Krassimira Angelova

A model for constitutive lutropin receptor activation based on molecular simulation and engineered mutations in transmembrane helices 6 and 7.

Many naturally occurring and engineered mutations lead to constitutive activation of the G protein-coupled lutropin receptor (LHR), some of which also result in reduced ligand responsiveness. To elucidate the nature of interhelical interactions in this heptahelical receptor and changes thereof accompanying activation, we have utilized site-directed mutagenesis on transmembrane helices 6 and 7 of rat LHR to prepare and characterize a number of single, double, and triple mutants. The potent constitutively activating mutants, D556(6.44)H and D556(6.44)Q, were combined with weaker activating mutants, N593(7.45)R and N597(7.49)Q, and the loss-of-responsiveness mutant, N593(7.45)A. The engineered mutants have also been simulated using a new receptor model based on the crystal structure of rhodopsin. The results suggest that constitutive LHR activation by mutations at Asp-556(6.44) is triggered by the breakage or weakening of the interaction found in the wild type receptor between Asp-556(6.44) and Asn-593(7.45). Whereas this perturbation is unique to the activating mutations at Asp-556(6.44), common features to all of the most active LHR mutants are the breakage of the charge-reinforced H-bonding interaction between Arg-442(3.50) and Asp-542(6.30) and the increase in solvent accessibility of the cytosolic extensions of helices 3 and 6, which probably participate in the receptor-G protein interface. Asn-593(7.45) and Asn-597(7.49) also seem to be necessary for the high constitutive activities of D556(6.44)H and D556(6.44)Q and for full ligand responsiveness. The new theoretical model provides a foundation for further experimental work on the molecular mechanism(s) of receptor activation.

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

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 cysteine-rich 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 Galphas (transducin), and the betagamma 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 Gsalpha 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.

Conserved amino acids participate in the structure networks deputed to intramolecular communication in the lutropin receptor

The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) particularly susceptible to spontaneous pathogenic gain-of-function mutations. Protein structure network (PSN) analysis on wild-type LHR and two constitutively active mutants, combined with in vitro mutational analysis, served to identify key amino acids that are part of the regulatory network responsible for propagating communication between the extracellular and intracellular poles of the receptor. Highly conserved amino acids in the rhodopsin family GPCRs participate in the protein structural stability as network hubs in both the inactive and active states. Moreover, they behave as the most recurrent nodes in the communication paths between the extracellular and intracellular sides in both functional states with emphasis on the active one. In this respect, non-conservative loss-of-function mutations of these amino acids is expected to impair the most relevant way of communication between activating mutation sites or hormone-binding domain and G protein recognition regions.

Structure‐Function Relationships of the Luteinizing Hormone Receptor

Abstract: 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 Gsα. 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 luteinizing hormone receptor: insights into structure-function relationships and hormone-receptor-mediated changes in gene expression in ovarian cancer cells.

The luteinizing hormone receptor (LHR), one of the three glycoprotein hormone receptors, is necessary for critical reproductive processes, including gonadal steroidogenesis, oocyte maturation and ovulation, and male sex differentiation. Moreover, it has been postulated to contribute to certain neoplasms, particularly ovarian cancer. A member of the G protein-coupled receptor family, LHR contains a relatively large extracellular domain responsible for high affinity hormone binding; transmembrane activation then leads to G protein coupling and subsequent second messenger production. This review deals with recent advances in our understanding of LHR structure and structure-function relationships, as well as hormone-mediated changes in gene expression in ovarian cancer cells expressing LHR. Suggestions are also made for critical gaps that need to be filled as the field advances, including determination of the three-dimensional structure of inactive and active receptor, elucidation of the mechanism by which hormone binding to the extracellular domain triggers the activation of Gs, clarification of the putative roles of LHR in non-gonadal tissues, and the role, if any, of activated receptor in the development or progression of ovarian cancer.

