Designer Gonadotropins and Receptors: The Prospect of Recombinant Technology

Puett, David; Narayan, Prema

doi:10.1007/978-0-387-21508-2_24

Cited by 2 publications

(3 citation statements)

References 48 publications

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“…With an available crystal structure of hCG (Lapthorn et al, 1994;Wu et al, 1994;Tegoni et al, 1999), a reasonable working model of the LHR-ECD, coupled with considerable experimental structure-function data, primarily from site-directed mutagenesis, on both the hormone and receptor (Puett and Narayan, 2000;Zeng et al, 2001;Song et al, 2001a,b;Fanelli and Puett, 2002;Vischer et al, 2003a,b;Smits et al, 2003;Vassart et al, 2004), an attempt was made to generate plausible structures for the hCG-LHR-ECD complex. The 3D DOCK program (Gabb et al, 1997) was utilized for rigid-body docking; the structures were then ranked by an evaluation based on electrostatic interactions and geometrical fit, and finally the structures were filtered using several criteria for distance restraints, e.g.…”

Section: The Lhr-ecd: Structure and Hormone Bindingmentioning

confidence: 99%

A functional transmembrane complex: The luteinizing hormone receptor with bound ligand and G protein

Puett

DeMars

et al. 2007

Molecular and Cellular Endocrinology

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The luteinizing hormone receptor (LHR) is one of eight members in a cluster of the rhodopsin family of the large G protein-coupled receptor (GPCR) superfamily that contains some 800-900 genes in the human genome. LHR, along with its paralogons, follicle stimulating hormone receptor (FSHR) and thyroid stimulating hormone receptor, form one of the three classes in this cluster; the two other classes contain the relaxin-binding GPCRs and orphan GPCRs. These GPCRs are characterized by a relatively large ectodomain (ECD) containing leucine-rich-repeats (LRRs); in the class of glycoprotein hormone receptors, the LRR region is capped by N-terminal and C-terminal cysteinerich regions. Binding of human chorionic gonadotropin (hCG) or luteinizing hormone to the LHR-ECD triggers a conformational change of the transmembrane region of the receptor facilitating binding and activation of Gs, followed by effector enzyme activation and subsequent intracellular signaling. Viewing LHR as a transmembrane anchoring protein that sequentially binds hCG and Gs to give the hCG-LHR-Gs complex, numerous interactions and conformational changes must be considered. There is, unfortunately, a paucity of structural data on LHR, but crystal structures exist for hCG, the homologous FSH-FSHR-ECD (N-terminal fragment) complex, rhodopsin (in the inactive state), an active form of Gαs (transducin), and the βγ heterodimer. Using a combined experimental (site-directed mutagenesis followed by characterization in transfected cells) and computational (homology modeling and molecular dynamics simulations) approach, good working models are being developed for the protein-protein interaction faces and, in some cases, the ensuing conformational changes induced by complex formation. hCG binding to the LHR-ECD appears to involve several LRRs; LHR activation can be described in terms of disrupting a network of H-bonds in the cytosolic halves of helices 1-3, 6, and 7; and binding of LHR to Gs involves, in large part, intracellular loop 2 binding, presumably to Gsα at its C-terminus. Major gaps exist in our understanding at the molecular level of the six-polypeptide chain complex, hCG-LHR-Gs, but considerable progress has been made in the past few years.

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Section: The Lhr-ecd: Structure and Hormone Bindingmentioning

confidence: 99%

A functional transmembrane complex: The luteinizing hormone receptor with bound ligand and G protein

Puett

DeMars

et al. 2007

Molecular and Cellular Endocrinology

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“…One paradigm, based on protein engineering to produce fusion proteins, was developed by our laboratory to give constitutive ligand-mediated receptor activation and involved yoking a single chain hCG to the full length receptor (Wu et al 1996, Puett & Narayan 2000, Narayan et al 2000a,b, Schubert et al 2003 and to the receptor ectodomain (Fralish et al 2001). The former approach was used in the present study with full length hLHR in order to permit an evaluation of receptor activation in transfected HEK 293 cells.…”

Section: Structures Of the Yoked Mini-hcgs And Yoked Mini-hcg-hlhr Comentioning

confidence: 99%

“…Using various techniques such as site-directed mutagenesis (Chen & Puett 1991a,b, Chen et al 1991, 1992, Bielinski & Boime 1992, Liu et al 1993, Yoo et al 1993, Puett et al 1994, Puett & Narayan 2000, protein chimeras (Campbell et al 1991, Dias et al 1994, Han et al 1996, Grossmann et al 1997, and synthetic peptides (Salesse et al 1990, Keutmann 1992, many of the regions of hCG important for heterodimer formation and receptor binding and activation have been determined, as well as the suggestion that both subunits are required for full activity. The combined findings from these studies concerning ligand-receptor interactions have indicated that the central portion of the -subunit and the determinant loop (Moore et al 1980) of hCG (residues 93-100), i.e.…”

Section: Introductionmentioning

confidence: 99%

Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit

Schubert

Puett²

2003

Journal of Molecular Endocrinology

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Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common α-subunit and distinct β-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGβ have been separately fused to the N-terminus of the α-subunit to give N-des[1-91]hCGβ-α-C and N-des[1-91,110-114]hCGβ-α-C, i.e. fusion proteins of the hCGβ determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the α-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-β-α cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-β-α was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-β-α fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGβ in addition to the determinant loop/seat belt.

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Designer Gonadotropins and Receptors: The Prospect of Recombinant Technology

Cited by 2 publications

References 48 publications

A functional transmembrane complex: The luteinizing hormone receptor with bound ligand and G protein

A functional transmembrane complex: The luteinizing hormone receptor with bound ligand and G protein

Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit

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