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.
Of the 800-900 genes in the human genome that appear to encode G-protein-coupled receptors (GPCRs), two are known to encode receptors that bind the three heterodimeric human gonadotropins, luteinizing hormone (LH), chorionic gonadotropin (CG), and follicle-stimulating hormone (FSH). LH and CG bind to a common receptor, LHR, and FSH binds to a paralogous receptor. These GPCRs contain a relatively large ectodomain (ECD), responsible for high-affinity ligand binding, and a transmembrane portion, as in the other GPCRs. The ECD contains nine leucine-rich repeats capped by N-terminal and C-terminal cysteine-rich regions. The overall goal of this research is to elucidate the molecular mechanisms by which CG and LH bind to and activate LHR and the latter, in turn, activates Gs alpha. A combination of molecular modeling and site-directed mutagenesis, coupled with binding and signaling studies in transiently transfected HEK 293 cells expressing wild-type and mutant forms of LHR, has been used to develop and test models for the LHR ECD, the CG-LHR ECD complex, and the structural changes in the transmembrane helices and intracellular loops, particularly loop 2, that accompany receptor activation. In addition, a single-chain CG-LHR complex was designed in which a fusion protein of the two subunits of human CG was linked to full-length LHR. This ligand-receptor complex was shown to be constitutively active in cellular models and in transgenic mice, the latter of which exhibit precocious puberty. From a combination of molecular modeling, site-directed mutagenesis, genetic/protein engineering, and receptor characterization in cellular and animal models, considerable insight is being developed on the mechanisms of normal and aberrant activation of LHR.
The mechanisms of G protein coupling to G protein-coupled receptors (GPCR) share general characteristics but may exhibit specific interactions unique for each GPCR/G protein partnership. The extreme C terminus (CT) of G protein α-subunits has been shown to be important for association with GPCR. Hypothesizing that the extreme CT of Gα(s) is an essential component of the molecular landscape of the GPCR, human LH receptor (LHR), and β(2)-adrenergic receptor (β(2)-AR), a model cell system was created for the expression and manipulation of Gα(s) subunits in LHR(+) s49 ck cells that lack endogenous Gα(s). On the basis of studies involving truncations, mutations, and chain extensions of Gα(s), the CT was found to be necessary for LHR and β(2)-AR signaling. Some general similarities were found for the responses of the two receptors, but significant differences were also noted. Computational modeling was performed with a combination of comparative modeling, molecular dynamics simulations, and rigid body docking. The resulting models, focused on the Gα(s) CT, are supported by the experimental observations and are characterized by the interaction of the four extreme CT amino acid residues of Gα(s) with residues in LHR and β(2)-AR helix 3, (including R of the DRY motif), helix 6, and intracellular loop 2. This portion of Gα(s) recognizes the same regions of the two GPCR, although with differences in the details of selected interactions. The predicted longer cytosolic extensions of helices 5 and 6 of β(2)-AR are expected to contribute significantly to differences in Gα(s) recognition by the two receptors.
Insects have extraordinary species richness: over a million species have been identified, and even more await discovery and classification. Given their abundance and diversity, insects are excellent teaching tools for science classrooms. However, accurate insect identification can be especially challenging for beginning students. Accordingly, we have developed a dichotomous key that both precollege and university instructors and students can use efficiently to correctly identify 18 taxonomic orders of insects. Our key was developed to target insects most commonly encountered throughout the coastal southeastern United States, but it can easily be adapted to other regions. This key is novel in that it incorporates not only adult insects but also their immature stages. In addition, we included insects that are likely to be collected in all seasons, facilitating implementation in the classroom throughout the academic year.
In human adrenarche during childhood, the secretion of dehydroepiandrosterone (DHEA) from the adrenal gland increases due to its increased synthesis and/or decreased metabolism. DHEA is synthesized by 17a-hydroxylase/17,20-lyase, and is metabolized by 3b-hydroxysteroid dehydrogenase type 2 (3bHSD2). In this study, the inhibition of purified human 3bHSD2 by the adrenal steroids, androstenedione, cortisone, and cortisol, was investigated and related to changes in secondary enzyme structure. Solubilized, purified 3bHSD2 was inhibited competitively by androstenedione with high affinity, by cortisone at lower affinity, and by cortisol only at very high, nonphysiologic levels. When purified 3bHSD2 was bound to lipid vesicles, the competitive K i values for androstenedione and cortisone were slightly decreased, and the K i value of cortisol was decreased 2.5-fold, although still at a nonphysiologic level. The circular dichroism spectrum that measured 3bHSD2 secondary structure was significantly altered by the binding of cortisol, but not by androstenedione and cortisone. Our import studies show that 3bHSD2 binds in the intermitochondrial space as a membraneassociated protein. Androstenedione inhibits purified 3bHSD2 at physiologic levels, but similar actions for cortisol and cortisone are not supported. In summary, our results have clarified the mechanisms for limiting the metabolism of DHEA during human adrenarche.
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