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• W2022253215 abstract "Although synthesized in the same pituitary gonadotropes, the secretion profiles of lutropin (LH) and follitropin (FSH) differ. LH is secreted through a regulated pathway and associated with a bolus release at mid-estrous cycle. In contrast, the majority of FSH is secreted constitutively with an incremental increase until ovulation. Both share an identicalα subunit, and thus theβ subunit contains determinants for sorting into the regulated pathway. Previously, we demonstrated that a hydrophobic carboxyl-terminal heptapeptide of the LHβ subunit (Leu-Ser-Gly-Leu-Leu-Phe-Leu), not found in the FSHβ subunit, influences the intracellular behavior of the LH dimer. To test the hypothesis that the peptide contributes to differential sorting, we monitored the fates of LH and LHΔT (LHβ subunit lacking the carboxyl-terminal seven amino acids) dimers in the rat somatotrope-derived GH3 cell line in which both the regulated and constitutive secretory pathways operate. Pulse-chase labeling demonstrated that the LHΔT dimer was diverted to the constitutive pathway, resulting in a significant decrease in the corresponding intracellular pool. Forskolin stimulated LH dimer release 3-fold, which was accompanied by a parallel decrease of intracellular LH; only marginal forskolin stimulation of LHΔT was seen. Immunofluorescence after cycloheximide treatment demonstrated decreased retention of LHΔT compared with LH, consistent with increased constitutive secretion of LHΔT. We also demonstrated that fusing the heptapeptide to the carboxyl terminus of the FSHβ subunit resulted in an increased regulated secretion of this FSH analog compared with wild-type FSH. These data are the first to identify a novel structural determinant responsible for the sorting of a member of the glycoprotein hormone family into the regulated secretory pathway. Although synthesized in the same pituitary gonadotropes, the secretion profiles of lutropin (LH) and follitropin (FSH) differ. LH is secreted through a regulated pathway and associated with a bolus release at mid-estrous cycle. In contrast, the majority of FSH is secreted constitutively with an incremental increase until ovulation. Both share an identicalα subunit, and thus theβ subunit contains determinants for sorting into the regulated pathway. Previously, we demonstrated that a hydrophobic carboxyl-terminal heptapeptide of the LHβ subunit (Leu-Ser-Gly-Leu-Leu-Phe-Leu), not found in the FSHβ subunit, influences the intracellular behavior of the LH dimer. To test the hypothesis that the peptide contributes to differential sorting, we monitored the fates of LH and LHΔT (LHβ subunit lacking the carboxyl-terminal seven amino acids) dimers in the rat somatotrope-derived GH3 cell line in which both the regulated and constitutive secretory pathways operate. Pulse-chase labeling demonstrated that the LHΔT dimer was diverted to the constitutive pathway, resulting in a significant decrease in the corresponding intracellular pool. Forskolin stimulated LH dimer release 3-fold, which was accompanied by a parallel decrease of intracellular LH; only marginal forskolin stimulation of LHΔT was seen. Immunofluorescence after cycloheximide treatment demonstrated decreased retention of LHΔT compared with LH, consistent with increased constitutive secretion of LHΔT. We also demonstrated that fusing the heptapeptide to the carboxyl terminus of the FSHβ subunit resulted in an increased regulated secretion of this FSH analog compared with wild-type FSH. These data are the first to identify a novel structural determinant responsible for the sorting of a member of the glycoprotein hormone family into the regulated secretory pathway. Lutropin (LH) 4The abbreviations used are: LH, lutropin (luteinizing hormone); FSH, follitropin (follicle-stimulating hormone); CG, chorionic gonadotropin; CHX, cycloheximide; ER, endoplasmic reticulum; CHO, Chinese hamster ovary. and follitropin (FSH) are synthesized and secreted by pituitary gonadotropes and are members of the glycoprotein hormone family, which also includes thyrotropin (TSH) and the placental hormone chorionic gonadotropin (CG). They are heterodimers that share a common α subunit but differ in their hormone-specific β subunits (1Pierce J.G. Parsons T.F. Annu. Rev. Biochem. 1981; 50: 465-495Crossref PubMed Scopus (1911) Google Scholar, 2Sairam, M. R. (1983) in Hormonal Proteins and Peptides: Gonadotropic Hormones (Li, C. H., ed) pp. 1-79, Academic Press, New YorkGoogle Scholar). Both subunits are glycosylated, containing asparagine (N)-linked oligosaccharides (3Green E.D. Boime I. Baenziger J.U. Mol. Cell. Biochem. 1986; 72: 81-100Crossref PubMed Scopus (57) Google Scholar, 4Kessler M.J. Mise T. Ghai R.D. Bahl O.P. J. Biol. Chem. 1979; 254: 7909-7914Abstract Full Text PDF PubMed Google Scholar). The mature carbohydrate structures are hormone-specific in that the terminal oligosaccharide is sulfate for LH (3Green E.D. Boime I. Baenziger J.U. Mol. Cell. Biochem. 1986; 72: 81-100Crossref PubMed Scopus (57) Google Scholar, 5Parsons T.F. Pierce J.G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7089-7093Crossref PubMed Scopus (90) Google Scholar), whereas FSH contains sialic acid (3Green E.D. Boime I. Baenziger J.U. Mol. Cell. Biochem. 1986; 72: 81-100Crossref PubMed Scopus (57) Google Scholar). LH and FSH play key roles in regulating reproductive function. In females, FSH stimulates follicular growth, maintaining a steady concentration during the early follicular stage, and is required to facilitate selection of follicles to the preovulatory phase. At this time, low levels of LH stimulate steroidogenesis in thecal cells by enhancing androgen synthesis, which in turn is converted to estradiol in the presence of FSH. The gradual increase in estradiol is essential to initiate the LH surge. The reciprocal relationship between FSH and estrogen concentrations during the follicular phase of the menstrual cycle is an exquisitely sensitive feedback pathway that governs the selection of the preovulatory follicle (6Luderer, U., and Schwartz, N. B. (1992) in Serono Symposium on FSH: Regulation of Secretion and Molecular Mechanisms of Action (Hunzicker-Dunn, M., and Schwartz, N. B., eds) pp. 1-25, Springer-Verlag, New YorkGoogle Scholar, 7Farnworth P.G. J. Endocrinol. 1995; 145: 387-395Crossref PubMed Scopus (100) Google Scholar). The modes of secretion for LH and FSH are linked to their function; LH is released in pulses via a regulated pathway (i.e. LH is stored in secretory granules) and associated with a bolus release at midcycle to rupture the follicle and form the corpus luteum (6Luderer, U., and Schwartz, N. B. (1992) in Serono Symposium on FSH: Regulation of Secretion and Molecular Mechanisms of Action (Hunzicker-Dunn, M., and Schwartz, N. B., eds) pp. 1-25, Springer-Verlag, New YorkGoogle Scholar, 7Farnworth P.G. J. Endocrinol. 1995; 145: 387-395Crossref PubMed Scopus (100) Google Scholar, 8Currie R.J. McNeilly A.S. J. Endocrinol. 1995; 147: 259-270Crossref PubMed Scopus (41) Google Scholar, 9Thomas S.G. Clarke I.J. Endocrinology. 1997; 138: 1347-1350Crossref PubMed Scopus (48) Google Scholar). By contrast, FSH is primarily constitutively secreted, tightly coupled to its synthesis rate with gradual incremental increases until ovulation (9Thomas S.G. Clarke I.J. Endocrinology. 1997; 138: 1347-1350Crossref PubMed Scopus (48) Google Scholar, 10Baenziger J.U. Kumar S. Brodbeck R.M. Smith P.L. Beranek M.