Evolution of the thyrotropin receptor: a G protein coupled receptor with an intrinsic capacity to dimerize

The rapidly escalating number of genome sequences has emphasized the basic tenants of the schema of life. By the same token comparisons according to specialized function or niche within nature expose genomic strategies to optimize the use of resources and ensure biological success. Increasing complexity may result from diversification, shuffling, and re-arrangement of an otherwise limited functional genomic complement. To further test the concept of relative structural plasticity of the TSH receptor we sequenced the TSHR gene of two Old World monkey species Macaca mulatta and Cercopithecus aethiops, evolutionary removed from Homo sapiens by >20Myr. Both genes encoded a protein of 764 residues. This structure was 99% homologous between the two species of Old World monkeys while C. aethiops was 97% and M. mulatta was 96% homologous to H. sapiens. TSHR sequence comparisons were sought for an additional eight mammals as well as four (two Salmon, Tilapia, and Sea Bass) from teleosts. The amino-acid sequences of the 14 TSH receptors were similar. The most variable sequences were those of the intracellular tail and the distal cysteine-rich C-terminus flanking region of the ectodomain, whereas the trans-membrane domain was most preserved. Some sequences were decidedly H. sapiens specific, while others were primate specific or showed the changes expected of evolutionary descent. Others, however, exhibited "cross-species polymorphism," sometimes at quite remarkable evolutionary distances. As opposed to H. sapiens the sequence differences may have subtle influences on TSHR function or may affect long-range compensation for radical changes in adducts. The two Old World monkeys share with other lower mammals the absence of a glycosylation site at 113-115. Sea Bass and Tilapia have four glycosylation sites, whereas the two salmon receptors have only three. Changes in some critical residues raise questions about variation in function: thus S281 is conserved in all mammals and an important determinant of negative agonist function of TSHR is replaced by R in Sea Bass. Likewise the K183, found at an important transitional region at LRR 6 conserved in all mammals, is represented by M in fish and may contribute to TSHR lutenization in fish. There is no evidence that evolutionary changes in primate receptors are more rapid than that in other mammals and the separation times of different mammals based on silent nucleotide changes of TSHR are closely parallel to archaeological estimates. Results of correlated mutation analysis, referenced to the rhodopsin crystal structure, affirms dimerization of TSHR transmembrane helices. In addition, it suggests the involvement of critical lipid-facing residues in the helices in receptor dimerization and oligomerization. We highlight the value of evolutionary informatics and set the stage for dissecting out potential subtle differences in TSHR function associated with structural variations.     

Thyrotropin-receptor-mediated diseases: a paradigm for receptor autoimmunity

Autoantibodies to the thyrotropin receptor (TSHR) can act as thyrotropin agonists or antagonists, or can cause thyroid hypertrophy. Neither the autoantibody-binding sites on the TSHR nor the intracellular mechanisms by which the autoantibodies mediate their u, erse functional effects are completely understood. This article reviews how cloning of the TSHR has contributed to our understanding of its structure and function, and has allowed induction of experimental autoimmunity to the TSHR.     

Structural basis of PSGL-1 binding to ERM proteins

P-selectin glycoprotein ligand-1 (PSGL-1), an adhesion molecule with O-glycosylated extracellular sialomucins, is involved in leukocyte inflammatory responses. On activation, ezrin-radixin-moesin (ERM) proteins mediate the redistribution of PSGL-1 on polarized cell surfaces to facilitate binding to target molecules. ERM proteins recognize a short binding motif, Motif-1, conserved in cytoplasmic tails of adhesion molecules, whereas PSGL-1 lacks Motif-1 residues important for binding to ERM proteins. The crystal structure of the complex between the radixin FERM domain and a PSGL-1 juxtamembrane peptide reveals that the peptide binds the groove of FERM subdomain C by forming a beta-strand associated with strand beta5C, followed by a loop flipped out towards the solvent. The Motif-1 3(10) helix present in the FERM-ICAM-2 complex is absent in PSGL-1 given the absence of a critical Motif-1 alanine residue, and PSGL-1 reduces its contact area with subdomain C. Non-conserved positions are occupied by large residues Met9 and His8, which stabilize peptide conformation and enhance groove binding. Non-conserved residues play an important role in compensating for loss of binding energy resulting from the absence of conserved residues important for binding.     

