Functional domains of the gonadotropin-releasing hormone receptor

Summary 1. The cloning of the mammalian gonadotropin-releasing hormone receptor sets the stage for rapid progress in understanding the structure of the receptor, its interaction with ligand, and its mechanisms of activation.2. The receptor is a 327 to 328-amino acid seven-transmembrane domain G protein-coupled receptor.3. Recent site-direct mutagenesis studies have provided considerable insight into glycosylation of the receptor, the arrangement of the helices, and the ligand binding domains.     

Functional impact of manipulation on the relative orientation of human prolactin receptor domains

Hormone binding creates active receptor dimers for class 1 cytokine receptors; however, the detailed molecular mechanism by which these receptors are activated by their ligands is not well characterized, and it is unknown if these receptors share common mechanisms. A rotation model has been proposed for the activation of human erythropoietin receptor and human growth hormone receptor and is supported by evidence showing that additions of alanine at the junction of the transmembrane (TM) and intracellular (IC) domains and/or within the TM domain influenced receptor activities. This evidence suggests that alanine additions changed the relative orientations of the IC domains and their subsequent activation. We wished to determine if a similar mechanism was at play with human prolactin receptor (hPRLr). Up to four alanines were added between the TM and either the IC or extracellular (EC) domains to extend the TM helix and to rotate the IC or EC domains. Also, up to four glycines were placed between the TM and IC domains to provide increased flexibility between these two domains. Wild-type hPRLr or various mutant receptors were expressed in human embryonic kidney 293T cells that express endogenous Janus kinase 2. In the absence of human prolactin (hPRL), none of the alanine or glycine additions increased the level of receptor phosphorylation above that of wild-type hPRLr. In the presence of hPRL, both wild-type hPRLr and each of the mutant receptors were successfully phosphorylated. These data do not support a rotation mechanism for hPRLr activation or a requirement of a fixed spatial relationship between the TM and IC domains for hPRLr activation. In a second set of experiments, both wild-type hPRLr and either alanine- or glycine-extended receptors were coexpressed in 293T cells. In the absence of hPRL, there was no detectable phosphorylation of hPRLr. Such data do not support a piston movement between the hPRLr pair in their activation.     

Functional analysis of the cytoplasmic domains of the human thyrotropin receptor by site-directed mutagenesis

The thyrotropin (TSH) receptor belongs to a family of guanine nucleotide protein-coupled receptors with seven transmembrane-spanning regions joined regulatory together by extracellular and intracellular loops. The cytoplasmic domain comprises three cytoplasmic loops and a cytoplasmic tail that are likely to be important in coupling of the receptor to the guanine nucleotide proteins. To address the question of which portions of the cytoplasmic domain of the TSH receptor are important in this process, we have altered groups of amino acids in the region of the TSH receptor by site-directed mutagenesis. Because of the low affinity of TSH binding to the TSH receptor mutated in the amino terminus of the second cytoplasmic loop and the amino terminus of the cytoplasmic tail, definitive conclusions cannot be made regarding the roles of these regions in signal transduction. However, our data indicate that the first cytoplasmic loop (residues 441-450), the carboxyl-terminal region of the second cytoplasmic loop (residues 528-537), and the carboxyl-terminal (but not the amino-terminal) region of the third cytoplasmic loop (residues 617-625) are important in the ability of the TSH receptor to mediate an increase in intracellular cAMP production. Furthermore, two-thirds of the carboxyl-terminal end of the cytoplasmic tail (residues 709-764; corresponding to the region not conserved between the TSH and lutropin/chorionic gonadotropin receptors) can be removed without functional impairment of the TSH receptor.     

