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T H Ji

Publications and source records attributed to T H Ji.

At least 37 records · Page 2Linked to original sources
PubMed1997

Substitution of the seat-belt region of the thyroid-stimulating hormone (TSH) beta-subunit with the corresponding regions of choriogonadotropin or follitropin confers luteotropic but not follitropic activity to chimeric TSH.

The region between the 10th and 12th cysteine (Cys88-Cys105 in human thyroid-stimulating hormone beta-subunit (hTSHbeta)) of the glycoprotein hormone beta-subunits corresponds to the disulfide-linked seat-belt region. It wraps around the common alpha-subunit and has been implicated in regulating specificity between human choriogonadotropin (hCG) and human follicle-stimulating hormone (hFSH), but determinants of hTSH specificity are unknown. To characterize the role of this region for hTSH, we constructed hTSH chimeras in which the entire seat-belt region Cys88-Cys105 or individual intercysteine segments Cys88-Cys95 and Cys95-Cys105 were replaced with the corresponding sequences of hCG and hFSH or alanine cassettes. Alanine cassette mutagenesis of hTSH showed that the Cys95-Cys105 segment of the seat-belt was more important for TSH receptor binding and signal transduction than the Cys88-Cys95 determinant loop region. Replacing the entire seat-belt of hTSHbeta with the hCG sequence conferred full hCG receptor binding and activation to the hTSH chimera, whereas TSH receptor binding and activation were abolished. Conversely, introduction of the hTSHbeta seat-belt sequence into hCGbeta generated an hCG chimera that bound to and activated the TSH receptor but not the CG/lutropin (LH) receptor. In contrast, an hTSH chimera bearing hFSH seat-belt residues did not possess any follitropic activity, and its thyrotropic activity was only slightly reduced. This may in part be due to the fact that the net charge of the seat-belt is similar in hTSH and hFSH but different from hCG. However, exchanging other regions of charge heterogeneity between hTSHbeta and hFSHbeta did not confer follitropic activity to hTSH. Thus, exchanging the seat-belt region between hTSH and hCG switches hormonal specificity in a mutually exclusive fashion. In contrast, the seat-belt appears not to discriminate between the TSH and the FSH receptors, indicating for the first time that domains outside the seat-belt region contribute to glycoprotein hormone specificity.

Amino Acid Sequence↗
PubMed1997

Roles of transmembrane prolines and proline-induced kinks of the lutropin/choriogonadotropin receptor.

The lutropin/choriogonadotropin receptor is a seven-helix transmembrane (TM) receptor. A unique feature of TM helices is the content of Pro, which generally is absent in alpha helices of globular proteins. Because Pro disrupts helices and introduces a approximately 26 degrees kink, it has been speculated that Pro plays a crucial role in the structure of TM helices, exoloops, and cytoloops of TM receptors. To examine the roles of the five TM Pros of the lutropin/choriogonadotropin receptor, these residues were individually substituted. Mutant receptors were examined for surface expression, hormone binding, and cAMP induction. Surface expression was monitored after introducing the flag epitope into the receptors. Flag epitopes slightly affected cAMP induction but not hormone binding or surface expression of receptors as monitored by immunofluorescence microscopy and 125I-anti-flag antibody. The results indicate that Pro479 in TM 4 and Pro598 in TM 7 play important yet contrasting roles. Pro479 is crucial for hormone binding at the cell surface but not after solubilization of the receptor. This is more likely due to the Pro side chain than the Pro-induced kink. Pro598 is important for surface expression. The kinks of Pro463 of TM 4, Pro562 of TM 6, or Pro591 of TM 7 are not important because the substitution of Phe for these residues did not significantly impact surface expression, hormone binding, and cAMP induction.

Amino Acid Sequence↗
PubMed1997

Interaction, signal generation, signal divergence, and signal transduction of LH/CG and the receptor.