Human NKG2F is expressed and can associate with DAP12

The NKG2 family of C-lectin type molecules is important for regulating the function of natural killer and subpopulations of T cells. NKG2A/B contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and accordingly functions as an inhibitory receptor, whereas NKG2C and -E/H associate with DAP12 via a positively charged residue in their transmembrane domains and function as activation receptors. Each of these molecules is paired with CD94 for expression and recognizes HLA-E as a ligand. NKG2F is an orphan gene within the NKG2 family whose translated product would contain both a positively charged residue in its transmembrane region, an intracellular ITIM-like sequence and an extracellular domain (62 residues) that is truncated relative to other NKG2 molecules. We show that NKG2F is expressed as a protein in NK cells. Expression appears to be confined to intracellular compartments probably due to its inability to associate with CD94. It can however associate with DAP12 thereby providing activation signaling potential. We were unable to demonstrate phosphorylation of the Tyr residue in the ITIM-like motif suggesting that it is a mock ITIM. NKG2F could be a receptor component with an as yet unidentified partner(s), could function to regulate cell activation through competition for DAP12 with other receptors, such as NKG2C and -E/H, or it could simply be a vestigial gene product.     

Characterization of the spike protein of human coronavirus NL63 in receptor binding and pseudotype virus entry

The spike (S) protein of human coronavirus NL63 (HCoV-NL63) mediates both cell attachment by binding to its receptor hACE2 and membrane fusion during virus entry. We have previously identified the receptor-binding domain (RBD) and residues important for RBD-hACE2 association. Here, we further characterized the S protein by investigating the roles of the cytoplasmic tail and 19 residues located in the RBD in protein accumulation, receptor binding, and pseudotype virus entry. For these purposes, we first identified an entry-efficient S gene template from a pool of gene variants and used it as a backbone to generate a series of cytoplasmic tail deletion and single residue substitution mutants. Our results showed that: (i) deletion of 18aa from the C-terminus enhanced the S protein accumulation and virus entry, which might be due to the deletion of intracellular retention signals; (ii) further deletion to residue 29 also enhanced the amount of S protein on the cell surface and in virion, but reduced virus entry by 25%, suggesting that residues 19-29 contributes to membrane fusion; (iii) a 29aa-deletion mutant had a defect in anchoring on the plasma membrane, which led to a dramatic decrease of S protein in virion and virus entry; (iv) a total of 15 residues (Y498, V499, V531, G534, G537, D538, S540, G575, S576, E582, W585, Y590, T591, V593 and G594) within RBD were important for receptor binding and virus entry. They probably form three receptor binding motifs, and the third motif is conserved between NL63 and SARS-CoV.     

A study on antigenicity and receptor-binding ability of fragment 450-650 of the spike protein of SARS coronavirus

The spike (S) protein of SARS coronavirus (SARS-CoV) is responsible for viral binding with ACE2 molecules. Its receptor-binding motif (S-RBM) is located between residues 424 and 494, which folds into 2 anti-parallel beta-sheets, beta5 and beta6. We have previously demonstrated that fragment 450-650 of the S protein (S450-650) is predominantly recognized by convalescent sera of SARS patients. The N-terminal 60 residues (450-510) of the S450-650 fragment covers the entire beta6 strand of S-RBM. In the present study, we demonstrate that patient sera predominantly recognized 2 linear epitopes outside the beta6 fragment, while the mouse antisera, induced by immunization of BALB/c mice with recombinant S450-650, mainly recognized the beta6 strand-containing region. Unlike patient sera, however, the mouse antisera were unable to inhibit the infectivity of S protein-expressing (SARS-CoV-S) pseudovirus. Fusion protein between green fluorescence protein (GFP) and S450-650 (S450-650-GFP) was able to stain Vero E6 cells and deletion of the beta6 fragment rendered the fusion product (S511-650-GFP) unable to do so. Similarly, recombinant S450-650, but not S511-650, was able to block the infection of Vero E6 cells by the SARS-CoV-S pseudovirus. Co-precipitation experiments confirmed that S450-650 was able to specifically bind with ACE2 molecules in lysate of Vero E6 cells. However, the ability of S450-510, either alone or in fusion with GFP, to bind with ACE2 was significantly poorer compared with S450-650. Our data suggest a possibility that, although the beta6 strand alone is able to bind with ACE2 with relatively high affinity, residues outside the S-RBM could also assist the receptor binding of SARS-CoV-S protein.     

