Influence of the Hinge Region and Its Adjacent Domains on Binding and Signaling Patterns of the Thyrotropin and Follitropin Receptor

Glycoprotein hormone receptors (GPHR) have a large extracellular domain (ECD) divided into the leucine rich repeat (LRR) domain for binding of the glycoprotein hormones and the hinge region (HinR), which connects the LRR domain with the transmembrane domain (TMD). Understanding of the activation mechanism of GPHRs is hindered by the unknown interaction of the ECD with the TMD and the structural changes upon ligand binding responsible for receptor activation. Recently, our group showed that the HinR of the thyrotropin receptor (TSHR) can be replaced by those of the follitropin (FSHR) and lutropin receptor (LHCGR) without effects on surface expression and hTSH signaling. However, differences in binding characteristics for bovine TSH at the various HinRs were obvious. To gain further insights into the interplay between LRR domain, HinR and TMD we generated chimeras between the TSHR and FSHR. Our results obtained by the determination of cell surface expression, ligand binding and G protein activation confirm the similar characteristics of GPHR HinRs but they also demonstrate an involvement of the HinR in ligand selectivity indicated by the observed promiscuity of some chimeras. While the TSHR HinR contributes to specific binding of TSH and its variants, no such contribution is observed for FSH and its analog TR4401 at the HinR of the FSHR. Furthermore, the charge distribution at the poorly characterized LRR domain/HinR transition affected ligand binding and signaling even though this area is not in direct contact with the ligand. In addition our results also demonstrate the importance of the TMD/HinR interface. Especially the combination of the TSHR HinR with the FSHR-TMD resulted in a loss of cell surface expression of the respective chimeras. In conclusion, the HinRs of GPHRs do not only share similar characteristics but also behave as ligand specific structural and functional entities.     

Functional divergence of glycoprotein hormone receptors

Two lamprey glycoprotein hormone receptors (lGpH-R I and II) highly similar with gnathostome GpH-Rs were cloned from sea lamprey testes and thyroid, respectively. Vertebrate glycoprotein protein receptors have a large extracellular domain (ED) containing a leu rich domain (LRD) linked to a rhodopsin-like transmembrane domain (TMD) through a highly divergent linker region (signal specificity domain, SSD or 'hinge' region) and a third major segment, the intracellular domain. To determine the potential roles of the different domains in the activation of the receptor following ligand-receptor binding, functional assays were performed on lGpH-R I/rat luteinizing hormone (LH)-R domain swapped chimeric receptors. These results show that the functional roles of the lamprey glycoprotein-receptor I (lGpH-R I) domains are conserved compared with its Gnathostome homologs. The ability of different glycoprotein hormones to activate chimeric lamprey/rat receptors suggests that the selectivity of the GpH-Rs in respect to their ligands is not controlled exclusively by a single domain but is the result of specific interactions between domains. We hypothesize that these interactions were refined during millions of years of co-evolution of the receptors with their cognate ligands under particular intramolecular, intermolecular and physiological constraints.     

Determinants of ligand binding specificity in the glial cell line-derived neurotrophic factor family receptor alpha S

The glial cell line-derived neurotrophic factor (GDNF) family comprise a subclass of cystine-knot superfamily ligands that interact with a multisubunit receptor complex formed by the c-Ret tyrosine kinase and a cystine-rich glycosyl phosphatidylinositol-anchored binding subunit called GDNF family receptor alpha (GFRalpha). All four GDNF family ligands utilize c-Ret as a common signaling receptor, whereas specificity is conferred by differential binding to four distinct GFRalpha homologues. To understand how the different GFRalphas discriminate ligands, we have constructed a large set of chimeric and truncated receptors and analyzed their ligand binding and signaling capabilities. The major determinant of ligand binding was found in the most conserved region of the molecule, a central domain predicted to contain four conserved alpha helices and two beta strands. Distinct hydrophobic and positively charged residues in this central region were required for binding of GFRalpha1 to GDNF. Interaction of GFRalpha1 and GFRalpha2 with GDNF and neurturin required distinct subsegments within this central domain, which allowed the construction of chimeric receptors that responded equally well to both ligands. C-terminal segments adjacent to the central domain are necessary and have modulatory function in ligand binding. In contrast, the N-terminal domain was dispensable without compromising ligand binding specificity. Ligand-independent interaction with c-Ret also resides in the central domain of GFRalpha1, albeit within a distinct and smaller region than that required for ligand binding. Our results indicate that the central region of this class of receptors constitutes a novel binding domain for cystine-knot superfamily ligands.     

