3 and 4) were grown on Transwell filters and metabolically labeled overnight with [35S] cysteine. Media from the apical (Ap) and basolateral (BL) compartments were immunoprecipitated with antiserum directed against the CGß subunit, which reacts equally with either the LHß or CGß subunits. Coprecipitation of the {alpha} subunit with the CGß antiserum indicates heterodimer formation. The proteins were analyzed by SDS-polyacrylamide gels and autoradiography. B) The relative percent secretion of LH114 dimer and LHß114 subunit into the apical (stippled bars) and basolateral (solid bars) compartments were determined by densitometry from autoradiographs (see Methods). The combined secretion of protein into the medium of apical and basolateral compartments was taken as 100%. The apical and basolateral ratios for dimer and subunits were determined from the percent of protein present in each compartment. Results are shown as the mean ± SEM (%). Asterisks indicate significant difference, P < 0.05. C) The basolateral (BL)/apical (Ap) distribution in media collected from MDCK cells expressing wild-type LH dimer (lanes 5 and 6) and LHß subunit (lanes 7 and 8). Media from the apical and basolateral compartments were analyzed as described in A. D) The relative percent secretion of wild-type LH dimer and LHß subunit were determined as described in B. Results are shown as the mean ± SEM (%). Asterisks indicate significant difference, P < 0.05

Secretion of LH114-CTP Dimer and the LHß114-CTP Subunit

The above data, together with our previous studies, show that the carboxy-terminal regions of the LHß and CGß subunits influence polarized secretion of subunits and their dimers from pituitary and placenta, respectively. That the carboxyl ends of the CGß subunit contain a determinant for the apical secretion predicts that an LHß chimera bearing the CTP should be preferentially released apically. To test this, the heptapeptide of LHß was replaced with the CTP domain (LHß114-CTP) (Fig. 1). The total secretion of LH114-CTP dimer (n = 6 experiments and N = 10 clones) and the uncombined LHß114-CTP subunit (n = 5 experiments and N = 10 clones) (Fig. 3A) was comparable with the secretion of LH114 dimer/LHß114 (Fig. 2), indicating that the CTP has a minimal effect on the overall secretion of LHß114. In contrast with LH114 dimer and LHß114, densitometric analyses of labeled proteins (Fig. 3B) show that secretion of LH114-CTP dimer or uncombined LHß114-CTP subunit displayed an apical preference (63% ± 1.1% and 66% ± 1.1%, respectively, P < 0.05) similar to that observed for CG (73% ± 2.5%; n = 3 experiments and N = 3 clones) (see also Jablonka-Shariff et al. [31]). Thus, the CTP redirected LH114 dimer/LHß114 subunit to the apical compartment. These results further confirm that the carboxy termini of LHß and CGß subunits play a role in the basolateral and apical secretion patterns, respectively, of the corresponding dimers.



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  		FIG. 3. Secretion of LH114-CTP dimer and the LHß114-CTP subunit. A) MDCK cells expressing LH114-CTP dimer (lanes 1 and 2) or LHß114-CTP subunit (lanes 3 and 4) were grown on Transwell filters and metabolically labeled overnight with [35S] cysteine. The media from the apical (Ap) and basolateral (BL) compartments were immunoprecipitated with CGß antiserum and analyzed as described in Figure 2. B) The relative percent secretion of LH114-CTP dimer and LHß114-CTP subunit into the apical (stippled bars) and basolateral (solid bars) compartments were quantitated by densitometry as described in Figure 2. Results are shown as the mean ± SEM (%). Asterisks indicate significant difference, P < 0.05.

The shift from basolateral to apical preference when the CTP is added to the LHß114 might be attributed to a nonspecific effect of adding peptide to the carboxyl end of LHß114. Because we showed previously that the apical activity of the CTP in CG dimer was due to the O-linked oligosaccharides [31], we created a chimeric LHß114-dgCTP subunit devoid of the O-linked oligosaccharides (Figs. 1 and 4). In this case, synthesis of uncombined LHß114-dgCTP (Fig. 4) displayed a basolateral preference (63% ± 1.4%; n = 4 experiments and N = 6 clones), similar to that observed for LHß114 subunit (Fig. 2). The polarity of LH114 dimer containing dgCTP (Fig. 4) was only slightly affected; apical = 45% ± 1.3% vs. basolateral 55% ± 1.3% (n = 4 experiments and N = 7 clones). These results suggest that merely substituting the carboxyl end does not dramatically alter polarity of the LH114 dimer to the apical compartment.



