Isolation and Characterization of the Putative Photoreceptor for Phototaxis in Amoebae of the Cellular Slime Mold,Dictyostelium discoideum

Amoebae of the cellular slime mold Dictyostelium discoideum (strain AX2) produce a pigment with an absorption spectrum that closely resembles the action spectrum for phototaxis. The protein-pigment complex was isolated and purified by sucrose gradient centrifugation, fast protein liquid chromatography (FPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). It is tightly membrane-bound and the bulk of it is located in the mitochondrial membrane fraction, while a small part is located in the cytoplasmic membrane fraction, as indicated by marker enzyme tests (succinate dehydrogenase for mitochondria and alkaline phosphatase for the cytoplasmic membrane). It is speculated that the pigment bound to the cytoplasmic membrane acts as photoreceptor and that bound to the mitochondria operates as a shading pigment in the light direction perception mechanism of Dictyostelium amoebae.     

Glucocorticoid receptors in lung. Comparison between nonactivated and activated forms of the cytoplasmic glucocorticoid binding protein and their relationship to the nuclear binding protein of fetal rabbit lung.

In the absence of salt the cytoplasmic glucocorticoid receptor of fetal rabbit lung sediments at 7 S while the nuclear receptor sediments at 4 S. However, if nuclear extracts are mixed with receptor-depleted cytosol preparations in dilute buffer solutions without added salt, the nuclear 4 S receptor sediments as a 7 S species similar to that observed for the cytoplasmic form under the same conditions suggesting an interaction of the nuclear receptor with other cytosol proteins rather than with itself. In addition, both cytoplasmic and nuclear receptors sediment at 4 S in 0.4 M KCl and a major fraction of the nuclear receptor has an agarose elution profile identical to that of the cytoplasmic receptor. Thus a major fraction of the nuclear receptors is indistinguishable from the cytoplasmic receptors by the methods used. Since the cytoplasmic receptor sediments at 4 S in 0.15 M KCl, it is suggested that in vivo the glucocorticoid receptor may exist as a 4 S species and that the 7 S form described previously may result from an interaction of the 4 S component with other cytosol proteins in hypotonic media. About 25% of the receptor present in nuclear extracts has an agarose elution profile different from that of the cytoplasmic receptor in 0.4 M KCl. This suggests that either the nuclear receptor associates with itself or other nuclear proteins or that more than one form of nuclear receptor exists. Earlier observations suggested that in the absence of hormone the glucocorticoid receptor is localized exclusively in the cytoplasm of lung cells and that the nuclear receptor is formed by a transfer of the cytoplasmic steroid-receptor complex into the nucleus. A prerequisite for this transfer seems to be a modification of the receptor to an active form which can bind to nuclei. This receptor transfomration, referred to in this paper as activation of the receptor, can occur in the absence of nuclei and is highly dependent on temperature and ionic strength. Cytoplasmic receptors activated either by heating or by exposure to high ionic strength are indistinguishable from nonactivated receptors by sucrose density gradient analysis or by agarose gel filtration in solutions containing 0.4 M KCl. Simiarly, no significant difference in the absence of salt is observed after activation by heating. These results suggest that activation of the cytoplasmic glucocorticoid receptor involves conformational changes which favor its transfer and/or binding to nuclear sites rather than conversion of a 4 S species to a faster-sedimenting form by dimerization or by addition of another protein unit as has been proposed for the activation of the estrogen receptor of the rat uterus.     

Process of attachment of phi X174 parental DNA to the host cell membrane

The phi X174-DNA membrane complex was isolated from Escherichia coli infected with phi X174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis. The phi X174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having bouyant densities of 1.195 and 1.150 g/ml, respectively. Immediately after infection with phi X147, replicating DNA was pulse-labeled and then the incorporated label was chased. The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction. The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI). The phi X174 DNA-membrane complex contained a speficic membrane-bound protein having a molecular weigth of 80,000 which is accumulated in the host DNA-membrane complex. These results suggest that when phi X174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.     

