Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells.

Corless, Christopher L.; Matzuk, Martin M.; Ramabhadran, Triprayar V.; Krichevsky, Alexander; Boime, Irving

doi:10.1083/jcb.104.5.1173

Cited by 113 publications

(60 citation statements)

References 60 publications

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“…The unmodified subunit was not observed in the culture media (Fig. 4A, lane 3), suggesting that it was retained in the cell as shown previously (42,43). The electrophoretic migrations of the intracellular forms of labeled LH␤CTP and LH␤boCTP were similar (apparent molecular mass of 20 -21 kDa, Fig.…”

Section: Resultsmentioning

confidence: 48%

The LHβ 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

Nakav

Jablonka‐Shariff

Kaner

et al. 2005

Journal of Biological Chemistry

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The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) ␤ to the ␤-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LH␤ gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LH␤ gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LH␤ gene (boCTP) would be sufficient to generate the LH␤ species of a ruminant with properties typical to the CG␤ subunit. The mutated bovine LH␤-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CG␤ CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CG␤ subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCG␤-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCG␤ subunit, a sorting function attributed to the Oglycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LH␤ to CG␤ evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LH␤ gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.The family of glycoprotein hormones includes lutropin (LH), 1 follitropin, and thyrotropin, as expressed by the pituitary, and chorionic gonadotropin (CG), which is produced by the placenta of primates and equids (1). Each hormone is a noncovalent heterodimer composed of a common ␣-subunit and a unique ␤-subunit that confers receptor specificity. The ␤-subunits have substantial sequence similarities, including conserved location of cysteine residues that suggests an origin from a common ancestral gene (2). The human (h) CG␤ genes have evolved from an ancestral LH␤ gene by frameshift mutations that resulted in a readthrough into a previously untranslated region and the extension of the reading frame (3, 4) (Fig. 1A). As a result, a short carboxyl-terminal sequence of hLH␤ is replaced by a longer carboxyl-terminal peptide (CTP) in hCG␤ (3-6). In the equine (e), both the placental CG␤ and pituitary LH␤ subunits are products of the same gene (eLH/CG␤) and contain a CTP that is presumed to have evolved by a distinct mechanism (7) (for further explanations see "Results"). Except for primates and equids, no other identified animal species b...

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Section: Resultsmentioning

confidence: 48%

The LHβ 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

Nakav

Jablonka‐Shariff

Kaner

et al. 2005

Journal of Biological Chemistry

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“…The glycoprotein hormone-specific quaternary structure, i.e. the heterodimer, signals key functional intracellular events such as efficient secretion and hormone-specific processing of the subunit N-linked oligosaccharides (23). Our data imply that analogs with different conformations can bind to the receptor and activate adenylate cyclase.…”

Section: Discussionmentioning

confidence: 89%

The Biologic Action of Single-chain Choriogonadotropin Is Not Dependent on the Individual Disulfide Bonds of the β Subunit

Ben-Menahem

Kudo

Pixley

et al. 1997

Journal of Biological Chemistry

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Disrupting disulfide loops in the human chorionic gonadotropin ␤ subunit (CG␤) inhibits combination with the ␣ subunit. Because the bioactivity requires a heterodimer, studies on the role of disulfide bonds on receptor binding/signal transduction have previously been precluded. To address this problem, we bypassed the assembly step and genetically fused CG␤ subunits bearing paired cysteine mutations to a wild-type ␣ (WT␣) subunit. The changes altered secretion of the single-chain mutants which parallel that seen for the CG␤ monomeric subunit. Despite conformational changes in CG disulfide bond mutants (assayed by gel electrophoresis and conformationally sensitive monoclonal antibodies), the variants bind to the lutropin/CG receptor and activated adenylate cyclase in vitro. The data show that the structural requirements for secretion and bioactivity are not the same. The results also suggest that the extensive native subunit interactions determined by the cystine bonds are not required for signal transduction.Moreover, these studies demonstrate that the singlechain model is an effective approach to structure-activity relationships of residues and structural domains associated with assembly of multisubunit ligands.Mutational and structural studies have revealed that small clusters of amino acids rather than large structural motifs often contribute to most of the energy involved in proteinprotein interactions such as hormone binding to its receptor (for review, see Refs. 1-3). However, it is not clear how the conformation of a peptide ligand contributes to the coupling of binding to signal transduction. This is especially an issue for the function of hormone-receptor complexes involving multisubunit ligands. A convenient model for studying the tertiary and quaternary determinants in signaling is the glycoprotein hormone family which include human chorionic gonadotropin (hCG), 1 lutropin (LH), follitropin (FSH), and thyrotropin. Each is a non-covalent heterodimer composed of a common ␣ and unique ␤ subunit which allows recognition of the corresponding G-protein-coupled receptor (for review, see Ref. 4).Recent crystallographic studies of hCG revealed a significant structural similarity to several growth factor families, e.g. transforming growth factor ␤, which contain a cystine knot motif composed of three pairs of bridged cysteine residues (5-8). Each hCG subunit contains a cystine knot configuring three disulfide-bonded loops which are the major structural motifs (7,8). Based on the crystallographic studies of hCG (7,8), the disulfide bonds in the CG␤ subunit are at positions 9 -57, 34 -88, 38 -90, 23-72, 93-100, and 26 -110 (Fig. 1A). The folding intermediates associated with the ordered formation of these bridges to acquire an assembly-competent form is well documented (9, 10). Current models of hCG action presume that the conformation of the dimer and the highly interactive contacts between the two subunits are critical for function (11)(12)(13)(14). One method to examine the functional role of the structural mo...

