Eleven amino acids (Lys-201 to Lys-211) and 9 amino acids (Gly-222 to Leu-230) in the human thyrotropin receptor are involved in ligand binding.

Our previous studies involving chimeric thyrotropin-lutropin/choriogonadotropin (TSH-LH/CG) receptors suggest that multiple segments spanning the entire extracellular domain of the human TSH receptor contribute to the TSH binding site. Nevertheless, the mid-region (segment C, amino acid residues 171-260) of the receptor extracellular domain is particularly important in TSH binding. In the present studies, we constructed seven new chimeric receptors in order to analyze segment C in further detail. Seven small segments spanning segment C of the TSH receptor were replaced with the counterpart of the rat LH/CG receptor. These mutant receptors were stably introduced into Chinese hamster ovary cells and were tested for hormone binding and cAMP responsiveness to hormone stimulation. The results indicate that 11 amino acids of the TSH receptor (Lys-201 to Lys-211) and the corresponding region of the LH/CG receptor (Thr-202 to Ile-212) are important for specific TSH and human CG binding, respectively. In addition, nine amino acids of the TSH receptor (Gly-222 to Leu-230) are also involved in TSH binding. A further conclusion from these data is that TSH and human CG bind to partially overlapping sites on their respective receptor molecules.     

Role of cysteine residues in the extracellular domain and exoplasmic loops of the transmembrane domain of the TSH receptor: effect of mutation to serine on TSH receptor activity and response to thyroid stimulating autoantibodies.

The extracellular domain of the thyrotropin (TSH) receptor is the primary site with which TSH and receptor autoantibodies interact. Cysteines 494 or 569 in the 1st and 2nd exoplasmic loops, respectively, of the transmembrane domain of the TSH receptor are important in this process or in coupling ligand binding to signal generation. Thus, when either is mutated to serine, a receptor results which has no detectable TSH binding and no cAMP response to TSH or thyroid stimulating autoantibodies after transfection, despite the fact the mutant receptor is normally synthesized, processed, and integrated in the membrane, as evidenced by Western blotting using a TSH receptor-specific antibody. Additional site directed mutagenesis studies are performed in order to identify cysteine residues in the extracellular domain of the receptor which, with cysteines 494 and 569, are important for tertiary structure and receptor bioactivity.     

Extracellular domain chimeras of the TSH and LH/CG receptors reveal the mid-region (amino acids 171-260) to play a vital role in high affinity TSH binding

We constructed a series of TSH-LH/CG receptor chimeras by homologous substitution of relatively small regions of the TSH receptor extracellular domain for the corresponding region of the extracellular domain of the LH/CG receptor. Constructs were stably expressed in Chinese hamster ovary cells. Of the five chimeric receptors, only TSH-LHR-14, which contains mid-region domain C (amino acid residues 171-260) of the extracellular component of the TSH receptor, exhibited TSH binding of relatively high affinity. Consistent with this TSH binding, chimera TSH-LHR-14 was the only one that demonstrated a functional response to TSH stimulation in terms of intracellular cAMP generation. These data indicate that domain C plays a vital role in TSH receptor function.     

Identification of separate determinants on the thyrotropin receptor reactive with Graves' thyroid-stimulating antibodies and with thyroid-stimulating blocking antibodies in idiopathic myxedema: these determinants have no homologous sequence on gonadotropin receptors.