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 334-338Crossref PubMed Scopus (160) Google Scholar). The N-linked oligosaccharides play a critical role in the extracellular stability of both hormones. Sulfated oligosaccharides lead to a rapid clearance of LH in vivo, regulating its pulsatile release (3Green E.D. Boime I. Baenziger J.U. Mol. Cell. Biochem. 1986; 72: 81-100Crossref PubMed Scopus (57) Google Scholar, 10Baenziger J.U. Kumar S. Brodbeck R.M. Smith P.L. Beranek M.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 334-338Crossref PubMed Scopus (160) Google Scholar, 11Baenziger J.U. Faseb. J. 1994; 8: 1019-1025Crossref PubMed Scopus (66) Google Scholar). Sialylation results in greater extracellular longevity of FSH as compared with LH. Although the structural motifs that govern the differential sorting of LH and FSH have yet to be identified, critical structural cues exist that might account for these intracellular events. LH and FSH are synthesized in the same cell and share an identical α subunit, and thus the β subunit must represent the key determinant for the specificity of carbohydrate processing and for the intracellular segregation of one or both hormones. Of all the human glycoprotein hormone β subunits, the LHβ subunit is the most hydrophobic, particularly in the region between residues 75 and 121 (12Talmadge K. Vamvakopoulos N.C. Fiddes J.C. Nature. 1984; 307: 37-40Crossref PubMed Scopus (274) Google Scholar, 13Matzuk M.M. Spangler M.M. Camel M. Suganuma N. Boime I. J. Cell Biol. 1989; 109: 1429-1438Crossref PubMed Scopus (50) Google Scholar). DNA sequences for the LHβ (12Talmadge K. Vamvakopoulos N.C. Fiddes J.C. Nature. 1984; 307: 37-40Crossref PubMed Scopus (274) Google Scholar) and thyrotropin β (14Hayashizaki Y. Miyai K. Kato K. Matsubara K. FEBS Lett. 1985; 188: 394-400Crossref PubMed Scopus (80) Google Scholar) subunits encode hydrophobic stretches of seven and six amino acids, respectively, at their carboxyl termini, but a similar heptapeptide is not observed at the carboxyl terminus of the FSHβ subunit. This difference points to the carboxyl end of the LHβ subunit as a potential candidate for a sorting determinant. Some important observations that might explain the unique secretion patterns discussed above have been obtained from transfected animal cell lines. Earlier studies from our laboratory and others demonstrated that the unassembled pituitary β subunits do not efficiently exit the ER in the absence of the α subunit (15Corless C.L. Matzuk M.M. Ramabhadran T.V. Krichevsky A. Boime I. J. Cell Biol. 1987; 104: 1173-1181Crossref PubMed Scopus (86) Google Scholar, 16Keene J.L. Matzuk M.M. Otani T. Fauser B.C. Galway A.B. Hsueh A.J. Boime I. J. Biol. Chem. 1989; 264: 4769-4775Abstract Full Text PDF PubMed Google Scholar, 17Kaetzel D.M. Virgin J.B. Clay C.M. Nilson J.H. Mol. Endocrinol. 1989; 3: 1765-1774Crossref PubMed Scopus (29) Google Scholar). Although co-expression with the α subunit rescues the β subunits, differences exist in the extent of assembly of the α/β subunit pairs. For example, in the case of LHβ, the amount of dimer formed in transfected CHO cells is less than 10% (12Talmadge K. Vamvakopoulos N.C. Fiddes J.C. Nature. 1984; 307: 37-40Crossref PubMed Scopus (274) Google Scholar, 15Corless C.L. Matzuk M.M. Ramabhadran T.V. Krichevsky A. Boime I. J. Cell Biol. 1987; 104: 1173-1181Crossref PubMed Scopus (86) Google Scholar, 18Muyan M. Furuhashi M. Sugahara T. Boime I. Mol. Endocrinol. 