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  		FIG. 4. Secretion of LH114-dgCTP dimer and the LHß114-dgCTP subunit. A) Cells expressing LH114-dgCTP dimer (lanes 1 and 2) or LHß114-dgCTP subunit (lanes 3 and 4) devoid of the O-linked oligosaccharides were grown on Transwell filters and metabolically labeled overnight with [35S] cysteine. Media from the apical (Ap) and basolateral (BL) compartments were immunoprecipitated with CGß or {alpha} antiserum. In the dimer, LHß114-dgCTP subunit comigrates with the {alpha} subunit on SDS gels. That the LHß114-dgCTP combines with the {alpha} subunits was confirmed by nondenatured Western blots using antisera against {alpha} and CGß subunits. B) The relative percent secretion of LH114-dgCTP dimers and LHß114-dgCTP subunit into the apical (stippled bar) or basolateral (solid bar) compartments was quantitated by densitometry as described in Figure 2. Results are shown as the mean ± SEM (%). Asterisk indicates significant difference, P < 0.05.

Across several animal species, both FSH and LH are synthesized in the same cell, i.e., the gonadotroph [3, 5–7, 43, 44]. However, it has been reported that FSH and LH reside in separate populations of gonadotrophs in bovine and chicken pituitary [44, 45]. Although FSH and LH are released into the circulation, the physiological cues regulating their release are not the same. Given the differences in the sequences between the LHß/CGß and FSH subunits, we examined the partition of FSH synthesized in the MDCK cells (Fig. 5A, lanes 1 and 2). It is evident that, in contrast with the polarized secretion behavior observed for the LH/CG dimers, the FSH dimer displayed no apparent polarized secretion (Fig. 5B), i.e., the dimer was observed equally in both compartments. This lack of polarity was also observed for the FSHß subunit (data not shown). Curiously, addition of CTP to the carboxyl terminus of the FSHß subunit did not significantly alter the polarity of the resulting heterodimer (Fig. 5A, lanes 3 and 4). Thus, despite the CTP in CG and LH, addition of the sequence to FSH did not significantly alter its polarity (Fig. 5B).



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  		FIG. 5. Secretion of FSH and FSH-CTP dimers. A) Cells expressing FSH dimer (lanes 1 and 2) or FSH-CTP dimer (lanes 3 and 4) were grown on Transwell filters and metabolically labeled overnight with [35S] cysteine. Media from the apical (Ap) and basolateral (BL) compartments were immunoprecipitated with FSHß or {alpha} antiserum. In the FSH dimer lanes (1 and 2), FSHß subunit comigrates with the {alpha} subunit on SDS gels (arrowhead). FSH-CTP dimer (lanes 3 and 4) migrates as two bands due to addition of the CTP and its O-linked oligosaccharides. B) The relative percent secretion of FSH and FSH-CTP dimers into the apical (stippled bars) and basolateral (solid bars) compartments were quantitated by densitometry as described in Figure 2. Results are shown as the mean ± SEM (%)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The LHß and CGß subunits are the most closely related ß subunits in the glycoprotein hormone family, having evolved from the same ancestral gene [17], and share more than 85% sequence identity. Moreover, LH and CG bind to the same receptor and elicit the same biological response [1, 16, 17]. A major difference between them is their carboxy terminal region. CGß bears a 31-amino acid hydrophilic terminal peptide that contains four O-glycans while LHß has a hydrophobic heptapeptide. Here we show that the heptapeptide in the LHß subunit encodes a basolateral determinant. There are several reports showing that cytoplasmic domains in polarized membrane proteins bear motifs consisting of a tyrosine residue near at least one hydrophobic amino acid, or a sequence bearing a leucine/leucine or a leucine/isoleucine pair, which are involved in their basolateral sorting [30, 46–49]. Although the heptapeptide would face the endoplasmic reticulum lumen—not the cytoplasm—it is intriguing that this carboxyl-terminal sequence in the LHß subunit also contains a dileucine motif (-leu-ser-gly-leu-leu-phe-leu).