Isolation and characterization of the subunits of bovine follitropin.

Highly purified bovine follitropin was dissociated into its alpha- and beta-subunits after treatment with 1 M-propionic acid. The dissociated subunits were fractionated by chromatography on DEAE-cellulose and further purified by gel filtration on Sephadex G-100. The isolated alpha- and beta-subunits were biologically inactive, but their recombinants regenerated 80% of the follitropin activity. The alpha-subunit of bovine follitropin recombined with the beta-subunits of bovine lutropin and thyrotropin to regenerate 70% of lutropin and 50% of thyrotropin activities respectively. The beta-subunit of bovine follitropin recombined with the alpha-subunit of either bovine lutropin or thyrotropin to regenerate about 75% of follitropin activity. Recombinations were monitored by specific radioligand-receptor assays and polyacrylamide-gel electrophoresis. The elution volumes of the alpha- and beta-subunits of bovine follitropin after gel filtration on Sephadex G-100 were almost identical. The amino acid composition of bovine follitropin-alpha was low in histidine, arginine, isoleucine and leucine, but relatively high in lysine, threonine and glutamic acid. The bovine follitropin-beta contained one methionine residue and low amounts of histidine and phenylalanine, but relatively high in aspartic acid, threonine and glutamic acid. The N-terminal residues of the alpha- and beta-subunits of bovine follitropin were identified to be phenylalanine and glycine respectively.     

A low molecular weight agonist signals by binding to the transmembrane domain of thyroid-stimulating hormone receptor (TSHR) and luteinizing hormone/chorionic gonadotropin receptor (LHCGR)

Many cognate low molecular weight (LMW) agonists bind to seven transmembrane-spanning receptors within their transmembrane helices (TMHs). The thienopyrimidine org41841 was identified previously as an agonist for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and suggested to bind within its TMHs because it did not compete for LH binding to the LHCGR ectodomain. Because of its high homology with LHCGR, we predicted that thyroid-stimulating hormone receptor (TSHR) might be activated by org41841 also. We show that org41841 is a partial agonist for TSHR but with lower potency than for LHCGR. Analysis of three-dimensional molecular models of TSHR and LHCGR predicted a binding pocket for org41841 in common clefts between TMHs 3, 4, 5, 6, and 7 and extracellular loop 2 in both receptors. Evidence for this binding pocket was obtained in signaling studies with chimeric receptors that exhibited improved responses to org41841. Furthermore, a key receptor-ligand interaction between the highly conserved negatively charged E3.37 and the amino group of org41841 predicted by docking of the ligand into the three-dimensional TSHR model was experimentally confirmed. These findings provide the first evidence that, in contrast to the ectodomain binding of cognate ligands, a LMW agonist can bind to and activate glycoprotein hormone receptors via interaction with their transmembrane domain.     

Complex chimeras to map ligand binding sites of GPCRs

Family 1a GPCRs are thought to bind small molecule ligands in a pocket comprising sequences from non-contiguous transmembrane helices. In this study, receptor-ligand binding determinants were defined by building a series of complex chimeras where multiple sequences were exchanged between related G-protein coupled receptors. Regions of P2Y(1), P2Y(2) and BLT(1) predicted to interact with nucleotide and leukotriene ligands were identified and receptors were engineered within their transmembrane helices to transpose the ligand binding site of one receptor on to another receptor. Ligand-induced activation of chimeras was compared with wild-type receptor activation in a yeast reporter gene assay. Binding of ligand to a P2Y(2)/BLT(1) chimera confirmed that the ligand binding determinants of BLT(1) are located in the upper regions of the helices and extracellular loops of this receptor and that they had been successfully transferred to a receptor that normally binds unrelated ligands.     

GRIS: glycoprotein-hormone receptor information system

The glycoprotein-hormone receptor information system (GRIS) presents a comprehensive view on all available molecular data for the lutropin/choriogonadotropin receptor, follitropin receptor, and thyrotropin receptor G protein-coupled receptors. It features a mutation database presently containing 696 point mutations, combined with all sequences and the associated homology models. The mutation information was automatically extracted from the literature and manually augmented with respect to constitutivity, surface expression, sensitivity to hormones, and binding affinity. All information in this integrated system is presented in a G protein-coupled receptor specialist-friendly way. A series of interactive tools such as rotamer analysis, mutation prediction, or cavity visualization aids with the design and interpretation of experiments. A universal residue numbering system has been introduced to ease database searches as well as the use of the information in conjunction with literature data from diverse origins. Users can upload new mutations. GRIS is freely accessible at http://gris.ulb.ac.be/.     