1996; 10: 1678-1687PubMed Google Scholar), whereas more than 80% of the steady-state FSHβ subunit is secreted as a component of the heterodimer (16Keene J.L. Matzuk M.M. Otani T. Fauser B.C. Galway A.B. Hsueh A.J. Boime I. J. Biol. Chem. 1989; 264: 4769-4775Abstract Full Text PDF PubMed Google Scholar). The terminal LHβ heptapeptide (Leu-Ser-Gly-Leu-Leu-Phe-Leu) accounts, in part, for this inefficient assembly (13Matzuk M.M. Spangler M.M. Camel M. Suganuma N. Boime I. J. Cell Biol. 1989; 109: 1429-1438Crossref PubMed Scopus (50) Google Scholar, 18Muyan M. Furuhashi M. Sugahara T. Boime I. Mol. Endocrinol. 1996; 10: 1678-1687PubMed Google Scholar). Based on these observations, we proposed that this sequence serves as a signal capable of governing the intracellular sorting and trafficking of LH (13Matzuk M.M. Spangler M.M. Camel M. Suganuma N. Boime I. J. Cell Biol. 1989; 109: 1429-1438Crossref PubMed Scopus (50) Google Scholar). The experiments described above were performed with CHO cells, which secrete proteins only by the constitutive route (13Matzuk M.M. Spangler M.M. Camel M. Suganuma N. Boime I. J. Cell Biol. 1989; 109: 1429-1438Crossref PubMed Scopus (50) Google Scholar, 16Keene J.L. Matzuk M.M. Otani T. Fauser B.C. Galway A.B. Hsueh A.J. Boime I. J. Biol. Chem. 1989; 264: 4769-4775Abstract Full Text PDF PubMed Google Scholar, 17Kaetzel D.M. Virgin J.B. Clay C.M. Nilson J.H. Mol. Endocrinol. 1989; 3: 1765-1774Crossref PubMed Scopus (29) Google Scholar, 18Muyan M. Furuhashi M. Sugahara T. Boime I. Mol. Endocrinol. 1996; 10: 1678-1687PubMed Google Scholar, 19Matzuk M.M. Krieger M. Corless C.L. Boime I. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6354-6358Crossref PubMed Scopus (77) Google Scholar) without intracellular accumulation of mature hormone dimers, thus precluding studies of glycoprotein hormone secretion by the regulated pathway. To investigate sorting of the LH/FSH dimers and free subunits, we have used the GH3 cell line (20Bielinska M. Rzymkiewicz D. Boime I. Mol. Endocrinol. 1994; 8: 919-928Crossref PubMed Scopus (17) Google Scholar, 21Muyan M. Rzymkiewicz D.M. Boime I. Mol. Endocrinol. 1994; 8: 1789-1797Crossref PubMed Google Scholar), which is derived from pituitary somatotropes and contain storage vesicles responsive to secretagogues (20Bielinska M. Rzymkiewicz D. Boime I. Mol. Endocrinol. 1994; 8: 919-928Crossref PubMed Scopus (17) Google Scholar, 21Muyan M. Rzymkiewicz D.M. Boime I. Mol. Endocrinol. 1994; 8: 1789-1797Crossref PubMed Google Scholar, 22Dannies P.S. Endocr. Rev. 1999; 20: 3-21PubMed Google Scholar). Importantly, we previously demonstrated that transfected GH3 cells secrete LH and FSH primarily through regulated and constitutive secretory pathways, respectively (21Muyan M. Rzymkiewicz D.M. Boime I. Mol. Endocrinol. 1994; 8: 1789-1797Crossref PubMed Google Scholar), and that the LH N-linked carbohydrates were sulfated (20Bielinska M. Rzymkiewicz D. Boime I. Mol. Endocrinol. 1994; 8: 919-928Crossref PubMed Scopus (17) Google Scholar). The above observations were in marked contrast with data obtained from transfected CHO cells in which no detectable responses to secretagogues or LH sulfation were observed (20Bielinska M. Rzymkiewicz D. Boime I. Mol. Endocrinol. 1994; 8: 919-928Crossref PubMed Scopus (17) Google Scholar, 21Muyan M. Rzymkiewicz D.M. Boime I. Mol. Endocrinol. 1994; 8: 1789-1797Crossref PubMed Google Scholar). Here we tested the requirement of the LHβ heptapeptide to direct LH to the regulated pathway of GH3 cells. The data indicate that the carboxyl-terminal heptapeptide contributes to the trafficking of LH dimer to the regulated secretory pathway. Cell Culture and Stable Transfection—GH3 cells were a gift from Dr. Dennis Shields (Albert Einstein College of Medicine, New York). The cells were grown (no more than 30 passages) at 37 °C in Ham's F-12 medium (Mediatech Inc., Herndon, VA) supplemented with 12.5% horse serum (Invitrogen), 2.5% fetal bovine serum (Harlan Bioproducts for Science, Inc., Indianapolis, IN), 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified 5% CO2 incubator. Transfections were performed using Lipofectamine 2000 (Invitrogen) on semiconfluent cells in 6-well plates. Cells were transfected with 4 μg of the α, LHβ, LHβ114 (designated LHβΔT), FSHβ, or FSHβ-LHβ chimera (designated FSHβ-L) subunit genes (Fig. 1) contained in the vector pM2HA (19Matzuk M.M. Krieger M. Corless C.L. Boime I. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6354-6358Crossref PubMed Scopus (77) Google Scholar) to obtain clones expressing the individual subunits and corresponding dimers. Stable clones were selected ∼16 days later with 0.25 mg/ml G418 (Research Product International, Mt. Prospect, IL). Single colonies were isolated and subsequently screened by immunoprecipitating the media and lysates of metabolically labeled cells (see below). Several clones (n = 5 per dimer) expressing the dimers LH, LHΔT, FSH, or FSH-L were maintained in culture in the presence of 0.125 mg/ml G418 and used for the experiments described below. The mutant LHβΔT described previously lacks a seven-amino acid extension (Leu-Ser-Gly-Leu-Leu-Phe-Leu) at the carboxyl terminus of the LHβ subunit (13Matzuk M.M. Spangler M.M. Camel M. Suganuma N. Boime I. J. Cell Biol. 1989; 109: 1429-1438Crossref PubMed Scopus (50) Google Scholar). To construct FSHβ-L, the heptapeptide sequence of LHβ subunit (plus the stop codon) was inserted in-frame at the 3′-end of the FSHβ subunit by using overlapping PCR mutagenesis. The PCR was performed using KlenTag DNA polymerase (Sigma) and GenAmp PCR system 2400 (PerkinElmer Life Sciences). The following primers were used in the construction of the FSHβ-L chimera: oligo 1 (universal primer for pM2HA); 5′-TTC TCC CCC GCA GCC CTA GAA GAC GTT CCA-3′; oligo 2; 5′-GAG GAG GCC TGA GAG TTC TTT CAT TTC ACC-3′; oligo 3; 5′-GGT GAA ATG AAA GAA CTC TCA GGC CTC CTC-3′; oligo 4 (universal primer for pM2HA); 5′-TTT TCA CTG CAT TCT AGT TGT GGT TTG TCC-3′. The universal primers (oligos 1 and 4) corresponded to the sequences in pM2HA vector located upstream and downstream of multiple cloning sites. In the first PCR reaction, the FSHβ-CTP-α gene was used as a template with primers 1 and 2 to amplify product A containing the entire FSHβ sequence and the beginning of the heptapeptide of LHβ subunit. A parallel reaction containing primers 3 and 4 and the LHβ subunit as a template generated PCR product B comprising part of FSHβ exon 3 and the heptapeptide sequence of LHβ with its stop codon. The overlapping PCR was performed using fragments A and B with primers 1 and 4, resulting in the final product FSHβ-L (FSHβ with the seven amino acids of LHβ subunit), which was sequenced to ensure no errors occurred during the PCR reactions. The FSHβ-L chimera was enzymatically digested by BamHI and inserted into the pM2HA vector. The pM2HA vector is a pSV2 neo derivative, which contains the ampicillin resistance and neomycin resistance genes and the Harvey murine sarcoma virus long terminal repeat. Metabolic Labeling and Immunoprecipitation—For continuous labeling experiments, cells were plated into 6- or 12-well dishes and grown for ∼4 days to near confluency. Cells were labeled for 16 h with 20 μCi/ml [35S]cysteine (specific activity > 1000 Ci/mmol, MP Biomedicals Inc., Irvine, CA) in Ham's F-12 medium minus cysteine and G418 but supplemented with 7.5% dialyzed fetal bovine serum, glutamine, and antibiotics. Labeling with inorganic sulfate was performed for 16 h in Ham's F-12 sulfate-free medium with 0.7 mCi/ml carrier-free sulfate (Na2[35S]O4; MP Biomedicals Inc.) supplemented with 7.5% dialyzed fetal bovine serum, glutamine, and antibiotics (20Bielinska M. Rzymkiewicz D. Boime I. Mol. Endocrinol. 