The LH/CG receptor is comprised of two structurally and functionally distinct domains, extracellular N-terminal exodomain and membrane-embedded endodomain. These two domains can separately be expressed and processed, including folding. The exodomain alone has the high-affinity hormone binding site but is not capable of generating hormonal signal. In contrast, the endodomain alone has the site for receptor activation. These two domains contact each other in holo-receptor and split receptor. This interaction, particularly through exoloops 2 and 3, constrains the high-affinity hormone binding at the exodomain. Conversely, the exodomain could be involved in receptor activation. Therefore, these two domains are not entirely independent, although they can be independently synthesized and processed. The existing evidence indicates that hCG and the receptor undergo multiple stages of interactions leading to receptor activation. Initial high-affinity binding of hCG to the exodomain results in conformational adjustments of the hCG/exodomain complex. This leads to the secondary, low-affinity contact of the hCG/exodomain complex with the endodomain. This secondary contact is responsible for generating signals. They are transduced through transmembrane domains (TM) to the cytoplasmic portion (cytoloops and the C-terminal tail) of the receptor and then transferred to cytoplasmic signaling molecules such as G protein. Mutations in the exodomain and endodomain (N-extension, exoloops, TM, cytoloops, and cytoplasmic tail) have the potential to interfere with receptor activation at different steps: signal generation, transduction, and transfer. Binding of hCG to the LH/CG receptor is known to induce two signals, one for adenylyl cyclase/ cAMP and the other for phospholipase C/inositol phosphate/diacylglycerol. The cAMP signal and IP signal diverge at the surface of the receptor. These independent signals are separately transduced through the transmembrane domains to the cytoplasmic part of the receptor, indicating the existence of the distinct transducers for each of the signals. Furthermore, it is likely that the divergent signals are separately transferred to cytoplasmic signal molecules such as G protein. In addition, each cAMP signal and IP signal consists of at least three separate subsignals: affinity signal, maximal production (efficacy) signal, and basal level signal. In heterodimeric hCG there are distinct parts responsible for high-affinity receptor binding and receptor activation. Particularly, the C-terminal residues of the alpha subunit play a crucial role in receptor activation. This alpha subunit is shared with other glycoprotein hormones, follicle-stimulating hormone, and thyroid-stimulating hormone. Interestingly, the alpha C-terminal residues play distinct roles in all three hormones, despite its common nature.

Amino Acid Sequence↗
PubMed1996

Molecular mechanism of LH/CG receptor activation.

It is known that the N-terminal half of the LH/CG receptor is responsible for high hCG binding whereas the C-terminal half is capable of receptor activation. Our results suggest that initial hCG binding at the high affinity site in the N-half receptor induces conformational adjustments. This leads to low affinity secondary contacts of the complex of hCG/the N-half receptor with the C-half receptor. This low affinity secondary contact is responsible for activating the receptor. This is based on the following observations. The C-terminal tail of hCG alpha is known to be involved in activation of the LH/CG receptor. In addition to hCG, we examined the C-terminal three residues (His90-Lys91-Ser92) of the common alpha subunit of FSH and TSH. The results show their differential roles in the three hormones. Ser92 is important for binding and cAMP induction of TSH but not for hCG and FSH. Lys91 is important for binding and cAMP induction of hCG, and cAMP induction but not binding of FSH. It is not important for binding or cAMP induction of TSH. His90 is important for all three hormones. When all three residues were truncated, FSH and TSH lose their affinity for binding and cAMP induction, whereas hCG is still capable of binding but not cAMP induction. Therefore, the three amino acids contribute differently in receptor binding and cAMP induction of hCG, FSH and TSH. Our data also indicate that the evolution of the alpha subunit has been constrained in order not to impair any of the hormones. This suggests that each hormone can be independently engineered to improve the potency. To chemically identify the contact site of the alpha C-tail of hCG in the LH/CG receptor, a decamer peptide corresponding to the alpha subunit sequence from His83 to Ser92 (peptide alpha 81-92) was derivatized with UV sensitive reagent, ABG and radio-iodinated. The resulting ABG-125I-peptide alpha 83-92 was capable of binding and activating the LH/CG receptor. Furthermore, it specifically photoaffinity-labeled the LH/CG receptor. In addition, the amino group of alpha Lys91 of peptide alpha 83-92 is crosslinked to a carboxyl group of the receptor, an indication of close association. Reciprocal mutagenesis of alpha Lys91 and Asp397 in exoloop 1 of the LH/CG receptor suggests the complementary of this pair in receptor activation but not the high affinity interaction of hCG and the receptor. In addition, Lys583 of exoloop 3 is also crucial for receptor activation. To test the conformational adjustment, ABG was attached to hCG alpha and reassociated with untreated beta to produce ABG-125I-alpha/beta. The extent of inter-subunit crosslinking of ABG-125I-alpha/beta bound to the receptor was two to three fold less than unbound ABG-125I-alpha/beta. This result indicates structural change at the subunit interface in response to hCG binding to the receptor.

