A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells.

Peters, Barry P.; Krzesicki, Raymond F.; Hartle, Richard J.; Perini, Fulvio; Ruddon, Raymond W.

doi:10.1016/s0021-9258(17)42523-3

Cited by 80 publications

(8 citation statements)

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“…LHfl that accumulates in transfected C12"/cells is sensitive to endoglycosidase H (11), suggesting that it is retained in the endoplasmic reticulum (ER). Because assembly normally occurs in the ER (18,43), and because =50% of the retained LH/3 can combine, the ER is the likely site of accumulation of uncombined LH/3. Also, the disappearance of unassembled LH/3, TSH/3, and FSH/3 subunits could occur via the recently described "ER degradation pathway" for the disposal of unassembled membrane proteins (30).…”

Section: Discussionmentioning

confidence: 99%

“…UMAN chorionic gonadotropin (CG), t lutropin (LH), follitropin (FSH), and thyrotropin (TSH) are a family of heterodimeric glycoprotein hormones that share a common t~ subunit but differ in their hormonespecific fl subunits (8,45,52). Combination of the ot and subunits begins in the endoplasmic reticulum (18,43) and for CG, dimerization is completed before the addition of the O-linked oligosaccharides in the Golgi (43). Although the fl subunits determine biological specificity of the hormones, there is a high degree of amino acid homology between these subunits (50), which is most apparent for LHfl and CGfl.…”

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confidence: 99%

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Mutagenesis and chimeric genes define determinants in the beta subunits of human chorionic gonadotropin and lutropin for secretion and assembly.

Matzuk

Spangler

Camel

et al. 1989

The Journal of Cell Biology

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Abstract. Chorionic gonadotropin (CG) and lutropin (LH) are members of a family of glycoprotein hormones that share a common a subunit but differ in their hormone-specific fl subunits. The glycoprotein hormone ~ subunits share a high degree of amino acid homology that is most evident for the LHfl and CGfl subunits having >80% sequence similarity. However, transfection studies have shown that human CGfl and ot can be secreted as monomers and can combine efficiently to form dimer, whereas secretion and assembly of human LHfl is less efficient. To determine which specific regions of the LHfl and CGfl subunits are responsible for these differences, mutant and chimeric LH/%CGfl genes were constructed and transfected into CHO cells. Expression of these subunits showed that both the hydrophobic carboxy-terminal seven amino acids and amino acids Trp s, Ile ~5, Met 42, and Asp 77 together inhibit the secretion of LHfl. The carboxy-terminal amino acids, along with Trp s, Ile ~, Met 42, and Thr 5s are implicated in the delayed assembly of LHfl. These unique features of LHfl may also play an important role in pituitary intracellular events and may be responsible for the differential glycosylation and sorting of LH and FSH in gonadotrophs. HUMAN chorionic gonadotropin (CG), t lutropin (LH), follitropin (FSH), and thyrotropin (TSH) are a family of heterodimeric glycoprotein hormones that share a common t~ subunit but differ in their hormonespecific fl subunits (8,45,52). Combination of the ot and subunits begins in the endoplasmic reticulum (18, 43) and for CG, dimerization is completed before the addition of the O-linked oligosaccharides in the Golgi (43). Although the fl subunits determine biological specificity of the hormones, there is a high degree of amino acid homology between these subunits (50), which is most apparent for LHfl and CGfl. They are 85 % homologous in the first 114 amino acids (51), and this relationship is responsible for the binding of CG and LH to a common gonadal receptor (8, 45, 52). However, CGfl and LHfl contain two prominent structural differences: (a) LHfl contains one N-linked oligosaccharide at position 30, whereas CGfl contains two N-linked units at sites 13 and 30; and (b) CGfl contains a 31-amino acid hydrophilic COOH-terminal extension with four O-linked oligosaccharides (3, 7, 21, 23) compared with a shorter, 7-amino acid, hydrophobic stretch on LH/~

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Mutagenesis and chimeric genes define determinants in the beta subunits of human chorionic gonadotropin and lutropin for secretion and assembly.