1994; 8: 919-928Crossref PubMed Scopus (17) Google Scholar). For pulse-chase experiments, confluent cells grown on 6-well plates were preincubated for 1.5 h with cysteine-free medium followed by a 20 min pulse in this medium containing 80 μCi/ml [35S]cysteine. At the end of the pulse, the medium was aspirated, and the cells were washed twice with prewarmed chase medium comprising Ham's F-12, 1 mm unlabeled l-cysteine (Sigma), 7.5% dialyzed fetal bovine serum, glutamine, and antibiotics and incubated in this medium for up to 24 h. All collected media and cell lysates were treated with iodoacetamide and phenylmethanesulfonyl fluoride to inhibit proteases. After centrifugation to remove cell debris, samples (2 ml) were precleared with 7.5 μl/ml normal rabbit serum and Pansorbin (EMD Biosciences Inc., La Jolla, CA). The supernates were divided into two aliquots and immunoprecipitated for 2 h at room temperature by anti-α or anti-CGβ (which cross-reacts with the LHβ subunit) rabbit polyclonal sera. The immune complexes were precipitated with Pansorbin and subjected to SDS-PAGE on 12.5 or 15% gels. For quantitative comparisons of LH and LHΔT, equal volumes of samples were always loaded on the gel. The gels were soaked in 1 m sodium salicylate for 15 min, dried, and exposed to x-ray film (18Muyan M. Furuhashi M. Sugahara T. Boime I. Mol. Endocrinol. 1996; 10: 1678-1687PubMed Google Scholar). Forskolin-stimulated Secretion—To examine gonadotropin storage and regulated secretion of dimers, five wells of LH and LHΔT cells were labeled for 16 h with [35S]sulfate or [35S]cysteine. Medium and lysate from one well were collected and served as the 0 time controls for the subsequent experiments. Cells in the remaining four wells were preincubated in chase medium (containing 1 mm unlabeled l-cysteine, 7.5% dialyzed fetal bovine serum, glutamine, and antibiotics) for 2 h to reduce the background of the constitutive pool. After the 2-h preincubation, media were collected and frozen for immunoprecipitation. During a second 2-h period, two wells received fresh chase medium with forskolin (25 μm final concentration; Sigma) (20Bielinska M. Rzymkiewicz D. Boime I. Mol. Endocrinol. 1994; 8: 919-928Crossref PubMed Scopus (17) Google Scholar), and the remaining two wells were incubated without secretagogue. Cells expressing LHΔT were also incubated in the presence or absence of forskolin immediately after a 16-h labeling without the 2-h preincubation period. All labeled culture media and cell lysates were then immunoprecipitated with α antiserum and analyzed by 12.5% SDS-PAGE. To examine the storage and secretion of FSH and FSH-L dimers, cells were labeled for 4 h with [35S]cysteine instead of sulfate, because the N-linked oligosaccharides of FSH are not sulfated as they are in LH. Medium and lysate from one well were collected and served as the 0 time controls for the subsequent experiment. Cells in one well received chase medium for 2 h with forskolin (25 μm final concentration) (20Bielinska M. Rzymkiewicz D. Boime I. Mol. Endocrinol. 1994; 8: 919-928Crossref PubMed Scopus (17) Google Scholar), and the remaining well was incubated without secretagogue. All labeled culture media and cell lysates were then immunoprecipitated with FSH dimer-specific antibody (FSH 54B) and analyzed by 15% SDS-PAGE. Immunofluorescence—GH3 cells expressing LH or LHΔT were grown on Fisherbrand Superfrost-Plus microscope slides (Fisher Scientific) in Petri dishes as described above. The cells were incubated at 37 °C for 4 h in the presence or absence of cycloheximide (CHX) (15 μg/ml; Sigma). Immediately following CHX treatment, all cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% Tween for 10 min. Cells were then incubated in 20% normal goat serum (Vector