Affinity Labels↗
PubMed1996

The luteinizing hormone/chorionic gonadotropin receptor has distinct transmembrane conductors for cAMP and inositol phosphate signals.

The luteinizing hormone/chorionic gonadotropin receptor is a member of the seven-transmembrane receptor family. It is coupled, presumably via Gs and Gq, to two signal pathways involving adenylyl cyclase/cAMP and phospholipase C/inositol phosphate (IP). Little is known about the events prior to G-protein coupling: for example, whether these signals are generated from a single or multiple independent origins and mechanisms, when and where they diverge, and how they are transduced. We report novel observations that the cAMP signal and the IP signal originate and diverge upstream of G-protein coupling. The generation of these two signals independently involves Lys583 in exoloop 3 of the rat receptor. For this study, Lys583 of the receptor was substituted with a panel of amino acids, and mutant receptors were assayed for hormone binding and induction of cAMP, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. No substitutions for Lys583 were permissible for cAMP induction, despite successful surface expression and hormone binding. In contrast, several substitutions were permissible for IP induction. Our results suggest two distinct transmembrane signal conductors for cAMP and inositol phosphate signals and imply particular models of receptor activation not previously suggested.

Animals↗
PubMed1996

Photoaffinity labeling of the lutropin receptor with synthetic peptide for carboxyl terminus of the human choriogonadotropin alpha subunit.

Human choriogonadotropin (hCG) consists of an alpha subunit and a beta subunit. The existing evidence from various studies using truncation, substitution, synthetic hormone peptides, and hCG crystals suggests that the C-terminal region of the alpha subunit contacts the luteinizing hormone/choriogonoadotropin (LH/CG) receptor and is involved in receptor activation. Despite a deluge of the speculation and the important role of the alpha C-terminal region, direct evidence for its interaction with the receptor has been elusive. Because of the significant biological activity, it is imperative to prove the interaction of the alpha C-terminal region. For this purpose, decamer peptides corresponding to the alpha subunit sequence from His83 to Ser92 (alpha 83-92) were derivatized with the N-hydroxysuccinimide ester of 4-azidobenzoylglycine (ABG) and radioiodinated. The resulting ABG-125I-alpha 83-92 was capable of binding and activating the LH/CG receptor. Furthermore, UV-sensitive ABG-125I-alpha 83-92 exclusively photoaffinity-labeled an approximately of 86-kDa molecule. This labeled molecule was shown to be the LH/CG receptor by various methods including immunoprecipitation by anti-LH/CG receptor antiserum. In addition, evidence is presented that the amino group of alpha Lys91 of alpha 83-92 is in such close proximity to a carboxyl group of the receptor that this pair is cross-linked to form an amide, a zero length cross-link. This low affinity contact of alpha 83-92 and the receptor is sufficient for receptor activation and is crucial for the full understanding of the mechanistics of the receptor activation steps.

Affinity Labels↗
PubMed1996

Exoloop 3 of the luteinizing hormone/choriogonadotropin receptor. Lys583 is essential and irreplaceable for human choriogonadotropin (hCG)-dependent receptor activation but not for high affinity hCG binding.

The luteinizing hormone/choriogonadotropin (CG) receptor belongs to a subfamily of glycoprotein hormone receptors within the seven-transmembrane receptor family. It is comprised of an extracellular N-terminal half of 341 amino acids and a membrane-associated C-terminal half of 303 amino acids. The N-terminal half is capable of high affinity hormone binding whereas the C-terminal half is capable of low affinity hormone binding and receptor activation. However, the precise location of the receptor activation site is currently unknown. We present evidence for the first time that Lys583 of exoloop 3 is crucial and irreplaceable for receptor activation to induce cAMP synthesis. Exoloop 3 is comprised of 11 amino acids and flanked by two Lys residues, Lys573 and Lys583, that are located at the boundaries with the transmembrane columns 6 and 7, respectively. All substitutions including Arg for Lys583 did not affect the high affinity human CG binding, but they resulted in the complete loss of cAMP synthesis induced by human CG. Ala substitutions of the other amino acids in exoloop 3 did not make such a dramatic impact on cAMP induction. The Ala scan revealed two distinct groups of amino acids in terms of their importance in cAMP induction, one group being more important than the other. Interestingly, these two groups of amino acids are arranged in an alternate sequence. This result suggests a specific structure similar to a beta-like structure for exoloop 3.

Amino Acid Sequence↗
PubMed1995

Differential roles of exoloop 1 of the human follicle-stimulating hormone receptor in hormone binding and receptor activation.