Matzuk

Spangler

Camel

et al. 1989

The Journal of Cell Biology

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“…It must be emphasized that just because exportable protein s normally dimerize in the ER , it does not necessarily follow that folde d monomers must be r estrained from transport (Hoshina and Boim e, 1982;Peters et al ., 1984 ;Singh et al, 1990 ). Thus, it sho uld not be surprising that a proinsulin mutant defective for homodimeri zat ion can in fact be transported through th e secretory pathway (Quin n et al ., 1991 ).…”

Section: Effects Of Chloroqu Ine and Zin C On Insulin Disulfide Accessibility Withi N The Secretory Granu Lementioning

confidence: 99%

Intracellular Transport of Proinsulin in Pancreatic β-Cells

Huang

Arvan

1995

Journal of Biological Chemistry

115

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In pancreatic islets, formation of beta-secretory granule cores involves early proinsulin homohexamerization and subsequent insulin condensation. We examined proinsulin conformational maturation by monitoring accessibility of protein disulfide bonds. Proinsulin disulfides are intact immediately upon synthesis, but are > or = 90% sensitive to in vivo reduction with 2 mM dithiothreitol; wash out of dithiothreitol leads to reoxidation, proinsulin transport, and conversion to insulin. With t1/2 approximately 10 min, newly synthesized proinsulin becomes resistant to disulfide reduction, correlating with endoplasmic reticulum (ER) export. However, inhibition of ER export with brefeldin A blocks acquisition of resistance to reduction, and once proinsulin arrives in the Golgi, it resists reduction despite brefeldin treatment. Moreover, in vivo, resistance of proinsulin disulfides is overcome after increasing [dithiothreitol] > 10-fold, or in vitro, in islets lysed in a zinc-free, but not a zinc-containing, medium. Employing 30 mM dithiothreitol in vivo, a further decrease in disulfide accessibility is observed following proinsulin conversion to insulin. Incubation of islets with chloroquine or zinc enhances and diminishes accessibility of insulin disulfides, respectively. We hypothesize that two major conformational changes culminating in granule core formation, proinsulin hexamerization and insulin condensation, are sensitive to zinc and occur upon ER exit and arrival in immature secretory granules, respectively.

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“…Previous work on Ig assembly in Xenopus oocytes has shown that Ig heavy chains are not secreted when synthesized in the absence of L chains but are stably maintained within the oocyte and can be rescued as a consequence of a subsequent injection of the mRNA coding for the complementary chain (Colman et al, 1982). In contrast, data obtained in mammalian cells have provided examples of a '~time elapsed after synthesis" dependent loss of ability to participate in an assembly process (Peters et al, 1984;Bergman and Kuehl, 1979). Here we have shown that HA monomers are maintained stably in the oocyte ER and that >50% of them are still available for trimerization and transport even 24 h after synthesis.…”

Section: Monomers Are Stable For Long Periods In Oocytesmentioning

confidence: 99%

Trimer formation determines the rate of influenza virus haemagglutinin transport in the early stages of secretion in Xenopus oocytes.

Ceriotti

Colman

1990

The Journal of Cell Biology

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Abstract.We have previously shown that influenza haemagglutinin (HA) acquires Endo H resistance en route to the cell surface after microinjection of its mRNA into Xenopus oocytes (Ceriotti, A. and A. Colman. 1989. J. Cell Biol. 109:1439-1444 In this paper we use the injection of varying amounts of mRNA (0.05-5 ng/oocyte) to effect a 30-fold change in HA protein synthesis within the oocyte. Using the Endo H assay as an indicator of protein movement from the ER to the medial Golgi we find that this movement is reduced, sometimes dramatically, when intracellular HA levels fall. This reduction in movement is closely correlated with a decreased rate of trimer formation as assessed both by trypsin resistance and sedimentation analysis, leading us to conclude that trimer formation is not only, as has been shown before essential for ER-Golgi complex movement, but is the major rate limiting step in this movement. Interestingly at least 50% of unassembled HA monomers that accumulate after low HA synthesis can be rescued into trimers over 24 h later, after a second injection of concentrated HA mRNA.In contrast when we repeated this experiment with another membrane protein, the human low density lipoprotein, or with murine secretory immunoglobulin we found that the rate of movement was insensitive to the protein concentration. This latter result seemed surprising since earlier work had shown that unassembled IgG heavy chains (like monomeric HA) remain in the oocyte ER; however in these present experiments we have been unable to detect any unassembled heavy chains even at the lowest expression levels, indicating that tetramerization of Ig is much faster than trimerization of HA.

show abstract

A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells.

Cited by 80 publications

References 58 publications

Mutagenesis and chimeric genes define determinants in the beta subunits of human chorionic gonadotropin and lutropin for secretion and assembly.

Mutagenesis and chimeric genes define determinants in the beta subunits of human chorionic gonadotropin and lutropin for secretion and assembly.

Intracellular Transport of Proinsulin in Pancreatic β-Cells

Trimer formation determines the rate of influenza virus haemagglutinin transport in the early stages of secretion in Xenopus oocytes.

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