Despite the decrease in the extent of basolateral release of the LHß114, it still exhibited preferential basolateral sorting. This implies that the heptapeptide acts in concert with other determinant(s) in the ß subunit or is one of a series of interdependent basolateral sorting signals. Given that the polarities of other proteins released from epithelial cells are influenced by the asparagine-linked oligosaccharides [50–52], a potential determinant that could contribute to the basolateral sorting is the N-linked oligosaccharide at asparagine 30 in the LHß subunit.

In a somewhat related issue, the LHß and CGß carboxyl termini also influence their assembly with the {alpha} subunit and secretion [20, 38]. Previous transfection studies in Chinese hamster ovary cells revealed that the CGß subunit is secreted as a monomer and combines efficiently with the {alpha} subunit, whereas secretion and assembly of the LHß subunit is much less efficient [38, 53]. It was shown by mutagenesis studies that the LHß heptapeptide and several other amino acids in the LHß subunit were implicated in this behavior [20, 38]. Thus, the carboxyl terminus of the LHß subunit is a hot spot for not only the intracellular pathways of assembly and secretion but also for polarized trafficking.

We previously demonstrated that CG is preferentially secreted apically from MDCK cells [31]. Deleting the CTP from CGß subunit redirected apical secretion of CG to the basolateral surface, similar to that observed for wild-type LH dimer, indicating that the CTP sequence is a major routing signal for the apical secretion of CG [31]. Consistent with these results is the current observation that the addition of the CTP to the LHß subunit promoted release of the chimera to the apical compartment. It is unclear, however, why the FSH-CTP analogue dimer displayed little apical sorting. The absence of significant polarity may preclude entrance in the apical/basolateral sorting pathway. Thus, such proteins like FSH would be refractory to the absence or presence of sorting signals. In the case of CG, the CTP serves as an apical determinant essential for directing steadily increasing levels of hormone in the maternal uterine blood during the first trimester of pregnancy. Trophoblasts are polarized epithelia, forming tight junctions and desmosomes [54, 55]. Although pituitary gonadotrophs are epithelial and exocytosis empties into a basal laminar blood supply, there is no apparent apical domain facing a luminal cavity; rather, the gonadotrophs abut against each other. Nevertheless, there are some intriguing observations that imply that the LH/FSH secretion behavior in MDCK cells reflects their intracellular disposition in vivo. As discussed earlier, studies in the sheep pituitary showed that LH-containing secretory granules are polarized near the vascular bed during the preovulatory surge, while FSH is more uniformly distributed throughout the cytoplasm [5, 6]. Secretory and membrane proteins exit the endoplasmic reticulum and are transported to the trans-Golgi network. At this site in polarized cells, proteins are sorted and routed to their respective basolateral or apical domains [29, 30]. That at the preovulatory surge a significant fraction of gonadotrophs polarize due to a redistribution of LH-containing granules [6] implies that the disposition of LH and FSH into the circulation occurs by different pathways/mechanisms. This is consistent with the data reported here, in which LH and FSH exhibit unique secretion patterns from the MDCK cells, which may reflect an FSH exit through central blood vessels in the pituitary, whereas LH exocytosis occurs through polarized granules adjacent to the capillaries in the basal laminar region of the gonadotroph.


   ACKNOWLEDGMENTS
 
We thank Ms. L. Lobos and Ms. D. Redmond for their assistance in the preparation of this manuscript. We are also grateful to Vicenta Garcia-Campayo for her comments regarding the manuscript.


   FOOTNOTES
 
1 Correspondence: FAX: 314 361 3560; iboime{at}molecool.wustl.edu Back

Received: 27 August 2003.

First decision: 16 September 2003.