Mapping out regions on the surface of the aspartate receptor that are essential for kinase activation

The aspartate receptor of bacterial chemotaxis is representative of a large family of taxis receptors widespread in prokaryotes. The homodimeric receptor associates with cytoplasmic components to form a receptor-kinase signaling complex. Within this complex the receptor is known to directly contact the histidine kinase CheA, the coupling protein CheW, and other receptor dimers. However, the locations and extents of the contact regions on the receptor surface remain ambiguous. The present study applies the protein-interactions-by-cysteine-modification (PICM) method to map out surfaces on the aspartate receptor that are essential for kinase stimulation in the assembled receptor-kinase complex. The approach utilizes 52 engineered cysteine positions scattered over the surface of the receptor periplasmic and cytoplasmic domains. When the bulky, anionic probe 5-fluorescein-maleimide is coupled to these positions, large effects on receptor-mediated kinase stimulation are observed at eight cytoplasmic locations. By contrast, no large effects are observed for probe attachment at exposed positions in the periplasmic domain. The results indicate that essential receptor surface regions are located near the hairpin turn at the distal end of the cytoplasmic domain and in the cytoplasmic adaptation site region. These surface regions include the docking sites for CheA, CheW, and other receptor dimers, as well as surfaces that transmit information from the receptor adaptation sites to the kinase. Smaller effects observed in the cytoplasmic linker or HAMP region suggest this region may also play a role in kinase regulation. A comparison of the activity perturbations caused by a dianionic, bulky probe (5-fluorescein-maleimide), a zwitterionic, bulky probe (5-tetramethyl-rhodamine-maleimide), and a nonionic, smaller probe (N-ethyl-maleimide) reveals the roles of probe size and charge in generating the observed effects on kinase activity. Overall, the results indicate that interactions between the periplasmic domains of different receptor dimers are not required for kinase activation in the signaling complex. By contrast, the observed spatial distribution of protein contact surfaces on the cytoplasmic domain is consistent with both (i) distinct docking sites for cytoplasmic proteins and (ii) interactions between the cytoplasmic domains of different dimers to form a trimer-of-dimers.     

Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling

Transforming growth factor beta (TGF-beta) signals through three high affinity cell surface receptors, TGF-beta type I, type II, and type III receptors. The type III receptor, also known as betaglycan, binds to the type II receptor and is thought to act solely by "presenting" the TGF-beta ligand to the type II receptor. The short cytoplasmic domain of the type III receptor is thought to have no role in TGF-beta signaling because deletion of this domain has no effect on association with the type II receptor, or with the presentation role of the type III receptor. Here we demonstrate that the cytoplasmic domains of the type III and type II receptors interact specifically in a manner dependent on the kinase activity of the type II receptor and the ability of the type II receptor to autophosphorylate. This interaction results in the phosphorylation of the cytoplasmic domain of the type III receptor by the type II receptor. The type III receptor with the cytoplasmic domain deleted is able to bind TGF-beta, to bind the type II receptor, and to enhance TGF-beta binding to the type II receptor but is unable to enhance TGF-beta2 signaling, determining that the cytoplasmic domain is essential for some functions of the type III receptor. The type III receptor functions by selectively binding the autophosphorylated type II receptor via its cytoplasmic domain, thus promoting the preferential formation of a complex between the autophosphorylated type II receptor and the type I receptor and then dissociating from this active signaling complex. These studies, for the first time, elucidate important functional roles of the cytoplasmic domain of the type III receptor and demonstrate that these roles are essential for regulating TGF-beta signaling.     

The selective isolation of the uterine oestradiol-receptor complex by binding to oligo(dT)-cellulose. The mediation of an essential activator in the transformation of cytosol receptor.

The [3H]oestradiol-receptor complex was selectively isolated from rat uterus cytosol by column chromatography on oligo(dT)-cellulose. Optimal conditions are described for the binding of the complex to oligo(dT)-cellulose, which is shown to be similar to its binding to DNA-cellulose. The cytosol complex has an apparent mol. wt. of 50,000-60,000 in high salt concentrations, as determined by Sephadex G-100 chromatography. This corresponds to the 4S cytoplasmic oestradiol receptor. In binding to oligo(dT)-cellulose the receptor is transformed into a form with an apparent mol.wt. of 100,000-120,000, corresponding to the 5S nuclear receptor complex. This transformation mimics the conversion in vivo of the cytoplasmic oestradiol receptor into the nuclear form. The binding of the complex to oligo(dT)-cellulose as a 5S nuclear form is unequivocally demonstrated to require the mediation of an activating present in the cytosol. The requirement for an activating factor is discussed in relation to reports that nuclear binding of the oestradiol-receptor complex is not dictated solely by the availability of the cytoplasmic oestradiol receptor.     

Rat ovarian lutropin receptor is a transmembrane protein. Evidence for an Mr-20,000 cytoplasmic domain.

