Contacts between extracellular loop two and transmembrane helix six determine basal activity of the thyroid-stimulating hormone receptor

A number of alanine mutations in extracellular loop two (ECL2) of the thyroid-stimulating hormone receptor (TSHR) were found to increase or decrease basal activity when compared with the wild type receptor. K565A was identified as a mutant with decreased basal activity, and strongly impaired hormone induced signaling activity. To gain insights into how ECL2 mutants affect basal activity, we focused on constitutively activating pathogenic mutant I568V in ECL2, which exhibits elevated basal activity. Because our molecular model suggests that Ile-568 is embedded in an environment of hydrophobic residues provided by transmembrane helix bundle, we tested mutants in this region to identify potential interaction partner(s) for Ile-568. Indeed, the double mutant I568V/I640L (ECL2/TMH6) suppresses the increased basal activity exhibited by I568V alone. We suggest a spatial and functional relationship between ECL2 and TMH6 in which side chain interaction between Ile-568 and Ile-640 constrains the receptor in a conformation with low basal activity. Although the single mutant I640L exhibits basal activity lower than wild type, its differently branched and bulkier side chain complements the reduced side chain bulk in I568V, restoring wild type basal activity to the double mutant. This scenario is confirmed by the reciprocal double mutant I640V/I568L. The combination of basally increased activity of I640V and basally decreased activity of mutant I568L also restores basal activity of wild type TSHR. These and other mutant phenotypes reported here support a dynamic interface between TMH6 and ECL2. Disruption of this critical interface for signaling by introduction of mutations in TSHR can either increase or decrease basal activity.     

Active conformation of the erythropoietin receptor: random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains

In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu(226) and Ile(227)) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.     

Activation of the luteinizing hormone receptor following substitution of Ser-277 with selective hydrophobic residues in the ectodomain hinge region

Glycoprotein hormone receptors are G protein-coupled receptors with ligand-binding ectodomains consisting of leucine-rich repeats. The ectodomain is connected by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Gain-of-function mutants of luteinizing hormone (LH) and thyroid-stimulating hormone receptors found in patients allowed identification of residues important for receptor activation. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the LH (S277I) and follicle-stimulating hormone receptors (S273I) and observed increased basal cAMP production and ligand affinity by mutant receptors. For the LH receptor, conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (Leu>Val>Met>Ile) confers constitutive receptor activation whereas serine deletion or substitution with charged Arg, Lys, or Asp led to defective receptor expression. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of glycoprotein hormone receptors constrain the TM region. Point mutations in the hinge region of these proteins, or ligand binding to these receptors, could cause conformational changes in the TM region that result in G(s) activation.     

Mutations in the mouse TSH receptor equivalent to human constitutively activating TSH receptor mutations also cause constitutive activity.

Constitutively activating mutations in the human thyroid-stimulating hormone (TSH) receptor (TSHr) have been identified as the most prevalent molecular cause of non-autoimmune hyperthyroidism. To investigate the feasibility of an animal model for non-autoimmune hyperthyroidism, we introduced two mutations in the mouse TSHr which had previously been identified in the human TSHr. The two human mutations showed strong differences in TSH binding, basal cAMP and IP accumulation. In the human TSHr, the Ile 486 Phe mutation causes a high increase of basal cAMP accumulation and also basal stimulation of the inositol phosphate pathway, whereas the Val 509 Ala mutation results in a low increase of basal cAMP accumulation without affecting IP signaling. RNA was isolated from mouse thyroid tissue and reverse transcribed. A 2.4 kb PCR product from the mouse TSHr was cloned into the pGEM-T vector system. Ile was substituted with Phe at codon 486 and Val with Ala at codon 509. These mutated mouse TSHrs were subcloned in the pSVL expression vector. After transient expression in COS-7 cells, basal and TSH-stimulated cAMP and IP accumulation, cell surface expression and TSH binding were determined and directly compared to the human TSHr. Whereas constitutively activating mutations of the human parathyroid hormone (PTH)/PTH-related peptide receptor showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor, these two mouse TSHr mutations resulted in constitutive activity similar to the homologous mutations in the human TSHr. Therefore, it should be possible to establish a mouse model for non-autoimmune hyperthyroidism by homologous recombination to study the pathogenetic mechanisms of non-autoimmune hyperthyroidism.     