Porcine follitropin. Isolation and characterization of the native hormone and its alpha and beta subunits

The properties of porcine follitropin and its subunits which have not yet been characterized are presented. The porcine follitropin obtained has a biological potency of 81 times the National Institutes of Health Porcine Follitropin P-1 preparation. Its contamination by lutropin and thyrotropin amounted to 1 and 0.5 percent by weight respectively, as measured by radioimmunoassay. The alpha and beta subunits of porcine follitropin were obtained by incubation in an acidic urea solution followed by anion exchange chromatography. The amino acid composition of porcine follitropin alpha subunit was found to be identical to that of alpha chain of porcine lutropin and thyrotropin. These porcine alpha chains differ, nevertheless, markedly in their carbohydrate composition particularly with respect to their mannose and galactose contents. The amino-terminal residue of the follitropin alpha subunit is threonyl. The carboxy-terminal end of the alpha chain is of variable length. Cysteyl residue was detected at the aminoterminal end of the follitropin beta chain with glutamic acid at its carboxy-terminal end. Cross-contamination of the alpha and beta subunit preparations was measured by specific radioimmunoassay and amounted to 0.5 and 0.1 percent by weight respectively.     

Asp383 in the second transmembrane domain of the lutropin receptor is important for high affinity hormone binding and cAMP production.

The lutropin (LH), follitropin, and thyrotropin receptors belong to the superfamily of G-protein coupled receptors and have some unique structural features. These glycoprotein hormone receptors comprise a C-terminal half and an N-terminal half of similar size. The C-terminal half is equivalent to the entire structure of other G-protein coupled receptors and has seven transmembrane domains, three cytoplasmic loops, three exoplasmic loops, and a C terminus. In contrast, the hydrophilic N-terminal half is exoplasmic and unique to the glycoprotein hormone receptors. This large N-terminal half of the LH receptor has recently been shown to be capable of binding the hormone. Therefore, these glycoprotein hormone receptors are structurally and functionally different from other G-protein coupled receptors. In an attempt to define the role of the membrane-associated C-terminal half of the LH receptor, we have prepared several mutant receptors in which an Asp or Glu in the seven transmembrane domains has been converted to Asn or Gln, respectively. These include Asp383----Asn in the second transmembrane domain, Glu410----Gln in the third transmembrane domain, and Asp556----Asn in the sixth transmembrane domain. All these mutant receptors were successfully expressed in Cos 7A cells. The Glu410----Gln and Asp556----Asn mutants maintained normal affinities for hormone binding and cAMP production, but the Asp383----Asn mutant showed significantly lower affinities. Although Asp383 of the LH receptor is conserved in all G-protein coupled receptors cloned to date except the substance P receptor, which has Glu in the place of the Asp residue, this is the first observation of the critical role of the Asp in hormone binding and subsequent stimulation of cAMP production.     

The posttranslational modification of thyrotropin receptor and thyroid diseases

The thyrotropin receptor (TSHR), lutropin receptor, and follitropin receptor are related members of the superfamily of leucine-rich repeats containing adenylate cyclase stimulating receptors. The unique posttranslational modification of the TSHR leads to the transformation of its monomeric form to the subunit structure where the subunits A and B are connected by disulphide bonds. This natural processing occurs with the release from the receptor of a short peptide C, and is followed by the release of the subunit A. Both monomeric and dimeric forms of the receptor are stimulated by TSH, so no clear functional significance of TSHR modifications have been found. We can speculated that the processing of TSHR with the release of its large fragments contributes to the development of autoimmune diseases and production anti-TSH receptor autoantibodies. The extrathyroidal manifestations of Graves disease may also be related to metastasis of the autoimmune reaction to extrathyroidal sites via the released A subunit. The TSHR processing may, to some extent, be connected to the hyperthyroidism since the release of the subunit A from the receptor augmented the adenylate cyclase activity in the absence of TSH. According to the recent model of receptors action the TSHR is in equilibrium between the inactive (closed) and active (opened) conformations. In opened conformation it can associate with Gs protein and trigger the intracellular signal. TSH and stimulating autoantibodies preferentially bind to opened receptors and stabilizes them.     