Laboratories, Burlingame, CA) for 1 h to block nonspecific binding and washed three times for 10 min in 2% bovine serum albumin (Sigma). After washing, cells were incubated in rabbit anti-CGβ antiserum (1:250; also used for immunoprecipitation experiments) for 30 min at room temperature. The cells were washed three times in 2% bovine serum albumin and incubated in goat anti-rabbit Alexa Fluor 488 (1:250; Invitrogen) for 20 min. Cells were then washed three times in 2% bovine serum albumin and once in phosphate-buffered saline and counterstained with TOPRO-iodide (Invitrogen) for 15 min. Cells were washed three times in phosphate-buffered saline for 10 min; coverslips were added using VectaShield mounting medium (Vector Laboratories), and cells were examined by confocal microscopy using an Olympus FV-500 microscope with a ×60 water objective. The experiment was repeated three times. Cells from at least two fields from each slide were counted by two individuals (one blind to treatment) and were scored as either negative or positive; ∼200 cells per slide were counted. Cells were considered positive if multiple discrete puncta were observed. Negative cells were devoid of puncta, similar to cells incubated with normal rabbit serum. Analysis of Data—The labeled bands from autoradiography were scanned using a GS-710 calibrated imaging densitometer, and the intensity of bands was quantitated by densitometric analysis using Quantity One software (Bio-Rad Laboratories). Equal exposure times for the autoradiograms were used when comparing the results of synthesis and protein secretion for LH, LHΔT, FSH, and FSH-L dimers. The secretion t½ for the dimers corresponds to the time when 50% of the labeled dimer, as determined from the presence of α subunit, accumulated in the medium when immunoprecipitating with CGβ antisera. Recovery of subunit (%) was determined as the amount of labeled subunit in medium as a fraction of the total. The sorting index of LH, LHΔT, FSH, and FSH-L dimer corresponds to the ratio of the band intensity on autoradiograms obtained with forskolin (+F) to the band intensity obtained without (-F) forskolin. For immunofluorescence, results are expressed as the percentage of positive cells. Each experiment was repeated three to eight times, analyzed using a paired t test, and the results are expressed as mean ± S.E., with p < 0.05 considered significantly different. Secretion of LH and LHΔT After Steady-state Labeling—Previously we showed in CHO cells that deleting the seven-amino acid hydrophobic carboxyl-terminal extension from the LHβ subunit enhances secretion and alters processing of the N-linked oligosaccharides of this truncated subunit (designated LHβΔT, Fig. 1) compared with the wild-type LHβ subunit (19Matzuk M.M. Krieger M. Corless C.L. Boime I. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6354-6358Crossref PubMed Scopus (77) Google Scholar). Thus, we suspected that, because this sequence affects the intracellular disposition of the subunit, it contributes to LH sorting in the regulated pathway. Because CHO cells lack a regulated secretory pathway, we examined the secretion of LH and the mutant LHΔT in GH3 cells, which contain both constitutive and regulated routes (20Bielinska M. Rzymkiewicz D. Boime I. Mol. Endocrinol. 1994; 8: 919-928Crossref PubMed Scopus (17) Google Scholar, 21Muyan M. Rzymkiewicz D.M. Boime I. Mol. Endocrinol. 