Antibody-induced activation of p185HER2 in the human lung adenocarcinoma cell line Calu-3 requires bivalency

In the present study we utilized two previously described monoclonal antibodies (mAb), and their respective Fab portions, directed against the extracellular domain of p185^HER2, a transmembrane glycoprotein with intrinsic tyrosine kinase activity coded by theHER2/neu oncogene, to study the mechanism of mAb-induced receptor internalization and phosphorylation. Fluorescence scan analysis and direct binding of radiolabelled mAb and their Fab fragments showed that entire MGR2 and MGR3 mAb were reactive with similar binding affinity on two cell lines (Calu-3 and Sk-Br-3) overexpressing the p185^HER2 receptor, and unreactive on unrelated cells. The corresponding Fab fragments were positive on the related cells, but bound with diminished intensity and affinity. Entire MGR2 and MGR3 induced internalization in both Calu-3 and Sk-Br-3 cells, whereas their Fab portions were not internalized. When the bivalency of the MGR2 Fab fragment was artificially reconstituted by incubation with rabbit anti-(mouse IgG), internalization was obtained. Monovalent binding of the entire labelled antibodies, obtained in the presence of a saturating amount of unlabelled antibody, decreased both the rate and the final amount of internalized antibody. Metabolic labelling and immunoblotting experiments showed that incubation with entire MGR3 amplified the basal phosphorylation of the p185^HER2 receptor in Calu-3 and Sk-Br-3 cells, whereas MGR3 Fab decreased the signal. Taken together, our data indicate that antibody-mediated activation of p185^HER2 in Calu-3 and Sk-Br-3 cells occurs through the dimerization of receptor molecules and that bivalency of the activating antibody is mandatory for induction of internalization and phosphorylation of the receptor. Our data support an allosteric model of activation for the p185^HER2 receptor.     

The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation: insight from structures of the prolactin receptor

The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has?a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane proximal domain of the human PRLR and find that the tryptophans of the motif adopt a T-stack conformation in the unbound state. By contrast, in the hormone bound state, a Trp/Arg-ladder is formed. The conformational change is hormone-dependent and influences the receptor-receptor dimerization site 3. In the constitutively active, breast cancer-related receptor mutant PRLR(I146L), we observed a stabilization of the dimeric state and a change in the dynamics of the motif. Here we demonstrate a structural link between the WSXWS motif, hormone binding, and receptor dimerization and propose it?as?a general mechanism for class 1 receptor activation.     

Evidence that constitutively active luteinizing hormone/human chorionic gonadotropin receptors are rapidly internalized.

The luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, which belongs to the family of G-protein coupled receptors, plays an important role in gonadal steroidogenesis. Substitution of aspartic acid 556 of the LH/hCG receptor with glycine (D556G) creates a constitutively active receptor that activates adenylyl cyclase in the absence of hormone. To examine receptor internalization, human embryonic kidney cells (293 T) expressing wild type (WT) or D556G mutant receptors were incubated with [125I]hCG and subsequently analyzed for cell surface bound and internalized radioactivity. Comparison of the rate constants of internalization of the D556G mutant and WT receptors revealed that the rate of internalization of the D556G mutant was five times greater than that of the WT receptor. Although the D556G receptor internalizes [125I]hCG rapidly, a corresponding increase in [125I]hCG degradation was not seen. The internalization of another constitutively active LH/hCG receptor (aspartic acid 556 to tyrosine) was also greater than that of the WT receptor. Internalization of receptor bound [125I]hCG was inhibited by a hypertonic sucrose solution, confirming that the ligand enters the cell by receptor-mediated endocytosis. Furthermore, the constitutively active D556G and D556Y LH/hCG receptors utilize the arrestin dependent internalization pathway. These results suggest that the active state conformation of the constitutively active receptor is conducive to rapid internalization.     

Identification of cysteine 530 as the covalent attachment site of an affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen receptor

Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain.     

Conformational Selection and Equilibrium Governs the Ability of Retinals to Bind Opsin

