Basolateral Localization and Transcytosis of Gonadotropin and Thyrotropin Receptors Expressed in Madin-Darby Canine Kidney Cells

Abstract

؊ and cholera toxin. G s activation provoked a similar effect on LH receptor distribution in MDCK cells, whereas it did not modify the compartmentalization of the TSH receptor. Hormone-specific transcytosis was observed in MDCK cells expressing the gonadotropin (FSH and LH) receptors and was increased after cholera toxin administration.The gonadotropin (LH 1 and FSH) and thyrotropin (TSH) receptors belong to the large family of G-protein-coupled receptors (1-4). They possess the distinctive seven transmembrane spanning domains. However, they form a specific subgroup characterized by the presence of a large extracellular domain constituted by the repetition of leucine-rich motif (5). This ectodomain is responsible for the high affinity hormone binding (6 -9). Gonadotropin and TSH receptors are mainly coupled to G s and thus activate adenylate cyclase. The same receptors are also able to activate phospholipase C at high hormone concentrations. Mutations of the receptors have been described leading either to their constitutive activation (10 -12) or in contrast to a loss of receptor function (13)(14)(15).Little is known about the cellular trafficking of G-proteincoupled receptors in general and of this subgroup of receptors in particular. Ultrastructural immunocytochemistry has been used to analyze the internalization mechanisms of some receptors (16 -18). LH receptor-driven hormone transcytosis was observed through endothelial cells of testicular blood vessels (19). Recently immunocytochemistry using specific monoclonal antibodies demonstrated the basolateral distribution of the TSH receptor in thyroid cells (20) and of the FSH receptor in Sertoli cells (21), whereas the LH receptor was present all over the cell surface of thecal, granulosa, and luteal cells in the ovary and Leydig cells in the testes (22,23).The question was thus raised as to whether these differences in cellular distribution were due to differences in the structure of receptors or simply secondary to the fact that thyroid follicular cells and Sertoli cells are polarized, whereas the LH receptor-expressing cells are not polarized. Another question raised was whether the mechanisms of receptor basolateral delivery were cell-specific or whether the receptors contained specific signals that could direct them to this membrane compartment in any polarized cell. To answer these questions we have established MDCK cell lines that express the FSH, LH, and TSH receptors. Using these models we have analyzed the distribution of the receptors in polarized monolayers. MATERIALS AND METHODSCell Culture-MDCK cells (type II) were seeded and grown on coverslips (Nunc) or filters (0.4-m polycarbonate, tissue culture-treated, Transwell costar) as described previously (24,25).Antireceptor Antibodies-Mouse monoclonal antibodies FSHR323 (21), LHR38 (26),27) recognize an epitope in the extracellular domain of the FSH, LH, and TSH receptors, respectively. Antibody T3-365 has been raised against the intracellular domain of the TSH receptor (20). The rabbit polyclonal TSHR...

The Basolateral Localization Signal of the Follicle-stimulating Hormone Receptor

“…After washing, the cells were mounted with fluorescent Montaing medium (DAKO) and observed with a Zeiss microscope (Axiovert 135M) in conjunction with a confocal laser scanning unit (Zeiss LSM 410). To open the tight junctions, MDCK cells were incubated for 1 h with Ca 2ϩ -free Dulbecco's modified Eagle's medium, washed twice with calcium and magnesium-free PBS, and then incubated with 10 mM EGTA in the same buffer (13). The cells were then fixed in 3% paraformaldehyde for 15 min and further processed as described above.…”

“…Receptors present on the cell surface were immunoprecipitated as described previously (13). Briefly, cells were pulse-labeled for 1 h with 1 mCi/ml Express 35 S (NEN Life Science Products) and chased for 3 h in the same medium containing unlabeled amino acids and 1% BSA.…”

“…Immunofluorescence and Confocal Microscopy-MDCK cells were grown to confluence on coverslips as described (13). The cells were washed with PBS ϩ (phosphate-buffered saline (Dulbecco's formulation) with 0.1 mM CaCl 2 and 1 mM MgCl 2 ) and fixed for 15 min in 3% paraformaldehyde in PBS ϩ .…”

“…Cell Culture and Expression of FSHR Mutants-MDCK cells (type II) were seeded and grown on coverslips (Nunc) or filters (0.4-m polycarbonate Transwell Costar Corp., Cambridge, MA) as described previously (13). The cells were cotransfected with expression vectors encoding either the wild-type or the mutated FSHR and with the plasmid pSV-neo, which confers resistance to the antibiotic G418.…”

“…This difference in receptor distribution could result from either a difference in the structure of the receptors or from differential expression in polarized or nonpolarized cells. To distinguish between these possibilities, we have transfected the three receptors into polarized MDCK cells (13). We observed that all three receptors were directly delivered to the basolateral membrane of these cells, suggesting that they all contain a basolateral targeting signal.…”