Overexpression and functional characterization of the extracellular domain of the human alpha1 glycine receptor

A novel truncated form (residues 1-214, with a randomized C-terminal tail) of the ligand-binding extracellular domain (ECD) of the human alpha1 glycine receptor (GlyR), with amino acids from the corresponding sequence of an acetylcholine binding protein (AChBP) substituted for two relatively hydrophobic membrane-proximal loops, was overexpressed using a baculovirus expression system. The mutant GlyR ECD, named GlyBP, was present in both soluble and membrane-associated fractions after cell lysis, though only the latter appeared to be in a native-like conformation capable of binding strychnine, a GlyR specific antagonist. The membrane-associated GlyBP was solubilized, and detergent/lipid/protein micelles were affinity purified. After detergent removal, GlyBP may be isolated in either aqueous or vesicular form. Binding assays and spectroscopic studies using circular dichroism and FRET are consistent with both forms adopting equivalent native-like conformations. Thus, GlyBP may be isolated as a soluble or membrane-associated assembly that serves as a structural and functional homologue of the ECD of GlyR.     

Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor

The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor alpha1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.     

Identification of proteins with high affinity for refolded and native PrPC

PrPC, the cellular prion protein, is widely expressed in most tissues, including brain, muscle and the gastrointestinal tract, but its physiological role remains unclear. During propagation of transmissible spongiform encephalopathies (TSEs), prion protein is converted to the pathological isoform, PrPSc, in a process believed to be mediated by as-yet-unknown host factors. The identification of proteins associated with PrP may provide information about the biology of prions and the pathogenesis of TSEs. In the present work, we report proteins identified from brain tissue based on their ability to bind to recombinant PrP (recPrP) or form multimolecular complexes with native PrPC in the presence of cross-linkers. Immobilized his-tagged recPrP was used as an affinity matrix to isolate PrP-interacting proteins from brain homogenates of normal individuals. In parallel, PrPC-associated proteins were characterized by cross-linking and co-immunoprecipitation assays. The unknown molecules were identified by MS and the results of LC-MS/MS analysis were subsequently verified by Western blot. Both techniques resulted in identification of proteins participating in the formation of cytoskeleton and signal transduction, further supporting the hypothesis that PrP is involved in the organization and function of receptors throughout the nervous system.     

Mammalian alpha 1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit

alpha 1-Adrenergic receptors from a cultured smooth muscle cell line (DDT1 MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl) pentanoyl]-1-piperazinyl]-quinazoline), an alpha 1 receptor-selective antagonist), a wheat germ agglutinin-agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent Mr = 80,000 that co-migrates with the peptide labeled by the specific alpha 1-adrenergic receptor photoaffinity probe 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[125I]iodophenyl)pentanoyl] -1-piperazinyl] quinazoline. The specific activity (approximately 13,600 pmol of ligand binding/mg of protein) of purified receptor preparations is consistent with that expected for a pure peptide of Mr = 80,000 containing a single ligand binding site. Overall yields approximate 14% of initial crude particulate binding. The purified receptor preparations bind agonist and antagonist ligands with appropriate alpha 1-adrenergic specificity, stereoselectivity, and affinity. Peptide maps of the pure alpha 1-adrenergic receptor and the pure human platelet alpha 2-adrenergic receptor (Regan, J.W., Nakata, H., DeMarinis, R.M., Caron, M.G., and Lefkowitz, R.J. (1986) J. Biol. Chem. 261, 3894-3900) using several different proteases suggest that these two receptors show little if any structural homology.     

Retention of ligand binding activity by the extracellular domain of the IL-1 receptor

The IL-1R on murine T cells is an 80-kDa cell surface glycoprotein which binds both IL-1 alpha and IL-1 beta. We have recently isolated a cDNA clone encoding this molecule. From the primary sequence mature receptor is predicted to be a 557 residue integral membrane protein with a 319 residue carbohydrate-rich extracellular region. We have constructed a cDNA clone encoding this region of the protein (residues 1 to 316). Expression of this cDNA in HeLa cells leads to secretion of a soluble IL-1 alpha binding protein into the culture medium. Quantitative binding experiments with the truncated receptor show that it possesses IL-1 binding properties which are indistinguishable from those of full length IL-1R. Gel filtration chromatography experiments show that a complex can be formed between a single truncated receptor molecule and a single IL-1 alpha molecule.     

The human alpha 2-macroglobulin receptor contains high affinity calcium binding sites important for receptor conformation and ligand recognition.

