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I Ji

Publications and source records attributed to I Ji.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1991

Conversion of lysine 91 to methionine or glutamic acid in human choriogonadotropin alpha results in the loss of cAMP inducibility.

Human choriogonadotropin (hCG) is a heterodimeric glycoprotein hormone. The alpha subunit comprises 92 amino acids, of which 6 are Lys residues (Morgan, F.G., Birken, S., and Canfield, R.E. (1975) J. Biol. Chem. 250, 5247-5258). Our photoaffinity-labeling studies indicate that several of these Lys residues make contact with the lutropin receptor and are covalently cross-linked to the receptor. Lys-91 of the alpha subunit is of interest because deletion of the two alpha C-terminal residues, Lys-91 and Ser-92, results in a significant reduction in the bioactivity of lutropin and thyrotropin (Cheng, K.-W., Glazer, A.N., and Pierce, J.G. (1973) J. Biol. Chem. 248, 7930-7937). To determine the importance of Lys-alpha 91, we substituted it with Arg, Met, or Glu. The resulting mutant alpha cDNA constructs were co-transfected into CHO cells with the wild type hCG beta cDNA construct. Secreted hCG dimers were assayed for binding to receptors on porcine granulosa cells and stimulation of cAMP synthesis in a murine Leydig tumor cell line. The natural hCG, wild type hCG, and all mutant hCGs recognized the receptor, although with somewhat divergent affinities. However, there was a striking difference in the ability of cAMP induction. The natural hCG, wild type hCG, and Lys-91----Arg mutant hCG induced cAMP synthesis, whereas the Lys-91----Met and Lys-91----Glu mutants did not. These results demonstrate that Lys-91 is important for receptor modulation in the stimulation of cAMP synthesis.

Animals↗
PubMed1991

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

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

Amino Acid Sequence↗
PubMed1991

Analyses of ovine corpora lutea for tumor necrosis factor mRNA and bioactivity during prostaglandin-induced luteolysis.

It has been suggested that tumor necrosis factor (TNF) participates in the mechanism of regression of the corpus luteum. We measured luteal expression of TNF alpha mRNA and biological activity during prostaglandin-induced luteolysis in sheep. Initiation of functional luteolysis was marked by a sharp decline in concentrations of progesterone in luteal tissue beginning 4 h after administration of luteolysin. Structural regression of corpora lutea was manifested by a reduction in glandular weight at 16 h. A luteal cytotoxic factor with TNF alpha-like bioactivity was isolated after the decrease in tissue progesterone had occurred, but before evidence of luteal resorption. We were unable to detect temporal alterations in TNF alpha mRNA in luteal samples by classical Northern blot or in situ hybridization analyses. These results imply that luteal TNF alpha is derived primarily as a preformed entity from an extraovarian source, such as infiltrating leukocytes. These results raise the possibility that this cytokine might not be involved in the early stages of luteal regression in the ewe, yet could play a secondary role, perhaps in the subsequent opsonization and removal of degenerating cells.

Animals↗
PubMed1991

Structure of the luteinizing hormone receptor gene and multiple exons of the coding sequence.

The genomic structure of the LH receptor is important to our understanding of its expression mechanisms, functional domains, relationships with other hormone receptors, and evolution. We have isolated four overlapping cosmid clones and six subgenomic clones of the rat LH receptor gene. They span a total of 95.6 kilobases (kb) and extend from 23 kb upstream of the translation start site to 13 kb down-stream of the stop codon. In addition, part of the human LH receptor gene has been isolated. The coding region of the rat hormone receptor gene spans over 60 kb and consists of 11 exons and 10 introns. Southern blots hybridized with exon 1 and exon 11 probes as well as gene dose analyses demonstrate that a single copy gene encodes the rat LH receptor. Sequence comparison suggests that the porcine and human LH receptor genes have similar, if not identical, exon-intron structures. There is no consensus cAMP-responsive element within 600 basepairs up-stream of the translation start site in spite of the cAMP responsiveness of the LH receptor gene. There are, however, unconventional cAMP-responsive elements in the region: one which is identical, several which are homologous to the activating protein-2-binding elements, CCCCAGGC, and several sequences which are similar to the G-rich cAMP-responsive element found in P450c21, a steroid 21-hydroxylase. The first 10 exons encode the N-terminal half of the molecule, while exon 11 encodes the C-terminal half of the molecule. This last exon is the same in the rat and human genes. The DNA and amino acid sequences of the first 10 exons show significant similarities and reveal repetitive sequence motifs. They have similar sizes which occur in the range of 69 and 183 bases; 8 of them are from 69-81 bases. Despite these remarkable similarities, structural predictions of exons 1-10 show a diversity of structures. The N-terminal half of the LH receptor appears to have a folded structure, with frequent turns and an extensive surface area. Part of the surface is predicted to be covered by amphiphilic helices and beta structures, types of secondary structure frequently found at the interfaces between subunits or between 2 interacting molecules. The introns dividing these exons also share many similarities.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Exons 1-10 of the rat LH receptor encode a high affinity hormone binding site and exon 11 encodes G-protein modulation and a potential second hormone binding site.

We have reported that the rat LH receptor is encoded by 11 exons of a single copy gene. Exons 1-10 encode the N-terminal half and exon 11 the C-terminal half. Since exon splice sites often mark structural transitions of multiexon molecules, we have attempted to define the function of the exons by generating mutant receptors with missing exons. As a first step, we have constructed two LH mutant receptors, one containing exons 1-10 (LH receptor (exon)1-10) and the other containing exon 1 and exon 11 (LH receptor(exon)1&11). These mutant receptors were functionally expressed in Cos 7A cells. The LH mutant receptor(exon)1-10, which lacks the membrane associated C-terminal half of the receptor, showed a high affinity for hCG. Surprisingly, the LH mutant receptor(exon)1&11 recognized hCG with a low affinity and stimulated G-proteins and cAMP production. The results demonstrate that exons 1-10 encode a high affinity hCG binding site and proves an important hypothesis that exon 11 encodes the site for receptor-modulation to activate G-proteins. Furthermore, the results raises an intriguing possibility of a second hormone binding site in the C-terminal half and multistep hormone binding.

Animals↗
PubMed1990

Differential interactions of human choriogonadotropin and its antagonistic aglycosylated analog with their receptor.

Human choriogonadotropin (hCG) is a heterodimeric hormone consisting of an alpha subunit and a beta subunit. hCG and aglycosylated hCG (aghCG) have similar receptor binding affinities but differ in their ability to activate hormone-responsive adenylate cyclase. aghCG is an effective antagonist. The mechanisms of this antagonism and interactions of antagonistic aghCG with the receptor are not understood. To address this critical question, we have examined the interaction of this hormone analog with the receptor. The hormone receptor on porcine granulosa cells is a glycoprotein of 86 kDa and thas three domains of 24 kDa, 28 kDa, and 34 kDa, which are disulfide-linked. They undergo proteolysis, particularly when bound to the hormones, to produce three polypeptide components. These three receptor components can readily be identified through the use of affinity labeling with the hormones. Affinity labeling with an amino-specific homobifunctional reagent and subsequent cleavage indicate that hCG is cross-linked directly to the 24-kDa receptor component. In contrast, aghCG is cross-linked directly to the 34-kDa component. The peptide map of the cross-linked aghCG-34-kDa receptor component produced by papain treatment is different from the peptide map of the cross-linked complex of hCG-24-kDa component. This difference in receptor binding may be a factor determining the success or failure of signal transduction from the receptor to the effector system, guanine nucleotide-binding regulatory protein, and adenylate cyclase.

Affinity Labels↗