The follicle-stimulating hormone (FSH) receptor is a member of the glycoprotein hormone receptor subfamily of the seven-transmembrane receptor superfamily. These receptors have an extracellular N-terminal half of approximately 350 amino acids and a membrane-associated C-terminal half of approximately 350 amino acids. The N-terminal halves have the high affinity hormone binding site. On the other hand, the C-terminal half of the lutropin/choriogonadotropin receptor has the receptor activation site. However, little is known about the activation site and mechanism of the FSH receptor, although the existing evidence indicates crucial differences in the activation of the FSH receptor and the lutropin/choriogonadotropin receptor. As a first step to resolve this issue, we examined the upstream juxtamembrane five amino acids, Asp405-Ile406-His407-Thr408-Lys409, of the exoloop 1. Ala scan and multi-substitutions show that the five amino acid sequence is important for both hormone binding and receptor activation to induce cAMP synthesis, despite its short length. Specifically, His407 is important for high affinity hormone binding, whereas Asp405, Thr408, and Lys409 are crucial for receptor activation. The data suggest that the five amino acids may form a turn of helix that is an extension of the transmembrane helix 2. In this helical arrangement, Asp405, Thr408, and Lys409 are grouped to form a hydrophilic face of the helix, suggesting a correlation between this arrangement and receptor activation. In addition, the diverse and differential roles of the five amino acids indicate that high affinity hormone binding and receptor activation are discernible functions. These novel observations will be helpful for understanding the activation mechanism of the FSH receptor.

Amino Acid Sequence↗
PubMed1995

Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis.

Environmentally friendly toxins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects. However, little is known about the insect receptor molecules that bind these toxins and the mechanism of insecticidal activity. We report here for the first time the cloning and expression of a cDNA that encodes a receptor (BT-R1) of the tobacco hornworm Manduca sexta for an insecticidal toxin of B. thuringiensis. The receptor is a 210-kDa membrane glycoprotein that specifically binds the cryIA(b) toxin of B. thuringiensis subsp. berliner and leads to death of the hornworm. BT-R1 shares sequence similarity with the cadherin superfamily of proteins.

Amino Acid Sequence↗
PubMed1995

Lys91 and His90 of the alpha-subunit are crucial for receptor binding and hormone action of follicle-stimulating hormone (FSH) and play hormone-specific roles in FSH and human chorionic gonadotropin.

Glycoprotein hormones, FSH, LH, CG, and TSH, consist of a common alpha-subunit and a hormone-specific beta-subunit. Both subunits are thought to interact with the hormone receptors. Although several C-terminal residues of hCG alpha are known to contact the LH/CG receptor, little is known about the roles of individual C-terminal residues of FSH alpha. In this report, substitutions of various amino acids for the penultimate Lys91 and the upstream His90 of the alpha-subunit demonstrate that these two residues of FSH alpha are important for high affinity receptor binding and hormone action to induce cAMP production. In contrast, the same residues of hCG alpha are more important for cAMP induction than for high affinity receptor binding. Some substitutions significantly improved receptor binding of FSH and hCG, whereas others were detrimental. Some had the same effect on both hormones, and others impacted differently. Particularly, the substitution of Val for alpha Lys91 resulted in an improved receptor binding of and a loss of cAMP induction by FSH and hCG. On the other hand, the substitution of Arg or Pro for alpha His90 abolished receptor binding of FSH, but not of hCG. These results allowed us to generate an antagonist to FSH. Our results indicate that alpha His90 and alpha Lys91 play roles in receptor binding and cAMP induction of FSH and hCG in strikingly different ways. They will be useful to elucidate the underlying mechanisms for the interaction of FSH and hCG with their complementary receptors as well as for receptor activation.

Animals↗
PubMed1995

Role of the carboxy-terminal residues of the alpha-subunit in the expression and bioactivity of human thyroid-stimulating hormone.