Membrane topography of the rat ovarian lutropin receptor was studied by two different approaches. Ovarian membrane preparation, labelled with 125I-labelled human choriogonadotropin in vivo, was subjected to extensive chymotryptic digestion. The soluble and membrane-bound radioactive complexes were cross-linked with glutaraldehyde, and analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. Chymotrypsin solubilized 70-75% of the radioactivity as Mr-96,000, Mr-74,000 and Mr-61,000 complexes, and decreased the size of the membrane-bound 125I-labelled human choriogonadotropin-receptor complex from Mr 130,000 to Mr 110,000. The Mr-110,000 complex was not observed when 0.1% Triton X-100 was present in the proteolytic digestion. Enrichment of inside-out-oriented plasma-membrane vesicles by concanavalin A affinity chromatography increased by 70% the fraction of radioactivity that remained in the membrane fraction after chymotrypsin treatment. Chymotrypsin also diminished the size of the membrane-bound unoccupied receptor from Mr 90,000 to Mr 70,000, as detected by ligand (125I-labelled human choriogonadotropin) blotting. These results suggest that the lutropin receptor is a transmembrane protein with a cytoplasmic domain of Mr 20,000 that is sensitive to proteolytic digestion in the inside-out-oriented plasma-membrane vesicles.     

Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.     

Studies on the nuclear binding of steroid hormone-receptor complex; characteristics of binding of nuclei from fetal rat liver to 3H-dexamethasone-liver cytoplasmic receptor complex

Binding of 3H-dexamethasone (Dex)-rat liver cytoplasmic receptor complex to nuclei from fetal rat livers in vitro exhibited a high-affinity and saturable nature (Kd=1.5 X 10- M, maximal binding sites=470 fmole/mg DNA), and the binding was inhibited competitively by prior injection of Dex in vivo. While binding of 3H-Dex-receptor complex to nuclei from adult rat liver was in low affinity and unsaturable, and injection of Dex prior to the sacrifice of animals did not influence the nuclear binding to 3H-Dex-receptor complex in vitro. Differential salt-extraction with KCl solution of the nuclear bound 3H-Dex receptor complex revealed the presence of salt-extractable and residual forms of bound receptors. The amount of the fraction extracted with 0.3 M KCl reached its maximum at 10 min after the start of incubation, while the 1.0 M KCl-extractable and residual fractions reached their maximum plateaus after 30 min of the incubation. Scatchard analysis revealed that the binding of the receptor complex to the 0.3M and 1.0M KCl fractions was saturable, while the residual fraction did not show any tendency of saturation under the experimental conditions employed in the present study. The results obtained in this work were compared to those which have been reported by other investigators.     

GM-CSF binding to its receptor induces oligomerisation of the common beta-subunit

The stoichiometry of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor complex is still unresolved. We have utilised a sensitive, functional assay for receptor homodimerisation to show that GM-CSF induces dimerisation of the common signalling subunit, hbeta(c). We generated a chimeric cytokine receptor in which the extracellular and transmembrane domains of hbeta(c)are fused to the cytoplasmic domain of erythropoietin receptor (EPO-R). Given that to induce EPO-R activation and mitogenic signalling there is a requirement for formation of a specific homodimeric complex, we reasoned that the cytoplasmic domain of EPO-R could be utilised as a highly sensitive reporter for functional homodimer formation. We show that, in the presence of a cytoplasmically truncated GM-CSF alpha-subunit, the hbetac-EPO receptor chimera transduces a mitogenic signal in BaF-B03 in response to GM-CSF. This is consistent with formation of a hbeta(c)homodimer following GM-CSF binding and implies that ligand stimulation induces formation of a higher order complex that contains the hbeta(c)homodimer.     

Signaling domains of the interleukin 2 receptor

Interleukin (IL-)2 and its receptor (IL-2R) constitute one of the most extensively studied cytokine receptor systems. IL-2 is produced primarily by activated T cells and is involved in early T cell activation as well as in maintaining homeostatic immune responses that prevent autoimmunity. This review focuses on molecular signaling pathways triggered by the IL-2/IL-2R complex, with an emphasis on how the IL-2R physically translates its interaction with IL-2 into a coherent biological outcome. The IL-2R is composed of three subunits, IL-2Ralpha, IL-2Rbeta and gammac. Although IL-2Ralpha is an important affinity modulator that is essential for proper responses in vivo, it does not contribute to signaling due a short cytoplasmic tail. In contrast, IL-2Rbeta and gammac together are necessary and sufficient for effective signal transduction, and they serve physically to connect the receptor complex to cytoplasmic signaling intermediates. Despite an absolute requirement for gammac in signaling, the majority of known pathways physically link to the receptor via IL-2Rbeta, generally through phosphorylated cytoplasmic tyrosine residues. This review highlights work performed both in cultured cells and in vivo that defines the functional contributions of specific receptor subdomains-and, by inference, the specific signaling pathways that they activate-to IL-2-dependent biological activities.     