A search within the IL-1 type I receptor reveals a peptide with hydropathic complementarity to the IL-1beta trigger loop which binds to IL-1 and inhibits in vitro responses

In previous research, we were able to demonstrate that a seven amino acid residue peptide (VITFFSL), designed as an antisense peptide of the beta-bulge trigger loop region of interleukin 1beta (IL-1beta) (QGEESND; residues 48-54 [mature protein sequence]), was able to interact with IL-1 specifically and inhibit the response to IL-1 in an in vitro bioassay. The evidence was consistent with a specific interaction ocurring between antisense peptide and the trigger loop region. On the basis that antisense peptides are able to interact with their corresponding sense peptide sequences as a result of their mutually complementary hydropathic profiles (Fassina G., Verdoliva, A., Cassani, G., Melli, M., 1994. Binding of type I IL-1 receptor fragment 151-162 to IL-1. Growth Factors 10, 99-106; Maier, C.C., Moseley, H.N.B., Zhou, S., Whitaker, J.N., Blalock, J.E., 1994. Indentification of interactive determinants on idiotypic-anti-idiotypic antibodies through comparison of their hydropathic profiles. Immunomethods 5, 107-113), we devised a computer program (FINDH) to search the amino acid residue sequence of interleukin-1 type 1 receptor (IL-1 R1) for peptide motifs possessing hydropathic complementarity to the trigger loop sequence. The most complementary "best-fit peptide" motif (LITVLNI) was located in the third extracellular domain of IL-1 R1. A best-fit peptide corresponding to this motif was synthesised and found to bind to IL-1beta as well as inhibit the response to IL-1 in two independent in vitro bioassays (monitoring IL-1 dependent serum amyloid A synthesis and IL-1 dependent alkaline phosphatase activity, respectively). A second peptide motif (VIEFITL) was identified and the corresponding peptide synthesised along with a reordered version (LTILINV) of the best fit peptide. Both failed to bind measurably with IL-1beta or inhibit the response to IL-1 in the two bioassays. This best fit peptide behaved very similarly, in terms of IL-1 binding and inhibition behaviour, to the original trigger loop antisense peptide. Reference to the recently released X-ray crystal structure of IL-1beta and the IL1-R1 extracellular domain shows that the best fit peptide motif in IL-1 R1 is not apparantly interacting with the IL-1 trigger loop, although both are close in space. The intriguing possibility exists that the best fit peptide motif could represent an alternative site for IL-1beta receptor interaction which has not thus far been identified.     

A novel TSHR gene mutation (Ile691Phe) in a Chinese family causing autosomal dominant non-autoimmune hyperthyroidism

Familial non-autoimmune hyperthyroidism is a rare autosomal dominant genetic disease resulting from activating mutations in the thyroid-stimulating hormone receptor (TSHR) gene. In this work a Chinese family with autosomal dominant non-autoimmune hyperthyroidism across four generations was collected. The strongest evidence for linkage in this study occurred on chromosome 14q24.2–31.3. By mutation scan of the TSHR gene located within the region of interest, a heterozygote substitution (A > T) at position 2071 of the TSHR was found, changing isoleucine 691 to phenylalanine. Our study identified the first germline mutation in the intracellular C-terminal domain of TSHR. Zheng Liu and Yuanming Sun contribute to this work equally.     

Intracellular entrapment of wild-type TSH receptor by oligomerization with mutants linked to dominant TSH resistance

TSH resistance is one of the causes of congenital hypothyroidism with thyroid gland in situ. We recently identified families with dominant transmission of partial TSH resistance due to heterozygous inactivating mutations in TSH receptor (TSHR) gene. Although we documented a poor routing of TSHR mutants to the cell membrane, the mechanism responsible for dominant inheritance of partial TSH resistance remained unexplained. We therefore co-transfected Cos-7 cells with wild-type TSHR and mutant receptors found in these patients. A variable impairment of cAMP response to bTSH stimulation was observed, suggesting that inactive TSHR mutants can exert a dominant negative effect on wild-type TSHR. We then generated chimeric constructs of wild-type or inactive TSHR mutants fused to different reporters. By fluorescence microscopy and immunoblotting, we documented an intracellular entrapment, mainly in the endoplasmic reticulum, and reduced maturation of wild-type TSHR in the presence of inactive TSHR mutants. Finally, fluorescence resonance energy transfer and co-immunoprecipitation experiments were performed to study the molecular interactions between wild-type and mutant TSHRs. The results are in agreement with the presence of oligomers formed by wild-type and mutant receptors in the endoplasmic reticulum. Such physical interaction represents the molecular basis for the dominant negative effect of inactive TSHR mutants. These findings provide an explanation for the dominant transmission of partial TSH resistance. This is the first report linking dominant negative mutations of a G protein-coupled receptor to an abnormal endocrine phenotype in heterozygous patients.     