Despite extensive study, how retinal enters and exits the visual G protein-coupled receptor rhodopsin remains unclear. One clue may lie in two openings between transmembrane helix 1 (TM1) and TM7 and between TM5 and TM6 in the active receptor structure. Recently, retinal has been proposed to enter the inactive apoprotein opsin (ops) through these holes when the receptor transiently adopts the active opsin conformation (ops*). Here, we directly test this “transient activation” hypothesis using a fluorescence-based approach to measure rates of retinal binding to samples containing differing relative fractions of ops and ops*. In contrast to what the transient activation hypothesis model would predict, we found that binding for the inverse agonist, 11-cis-retinal (11CR), slowed when the sample contained more ops* (produced using M257Y, a constitutively activating mutation). Interestingly, the increased presence of ops* allowed for binding of the agonist, all-trans-retinal (ATR), whereas WT opsin showed no binding. Shifting the conformational equilibrium toward even more ops* using a G protein peptide mimic (either free in solution or fused to the receptor) accelerated the rate of ATR binding and slowed 11CR binding. An arrestin peptide mimic showed little effect on 11CR binding; however, it stabilized opsin·ATR complexes. The TM5/TM6 hole is apparently not involved in this conformational selection. Increasing its size by mutagenesis did not enable ATR binding but instead slowed 11CR binding, suggesting that it may play a role in trapping 11CR. In summary, our results indicate that conformational selection dictates stable retinal binding, which we propose involves ATR and 11CR binding to different states, the latter a previously unidentified, open-but-inactive conformation.     

Deciphering conformational selectivity in the A2A adenosine G protein-coupled receptor by free energy simulations

Transmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G-protein) conformation. Receptor activation is normally modulated by extracellular ligand binding, but mutations in the receptor can also shift this equilibrium by stabilizing different conformational states. In this work, we built structure-energetic relationships of receptor activation based on original thermodynamic cycles that represent the conformational equilibrium of the prototypical A_2A adenosine receptor (AR). These cycles were solved with efficient free energy perturbation (FEP) protocols, allowing to distinguish the pharmacological profile of different series of A_2AAR agonists with different efficacies. The modulatory effects of point mutations on the basal activity of the receptor or on ligand efficacies could also be detected. This methodology can guide GPCR ligand design with tailored pharmacological properties, or allow the identification of mutations that modulate receptor activation with potential clinical implications.     

The hinge region of human thyroid-stimulating hormone (TSH) receptor operates as a tunable switch between hormone binding and receptor activation

The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of Thyroid stimulating hormone (TSH) binding. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. The hinge region, probably in close proximity with the α-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation.     

Intracellular cysteine residues in the tail of MHC class I proteins are crucial for extracellular recognition by leukocyte Ig-like receptor 1

The activity of NK cells is regulated by activating receptors that recognize mainly stress-induced ligands and by inhibitory receptors that recognize mostly MHC class I proteins on target cells. Comparing the cytoplasmic tail sequences of various MHC class I proteins revealed the presence of unique cysteine residues in some of the MHC class I molecules which are absent in others. To study the role of these unique cysteines, we performed site specific mutagenesis, generating MHC class I molecules lacking these cysteines, and demonstrated that their expression on the cell surface was impaired. Surprisingly, we demonstrated that these cysteines are crucial for the surface binding of the leukocyte Ig-like receptor 1 inhibitory receptor to the MHC class I proteins, but not for the binding of the KIR2DL1 inhibitory receptor. In addition, we demonstrated that the cysteine residues in the cytoplasmic tail of MHC class I proteins are crucial for their egress from the endoplasmic reticulum and for their palmitoylation, thus probably affecting their expression on the cell surface. Finally, we show that the cysteine residues are important for proper extracellular conformation. Thus, although the interaction between leukocyte Ig-like receptor 1 and MHC class I proteins is formed between two extracellular surfaces, the intracellular components of MHC class I proteins play a crucial role in this recognition.     

The formation of a salt bridge between helices 3 and 6 is responsible for the constitutive activity and lack of hormone responsiveness of the naturally occurring L457R mutation of the human lutropin receptor

The human lutropin receptor (hLHR) plays a pivotal role in reproductive endocrinology. A number of naturally occurring mutations of the hLHR have been identified that cause the receptor to become constitutively active. To gain further insights into the structural basis for the activation of the hLHR by activating mutations, we chose to examine a particularly strong constitutively activating mutation of this receptor, L457R, in which a leucine that is highly conserved among rhodopsin-like G protein-coupled receptors in helix 3 has been substituted with arginine. Using both disruptive as well as reciprocal mutagenesis strategies, our studies demonstrate that the ability of L457R to stabilize an active form of the hLHR is because of the formation of a salt bridge between the replacing amino acid and Asp-578 in helix 6. Such a lock between the transmembrane portions of helices 3 and 6 is concurrent with weakening the connections between the cytosolic ends of the same helices, including the interaction found in the wild-type receptor between Arg-464, of the (E/D)R(Y/W) motif, and Asp-564. This structural effect is properly marked by the increase in the solvent accessibility of selected amino acids at the cytosolic interfaces between helices 3 and 6. The integrity of the conserved amino acids Asn-615 and Asn-619 in helix 7 is required for the transfer of the structural change from the activating mutation site to the cytosolic interface between helices 3 and 6. The results of in vitro and computational experiments further suggest that the structural trigger of the constitutive activity of the L457R mutant may also be responsible for its lack of hormone responsiveness.     