Unoccupied binding sites for oestrogen in nuclei of a breast tumour cell line (MCF-7).

1. A method to measure both occupied and unoccupied oestrogen receptors directly in the crude nuclear fraction of the MCF-7 cells was developed. The receptors had high affinity for oestradiol (Kd approx. 0.7 nM) and binding specificity characteristics of oestrogen receptors. 2. A substantial amount of the unoccupied receptors were found in the crude nuclear fraction. 3. Several experiments excluded the possibility that the unoccupied nuclear receptor might be a cytoplasmic contaminant. (a) Multiple extractions with Tris buffer released about 75% of the total receptor content, leaving the rest unextractable in the crude nuclear fraction. (b) Nuclei purified by centrifugation through 1.8M-sucrose and treatment with 0.7% Triton X-100, or by centrifugation through 50% glycerol with 0.1% Triton X-100 contained similar amounts of unoccupied receptors to that found in the crude nuclear fraction. (c) In cells cultured during 5 days after preconfluency a 3-fold increase in the amount of unoccupied cytoplasmic receptors occurred, whereas the amount of unoccupied nuclear receptors did not change significantly and conversely in cells exposed to increasing concentrations of oestradiol the unoccupied cytoplasmic receptor was continuously depleted but no considerable change in the unoccupied nuclear receptor was found.     

Steroid-binding properties and stabilization of cytoplasmic glucocorticoid receptors from rat thymus cells

1. A competitive binding assay was adapted for determination of the specific binding of glucocorticoids to cytoplasmic receptors from rat thymus cells. The steroid–receptor complexes prepared by incubation of a cytoplasmic fraction from rat thymus cells with [1,2-³H₂]cortisol or with [1,2,4-³H₃]triamcinolone acetonide had rates of dissociation at 37°C similar to those from intact cells. 2. The cytoplasmic receptor was unstable at 3°C, but the rate of inactivation was decreased in the presence of 2.5mm-EDTA. The steroid–receptor complex was stable. 3. Rate constants for association and for dissociation, and association constants, were determined for the interactions of cortisol, cortexolone, dexamethasone and triamcinolone acetonide with the cytoplasmic receptor at 3°C. Differences in the association constants for different steroids could largely be accounted for by the differences in the rate constants for dissociation, but the rate constants for association did not vary greatly; the implications of these findings for the nature of the steroid-binding site are discussed. 4. A cytoplasmic fraction prepared from cells which had been incubated at 37°C under anaerobic conditions bound much less [1,2-³H₂]cortisol than did a fraction from aerobic cells, but the binding capacity was restored after exposure of the anaerobic cells to O₂. 5. The specific binding of [1,2-³H₂]-cortisol to intact thymus cells incubated aerobically was not affected by the presence of 0.1mm-cycloheximide, nor did this concentration of cycloheximide inhibit the recovery of specific binding observed when anaerobic cells were transferred to an aerobic atmosphere. 6. The energy dependence of specific binding of cortisol to the receptor is discussed with reference to possible mechanisms.     

Rat liver chromatin non-histone proteins and glucocorticoid binding

We have previously characterized a specific corticosterone binding protein in chromosomal non histone proteins (NHP) from rat liver. In this paper, we present evidence that a relationship exists between this protein and the cytoplasmic glucocorticoid receptor. The binding capacity of NHP is reduced by 40 p. cent when this fraction is isolated from adrenalectomized animals. Incubation of isolated nuclei with the glucocorticoid hormone receptor complex results in a decrease in the specific radioactivity of the cytoplasmic proteins and simultaneously in a rapid uptake of the isotope by the nucleus; radioactive hormone was extracted along with the NHP. Evidence is presented that the NHP component binding the hormone is closely related or identical to the cytoplasmic receptor-proteins. Progesterone and corticosterone compete similarly for the binding of dexamethasone to nuclear and cytoplasmic forms of the receptor. However the nuclear form of the receptor has a higher affinity for corticosterone (K_a : 6 × 10⁹ M⁻¹) than for dexamethasone (K_A : 10⁸ M⁻¹) in vitro.A mixture of rat liver NHP and cytosol was shown to bind specifically more corticosterone than when the two proteins were incubated separately with the hormone. The Scatchard analysis shows that the enhancement of binding is due to an interaction of nuclear and cytoplasmic proteins leading to the appearance of a stable protein-protein complex which has a high affinity for the hormone (K_a : 2 × 10⁸ M⁻¹). KCl prevented this interaction. Complex formation does not require the presence of the hormone. The experiments presented here favor the hypothesis of the existence of a regulatory protein in the nucleus. This protein associated with the binding protein to reveal or enhance the active form of the receptor.     