The glycoprotein hormones TSH, CG, LH, and FSH are heterodimers consisting of a hormone-specific beta-subunit and a common alpha-subunit. The aim of the present study was to investigate the role of the carboxy terminus of the common alpha-subunit (amino acids Tyr89-His90-Lys91-Ser92), which has been shown to be important for human (h) CG and hFSH, for the activity of hTSH. Successive truncations of the alpha-carboxy terminus by site-directed mutagenesis revealed a stepwise reduction of bioactivity occurring at residues alpha Ser92 and alpha His90 to 64% and 13%, respectively. This contrasts with previous findings for hCG and hFSH, where loss of bioactivity occurred in a single step with the deletion of alpha Lys91 but alpha Ser92 was not important. The decreased bioactivities of the hTSH alpha-truncation mutants were reflected by concomitant reductions of cAMP production, thyroid hormone synthesis and cell growth and were accompanied by a loss of receptor binding. Substitution of residues alpha Lys91 or alpha His90 with either a hydrophobic or a bulkier residues resulted in a reduction of receptor binding and signal transduction, indicating that the alpha-carboxy terminus of hTSH may interact with the TSH receptor in a tight contact area. Conversely, substitution of alpha His90 with smaller residues enhanced bioactivity. In addition, the integrity of the alpha-carboxy terminus was essential for hTSH expression. Thus, we showed common and different roles of the alpha-carboxy-terminal residues for the glycoprotein hormones. The unique role of alpha Ser92 in hTSH activity explains the evolutionary constraint to preserve the alpha-carboxy-terminal Ser92 in all glycoprotein hormones.

Amino Acid Sequence↗
PubMed1994

Characterization of different sizes of rat luteinizing hormone/chorionic gonadotropin receptor messenger ribonucleic acids.

The LH/CG receptor is uniquely expressed in the gonads of both sexes at specific stages of development. In ovaries, its expression marks particular steps of the ovulation cycle. An enigmatic aspect of expression of the LH/CG receptor is the dramatically diverse transcript sizes [from 7 to < 1 kilobase (kb)] and development-dependent expression of different sizes of mRNAs. It has been thought that mRNAs larger than 2.1 kb encode full-length receptors, whereas those smaller than 2.1 kb encode truncated receptor, because the full-length coding sequence is 2.1 kb. As a first step in elucidation of these diverse mRNAs and corresponding proteins, we have produced a series of cDNA clones and determined their DNA sequences and deduced the amino acid sequences of the resulting proteins. Our data demonstrate that variant mRNAs are produced by alternate splicing and polyadenylation, and they encode significantly shorter truncated receptor peptides. Surprisingly, many of these variant mRNAs are larger than 2.1 kb, and some are 4.2 kb. Some of them are polyadenylated in introns 3, 4, and 10. These alternate mRNAs were successfully expressed in 293 cells to produce receptor peptides 81, 116, and 294 amino acids in length compared to the wild-type receptor, which consists of 674 amino acids. Although these receptor peptides are not secreted, they are capable of binding the hormone, indicating the presence of a hormone contact site(s) in the short peptide fragments, particularly the N-terminal 81-amino acid segment. The data presented here will be helpful for understanding the functions of different sizes of mRNAs and also be valuable in studies designed to investigate whether individual cells express a specific message or multiple messages and how different classes of LH/CG receptor mRNAs are selectively expressed dependent on differentiation and development of the gonads.

Animals↗
PubMed1993

Receptor activation of and signal generation by the lutropin/choriogonadotropin receptor. Cooperation of Asp397 of the receptor and alpha Lys91 of the hormone.

We have developed a novel method of reciprocal substitution mutation to identify pairing of amino acids within a receptor and its ligand. Using this method, we demonstrate for the first time that a pair of counterionic amino acids, one from the lutropin/choriogonadotropin receptor and the other from the ligand (human choriogonadotropin), cooperate and perhaps interact with each other to activate the receptor and to generate hormonal signal. In this study, Asp397 of the receptor was converted to Lys while Lys91 of the alpha subunit of human choriogonadotropin was substituted with Asp, thus maintaining the counterionic nature of this pair of amino acids. Mutation at each of these positions does not affect the hormone-receptor interaction but results in the significant or complete loss of the bioactivity of both the receptor and the hormone. However, when the impotent mutant receptor and mutant hormone were paired to interact together, they induced cAMP synthesis, resulting in a potent receptor-hormone couple. Substitutions with other amino acids that eliminated the counterionic nature failed to induce cAMP synthesis. Our results shed light on the molecular mechanisms of receptor activation and will serve as a model for other G-protein-coupled peptide receptors, particularly glycoprotein hormone receptors.

Aspartic Acid↗
PubMed1993

Receptor activation is distinct from hormone binding in intact lutropin-choriogonadotropin receptors and Asp397 is important for receptor activation.