Analysis of the glucagon receptor first extracellular loop by the substituted cysteine accessibility method

Glucagon is an important hormone for the prevention of hypoglycemia, and contributes to the hyperglycemia observed in diabetic patients, yet very little is known about its receptor structure and the receptor-glucagon interaction. In related receptors, the first extracellular loop, ECL1, is highly variable in length and sequence, suggesting that it might participate in ligand recognition. We applied a variant of the SCAM (Substituted Cysteine Accessibility Method) to the glucagon receptor ECL1 and sequentially mutated positions 197 to 223 to cysteine. Most of the mutations (15/27) affected the glucagon potency, due either to a modification of the glucagon binding site, or to the destabilization of the active receptor conformation. We reasoned that side chains accessible to glucagon must also be accessible to large, hydrophilic cysteine reagents. We therefore evaluated the accessibility of the introduced cysteines to maleimide-PEO₂-biotin ((+)-biotinyl-3-maleimido-propionamidyl-3,6-dioxa-octanediamine), and tested the effect of pretreatment of intact cells with a large cationic cysteine reagent, MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide), on glucagon potency. Our results suggest that the second and third transmembrane helices (TM2 and TM3) are extended to position 202 and from position 215, respectively, and separated by a short β stretch (positions 203-209). Glucagon binding induced a conformational change close to TM2: L198C was accessible to the biotin reagent only in the presence of glucagon. Most other mutations affected the receptor activation rather than glucagon recognition, but S217 and D218 (at the top of TM3) were good candidates for glucagon recognition and V221 was very close to the binding site.     

Reduced activity of the NPR-A kinase triggers dephosphorylation and homologous desensitization of the receptor

NPR-A, the receptor for the atrial natriuretic peptide (ANP), is a 130-kDa protein presenting an extracellular ANP-binding domain, a single transmembrane domain, an intracellular regulatory kinase homology domain (KHD), and a guanylyl cyclase catalytic domain. Upon stimulation, NPR-A receptors are activated to produce cyclic guanosine monophosphate (cGMP) and are subsequently desensitized through dephosphorylation of residues at their KHD. We used wild-type rat (r) NPR-A (WT) and a disulfide-bridged mutant (C423S) expressed in human embryonic kidney (HEK) 293 cells to study receptor phosphorylation. We have previously characterized the C423S receptor as constitutively active and desensitized. At basal state, 32P incorporation in the rNPR-A(C423S) covalent dimer is about 24 times less efficient than incorporation in the WT rNPR-A. When membranes from WT and rNPR-A(C423S) are incubated with [35S]ATPgammaS, the mutant dimer receptor displays 3.5% of the thiophosphate incorporation found for WT rNPR-A. Since the rNPR-A(C423S) dimer is already extensively dephosphorylated, we then used the WT rNPR-A to study dephosphorylation. As previously documented, adding ANP globally induces time-dependent dephosphorylation of the receptor. However, in pulse-chase experiments with the WT rNPR-A, adding ANP during the chase does not lead to a significant effect on receptor dephosphorylation. On the other hand, thiophosphorylation of the WT rNPR-A previously desensitized with ANP is reduced to 8.3% of the incorporation for untreated receptor, similar to results found with the rNPR-A(C423S) at basal state. These results demonstrate that ANP-induced rNPR-A desensitization is modulated by a significant reduction in the activity or affinity of the rNPR-A kinase that contributes to the low phosphorylation level after induction. Moreover, we further document a close relationship between tight dimerization, dephosphorylation, and desensitization.     

Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRα1 ECD, we generated a model of the human GlyRα1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRα1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.     