Thyrotropin receptor mutations as a tool to understand thyrotropin receptor action

A large number of mutations have been identified in the thyrotropin (TSH) receptor (TSHR) gene causing human diseases. Toxic thyroid nodules are frequently associated with somatic constitutively activating TSHR mutations. Autosomal dominant non-autoimmune hyperthyroidism is caused by activating TSHR germline mutations. Inactivating germline mutations cause TSH unresponsiveness. Discovery of the different TSHR mutations in various regions of the receptor molecule has led to the identification of important domains for intramolecular TSHR signal transduction. However, despite the functional characterization of the naturally occurring mutations the precise molecular mechanisms of receptor activation including the processes of hormone binding, intramolecular signaling between the different TSHR domains and of G protein coupling are not completely understood. This review discusses the importance of the various receptor domains for TSHR activation identified on the basis of the naturally occurring gain or loss of function mutations and in vitro investigations performed with site-directed mutagenesis, synthetic peptides, or antibodies. Several in vitro studies have provided new insights into structure-function relationships by site-directed mutagenesis in combination with molecular modeling. These in vitro investigations have often been guided by naturally occurring mutations and have provided new insights into intramolecular changes during receptor activation. This has led to progress in understanding the mechanism of TSHR activation.     

Graves' disease: a host defense mechanism gone awry

In this report we summarize evidence to support a model for the development of Graves' disease. The model suggests that Graves' disease is initiated by an insult to the thyrocyte in an individual with a normal immune system. The insult, infectious or otherwise, causes double strand DNA or RNA to enter the cytoplasm of the cell. This causes abnormal expression of major histocompatibility (MHC) class I as a dominant feature, but also aberrant expression of MHC class II, as well as changes in genes or gene products needed for the thyrocyte to become an antigen presenting cell (APC). These include increased expression of proteasome processing proteins (LMP2), transporters of antigen peptides (TAP), invariant chain (Ii), HLA-DM, and the co-stimulatory molecule, B7, as well as STAT and NF-kappaB activation. A critical factor in these changes is the loss of normal negative regulation of MHC class I, class II, and thyrotropin receptor (TSHR) gene expression, which is necessary to maintain self-tolerance during the normal changes in gene expression involved in hormonally-increased growth and function of the cell. Self-tolerance to the TSHR is maintained in normals because there is a population of CD8- cells which normally suppresses a population of CD4+ cells that can interact with the TSHR if thyrocytes become APCs. This is a host self-defense mechanism that we hypothesize leads to autoimmune disease in persons, for example, with a specific viral infection, a genetic predisposition, or even, possibly, a TSHR polymorphism. The model is suggested to be important to explain the development of other autoimmune diseases including systemic lupus or diabetes.     

Molecular mapping of a site for Cd2+-induced modification of human ether-à-go-go-related gene (hERG) channel activation

Cd²⁺ slows the rate of activation, accelerates the rate of deactivation and shifts the half-points of voltage-dependent activation ( V _0.5,act) and inactivation ( V _0.5,inact) of human ether-à-go-go -related gene (hERG) K⁺ channels. To identify specific Cd²⁺-binding sites on the hERG channel, we mutated potential Cd²⁺-coordination residues located in the transmembrane domains or extracellular loops linking these domains, including five Cys, three His, nine Asp and eight Glu residues. Each residue was individually substituted with Ala and the resulting mutant channels heterologously expressed in Xenopus oocytes and their biophysical properties determined with standard two-microelectrode voltage-clamp technique. Cd²⁺ at 0.5 m m caused a +36 mV shift of V _0.5,act and a +18 mV shift of V _0.5,inact in wild-type channels. Most mutant channels had a similar sensitivity to 0.5 m m Cd²⁺. Mutation of single Asp residues located in the S2 (D456, D460) or S3 (D509) domains reduced the Cd²⁺-induced shift in V _0.5,act, but not V _0.5,inact. Combined mutations of two or three of these key Asp residues nearly eliminated the shift induced by 0.5 m m Cd²⁺. Mutation of D456, D460 and D509 also reduced the comparatively low-affinity effects of Ca²⁺ and Mg²⁺ on V _0.5,act. Extracellular Cd²⁺ modulates hERG channel activation by binding to a coordination site formed, at least in part, by three Asp residues.     