Functional domains of the 67-kDa laminin receptor precursor

We report the characterization of two functional domains of the metastasis-associated 67-kDa laminin receptor (67-LR). Using synthetic peptides deduced from the cDNA sequence of the 37-kDa precursor of the laminin receptor (37-LRP) as well as their corresponding affinity-purified polyclonal antibodies, we identified a unique laminin binding site as well as a membrane-associated domain of the receptor. In laminin dot blot and solid phase radioligand assays, a 20 amino acid synthetic peptide (IPCNNKGAHSVGLMWWMLAR, amino acid residues 161-180, designated peptide G) specifically bound to laminin with high affinity (Kd = 5 x 10(-8) M). Peptide G also specifically eluted the 67-LR from a laminin affinity column. Peptide G and laminin reacted with a 1:1 stoichiometry, suggesting that there is one recognition site on laminin for the peptide G domain. Immunofluorescence studies, performed on permeabilized and nonpermeabilized human A2058 melanoma cells using 10 different affinity-purified antibodies to distinct regions of the 37-LRP, identified an unusually short membrane-associated domain that was consistent with a computer predicted transmembrane domain (residues 86-101). Our data demonstrate for the first time that the 37-LRP has two functional domains consistent with the characteristics of the mature 67-LR. Furthermore, we propose peptide G as a potential inhibitor of tumor cell interactions with laminin.     

Functional domains of the granulocyte colony-stimulating factor receptor.

The granulocyte colony-stimulating factor (G-CSF) receptor has a composite structure consisting of an immunoglobulin(Ig)-like domain, a cytokine receptor-homologous (CRH) domain and three fibronectin type III (FNIII) domains in the extracellular region. Introduction of G-CSF receptor cDNA into IL-3-dependent murine myeloid cell line FDC-P1 and pro-B cell line BAF-B03, which normally do not respond to G-CSF, enabled them to proliferate in response to G-CSF. On the other hand, expression of the G-CSF receptor cDNA in the IL-2-dependent T cell line CTLL-2 did not enable it to grow in response to G-CSF, although G-CSF could transiently stimulate DNA synthesis. Mutational analyses of the G-CSF receptor in FDC-P1 cells indicated that the N-terminal half of the CRH domain was essential for the recognition of G-CSF, but the Ig-like, FNIII and cytoplasmic domains were not. The CRH domain and a portion of the cytoplasmic domain of about 100 amino acids in length were indispensable for transduction of the G-CSF-triggered growth signal.     

Different estrogen receptor structural domains are required for estrogen- and tamoxifen-dependent anti-proliferative activity in human mammary epithelial cells expressing an exogenous estrogen receptor

Estrogen (E) inhibits the growth of both non-tumorigenic, immortal human mammary epithelial cells (HMEC) and breast cancer cells which stably express exogenous estrogen receptors (ER). The anti-estrogenic compounds 4-hydroxy-tamoxifen (HT) and ICI 164384 (ICI) have different effects on the growth of the ER-transfectants. HT is a potent growth inhibitor, while ICI has no effect by itself but is able to block the anti-proliferative effects of E and HT. In order to elucidate the mechanism by which E or HT-bound ER inhibit cell growth, we have evaluated the effects of these compounds on the growth of HMEC stably expressing ER with mutations or deletions in the N-terminal A/B domain, the DNA-binding domain (DBD), and the C-terminal ligand-binding domain. These studies revealed that E and HT require different structural domains of the ER for their anti-proliferative activities. The N-terminal A/B domain is required for HT-, but not E-dependent growth inhibition. The DNA-binding domain of the ER is not essential for HT-mediated anti-proliferative effects, but is important for E-dependent activity. The effect of ER mutations on the ligand-inducible expression of the endogenous progesterone receptor (PR) and pS2 genes was also evaluated. Neither gene was induced in the cells containing the ER mutated in the DBD, even though cell growth was inhibited. These results suggest that E and HT use different pathways to elicit their anti-proliferative effects and that this occurs via modulation of genes that are controlled by mechanisms different from those important for activation of the PR and pS2 genes.     

Functional domains of the human epididymal protease inhibitor, eppin

Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram-negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin's domains are involved in the protein's antibacterial activity, only the Kunitz domain is required for selective protease inhibition.     

Functional properties of extracellular domains of transducer receptor gp130

Cytokine receptor molecules have been shown to have extracellular domains of complex structure and a multistep activation system. Glycoprotein gp130 is a typical transducer of cytokine signal; it functions by forming multicomponent receptor complexes and transferring signals of tens of cytokines from the IL-6 family. Structural organization and basic functioning principles of gp130 are well known, as well as related signal pathways, which function during normal differentiation and are involved in pathogenesis of many tumors. The role of gp130 in IL-6-dependent tumors is best studied. In this review, based on extensive accumulated data, we examine the functional significance of certain parts of gp130 extracellular domains. Potentials of a recently developed method for estimation of receptor activation at the level of epitope structure are discussed.     