Structural requirements for mutational lutropin/choriogonadotropin receptor activation

Different activation mechanisms of glycoprotein hormone receptors, which are members of the G protein-coupled receptor superfamily, have been proposed. For example, the large ectodomain of glycoprotein hormone receptors may function as an inverse agonist keeping the transmembrane domain in an inactive conformation. To provide support for this hypothesis, we have generated different lutropin/choriogonadotropin receptor (LHR) constructs lacking the ectodomain. Although some ectodomain-deficient LHR constructs were targeted to the cell surface, cAMP levels remained unchanged under basal conditions and agonist application but could be increased by a mutation within the transmembrane domain 6 (D578H). Taking advantage of a constitutive activating mutation (S277N) located in the extracellular domain, we showed that the intact leucine-rich repeat-containing ectodomain is essential for constitutive activation of the LHR by mutation of the hinge region. Our findings support an activation scenario in which agonist binding or mutational alterations expose a structure within the ectodomain, which then activates the transmembrane core.     

Hormone-induced conformational changes in the hepatic insulin receptor

The insulin receptor can exist in either a lower or a higher affinity state. Hormone binding alters the equilibrium between the two states of the insulin receptor, favoring the formation of that of higher affinity (Corin, R.E., and Donner, D.B. (1982), J. Biol. Chem. 257, 104-110). After brief or extended incubations with hormone, during which the fraction of higher affinity receptors increased, 125I-insulin was covalently coupled to the alpha subunits of its receptor using disuccinimidyl suberate. Some 125I-insulin remained bound to higher affinity receptors after dissociation of hormone from lower affinity sites. This hormone could also be covalently coupled to the alpha subunit of the receptor. During extended incubations between 125I-insulin and liver plasma membranes, components of the receptor were cleaved to yield degradation products of 120,000 and 23,000 Da. The significance of this process remains undetermined. Unoccupied insulin receptors were cleaved by trypsin to produce fragments of 94,000 and 37,000 Da which remained membrane-bound and could be covalently coupled to 125I-insulin. Trypsin treatment after binding yielded an additional receptor fragment of 64,000 Da. As the incubation time between 125I-insulin and membranes was lengthened, components of the receptor became progressively less sensitive to trypsin. Higher affinity binding sites isolated after release of rapid dissociating insulin were less sensitive to trypsin than were mixtures of higher and lower affinity receptors. These observations suggest that hormone binding produces two conformational changes (alterations of tryptic lability) in the hepatic insulin receptor. The first change is rapid and exposes parts of the receptor to tryptic degradation. The second, slower conformational change renders the receptor less sensitive to trypsin and occurs with the same time course as the increase of receptor affinity mediated by site occupancy.     

Monoclonal antibodies to ligand-occupied conformers of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function

Occupancy of integrin receptors induces conformational changes in the receptor, resulting in exposure of novel interactive sites termed ligand-induced binding sites (LIBS). We report here that Fab fragments of certain antibodies against LIBS on integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) block platelet aggregation. Thus, certain LIBS or the regions surrounding them may participate in events required for platelet aggregation. In addition, certain anti-alpha IIb beta 3 LIBS Fab fragments stimulated platelet aggregation. This was due to induction of fg binding to alpha IIb beta 3, apparently by shifting a conformational equilibrium between a "resting" and an "activated" state of alpha IIb beta 3. Some of the activating anti-LIBS Fab fragments also induced high affinity fibronectin binding to alpha IIb beta 3, whereas others did not. Thus, changes in the conformation of this integrin modulate both the specificity and affinity of ligand recognition.     

Monte Carlo loop refinement and virtual screening of the thyroid-stimulating hormone receptor transmembrane domain

Metropolis Monte Carlo (MMC) loop refinement has been performed on the three extracellular loops (ECLs) of rhodopsin and opsin-based homology models of the thyroid-stimulating hormone receptor transmembrane domain, a class A type G protein-coupled receptor. The Monte Carlo sampling technique, employing torsion angles of amino acid side chains and local moves for the six consecutive backbone torsion angles, has previously reproduced the conformation of several loops with known crystal structures with accuracy consistently less than 2?Å. A grid-based potential map, which includes van der Waals, electrostatics, hydrophobic as well as hydrogen-bond potentials for bulk protein environment and the solvation effect, has been used to significantly reduce the computational cost of energy evaluation. A modified sigmoidal distance-dependent dielectric function has been implemented in conjunction with the desolvation and hydrogen-bonding terms. A long high-temperature simulation with 2?kcal/mol repulsion potential resulted in extensive sampling of the conformational space. The slow annealing leading to the low-energy structures predicted secondary structure by the MMC technique. Molecular docking with the reported agonist reproduced the binding site within 1.5?Å. Virtual screening performed on the three lowest structures showed that the ligand-binding mode in the inter-helical region is dependent on the ECL conformations.