Assessing the role of Asp 194 in the transmembrane domains of the α-chain of the high-affinity receptor complex for immunoglobulin E in signal transduction

The high-affinity receptor complex for IgE plays a pivotal role in allergic responses since cross-linking of the high-affinity IgE receptor (Fc?RI) on target cells initiates a signaling cascade facilitating release of inflammatory mediators causing allergic responses. The transmembrane regions of the ligand binding domains of the high-affinity IgE and low-affinity IgG receptors share an invariant motif (LFAVDTGL) containing a polar aspartate within a predominantly non-polar setting. The functional importance of this aspartate residue (D194) in Fc?RI-mediated receptor signaling was assessed by site-directed mutagenesis. Rat basophilic leukemia cells (RBL-2H3) transfected with the human IgE binding subunit (Fc?RIα) incorporating polar substitutions like asparagine (D194N) or threonine (D194T) resulted in the formation of a functional rat/human chimeric receptor complex. When activated via huIgE and antigen, cells transfected with these variant receptor subunits supported mediator release, intracellular calcium mobilisation and tyrosine phosphorylation of γ-chain and Syk kinase while a non-polar substitution (D194L) gave rise to cell surface expression of the mutated receptor subunit but failed to initiate downstream signaling. No cell surface expression of huFc?RIα gene constructs was observed when D194 was replaced with the non-polar Ile (D194I) residue of similar size, the larger positively charged Arg (D194R) or lysine (D194K) residues, or the negatively charged glutamate (D194E) and smaller polar Ser (D194S) non-polar Ala (D194A) and V (D194V). These observations highlight importance of the size and charge of amino acid residue at position 194 in determining IgE receptor subunit interactions, cell surface localization, and initiation of downstream signaling events.     

Redistribution of critical major histocompatibility complex and T cell receptor-binding functions of residues in an antigenic sequence after biterminal substitution

Residues critical for establishing a trimolecular interaction with a major histocompatibility complex (MHC)-encoded receptor and a T cell antigen receptor (TcR) were determined for an antigenic nonapeptide. The N-terminal residue proved to be involved in binding of the peptide to both receptors and the C-terminal residue was essential for MHC binding. While substitution of either of these critical terminal residues by alanine resulted in an almost complete loss of peptide antigenicity, simultaneous substitution of both created a new functional ligand for the same MHC molecule and the same TcR. Notably, in the biterminally substituted peptide, the core residues took on new roles in the trimolecular interaction in that a residue critical in the authentic nonapeptide for TcR binding became critical for MHC binding and former spacer residues became essential to various degrees for the interaction with either receptor or both. Thus, apparently, the loss of the terminal residues' contribution was at least partially compensated by a redistribution of the roles among the remaining residues. These results reflect a cooperative contribution of all residues of an antigenic peptide to its binding to both receptors and thus challenge a static definition of agretope and epitope as MHC and TcR binding sites.     

Graves’ Disease: A Host Defense Mechanism Gone Awry

In this report we summarize evidence to support a model for the development of Graves’ disease. The model suggests that Graves’ disease is initiated by an insult to the thyrocyte in an individual with a normal immune system. The insult, infectious or otherwise, causes double strand DNA or RNA to enter the cytoplasm of the cell. This causes abnormal expression of major histocompatibility (MHC) class I as a dominant feature, but also aberrant expression of MHC class II, as well as changes in genes or gene products needed for the thyrocyte to become an antigen presenting cell (APC). These include increased expression of proteasome processing proteins (LMP2), transporters of antigen peptides (TAP), invariant chain (Ii), HLA-DM, and the co-stimulatory molecule, B7, as well as STAT and NF-_kB activation. A critical factor in these changes is the loss of normal negative regulation of MHC class I, class II, and thyrotropin receptor (TSHR) gene expression, which is necessary to maintain self-tolerance during the normal changes in gene expression involved in hormonally-increased growth and function of the cell. Self-tolerance to the TSHR is maintained in normals because there is a population of CD8⁺ cells which normally suppresses a population of CD4⁺ cells that can interact with the TSHR if thyrocytes become APCs. This is a host self-defense mechanism that we hypothesize leads to autoimmune disease in persons, for example, with a specific viral infection, a genetic predisposition, or even, possibly, a TSHR polymorphism. The model is suggested to be important to explain the development of other autoimmune diseases including systemic lupus or diabetes.