1994; 8: 1789-1797Crossref PubMed Google Scholar). Fig. 2 shows the secretion of two sets of representative clones (Groups 1 and 2) expressing either LH or LHΔT heterodimers after 16 h of labeling with [35S]cysteine. Immunoprecipitation of LH dimer secreting cells with α antiserum co-precipitates the β subunit, reflecting the extent of heterodimer formation (Fig. 2, A and B, lane 2), and the combined and free (asterisks) forms of α subunit. The unassembled α subunit is unique in that it undergoes a post-translational modification resulting in a more heterogeneous form, which clearly distinguishes it from the dimer species (Fig. 2, A and B, lane 2). The free α subunit is rapidly secreted with no detectable intracellular accumulation (Fig. 2, A and B, lane 1). As reported previously (18Muyan M. Furuhashi M. Sugahara T. Boime I. Mol. Endocrinol. 1996; 10: 1678-1687PubMed Google Scholar, 20Bielinska M. Rzymkiewicz D. Boime I. Mol. Endocrinol. 1994; 8: 919-928Crossref PubMed Scopus (17) Google Scholar), after overnight labeling the secreted heterodimer precipitated with α antiserum contains labeled α subunit associated with only a fraction of the total labeled β subunit seen in the lysate (Fig. 2, A and B, compare lanes 2 and 3). This is presumably due to the presence of a stable, nonlabeled intracellular pool of assembly-competent LHβ subunit (see “Discussion”). Precipitation of LH dimer by β subunit antiserum co-precipitates α subunit from the lysate and medium (Fig. 2, A and B, lanes 3 and 4). Note that the α subunit is only weakly observed in the lysate (Fig. 2, A and B, lane 3), reflecting the extent of heterodimer accumulating after steady state labeling. Note also the absence of the heterogeneous free α subunit in the media (Fig. 2, A and B, compare lanes 2 and 4). In contrast to the free α subunit, a significant fraction of the unassembled β subunit accumulates intracellularly (Fig. 2, A and B, lane 3), despite the presence of excess α subunit, and is confined to the ER (12Talmadge K. Vamvakopoulos N.C. Fiddes J.C. Nature. 1984; 307: 37-40Crossref PubMed Scopus (274) Google Scholar, 14Hayashizaki Y. Miyai K. Kato K. Matsubara K. FEBS Lett. 1985; 188: 394-400Crossref PubMed Scopus (80) Google Scholar). This result agrees with earlier studies showing that in transfected cell lines the LHβ subunit inefficiently heterodimerizes with the α subunit (18Muyan M. Furuhashi M. Sugahara T. Boime I. Mol. Endocrinol. 1996; 10: 1678-1687PubMed Google Scholar, 19Matzuk M.M. Krieger M. Corless C.L. Boime I. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6354-6358Crossref PubMed Scopus (77) Google Scholar) and the unassembled β subunit is not secreted efficiently. As expected from our previous work in CHO cells (18Muyan M. Furuhashi M. Sugahara T. Boime I. Mol. Endocrinol. 1996; 10: 1678-1687PubMed Google Scholar), the extent of heterodimer formation is significantly increased when the heptapeptide is deleted (Fig. 2, A and B, lanes 5-8). This is reflected in the 3.1 ± 0.4-fold reduction (p < 0.05) in the lysate fraction of the unassembled LHβΔT subunit immunoprecipitated with β antiserum compared with the LHβ subunit (Fig. 2, A and B, lanes 3 and 7). In addition, immunoprecipitation with α antiserum demonstrated a 2.6 ± 0.1-fold decrease (p < 0.05) in the amount of intracellular LHΔT (Fig. 2, A and B, lane 5) and 2.3 ± 0.2-fold increase (p < 0.05) in the secretion of LHΔT compared with LH dimer (lanes 2 and 6). This is associated with an increase in the incorporation of the labeled LHβΔT subunit in heterodimer compared with the wild-type LHβ subunit. It is curious that the truncated LHβΔT subunit migrates slower in the gel compared with the wild-type LHβ subunit. It is unclear whether this is due to the unmasking of a site for post-translational change or an effect on the mobility of the subunit on the gel. These data show that the total LHΔT secreted from GH3 cells is greater than LH dimer. Pulse-Chase Kinetics—The steady-state labeling suggested that less of the LHΔT dimer is retained in" @default.
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