Deletions, substitutions, or mutations of the rat TSH receptor extracellular domain between residues 20 and 107 (all residue numbers are determined by counting from the methionine start site) have been made by site-directed mutagenesis of receptor cDNA. After transfection in Cos-7 cells, constructs were evaluated for their ability to bind [125I]TSH or respond to TSH and thyroid-stimulating antibodies (TSAbs) from Graves' patients in assays measuring cAMP levels of the transfected cells. Assay results were compared to results from Cos-7 cells transfected with wild-type receptor constructs or vector alone. We identify threonine-40 as a TSAb-specific site whose mutation to asparagine, but not alanine, reduces TSAb activity 10-fold, but only minimally affects TSH-increased cAMP levels. We show that thyroid-stimulating blocking antibodies (TSBAbs), which block TSH or TSAb activity and are found in hypothyroid patients with idiopathic myxedema, continue to inhibit TSH-stimulated cAMP levels when threonine-40 is mutated to asparagine or alanine, suggesting that TSBAbs interact with different TSH receptor epitopes than the TSAb autoantibodies in Graves' patients. This is confirmed by the demonstration that these TSBAbs interact with high affinity TSH-binding sites previously identified at tyrosine-385 or at residues 295-306 of the extracellular domain of the TSH receptor. This is evidenced by a loss in the ability of TSBAbs to inhibit TSAb activity when these residues are mutated or deleted, respectively. Since the TSAb and TSBAb epitopes are in regions of the extracellular domain of the TSH receptor that have no homology in gonadotropin receptors, these data explain at least in part the organ-specific nature of TSH receptor autoantibodies in autoimmune thyroid disease. Data are additionally provided which indicate that residues 30-37 and 42-45, which flank the TSAb epitope at threonine-40, appear to be ligand interaction sites more important for high affinity TSH binding than for the ability of TSH to increase cAMP levels and that cysteine-41 is critical for TSH receptor conformation and expression on the surface of the cell. Thus, despite unchanged maximal values for TSH-increased cAMP levels, substitution of residues 42-45 or deletion of residues 30-37 results in receptors, which, by comparison to wild-type constructs, exhibit significantly worsened Kd values for TSH binding than EC50 values for TSH- or TSAb-increased cAMP activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gating mechanisms in Cys-loop receptors

The Cys-loop receptor superfamily of ligand-gated ion channels has a prominent role in neuronal signalling. These receptors are pentamers, each subunit containing ten β-strands in the extracellular domain and four α-helical transmembrane domains (M1–M4). The M2 domain of each subunit lines the intrinsic ion channel pore and residues within the extracellular domain form ligand binding sites. Ligand binding initiates a conformational change that opens the ion-selective pore. The coupling between ligand binding in the extracellular domain and opening of the intrinsic ion channel pore located in the membrane is not fully understood. Several loop structures, such as loop 2, the Cys-loop, the pre-M1 region and the M2–M3 loop have been implicated in receptor activation. The current “conformational change wave” hypothesis suggests that binding of a ligand initiates a rotation of the β-sheets around an axis that passes through the Cys-loop. Due to this rotation, the Cys-loop and loop 2 are displaced. Movement of the M2–M3 loop then twists the M2 domain leading to a separation of the helices and opening of the pore. The publication of a crystal structure of an acetylcholine binding protein and the refined structure of the Torpedo marmorata acetylcholine receptor have improved the understanding of the mechanisms and structures involved in coupling ligand binding to channel gating. In this review, the most recent findings on some of these loop structures will be reported and discussed in view of their role in the gating mechanism.     

Molecular dynamics simulations on SDF-1alpha: binding with CXCR4 receptor

Insights into the interacting mode of CXCR4 with SDF-1alpha are crucial in understanding the structural and functional characteristics of CXCR4 receptor. In this paper a computational pipeline, integrating protein structure prediction, molecular dynamics simulations, automated molecular docking, and Brownian dynamics simulations were employed to investigate the dynamic and energetic aspects of CXCR4 associating with SDF-1alpha. The entire simulation revealed the surface distribution feature of electrostatic potentials and conformational "open-close" process of the receptor. The possible binding conformation of CXCR4 was identified, and the CXCR4-SDF-1alpha binding complex was generated. Arg188-Glu277 salt bridge plays an important role for both the extracellular domain conformational change and SDF-1alpha binding. Two binding sites were mapped at the extracellular domain (Site 1) and inside the transmembrane domain (Site 2), which are composed of conserved residues. Sites 1 and 2 contribute approximately 60% and 40% to the binding affinity with SDF-1alpha, respectively. The binding model is in agreement with most of the experimental data. Transmembrane VI has more significant motion in the harmonious conformational transition of CXCR4 during SDF-1alpha binding, which may be possibly associated with signal transduction. Based on the modeling and simulation, a binding mechanism hypothesis between CXCR4 and SDF-1alpha and its relationship to the signal transduction has been proposed.     