Accepted: 21 October 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pierce JG, Parsons TF. Glycoprotein hormones: structure and function. Annu Rev Biochem 1981 50:465-495[CrossRef][Medline]
  2. Lloyd JM, Childs GV. Differential storage and release of luteinizing hormone and follicle-releasing hormone from individual gonadotropes separated by centrifugal elutriation. Endocrinology 1988 122:1282-1290[Abstract]
  3. Taragnat C, Bernier A, Fontaine J. Gonadotrophin storage patterns in the ewe during the oestrous cycle or after long-term treatment with a GnRH agonist. J Endocrinol 1998 156:149-157[Abstract]
  4. Conn PM, McArdle CA, Andrews WV, Huckle WR. The molecular basis of gonadotropin-releasing hormone (GnRH) action in the pituitary gonadotrope. Biol Reprod 1987 36:17-35[CrossRef][Medline]
  5. Currie RJ, McNeilly AS. Mobilization of LH secretory granules in gonadotrophs in relation to gene expression, synthesis and secretion of LH during the preovulatory phase of the sheep oestrous cycle. J Endocrinol 1995 147:259-270[Abstract]
  6. Thomas SG, Clarke IJ. The positive feedback action of estrogen mobilizes LH-containing, but not FSH-containing secretory granules in ovine gonadotropes. Endocrinology 1997 138:1347-1350[Abstract/Free Full Text]
  7. Crawford JL, Currie RJ, McNeilly AS. Replenishment of LH stores of gonadotrophs in relation to gene expression, synthesis and secretion of LH after the preovulatory phase of the sheep oestrous cycle. J Endocrinol 2000 167:453-463[Abstract]
  8. Luderer U, Schwartz NB. An overview of FSH regulation and action. In: Serono Symposium on FSH: Regulation of Secretion and Molecular Mechanisms of Action. 1992;1–25
  9. Bielinska M, Rzymkiewicz D, Boime I. Human luteinizing hormone and chorionic gonadotropin are targeted to a regulated secretory pathway in GH3 cells. Mol Endocrinol 1994 8:919-928[Abstract]
  10. Muyan M, Rzymkiewicz DM, Boime I. Secretion of lutropin and follitropin from transfected GH3 cells: evidence for separate secretory pathways. Mol Endocrinol 1994 8:1789-1797[Abstract]
  11. Thiede HA, Choate JW. Chorionic gonadotropin localization in the human by immunofluorescent staining. II. Demonstration of HSC in trophoblast and amnion epithelium of immature and mature placentas. Obstet Gynecol 1963 22:433-443[Free Full Text]
  12. Boyd JD, Hamilton WJ. The Human Placenta. Cambridge, England: Heffer and Sons, Ltd.; 1970
  13. Wynn RM. Cytotrophoblastic specializations: an ultrastructural study of the human placenta. Am J Obstet Gynecol 1972 114:339-355[Medline]
  14. Suemizu H, Osamura Y, Watanabe K. Ultrastructural localization of hCG and subunits in the human placenta and choriocarcinoma cell lines—morphological approach to secretory pathway. Acta Histochem Cytochem 1988 21:265-271
  15. Fiddes JC, Talmadge K. Structure, expression, and evolution of the genes for the human glycoprotein hormones. Recent Prog Horm Res 1984 40:43-78[Medline]
  16. Shome B, Parlow AF. The primary structure of the hormone-specific, beta subunit of human pituitary luteinizing hormone (hLH). J Clin Endocrinol Metab 1973 36:618-621[Medline]
  17. Talmadge K, Vamvakopoulos NC, Fiddes JC. Evolution of the genes for the beta subunits of human chorionic gonadotropin and luteinizing hormone. Nature 1984 307:37-40[CrossRef][Medline]
  18. Birken S, Canfield RE. Isolation and amino acid sequence of COOH-terminal fragments from the beta subunit of human choriogonadotropin. J Biol Chem 1977 252:5386-5392[Abstract/Free Full Text]
  19. Matzuk MM, Hsueh AJ, Lapolt P, Tsafriri A, Keene JL, Boime I. The biological role of the carboxyl-terminal extension of human chorionic gonadotropin beta-subunit. Endocrinology 1990 126:376-383[Abstract]