The twin-arginine leader-binding protein,DmsD, interacts with the TatB and TatC subunits of the Escherichia coli twin-arginine translocase

The twin-arginine translocase (Tat) pathway is involved in the targeting and translocation of fully folded proteins to the inner membrane and periplasm of bacteria. Proteins that use this pathway contain a characteristic twin-arginine signal sequence, which interacts with the receptor complex formed by the TatBC subunits. Recently, the DmsD protein was discovered, which binds to the twin-arginine signal sequences of the anaerobic respiratory enzymes dimethylsulfoxide reductase (DmsABC) and trimethylamine N-oxide (TMAO) reductase. In this work, the targeting of DmsD within Escherichia coli was investigated. Using cell fractionation and Western blot analysis, DmsD is found to be associated with the inner membrane of wild-type E. coli and a dmsABC mutant E. coli under anaerobic conditions. In contrast, DmsD is predominantly found in the cytoplasmic fraction of a Delta tatABCDE strain, which suggests that DmsD interacts with the membrane-associated Tat complex. Under aerobic conditions DmsD was also found primarily in the cytoplasmic fraction of wild-type E. coli, suggesting that physiological conditions have a significant effect upon the targeting of DmsD to the inner membrane. Size exclusion chromatography data and membrane washing studies indicate that DmsD is interacting tightly with an integral membrane protein and not with the lipid component of the E. coli inner membrane. Additional investigation into the nature of this interaction revealed that the TatB and TatC subunits of the translocase are important for the interaction of DmsD with the E. coli inner membrane.     

The effect of antiestrogens on the nuclear binding of the estrogen receptor

Experiments were designed to determine whether or not various antiestrogens in direct competition with estradiol-17β (E₂) would inhibit the translocation of the estrogen receptor complex from the cytoplasm to nuclei in rat uterine tissue. Incubation of the antiestrogens CI-628, $\text{cis}$-clomiphene, U-11,100A and MER-25 with rat uteri caused the nuclear uptake of the antiestrogen receptor complex which was greatest for most antiestrogens at concentrations of 1 × 10^?6 to 1 × 10^?5M. At higher concentrations of CI-628, $\text{cis}$-clomiphene, and U-11,100A the nuclear binding of the antiestrogen receptor complex was greatly decreased. Incubation of the antiestrogens with E₂ resulted in a dramatic inhibition of the nuclear uptake of the estrogen receptor. $\text{Trans}$-clomiphene, a weak estrogen, did not inhibit the movement of the uterine cytoplasmic receptor into the nuclear fraction.     

Conserved glycine residues in the cytoplasmic domain of the aspartate receptor play essential roles in kinase coupling and on-off switching

The aspartate receptor of the bacterial chemotaxis pathway serves as a scaffold for the formation of a multiprotein signaling complex containing the receptor and the cytoplasmic pathway components. Within this complex, the receptor regulates the autophosphorylation activity of histidine kinase CheA, thereby controlling the signals sent to the flagellar motor and the receptor adaptation system. The receptor cytoplasmic domain, which controls the on-off switching of CheA, possesses 14 glycine residues that are highly conserved in related receptors. In principle, these conserved glycines could be required for static turns, bends, or close packing in the cytoplasmic domain, or they could be required for conformational dynamics during receptor on-off switching. To determine which glycines are essential and to probe their functional roles, we have substituted each conserved glycine with both alanine and cysteine, and then measured the effects on receptor function in vivo and in vitro. The results reveal a subset of six glycines which are required for receptor function during cellular chemotaxis. Two of these essential glycines (G388 and G391) are located at a hairpin turn at the distal end of the folded cytoplasmic domain, where they are required for the tertiary fold of the signaling subdomain and for CheA kinase activation. Three other essential glycines (G338, G339, and G437) are located at the border between the adaptation and signaling subdomains, where they play key roles in CheA kinase activation and on-off switching. These three glycines form a ring around the four-helix bundle that comprises the receptor cytoplasmic domain, yielding a novel architectural feature termed a bundle hinge. The final essential glycine (G455) is located in the adaptation subdomain where it is required for on-off switching. Overall, the findings confirm that six of the 14 conserved cytoplasmic glycines are essential for receptor function because they enable helix turns and bends required for native receptor structure, and in some cases for switching between the on and off signaling states. An initial working model proposes that the novel bundle hinge enables the four-helix bundle to bend, perhaps during the assembly of the receptor trimer of dimers or during on-off switching. More generally, the findings predict that certain human disease states, including specific cancers, could be triggered by lock-on mutations at essential glycine positions that control the on-off switching of receptors and signaling proteins.