Novel information on the epitope of an inverse agonist monoclonal antibody provides insight into the structure of the TSH receptor

The TSH receptor (TSHR) comprises an extracellular leucine-rich domain (LRD) linked by a hinge region to the transmembrane domain (TMD). Insight into the orientation of these components to each other is required for understanding how ligands activate the receptor. We previously identified residue E251 at the LRD-hinge junction as contributing to coupling TSH binding with receptor activation. However, a single residue cannot stabilize the LRD-hinge unit. Therefore, based on the LRD crystal structure we selected for study four other potential LRD-hinge interface charged residues. Alanine substitutions of individual residues K244, E247, K250 and R255 (as well as previously known E251A) did not affect TSH binding or function. However, the cumulative mutation of these residues in varying permutations, primarily K250A and R255A when associated with E251A, partially uncoupled TSH binding and function. These data suggest that these three residues, spatially very close to each other at the LRD base, interact with the hinge region. Unexpectedly and most important, monoclonal antibody CS-17, a TSHR inverse agonist whose epitope straddles the LRD-hinge, was found to interact with residues K244 and E247 at the base of the convex LRD surface. These observations, together with the functional data, exclude residues K244 and E247 from the TSHR LRD-hinge interface. Further, for CS-17 accessibility to K244 and E247, the concave surface of the TSHR LRD must be tilted forwards towards the hinge region and plasma membrane. Overall, these data provide insight into the mechanism by which ligands either activate the TSHR or suppress its constitutive activity.     

The thyrotropin receptor hinge region is not simply a scaffold for the leucine-rich domain but contributes to ligand binding and signal transduction

The glycoprotein hormone receptor hinge region connects the leucine-rich and transmembrane domains. The prevalent concept is that the hinge does not play a significant role in ligand binding and signal transduction. Portions of the hinge are redundant and can be deleted by mutagenesis or are absent in certain species. A minimal hinge will be more amenable to future investigation of its structure and function. We, therefore, combined and progressively extended previous deletions (Delta) in the TSH receptor (TSHR) hinge region (residues 277-418). TSHRDelta287-366, Delta287-371, Delta287-376, and Delta287-384 progressively lost their response to TSH stimulation of cAMP generation in intact cells, consistent with a progressive loss of TSH binding. The longest deletion (TSHRDelta287-384), reducing the hinge region from 141 to 43 amino acids, totally lost both functions. Surprisingly, however, with deletions extending from residues 371-384, constitutive (ligand-independent) activity increased severalfold, reversing the suppressive (inverse agonist) effect of the TSHR extracellular domain. TSHR-activating point mutations I486F and I568T in the first and second extracellular loops (especially the former) had reduced activity on a background of TSHRDelta287-371. In summary, our data support the concept that the TSHR hinge contributes significantly to ligand binding affinity and signal transduction. Residues within the hinge, particularly between positions 371-384, appear involved in ectodomain inverse agonist activity. In addition, the hinge is necessary for functionality of activating mutations in the first and second extracellular loops. Rather than being an inert linker between the leucine-rich and transmembrane domains, the TSHR hinge is a signaling-specificity domain.     

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.     