  20. Muyan M, Furuhashi M, Sugahara T, Boime I. The carboxy-terminal region of the beta-subunits of luteinizing hormone and chorionic gonadotropin differentially influence secretion and assembly of the heterodimers. Mol Endocrinol 1996 10:1678-1687[Abstract]
  21. Muyan M, Boime I. The carboxyl-terminal region is a determinant for the intracellular behavior of the chorionic gonadotropin beta subunit: effects on the processing of the Asn-linked oligosaccharides. Mol Endocrinol 1998 12:766-772[Abstract/Free Full Text]
  22. Catt KJ, Pierce JG. Gonadotropin hormones of the adenohypophysis. In: Yean SSC, Jaffe RB (eds.), Reproductive Endocrinology. Philadelphia: WB Saunders Co.; 1986:75–114
  23. Fox KM, Dias JA, Van Roey P. Three-dimensional structure of human follicle-stimulating hormone. Mol Endocrinol 2001 15:378-389[Abstract/Free Full Text]
  24. Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, Morgan FJ, Isaacs NW. Crystal structure of human chorionic gonadotropin. Nature 1994 369:455-461[CrossRef][Medline]
  25. Le Gall AH, Yeaman C, Muesch A, Rodriguez-Boulan E. Epithelial cell polarity: new perspectives. Semin Nephrol 1995 15:272-284[Medline]
  26. Yeaman C, Grindstaff KK, Nelson WJ. New perspectives on mechanisms involved in generating epithelial cell polarity. Physiol Rev 1999 79:73-98[Abstract/Free Full Text]
  27. Rodriguez-Boulan E, Paskiet KT, Salas PJ, Bard E. Intracellular transport of influenza virus hemagglutinin to the apical surface of Madin-Darby canine kidney cells. J Cell Biol 1984 98:308-319[Abstract/Free Full Text]
  28. Boll W, Partin JS, Katz AI, Caplan MJ, Jamieson JD. Distinct pathways for basolateral targeting of membrane and secretory proteins in polarized epithelial cells. Proc Natl Acad Sci U S A 1991 88:8592-8596[Abstract/Free Full Text]
  29. Keller P, Simons K. Post-Golgi biosynthetic trafficking. J Cell Sci 1997 110:pt 243001-3009[Abstract]
  30. Matter K, Mellman I. Mechanisms of cell polarity: sorting and transport in epithelial cells. Curr Opin Cell Biol 1994 6:545-554[CrossRef][Medline]
  31. Jablonka-Shariff A, Garcia-Campayo V, Boime I. Evolution of lutropin to chorionic gonadotropin generates a specific routing signal for apical release in vivo. J Biol Chem 2002 277:879-882[Abstract/Free Full Text]
  32. Thorneycroft IH, Mishell DR Jr, Stone SC, Kharma KM, Nakamura RM. The relation of serum 17-hydroxyprogesterone and estradiol-17-beta levels during the human menstrual cycle. Am J Obstet Gynecol 1971 111:947-951[Medline]
  33. Shupnik MA. Gonadotropin gene modulation by steroids and gonadotropin-releasing hormone. Biol Reprod 1996 54:279-286[Abstract]
  34. Chappel SC, Howles C. Reevaluation of the roles of luteinizing hormone and follicle-stimulating hormone in the ovulatory process. Hum Reprod 1991 6:1206-1212[Abstract/Free Full Text]
  35. Childs GV, Unabia G, Miller BT. Cytochemical detection of gonadotropin-releasing hormone-binding sites on rat pituitary cells with luteinizing hormone, follicle-stimulating hormone, and growth hormone antigens during diestrous up-regulation. Endocrinology 1994 134:1943-1951[Abstract]
  36. Simons K, Virta H. Growing Madin-Darby canine kidney cells for studying epithelial cell biology. In: Celis JE (ed.), Cell Biology: A Laboratory Handbook. New York: Academic Press; 1998:101–106
  37. Matzuk MM, Krieger M, Corless CL, Boime I. Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells. Proc Natl Acad Sci U S A 1987 84:6354-6358[Abstract/Free Full Text]
  38. Matzuk MM, Spangler MM, Camel M, Suganuma N, Boime I. Mutagenesis and chimeric genes define determinants in the beta subunits of human chorionic gonadotropin and lutropin for secretion and assembly. J Cell Biol 1989 109:1429-1438[Abstract/Free Full Text]
  39. Fares FA, Suganuma N, Nishimori K, LaPolt PS, Hsueh AJ, Boime I. Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit. Proc Natl Acad Sci U S A 1992 89:4304-4308[Abstract/Free Full Text]