The receptor for alpha 2-macroglobulin-proteinase complexes (alpha 2MR) was purified recently, and its binding of ligand was shown to depend on calcium ions (Moestrup, S. K., and Gliemann, J. (1989) J. Biol. Chem. 264, 15574-15577). This paper shows that the 440-kDa human placental alpha 2MR is a cysteine-rich glycoprotein with high affinity calcium binding sites important for receptor conformation; and the relationship between Ca2+ concentration and receptor function is presented. Autoradiography showed 45Ca2+ binding to the 440-kDa alpha 2MR blotted onto nitrocellulose from a sodium dodecyl sulfate-polyacrylamide gel. alpha 2MR immobilized on nitrocellulose in the absence of sodium dodecyl sulfate bound 45Ca2+ in the presence of 5 mM Mg2+, and 2-3 microM unlabeled Ca2+ was required to displace half of the bound 45Ca2+. The calcium concentration dependence showed upward concave Scatchard plots, and the number of binding sites was estimated to be approximately eight/alpha 2MR molecule. Binding of calcium did not change in the pH range 6.5-8.0 but decreased at lower pH values. Addition of Ca2+ to the medium was necessary for receptor binding of the alpha 2-macroglobulin-trypsin complex, and half of the maximal binding capacity was obtained with about 16 micrograms Ca2+ at pH 7.8. The requirement for calcium was increased at lower pH values, and half of the maximal 125I-alpha 2M-trypsin binding was obtained with about 30-40 microM Ca2+ at pH 7.0. Monoclonal antibodies were produced against alpha 2MR, and one of them distinguished between the Ca2(+)-occupied and nonoccupied forms. Like Ca2+, Sr2+ and Ba2+ elicited ligand binding affinity and competed for binding with 45Ca2+ in the order Ca2+ greater than Sr2+ greater than Ba2+. In conclusion, calcium ions bind specifically to alpha 2MR with high affinity, and it is likely that several sites on the alpha 2MR molecule have to be occupied to elicit the conformation recognizing the ligand.     

Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet alpha 2-adrenergic receptor

The human platelet alpha 2-adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known [Kobilka et al. (1987) Science 238, 650-656]. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, we have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [3H]SKF 102229 (an antagonist) or p-azido[3H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [3H]SKF 102229 labeled receptor yielded one peptide of Mr 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of Mr 4000, which was further digested to the Mr 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[3H]clonidine-labeled receptor, a similar Mr 2400 peptide was obtained by lysylendopeptidase cleavage. This Mr 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet alpha 2-adrenergic receptor.     

Homology modeling and molecular dynamics simulations of the glycine receptor ligand binding domain

We present a homology based model of the ligand binding domain (LBD) of the homopentameric alpha1 glycine receptor (GlyR). The model is based on multiple sequence alignment with other members of the nicotinicoid ligand gated ion channel superfamily and two homologous acetylcholine binding proteins (AChBP) from the freshwater (Lymnaea stagnalis) and saltwater (Aplysia californica) snails with known high resolution structure. Using two template proteins with known structure to model three dimensional structure of a target protein is especially advantageous for sequences with low homology as in the case presented in this paper. The final model was cross-validated by critical evaluation of experimental and published mutagenesis, functional and other biochemical studies. In addition, a complex structure with strychnine antagonist in the putative binding site is proposed based on docking simulation using Autodock program. Molecular dynamics (MD) simulations with simulated annealing protocol are reported on the proposed LBD of GlyR, which is stable in 5 ns simulation in water, as well as for a deformed LBD structure modeled on the corresponding domain determined in low-resolution cryomicroscopy structure of the alpha subunit of the full-length acetylcholine receptor (AChR). Our simulations demonstrate that the beta-sandwich central core of the protein monomer is fairly rigid in the simulations and resistant to deformations in water.     

Biochemical characterization of the high affinity binding between the glycine receptor and gephyrin

Gephyrin is an essential and instructive molecule for the formation of inhibitory synapses. Gephyrin binds directly to the large cytoplasmic loop located between transmembrane helices three and four of the beta-subunit of the glycine receptor and to microtubules, thus promoting glycine receptor (GlyR) anchoring to the cytoskeleton and clustering in the postsynaptic membrane. Besides its structural role, gephyrin is involved in the biosynthesis of the molybdenum cofactor that is essential for all molybdenum-dependent enzymes in mammals. Gephyrin can be divided into an N-terminal trimeric G domain and a C-terminal E domain, which are connected by a central linker region. Here we have studied the in vitro interaction of gephyrin and its domains with the large cytoplasmic loop of the GlyR beta-sub-unit (GlyRbeta-loop). Binding of gephyrin to the GlyR is exclusively mediated by the E domain, and the binding site was mapped to one of its sub-domains (residues 496-654). By using isothermal titration calorimetry, a high affinity (K(d) = 0.2-0.4 microm) and low affinity (K(d) = 11-30 microm) binding site for the GlyRbeta-loop was found on holo-gephyrin and the E domain, respectively, with a binding stoichiometry of two GlyRbeta-loops per E domain in both cases. Binding of the GlyRbeta-loop does not change the oligomeric state of either full-length gephyrin or the isolated E domain.     

Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor

STRA6 is a multitransmembrane domain protein not homologous to any other proteins with known function. It functions as the high-affinity receptor for plasma retinol binding protein (RBP) and mediates cellular uptake of vitamin A from the vitamin A-RBP complex. Consistent with the diverse roles of vitamin A and the wide tissue expression pattern of STRA6, mutations in STRA6 are associated with severe pathological phenotypes in humans. The structural basis for STRA6's biochemical function is unknown. Although computer programs predict 11 transmembrane domains for STRA6, its topology has never been studied experimentally. Elucidating the transmembrane topology of STRA6 is critical for understanding its structure and function. By inserting an epitope tag into all possible extracellular and intracellular domains of STRA6, we systematically analyzed the accessibility of each tag on the surface of live cells, the accessibility of each tag in permeabilized cells, and the effect of each tag on RBP binding and STRA6-mediated vitamin A uptake from the vitamin A-RBP complex. In addition, we used a new lysine accessibility technique combining cell-surface biotinylation and tandem-affinity purification to study a region of the protein not revealed by the epitope tagging method. These studies not only revealed STRA6's extracellular, transmembrane, and intracellular domains but also implicated extracellular regions of STRA6 in RBP binding.     

Ligand-induced conformational change in the alpha7 nicotinic receptor ligand binding domain

Molecular dynamics simulations of a homology model of the ligand binding domain of the alpha7 nicotinic receptor are conducted with a range of bound ligands to induce different conformational states. Four simulations of 15 ns each are run with no ligand, antagonist d-tubocurarine (dTC), agonist acetylcholine (ACh), and agonist ACh with potentiator Ca(2+), to give insight into the conformations of the active and inactive states of the receptor and suggest the mechanism for conformational change. The main structural factor distinguishing the active and inactive states is that a more open, symmetric arrangement of the five subunits arises for the two agonist simulations, whereas a more closed and asymmetric arrangement results for the apo and dTC cases. Most of the difference arises in the lower portion of the ligand binding domain near its connection to the adjacent transmembrane domain. The transfer of the more open state to the transmembrane domain could then promote ion flow through the channel. Variation in how subunits pack together with no ligand bound appears to give rise to asymmetry in the apo case. The presence of dTC expands the receptor but induces rotations in alternate directions in adjacent subunits that lead to an asymmetric arrangement as in the apo case. Ca(2+) appears to promote a slightly greater expansion in the subunits than ACh alone by stabilizing the C-loop and ACh positions. Although the simulations are unlikely to be long enough to view the full conformational changes between open and closed states, a collection of different motions at a range of length scales are observed that are likely to participate in the conformational change.     

Mapping of the glycine receptor alpha 2-subunit gene and the GABAA alpha 3-subunit gene on the mouse X chromosome.

We have mapped the gene for the alpha 2-subunit of the inhibitory glycine receptor (Glra2) to the telomeric end of the mouse X chromosome by backcross analysis of a Mus musculus/Mus spretus interspecific cross. In addition, we have extended the mapping of the GABAA alpha 3-subunit receptor gene (Gabra3). A deduced gene order of cen-Cybb-Hprt-DXPas6-Gabra3-Rsvp-Gdx/Cf-8- Dmd-Pgk-1-DXPas2-Plp-DXPas1-Glra2-tel places Gabra3 proximal to the visual pigment gene Rsvp and Glra2 in the region of loci for hypophosphatemia (Hyp), steroid sulfatase (Sts), and the E1 alpha-subunit of pyruvate dehydrogenase (Pdha1). This establishes the XF region of the mouse X chromosome as homologous with the Xp22.1-p22.3 region of the human X chromosome and indicates the presence of an evolutionary breakpoint in the region of Xp21.3.     

EGF induces increased ligand binding affinity and dimerization of soluble epidermal growth factor (EGF) receptor extracellular domain.