173 Role of free cysteines in leptin receptor

Leptin, a 16-kDa adipocytic peptide hormone (product of ob gene), is known to play a key role in the control of body weight and exerts its influence by binding to its long-form receptor (Ob-Rb). Ob-Rb belongs to class I cytokine receptor superfamily and consists of an extracellular, transmembrane, and an intracellular domain. Cysteines including free and disulphide-bonded are known to play a significant role in recognition of leptin by its receptor and are known to be highly conserved in different organisms including human, macaca, mouse, dog, sheep, zebrafish, and medaca. Recently, the crystal structure of leptin-binding domain of human leptin receptor has been determined (1). Using the structural data, we analyzed the role of free cysteines in leptin-binding domain of leptin receptor through docking studies using Rosettadock. The conserved free cysteines namely Cys-604 and Cys-613 were mutated to alanines and this resulted in drastic change in the binding orientation of leptin and its receptor. Based on computational analysis, we propose that cysteines either free or involved in disulphide bridges might play a crucial role during signaling and might be the primary determinant of leptin-receptor interactions, the details of which will be discussed. Currently, understanding the structural basis of leptin and its binding to leptin receptor gains much significance since it might pave the way for designing inhibitors that might be used in controlling obesity.     

A Tyrosine Residue on the TSH Receptor Stabilizes Multimer Formation

Background

The thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post –translational modifications including intramolecular cleavage and receptor multimerization. Since monomeric and multimeric receptors coexist in cells, understanding the functional role of just the TSHR multimers is difficult. Therefore, to help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. Here, we have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction.

Methodology/Principal Findings

We studied ectodomain multimer formation by expressing the extracellular domain of the TSHR linked to a glycophosphotidyl (GPI) anchor in both stable and transient expression systems. Using co-immunoprecipitation and FRET of tagged receptors, we established that the TSH receptor ectodomain was capable of multimerization even when totally devoid of the transmembrane domain. Further, we studied the effect of two residues that likely made critical contact points in this interaction. We showed that a conserved tyrosine residue (Y116) on the convex surface of the LRR3 was a critical residue in ectodomain multimer formation since mutation of this residue to serine totally abrogated ectodomain multimers. This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation. Additionally, the Y116 mutation in the intact wild type receptor enhanced receptor degradation.

Conclusions/Significance

These data establish the TSH receptor ectodomain as one site of multimerization, independent of the transmembrane region, and that this interaction was primarily via a conserved tyrosine residue in LRR3.     

Emerging structure of the nicotinic acetylcholine receptors

The conversion of acetylcholine binding into ion conduction across the membrane is becoming more clearly understood in terms of the structure of the receptor and its transitions. A high-resolution structure of a protein that is homologous to the extracellular domain of the receptor has revealed the binding sites and subunit interfaces in great detail. Although the structures of the membrane and cytoplasmic domains are less well determined, the channel lining and the determinants of selectivity have been mapped. The location and structure of the gates, and the coupling between binding sites and gates, remain to be established.     

A deletion in the extracellular domain of the alpha platelet-derived growth factor (PDGF) receptor differentially impairs PDGF-AA and PDGF-BB binding affinities.

32D cells transfected with the human alpha platelet-derived growth factor receptor (alpha PDGFR) bind PDGF-AA, -AB, and -BB isoforms with high affinity, and the binding of each can be efficiently competed by all three isoforms. In an effort to develop better understanding of spatial relationships of binding sites for PDGF-AA and -BB, we constructed an alpha PDGFR mutant which deleted amino acids 150-189 within its extracellular domain. This mutant showed a marked decrease in high affinity binding sites for PDGF-AA without comparable alteration in affinity for PDGF-BB. These findings imply that the high affinity binding sites for PDGF-AA and PDGF-BB in the alpha PDGFR extracellular domain are not structurally coincident.     

Ligand-induced structural changes of the CD44 hyaluronan-binding domain revealed by NMR

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.     