Functional domains of the subtype 1 neurotensin receptor (NTS1)

The subtype 1 neurotensin receptor (NTS1) belongs to the family of G protein coupled receptors with seven transmembrane domains and mediates most of the known effects of neurotensin. In the past years, mutagenesis studies have allowed to delineate functional regions of the receptor involved in agonist and antagonist binding, G protein coupling, sodium sensitivity of agonist binding, and agonist-induced receptor internalization. These data are reviewed and discussed in the present paper.     

Expression of the estrogen receptor during differentiation of human osteoclasts

Estrogens play a key role in bone structural integrity, which is maintained by the opposing activity of bone forming osteoblasts and bone resorbing osteoclasts. The cellular effects of estrogens are mediated by estrogen receptors, however, the detailed molecular mechanism of ER regulation in osteoclasts has not yet been elucidated. We provide here a detailed analysis of the expression profile and functionality of ER during osteoclast differentiation. We employed a human primary osteoclast cell culture model to evaluate the regulation of estrogen receptor (ER) variant expression. We characterized the expression profile of estrogen receptors and studied the regulation of the predominant estrogen receptor-alpha (ER-alpha) during differentiation into osteoclasts. In addition to the full-length ER-alpha, a shorter ER-alpha mRNA variant is expressed and both ER-alpha variants are regulated during osteoclastogenesis. Furthermore, we show that the pS2 gene is an estrogen-regulated gene in osteoclasts. Analysis of the activity of the pS2 gene throughout differentiation, using chromatin immunoprecipitation (ChIP), revealed the functionality of ER-alpha during differentiation and shows that the occupancy of ER-alpha and activated polymerase II on the pS2 promoter decrease with time and can be blocked by the ER antagonist ICI 182780. These results help to dissect the molecular events relevant to estrogen signaling and provide a better understanding of the role of ER-alpha regulation during bone resorption mediated by osteoclasts.     

Multiple and cooperative trans-activation domains of the human glucocorticoid receptor

A 30 amino acid peptide (referred to as tau 2) that functions as an activation domain has been localized in the carboxyl terminus of the human glucocorticoid receptor. This sequence, when fused to yeast GAL4 as part of the ligand binding domain, generates a hormone-inducible activator. Tau 2 functions in a position-independent fashion and leads to a progressive gain of function when multimerized. A similar and independent activity has also been identified in the amino terminus of the receptor. These two sequences, although structurally unrelated, are both acidic in character and thus may have certain properties in common with yeast activator sequences.     

Functional dissection of structural domains in the receptor for colony-stimulating factor-1.

Receptor tyrosine kinases (RTKs) transduce external signals to the interior of the cell via a cytoplasmic kinase domain. We demonstrated previously that ligand-induced kinase activation of the colony-stimulating factor-1 receptor (CSF-1R) occurs via receptor oligomerization without propagation of conformational changes through the transmembrane (TM) domain (Lee, A. W., and Nienhuis, A. W. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7270-7274). We have now examined the role of the different subdomains in the metabolic and signaling properties of CSF-1R. Two types of chimeric receptors have been utilized, Glyfms A, with the extracellular and TM domains of glycophorin A (GpA) and the cytoplasmic domain of CSF-1R, and Glyfms B, where only the extracellular domain originates from GpA. Glyfms A was found to exhibit a higher basal level of in vitro kinase activity, an increased associated phosphatidylinositol (PtdIns) 3-kinase activity and to support enhanced cellular mitogenesis, compared with wild-type CSF-1R or to Glyfms B. The constitutive activation of Glyfms A is consistent with the hypothesis that the TM domain may play a role in receptor oligomerization. Cross-linking with anti-GpA antibodies activated the kinase function of Glyfms B leading to an increase in PtdIns 3-kinase association and to the transmission of a mitogenic signal. Our results indicate that an activated kinase domain contains the major determinant for coupling with PtdIns 3-kinase, independent of extracellular and TM sequences and of ligand binding. Both chimeric receptors underwent internalization in the presence of anti-GpA antibodies but were not degraded, including the tyrosine-phosphorylated and kinase-active population. These results suggest that structural determinants in the extracellular domain must be important for targeting internalized receptors for lysosomal degradation.