The binding of epidermal growth factor (EGF) to its cell surface receptor (EGF-R) results in a number of intracellular responses including the activation of the receptor intracellular tyrosine kinase. Receptor oligomerization induced by ligand binding has been suggested to play an important role in signal transduction. However, the mechanisms involved in oligomerization and signal transduction are poorly understood. We have produced and purified several milligrams of recombinant extracellular domain of the EGF receptor (EGF-Rx) using the baculovirus/insect cell expression system. The baculovirus-generated EGF-Rx is glycosylated, has had its signal peptide correctly cleaved, and exhibits a dissociation constant for EGF similar to that for solubilized full-length receptor, of about 100 nM. The binding of EGF to EGF-Rx leads to the formation of receptor dimers and higher oligomerization states which are irreversibly captured using the covalent cross-linking agent disuccinimidyl suberate. Interestingly, purified receptor monomers and dimers, stabilized by the cross-linker in the presence of EGF, exhibit increased binding affinity toward EGF as compared with receptor monomers which have not been exposed to EGF. It appears that the high affinity state of receptor can be maintained by the covalent cross-linking agent. These results indicate that in addition to ligand binding, the extracellular domain of EGF receptor possesses the inherent ability to undergo ligand-induced dimerization and that the low affinity state is converted to a high affinity state by EGF.     

The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily.     

Conserved features in the extracellular domain of human toll-like receptor 8 are essential for pH-dependent signaling

Toll-like receptor (TLR) 8 has an important role in initiating immune responses to viral single-stranded RNA and the antiviral compound resiquimod. Together with TLR3, -7, and -9, it forms a subgroup of the TLRs that are localized intracellularly and signal in response to pathogen-derived nucleic acids. In this work, we have used site-directed mutagenesis to identify regions of the TLR8 extracellular domain that are required for stimulus-induced signal transduction. We have shown that a cysteinerich sequence predicted to form a loop projecting from the solenoidal ectodomain structure at leucine-rich repeat 8 is essential for signaling in response to both single-stranded RNA and resiquimod. A second region, centered on an aspartic acid residue in leucine-rich repeat 17, is also required for TLR8 function. The corresponding residue in TLR9 is known to be important for pH-dependent binding and signaling in response to unmethylated CpG DNA, suggesting that the TLR7/8/9 subgroups share a common signaling mechanism. We have also shown that TLR8 is localized predominantly in the endoplasmic reticulum but that signaling is completely abolished by an inhibitor of vesicle-type H+ ATPases. This indicates that TLR8 is present at low levels in an acidified compartment and that a lowered pH is required for receptor function. We propose that pH-dependent changes in the ligand facilitate activation of the receptor. The protonated form of resiquimod, a cell-permeable weak base, is likely to concentrate significantly (approximately 100x) in acidified compartments, and this may potentiate low affinity interactions with either the receptor or a specific binding protein.     

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.     

The alpha subunit of the human IgE receptor (FcERI) is sufficient for high affinity IgE binding

The alpha subunit of the FcERI binds IgE with high affinity. Previous studies have demonstrated that alpha subunit expression requires the presence of beta and/or gamma subunits, and it is not known how these two subunits contribute to the ability of the alpha subunit to bind IgE. In this report, we describe the expression and characterization of a human chimeric alpha subunit. The data demonstrate that high affinity IgE binding does not require the presence of the beta and/or gamma subunits and that this activity is localized to the extracellular domain (residues 26-201) of the human alpha subunit. Permanent cell lines expressing the chimeric receptor were used to characterize the binding parameters of the alpha subunit. These cell lines provide a means of identifying therapeutic agents which may be effective in the treatment/management of allergic diseases.     

Expression of human IL-13 receptor alpha2 extracellular domain in Pichia pastoris

Interleukin-13 receptor alpha2 (IL-13Ralpha2) binds IL-13 with high affinity and plays an important role in IL-13 signaling as a decoy receptor. We expressed the extracellular domain of human IL-13Ralpha2 (1-313) in methylotrophic yeast Pichia pastoris. SDS-PAGE analysis by PAS staining and Western blot analysis detected the product of the extracellular domain of human IL-13Ralpha2 as glycoprotein from P. pastoris. The yield of purified extracellular domain of human IL-13Ralpha2 was 2mg from 1L of culture. From CD analysis, the 2D structure of the purified IL-13Ralpha2 showed the typical beta-sheet. ELISA of the purified IL-13Ralpha2 detected the binding activity for human IL-13. Thus, it was found that the active extracellular domain of human IL-13Ralpha2 was expressed from P. pastoris.