A molecular dissection of the glycoprotein hormone receptors

In glycoprotein hormone receptors, a subfamily of rhodopsin-like G protein-coupled receptors, the recognition and activation steps are carried out by separate domains of the proteins. Specificity of recognition of the hormones thyrotropin (TSH), lutropin (LH), human chorionic gonadotropin (hCG) and follitropin (FSH) involves leucine-rich repeats (LRRs) present in an N-terminal ectodomain, and can be associated with a limited number of residues at key positions of the LRRs. The mechanism by which binding of the hormones results in activation is proposed to involve switching of the ectodomain from a tethered inverse agonist to a full agonist of the serpentine, rhodopsin-like region of the receptor. Unexpectedly, the picture is complicated by the observation that promiscuous activation of one of the receptors (FSHr) by hCG or TSH can result from activating mutations affecting the serpentine region of the receptors.     

Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor

The thyrotropin receptor (TSHR) is a G protein-coupled receptor (GPCR) that is member of the leucine-rich repeat subfamily (LGR). In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM) 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors.     

Purification and properties of bovine pituitary follitropin.

A reproducible procedure was developed for the purification of follitropin from frozen bovine pituitary glands. The method involved precipitation with (NH4)2SO4 and acetone, followed by ion-exchange column chromatography on CM-cellulose and DEAE-cellulose and gel filtration on Sephadex G-100. A specific radioligand-receptor assay for follitropin was used to locate the activity in eluates after column chromatography and gel filtration. The potency of the highly purified bovine follitropin as measured by Steelman-Pohley bioassay was 164 times that of NIH-FSH-S1 standard preparation. They yield of bovine follitropin was 2.9 mg/kg of frozen pituitary glands. Electrophoretically, bovine follitropin was more acidic in nature and migrated further towards the anode than lutropin and thyrotropin. The elution volume of bovine follitropin by gel filtration on Sephadex G-100 was very similar to that of bovine lutropin. The amino acid composition of bovine follitropin was similar to that of sheep and human follitropin, being rich in lysine, aspartic acid, threonine, serine, glutamic acid and half-cystine.     

Role of the third intracellular loop of the angiotensin II receptor subtype AT2 in ligand-receptor interaction

Angiotensin II (Ang II) receptor subtypes AT1 and AT2 share 34% overall homology, but the least homology is in their third intracellular loop (3rd ICL). In an attempt to elucidate the role of the 3rd ICL in determining the similarities and differences in the functions of the AT1 and the AT2 receptors, we generated a chimeric receptor in which the 3rd ICL of the AT2 receptor was replaced with that of the AT1 receptor. Ligand-binding properties and signaling properties of this receptor were assayed by expressing this receptor in Xenopus oocytes. Ligand-binding studies using [125I-Sar1-Ile8] Ang II, a peptidic ligand that binds both the AT1 and the AT2 receptor subtypes, and 125I-CGP42112A, a peptidic ligand that is specific for the AT2 receptor, showed that the chimeric receptor has lost affinity to both ligands. However, IP3 levels of the oocytes expressing the chimeric receptor were comparable to the IP3 levels of the oocytes expressing the AT1 receptor, suggesting that the chimeric receptors could couple to phospholipase C pathway in response to Ang II. We have shown previously that the nature of the amino acid present in the position 215 located in the fifth transmembrane domain (TMD) of the AT2 receptor plays an important role in determining its affinity to different ligands. Our results from the ligand-binding studies of the chimeric receptor further support the idea that the structural organization of the region spanning the 5th TMD and the 3rd ICL of the AT2 receptor has an important role in determining the ligand-binding properties of this receptor.     

Swing-Out of the β3 Hybrid Domain Is Required for αIIbβ3 Priming and Normal Cytoskeletal Reorganization,but Not Adhesion to Immobilized Fibrinogen

Structural and functional analyses of integrin αIIbβ3 has implicated swing-out motion of the β3 hybrid domain in αIIbβ3 activation and ligand binding. Using data from targeted molecular dynamics (TMD) simulations, we engineered two disulfide-bonded mutant receptors designed to limit swing-out (XS-O). XS-O mutants cannot bind the high Mr ligand fibrinogen in the presence of an activating mAb or after introducing mutations into the αIIb subunit designed to simulate inside-out signaling. They also have reduced capacity to be “primed” to bind fibrinogen by pretreatment with eptifibatide. They can, however, bind the small RGD venom protein kistrin. Despite their inability to bind soluble fibrinogen, the XS-O mutants can support adhesion to immobilized fibrinogen, although such adhesion does not initiate outside-in signaling leading to normal cytoskeletal reorganization. Collectively, our data further define the biologic role of β3 hybrid domain swing-out in both soluble and immobilized high Mr ligand binding, as well as in priming and outside-in signaling. We also infer that swing-out is likely to be a downstream effect of receptor extension.     

