Tag Archives: Biologically Active Single-Chain Human

The Tie That Binds: CONCLUSIONS


Studies are underway on yoked forms of the other glycoprotein hormones, as well as intersubunit disulfide-linked LH and FSH, and it will be interesting to ascertain similarities and differences. Likewise, studies on yoked LH-LHR, FSH-FSHR, and thyroid-stimulating hormone (TSH)-TSHR, along with the various heterologous forms, e.g., yoked hCG-FSHR, should prove highly informative in elucidating structure-function relationships of the various members of the glycoprotein hormones and then-cognate receptors. buy flovent inhaler

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The Tie That Binds: YOKED hCG-LHR(2)

Unfortunately, we do not have accurate measures of receptor density for the yoked human hCG-LHR complex expressed on HEK 293 cells, and thus it is impossible to estimate a potency based, for example, on the basal cAMP expression levels and numbers of complex per cell. Moreover, there is no information on turnover, internalization rates, desensitization, etc. of the yoked hCG-LHR complex.

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The Tie That Binds: YOKED hCG-LHR(1)


In view of the success in preparing a single-chain hCG of the form N-fi-a-C, we then designed a single-chain hCG-LHR, i.e., yoked hCG-LHR (Fig. 2), with a C-terminal peptide (CTP) and Factor Xa cleavage site serving as linker between a and receptor. buy ortho tri-cyclen online

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The maximum amount of cAMP produced at high gonadotropin concentrations is less in the expressed forms than in urinary hCG, and yoked hCG appears more potent than wild-type hCG. The differences between expressed wild-type hCG and urinary hCG are attributed primarily to differences in the carbohydrate moieties. When expressed in mammalian (CHO) cells, the full-length hCG^-a yoked (or tethered) hormone is several-fold less potent than wild-type hCG in binding and several-fold more potent in cAMP stimulation when assayed in HEK 293 cells expressing LHR. Of considerable interest was the observation that the singlechain, full-length mammalian cell-expressed hCG was more potent than wild-type hCG in an in vivo assay.

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A number of single-chain hCG agonists have been expressed and characterized, including 0(l-145)-a(l-92), 0(l-123)-a(l-92), and 0-ct(l-92), as well as several with a Ser-Gly spacer—0-(Ser-Gly)5-a(l-92), 0(1-1 ll)-(Ser-Gly)6-a, and 0(l-lO2)-(Ser-Gly)4-a. Also, various unnatural disulfide-linked subunits obtained by replacing two amino acid residues located in close proximity in the structure of HF-treated hCG, one in a and one in (3, have been reported: a-Gln5/(3-Arg8, a-Met29/(3-Met41, ct-Arg35/(B-Ala35, a-Tyr37/p-Ile38, and a-Lys51/0-Asp”. Several of these intersubunit disulfide-linked hCGs exhibit increased thermal stability relative to wild-type hCG, as does the single-chain p(l-145)-a(l-92).

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The Tie That Binds: Design of Biologically Active Single-Chain Human Chorionic Gonadotropins(4)

Some of the mutations were naturally occurring and resulted in either constitutive activation or loss-of-function of LHR, while many of the loss-of-function mutants have been identified by site-directed mutagenesis. buy ortho tri-cyclen

Elucidation of the genomic structure of the LHR gene led to the suggestion of a possible origin of the putative ancestral glycoprotein hormone receptor gene. The LHR gene contains 10 exons that encode most of the ECD and one exon (no. 11) that encodes a small portion of the ECD, the seven transmembrane helices, the six connecting loops, and the cytoplasmic tail. It appears that exons 1-10, with the corresponding introns, were added to an intronless GPCR gene, probably of the adrenergic family, to form an early gene for a heptahelical receptor with a relatively large ECD.

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The Tie That Binds: Design of Biologically Active Single-Chain Human Chorionic Gonadotropins(3)

Chorionic Gonadotropins(3)

From the observation that much of the ECD of LHR is composed of pseudoleucine-rich repeats, several groups have modeled this region of the receptor with the structure of the leucine-rich repeat protein, porcine ribo-nuclease inhibitor, as a template. The resulting models exhibit a cusp-shaped structure, although there are specific differences in several of the proposed folding patterns, attributable in large part to the number of leucine-rich repeats chosen. Figure lb shows a model based on nine leucine-rich repeats developed by our laboratory in collaboration with Drs. Lapthom and Isaacs.

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The Tie That Binds: Design of Biologically Active Single-Chain Human Chorionic Gonadotropins(2)

The availability of a three-dimensional structure permitted the results of previous studies on hCG using chemical modifications, synthetic peptides, limited proteolysis, protein engineering to produce hormone chimeras, and site-directed mutagenesis to be interpreted on a molecular basis. Also, the availability of a structure for the heterodimer has spawned many new investigations, with emphasis on site-directed mutagenesis and hormone chimeras, aimed at probing hor-mone-receptor contact sites. These findings have resulted in the identification of many amino acid residues on both subunits that appear to contribute to receptor interaction. While there is controversy and there are many gaps to be filled in, the data indicate that the middle portion and C-terminal region of a and the determinant loop of hCG(3, extending beyond Cys100, are important in receptor recognition and activation. levitra super active plus

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The Tie That Binds: Design of Biologically Active Single-Chain Human Chorionic Gonadotropins(1)

Chain Human Chorionic Gonadotropins(1)

Recent major advances in the elucidation of structure-function relationships of the gonadotropins, hCG and LH, and their common receptor (LHR) include determination of the crystal structure of hydrogen fluoride (HF)-treated hCG, characterization of LHR cDNAs from several species (cf. for review), and determination of the genomic organization of the LHR gene. Coupled with the results of earlier and more recent gonadotropin/receptor investigations, these breakthroughs have placed the field on a firm molecular foundation and have opened up numerous new avenues of study. ventolin inhaler

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