Structural Basis of Activation of Cys-Loop Receptors: the Extracellular–Transmembrane Interface as a Coupling Region

Cys-loop receptors mediate rapid transmission throughout the nervous system by converting a chemical signal into an electric one. They are pentameric proteins with an extracellular domain that carries the transmitter binding sites and a transmembrane region that forms the ion pore. Their essential function is to couple the binding of the agonist at the extracellular domain to the opening of the ion pore. How the structural changes elicited by agonist binding are propagated through a distance of 50?Å to the gate is therefore central for the understanding of the receptor function. A step forward toward the identification of the structures involved in gating has been given by the recently elucidated high-resolution structures of Cys-loop receptors and related proteins. The extracellular–transmembrane interface has attracted attention because it is a structural transition zone where β-sheets from the extracellular domain merge with α-helices from the transmembrane domain. Within this zone, several regions form a network that relays structural changes from the binding site toward the pore, and therefore, this interface controls the beginning and duration of a synaptic response. In this review, the most recent findings on residues and pairwise interactions underlying channel gating are discussed, the main focus being on the extracellular–transmembrane interface.     

Human aryl-hydrocarbon receptor and its interaction with dioxin and physiological ligands investigated by molecular modelling and docking simulations

Molecular structure of the ligand binding domain of hAhR has been modelled by homology modelling techniques and used for docking simulations with dioxin and nine more xenobiotics and endogenous ligands. The study evidences that different sites may bind these ligands, whereas only one binding site has been previously indicated by past studies on the mouse homologous receptor. The differences in the sequence of mouse and human AhR ligand binding domain may explain this observation, being most of them in the additional sites observed. Preferences of the evaluated ligands for the different sites are reported and discussed in view of their functional role.     

The insulin receptor: a prototype for dimeric, allosteric membrane receptors?

The recent crystallographic structure of the insulin receptor (IR) extracellular domain has brought us closer to ending several decades of speculation regarding the stoichiometry and mechanism of insulin-receptor binding and negative cooperativity. It supports a bivalent crosslinking model whereby two sites on the insulin molecule alternately crosslink two partial-binding sites on each insulin-receptor half. Ligand-induced or -stabilized receptor dimerization or oligomerization is a general feature of receptor tyrosine kinases (RTKs), in addition to cytokine receptors, but the kinetic consequences of this mechanism have been less well studied in other RTKs than in the IR. Surprisingly, recent studies indicate that constitutive dimerization and negative cooperativity are also ubiquitous properties of G-protein-coupled receptors (GPCRs), which show allosteric mechanisms similar to those described for the IR.     

Site-directed mutagenesis of the human thyrotropin receptor: role of asparagine-linked oligosaccharides in the expression of a functional receptor

We studied the role of glycosylation in the expression of a functional human TSH receptor. Oligonucleotide-directed mutagenesis was used to replace, separately or together, the Asn codons with Gln in each of the six potential glycosylation sites in the receptor. Recombinant wild-type and mutated TSH receptors were stably expressed in Chinese hamster ovary cells. High affinity TSH binding and the cAMP response to TSH stimulation were abolished in the receptor mutated at Asn77 as well as in the receptor mutated at all six potential glycosylation sites. In the receptor mutated at Asn113, the affinity of TSH binding was markedly decreased (Kd, 2.6 x 10(-8) 3.3 x 10(-10) M in the wild-type receptor). This affinity was too low to permit the transduction of a signal, as measured by an increase in intracellular cAMP generation. Substitution of Asn at positions 99, 177, 198, and 302 did not appreciably affect the affinity of the TSH receptor for TSH binding or its ability to mediate an increase in intracellular cAMP levels. Therefore, either these four potential glycosylation sites are not glycolysated, or alternatively, oligosaccharide chains at these positions do not play a major role in the folding, intracellular trafficking, stability, or expression of a functional receptor on the cell surface. Conversely, our data suggest that N-linked glycosylation of Asn77 and Asn113 does play a role in the expression of a biologically active TSH receptor on the cell surface.     

Antibody Protection Reveals Extended Epitopes on the Human TSH Receptor

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.