The position of the alpha and beta subunits in a single chain variant of human chorionic gonadotropin affects the heterodimeric interaction of the subunits and receptor-binding epitopes

The glycoprotein hormone family represents a class of heterodimers, which include the placental hormone human chorionic gonadotropin (CG) and the anterior pituitary hormones follitropin, lutropin, and thyrotropin. They are composed of common alpha subunit and a hormone-specific beta subunit. Based on the CG crystal structure, it was suggested that the quaternary subunit interactions are crucial for biological activity. However, recent observations using single chain glycoprotein hormone analogs, where the beta and alpha subunits are linked (NH(2)-CGbeta-alpha; CGbetaalpha orientation), implied that the heterodimeric-like quaternary configuration is not a prerequisite for receptor binding/signal transduction. To study the heterodimeric alignment of the two subunit domains in a single chain and its role in the intracellular behavior and biological action of the hormone, a single chain CG variant was constructed in which the carboxyl terminus of alpha was fused to the CGbeta amino terminus (NH(2)-alpha-CGbeta; alphaCGbeta orientation). The secretion rate of alphaCGbeta from transfected Chinese hamster ovary cells was less than that seen for CGbetaalpha. The alphaCGbeta tether was not recognized by dimer-specific monoclonal antibodies and did not bind to lutropin/CG receptor. To define if one or both subunit domains were modified in alphaCGbeta, it was co-transfected with a monomeric alpha or CGbeta gene. In each case, alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes were formed indicating that CG dimer-specific epitopes were established. The alphaCGbeta/alpha complex bound to receptor indicating that the beta domain in the alphaCGbeta tether was still functional. In contrast, no significant receptor binding of alphaCGbeta/CGbeta was observed indicating a major perturbation in the alpha domain. These results suggest that although dimeric-like determinants are present in both alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes, the receptor binding determinants in the alpha domain of the tether are absent. These results show that generating heterodimeric determinants do not necessarily result in a bioactive molecule. Our data also indicate that the determinants for biological activity are distinct from those associated with intracellular behavior.     

Static and dynamic roles of extracellular loops in G-protein-coupled receptors: a mechanism for sequential binding of thyrotropin-releasing hormone to its receptor.

Small ligands generally bind within the seven transmembrane-spanning helices of G-protein-coupled receptors, but their access to the binding pocket through the closely packed loops has not been elucidated. In this work, a model of the extracellular loops of the thyrotropin-releasing hormone (TRH) receptor (TRHR) was constructed, and molecular dynamics simulations and quasi-harmonic analysis have been performed to study the static and dynamic roles of the extracellular domain. The static analysis based on curvature and electrostatic potential on the surface of TRHR suggests the formation of an initial recognition site between TRH and the surface of its receptor. These results are supported by experimental evidence. A quasi-harmonic analysis of the vibrations of the extracellular loops suggest that the low-frequency motions of the loops will aid the ligand to access its transmembrane binding pocket. We suggest that all small ligands may bind sequentially to the transmembrane pocket by first interacting with the surface binding site and then may be guided into the transmembrane binding pocket by fluctuations in the extracellular loops.     

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.     

Notch ligand endocytosis: mechanistic basis of signaling activity

Regulation of Notch signaling is critical to development and maintenance of most eukaryotic organisms. The Notch receptors and ligands are integral membrane proteins and direct cell-cell interactions are needed to activate signaling. Ligand-expressing cells activate Notch signaling through an unusual mechanism involving Notch proteolysis to release the intracellular domain from the membrane, allowing the Notch receptor to function directly as the downstream signal transducer. In the absence of ligand, the Notch receptor is maintained in an autoinhibited, protease resistant state. Genetic studies suggest that Notch ligands require ubiquitylation, epsin endocytic adaptors and dynamin-dependent endocytosis for signaling activity. Here we discuss potential models and supporting evidence to account for the absolute requirement for ligand endocytosis to activate signaling in Notch cells. Specifically, we focus on a role for ligand-mediated endocytic force to unfold Notch, override the autoinhibited state, and activate proteolysis to direct Notch-specific cellular responses.