Endothelin degradation by vascular smooth muscle cells

The mechanism(s) of degradation of the potent vasoconstrictor endothelin-1 (ET-1) by rat vascular smooth muscle A-10 cells, which possess the ETA receptor subtype, was investigated by incubating [125I]ET-1 (0.1 nM) with cells for 0-4 h at 37 degrees C in the presence and absence of lysosomal enzyme inhibitors, NH4Cl and chloroquine, and a neutral endopeptidase inhibitor, phosphoramidon. The assay buffer and cell extracts were analyzed by reverse-phase HPLC, and the radioactivity in the fractions was measured. In the absence of inhibitors, most of the radioactivity in the medium was in the form of [125I]Tyr after a 4 h incubation. When [125I]ET-1 was incubated with A-10 cells at 4 degrees C, six radiolabeled peaks, including some [125I]Tyr and about 30% of the original [125I]ET-1, were present in the medium. In the presence of 5 microM chloroquine there was no [125I]Tyr peak in the medium, indicating that internalization and putative lysosomal degradation of ET-1 were blocked. NH4Cl (50 and 100 mM) also reduced the amount of [125I]Tyr formed. The presence of ET-1 fragments indicated that, in addition to lysosomal degradation, some of the ligand is metabolized by enzymes located on the cell membrane; we demonstrated, however, that secreted proteases from A-10 cells are not involved in the degradation of ET-1. The neutral endopeptidase inhibitor, phosphoramidon, did not completely inhibit the metabolism of [125I]ET-1 to [125I]Tyr. These results establish that various cell-associated enzymes are capable of degrading ET-1 in A-10 cells. Moreover, analysis of the cell lysates indicated the presence of a relatively stable pool of ET-1-occupied receptors or compartmentalized ET-1, protected from cell proteases, which may contribute to the potent contractility of ET-1.

Functional Differences of Invariant and Highly Conserved Residues in the Extracellular Domain of the Glycoprotein Hormone Receptors

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in β-strands 2 (Lys74), 4 (Tyr124, Asn129, and Thr130), and 5 (Asp150 and Asp153) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in β-strands 2 (Ser78) and 8 (Asp224 and Ser226) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.

Metabolism of endothelin-1 by neuroblastoma x glioma hybrid (NG108-15) cells

The endothelin (ET) peptides, ET-1, ET-2, and ET-3, as well as the ETA and ETB receptor subtypes, are known to occur in brain, but there is a dearth of information on the metabolism of these peptides by the central nervous system (CNS). In this study we have investigated the kinetics of ET-1 binding to and dissociation from the hybrid neuroblastoma x glioma cell line, NG108-15, which is known to contain functional ET receptors, and metabolism of bound ET-1. [125I]ET-1 was incubated with cells for various periods of time up to 6 h, and the nature of the radioactivity in the cell medium and lysate was analyzed by reverse phase high performance liquid chromatography (HPLC). It was found that NG108-15 cells are capable of degrading [125I]ET-1 to [125I]Tyr and several fragments of intermediate hydrophobicity; however, a portion of the cell-associated [125I]ET-1 was protected from degradation for several hours.

IDENTIFICATION OF CONVENTIONAL AND NOVEL ENDOTHELIN RECEPTORS IN SHEEP CHOROID PLEXUS CELLS

Previous studies have demonstrated the presence of super-high affinity endothelin receptors with apparent Kds on the order of pM in different brain tissues. This study was designed to characterize, in detail, the receptors present in SCP cells, a non-transformed sheep choroid plexus cell line. Competitive binding assays with receptor-selective ligands indicated the presence of at least three classes of binding sites: a conventional receptor of the ETA subtype with a Kd = 0.4 nM that mediates an increase in intracellular levels of inositol 1,4,5-trisphosphate (IP3) in response to ET-1 and two additional sites with much higher binding affinities. The latter two sites are not coupled to the common signal transduction pathways of IP3, cAMP and cGMP. Northern blot analysis confirmed the presence of only the ETA subtype mRNA in SCP cells. It remains to determined if the multiple binding sites are distinct gene products, multiple affinity states of a single receptor molecule or a result of cooperative association of one site with either the ligand or with other proteins.

Identification of endothelin receptor subtypes in sheep choroid plexus

Endothelin (ET) and its G-protein-coupled receptors are distributed in a wide variety of tissues, including the brain. In this study, we have identified and characterized the endothelin receptor subtypes in sheep choroid plexus. Competitive binding experiments using [125I]ET-1 and the receptor subtype-selective ligands, ET-1, ET-3, BQ-123, Sarafotoxin 6c, and [Ala1,3,11,15]ET-1 demonstrated the presence of both ETA and ETB receptor subtypes in the ratio of 30:70. In addition, a small fraction of the total binding sites exhibited affinities for ET-1 in the subpicomolar range. Chemical crosslinking of [125I]ET-1 withbis(sulfosuccinimidyl)-suberate (BS3) to choroid plexus membranes revealed the presence of two bands, with apparent molecular masses of 89 and 45 kDa, corresponding to the ETA receptor, and three bands, with apparent molecular masses of 75, 58, and 33 kDa, corresponding to the ETB receptor. Of considerable interest was the finding that dimers of the [125I]ET-1-occupied ETA receptor could be identified by crosslinking, as could apparent dimers and tetramers of [125I]ET-1, but only when bound to receptor. In addition to mapping the distribution of ET receptors in sheep choroid plexus, our results strongly suggest that ET-1 binding to the ETA receptor leads to dimer formation.