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“…Since no bioactivity was detected in the media, secretion of a bioactive 35-kDa subunit by itself could be very inefficient; alternatively, similar to the 40-kDa subunit, the protein could be much less active or inactive altogether. Intracellular 35-kDa protein in the absence of the other subunit could be inherently unstable; there is precedence for this phenomenon, since it has been reported that 90% of the (3 chains of lutropin (LH), when expressed in the absence of a chains, are retained in the endoplasmic reticulum and are slowly degraded (16 carefully controlled renaturation and oxidation conditions would allow the disulfide bond formation required for generation of bioactive CLMF.…”

Section: Resultsmentioning

confidence: 99%

Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor.

Gubler

Chua

Schoenhaut

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

530

322

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Cytotoxic lymphocyte maturation factor (CLMF) is a disulfide-bonded heterodimeric lymphokine that (i) acts as a growth factor for activated T cells independent of interleukin 2 and (ii) synergizes with suboptimal concentrations of interleukin 2 to induce lymphokine-activated killer cells. We now report the cloning and expression of both human CLMF subunit cDNAs from a lymphoblastoid B-cell line, NC-37. The two subunits represent two distinct and unrelated gene products whose mRNAs are coordinately induced upon activation of NC-37 cells. Coexpression of the two subunit cDNAs in COS cells is necessary for the secretion of biologically active CLMF; COS cells transfected with either subunit cDNA alone do not secrete bioactive CLMF. Recombinant CLMF expressed in mammalian cells displays biologic activities essentially identical to natural CLMF, and its activities can be neutralized by monoclonal antibodies prepared against natural CLMF. Since this heterodimeric protein displays the properties of an interleukin, we propose that CLMF be given the designation interleukin 12.The molecular cloning and expression of recombinant cytokines has made possible both significant advances in our understanding of the molecular basis of immune responses and the development of new approaches to the treatment of disease states. As an example, recombinant interleukin 2 (recombinant IL-2) has been shown to be capable of causing regression of established tumors in both experimental animals (1) and in man (2); however, its clinical use has been associated with significant toxicity (2). One potential approach to improving the therapeutic utility of recombinant cytokines is to use them in combination (3,4 MATERIALS AND METHODScDNA Cloning. A subline of NC-37 cells selected for its ability to produce high levels of CLMF (7), NC-37.98, was induced with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 for 16 hr. Poly(A)+ RNA was isolated, and random hexamer-primed cDNA libraries were established in phage AgtlO by standard procedures. Mixedprimer polymerase chain reaction (PCR) using controlled ramp times (8) was performed as follows. PCR primers contained all possible codons and were 14 or 15 nucleotides long ( Fig. 1) with a 5' extension of 9 nucleotides containing an EcoRI site for subcloning. Degeneracies varied from 1 in 32 to 1 in 4096; 0.5-4 pmol per permutation of forward and reverse primer was used in a 50-to 100-,lI PCR mixture with 40 ng of cDNA made from NC-37.98 cells that had been activated by culture with 10 ng of PMA and 25 ng of calcium ionophore A23187 per ml for 16 hr (40-kDa subunit) or with 3 ,tg of human genomic DNA (35-kDa subunit). PCR cycling parameters were as follows. Initial denaturation was at 95°C for 7 min. Low-stringency annealing was performed by cooling to 37°C over 2 min, incubating 2 min at 37°C, heating to 72°C over 2.5 min, extending at 72°C for 1.5 min, heating to 95°C over 1 min, and denaturing at 95°C for 1 min. This cycle was repeated once. Thirty standard cycles (40-kDa subun...

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Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells.

Cited by 113 publications

References 60 publications

The LHβ 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

The LHβ 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

The Biologic Action of Single-chain Choriogonadotropin Is Not Dependent on the Individual Disulfide Bonds of the β Subunit

Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor.

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