  40. Garcia-Campayo V, Sato A, Hirsch B, Sugahara T, Muyan M, Hsueh AJ, Boime I. Design of stable biologically active recombinant lutropin analogs. Nat Biotechnol 1997 15:663-667[CrossRef][Medline]
  41. Garcia-Campayo V, Boime I. Independent activities of FSH and LH structurally confined in a single polypeptide: selective modification of the relative potencies of the hormones. Endocrinology 2001 142:5203-5211[Abstract/Free Full Text]
  42. Garcia-Campayo V, Sugahara T, Boime I. Unmasking a new recognition signal for O-linked glycosylation in the chorionic gonadotropin beta subunit. Mol Cell Endocrinol 2002 194:63-70[CrossRef][Medline]
  43. Dacheux F. Subcellular localization of gonadotropic hormones in pituitary cells of the castrated pig with the use of pre- and postembedding immunocytochemical methods. Cell Tissue Res 1984 236:153-160[CrossRef][Medline]
  44. Proudman JA, Vandesande F, Berghman LR. Immunohistochemical evidence that follicle-stimulating hormone and luteinizing hormone reside in separate cells in the chicken pituitary. Biol Reprod 1999 60:1324-1328[Abstract/Free Full Text]
  45. Bastings E, Beckers A, Reznik M, Beckers JF. Immunocytochemical evidence for production of luteinizing hormone and follicle-stimulating hormone in separate cells in the bovine. Biol Reprod 1991 45:788-796[Abstract]
  46. Hunziker W, Fumey C. A di-leucine motif mediates endocytosis and basolateral sorting of macrophage IgG Fc receptors in MDCK cells. Embo J 1994 13:2963-2967[Medline]
  47. Gabilondo AM, Hegler J, Krasel C, Boivin-Jahns V, Hein L, Lohse MJ. A dileucine motif in the C terminus of the beta2-adrenergic receptor is involved in receptor internalization. Proc Natl Acad Sci U S A 1997 94:12285-12290[Abstract/Free Full Text]
  48. Sandoval IV, Martinez-Arca S, Valdueza J, Palacios S, Holman GD. Distinct reading of different structural determinants modulates the dileucine-mediated transport steps of the lysosomal membrane protein LIMPII and the insulin-sensitive glucose transporter GLUT4. J Biol Chem 2000 275:39874-39885[Abstract/Free Full Text]
  49. Gu HH, Wu X, Giros B, Caron MG, Caplan MJ, Rudnick G. The NH2-terminus of norepinephrine transporter contains a basolateral localization signal for epithelial cells. Mol Biol Cell 2001 12:3797-3807[Abstract/Free Full Text]
  50. Kitagawa Y, Sano Y, Ueda M, Higashio K, Narita H, Okano M, Matsumoto S, Sasaki R. N-glycosylation of erythropoietin is critical for apical secretion by Madin-Darby canine kidney cells. Exp Cell Res 1994 213:449-457[CrossRef][Medline]
  51. Scheiffele P, Peranen J, Simons K. N-glycans as apical sorting signals in epithelial cells. Nature 1995 378:96-98[CrossRef][Medline]
  52. Rodriguez-Boulan E, Gonzalez A. Glycans in postGolgi apical targeting: sorting signals or structural props?. Trends Cell Biol 1999 9:291-294[CrossRef][Medline]
  53. Corless CL, Matzuk MM, Ramabhadran TV, Krichevsky A, Boime I. Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells. J Cell Biol 1987 104:1173-1181[Abstract/Free Full Text]
  54. Ramsey E. The Placenta. New York: Praeger Publishing; 1982
  55. Daniels-McQueen S, Krichevsky A, Boime I. Isolation and characterization of human cytotrophoblast cells. In: Miller RK (ed.), Trophoblast Research, vol. 2. New York: Plenum Press; 1987:423.



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S. Nakav, A. Jablonka-Shariff, S. Kaner, P. Chadna-Mohanty, H. E. Grotjan, and D. Ben-Menahem
The LH{beta} Gene of Several Mammals Embeds a Carboxyl-terminal Peptide-like Sequence Revealing a Critical Role for Mucin Oligosaccharides in the Evolution of Lutropin to Chorionic Gonadotropin in the Animal Phyla
J. Biol. Chem., April 29, 2005; 280(17): 16676 - 16684.
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