Hormone binding to receptors on the cell surface triggers a sequence of events (receptor activation and signal generation) leading to activation of effectors in the cytoplasm. Receptor activation and signal generation are difficult to study as both are intimately associated with hormone binding. The lutropin-choriogonadotropin (LH/CG) receptor offers a unique model to differentiate and examine receptor activation and signal generation from hormone binding. It belongs to a subfamily of glycoprotein hormone receptors within the G-protein-coupled receptor family. This receptor subfamily has several structural features different from the structures of other G-protein-coupled receptors. These receptors consist of a large extracellular N-terminal half and membrane-associated C-terminal half of similar size. The truncated N-terminal half alone is capable of high affinity hormone binding, whereas the truncated C-terminal half alone is capable of low affinity hormone binding and cAMP induction. However, this distinction between the high affinity hormone binding and low affinity hormone binding associated with cAMP induction has not been established in intact receptors. As a step to identify a structural element which is responsible for receptor activation and signal generation, we have identified an extracellular Asp of the C-terminal half of the LH/CG receptor which is unique and common to the glycoprotein hormone receptors. Evidence is presented for the first time that Asp397 is important for induction of cAMP synthesis but not essential for hormone binding. Since extracellular Asp397 cannot interact with G-protein in the cytoplasm, the inability of the mutant LH/CG receptors with an Asp397 substitution to induce cAMP synthesis is likely to be caused by a defect in the intermediate steps (receptor activation and signal generation) between hormone binding and activation of G-protein. Therefore, our results not only demonstrate that receptor activation and signal generation are distinct from high affinity hormone binding in intact LH/CG receptors, but they also identify an amino acid important for the processes.

Amino Acid Sequence↗
PubMed1993

COOH-terminal amino acids of the alpha subunit play common and different roles in human choriogonadotropin and follitropin.

Human choriogonadotropin (hCG) and follitropin (FSH) belong to the glycoprotein hormone family. These hormones are heterodimers and composed of a common alpha subunit and a distinct beta subunit which confers receptor-binding specificities. In addition to this structural similarity, they share a similar signal pathway involving G protein, adenylyl-cyclase and induction of cAMP synthesis. Therefore, a presumptive relationship of these common structure and function has been the subject of extensive past investigations, but a definitive clue has been elusive. As a step to address this important issue, a series of recombinant mutants of hCG and human FSH were generated in which the COOH-terminal amino acids of the alpha subunit were successively removed or substituted. Furthermore, a set of peptides were synthesized with sequences corresponding to different regions of the alpha subunit. Deletion of the alpha COOH-terminal Ser92 had no effect on receptor-binding or cAMP induction by FSH and hCG. Truncation of alpha Lys91-Ser92 or alpha His90-Lys91-Ser92 abolished the ability of both hormones to induce cAMP synthesis. It significantly reduced receptor binding of FSH but not hCG. The different functions of the alpha COOH-terminal region are further noticed with a peptide corresponding to the last 10 amino acids of alpha. It failed to bind to the FSH receptor but was capable of binding to the LH/CG receptor and stimulating cAMP synthesis. These results are the first conclusive evidence that alpha His90-Lys91 play an essential role in cAMP induction of both hormones. In contrast to this common role, they are necessary for FSH binding to the FSH receptor but not for hCG binding to the LH/CG receptor. The hCG alpha COOH-terminal region makes direct contact with the LH/CG receptor, and this low affinity contact is necessary and sufficient to activate the receptor for signal generation. This conclusion is supported by the study using mutant hCGs in which either alpha His90 or Lys91 was substituted.

Amino Acid Sequence↗
PubMed1993

A specific binding protein from Manduca sexta for the insecticidal toxin of Bacillus thuringiensis subsp. berliner.

Biopesticides based on the bacterium Bacillus thuringiensis have attracted wide attention as safe alternatives to chemical pesticides. In this paper, we report, for the first time, the identification and purification of a single binding protein from a lepidopteran insect, Manduca sexta, that is specific for a cryIA toxin of B. thuringiensis. The purified protein appeared as a single band of 210 kDa on a two-dimensional gel, had a pI of approximately 5.5, and stained with Schiff's reagent. The band material was sensitive to proteolytic digestion and was rich with acidic amino acids, indicating its protein nature. Radiolabeled toxin bound to the protein with a Kd value of 708 pM and could be specifically blocked by unlabeled toxin but not by toxins from other subspecies of B. thuringiensis. This study lays the groundwork to clone the toxin binding protein and to determine the molecular mechanism(s) of toxin action.

Animals↗