Correlation between intraoperative proximal segment rotation and post-sagittal split ramus osteotomy relapse: a three-dimensional cone beam computed tomography study.

This study evaluated the effects of proximal segment rotation and the extent of mandibular setback on post-sagittal split ramus osteotomy (SSRO) relapse using three-dimensional (3D) analysis. Thirty-one patients diagnosed with a skeletal class III malocclusion who underwent SSRO alone were enrolled in this study. The movements of the mandibular condyles were assessed using cone beam computed tomography (CBCT) and a 3D imaging program at ≤1 month before the operation (T0), 1 week after the operation (T1), and 6 months (T2) and 1year (T3) postoperative. Yaw and roll were increased at T1 as compared to T0. However, the proximal segments reverted to their original positions between T2 and T3. There was a positive correlation between the extent of the posterior movement of the mandible and relapse at 6 months and 1year postoperative. Although the proximal bone segments showed displacement in three dimensions at T1, they reverted to their original positions over time. In addition, although there was a positive correlation between the extent of the posterior movement of the mandible and the occurrence of post-surgical relapse at 6 months and 1year post-surgery, the rotation of the proximal bone segment during surgery had no relationship with postoperative relapse.

Diagnostic value of CA125 as a predictor of recurrence in advanced ovarian cancer.

PURPOSE: The aim of this study was to establish the guidelines for detecting early recurrences of advanced epithelial ovarian cancer by use of the CA-125 level. MATERIALS AND METHODS: Eighty-five of the patients who met the inclusion criteria were enrolled in this study. The authors examined 25 incremental changes of CA125 from one to 25 IU/ml, and compared the CA-125 value with other prognostic factors. Increases in the CA-125 level from the nadir level were expressed as CA-125- increments. RESULTS: Among the 25 increments, a CA-125-8 (eight IU/ml) was selected as the predictor that was the most efficient and time-effective. CA-125-8 had a sensitivity of 91.5%, a specificity of 84.6%, a positive predictive value of 93.1%, a negative predictive value of 81.5%, an efficiency of 89.4%. and a median lead-time of 68.5 days (p <0.0001). CONCLUSION: The authors suggest the incremented CA-125-8 as a predictor of recurrent advanced ovarian cancer.

Effect of proanthocyanidin-rich extract from Pinus radiata bark on immune response of specific-pathogen-free White Leghorn chickens.

Proanthocyanidins are naturally occurring compounds that are widely found in fruits, vegetables, nuts, seeds, flowers, and bark. We evaluated the immunomodulatory effects of proanthocyanidin-rich extract (PAE) from Pinus radiata bark in specific-pathogen-free White Leghorn chickens. Proliferation of peripheral blood mononuclear cells was significantly enhanced in chickens treated for 2 wk with 20 mg/kg of PAE. Proliferation of splenocytes and bursal cells was significantly enhanced in chickens treated for 5 wk with 5, 10, and 20 mg/kg of PAE. Thymocyte proliferation was significantly enhanced in chickens treated for 5 wk with 5 and 10 mg/kg of PAE. These effects were markedly enhanced by the presence of lipopolysaccharide, which acted on B cells responsible for humoral immunity, and concanavalin A, which acted directly on T cells involved in cell-mediated immunity. The PAE significantly promoted the expression of T helper 1 cytokine (interferon-γ) and decreased the expression of T helper 2 cytokine (IL-6). Thus, P. radiata PAE has immunomodulatory effects in specific-pathogen-free White Leghorn chickens.

Surgical outcome of endoscopic carpal tunnel release in 100 patients with carpal tunnel syndrome.

The purpose of this study is to present the surgical outcome of endoscopic carpal tunnel release (ECTR) for the treatment of carpal tunnel syndrome (CTS). One hundred and thirty-one procedures (36 right hands, 33 left hands and 31 bilateral hands) of single portal ECTR were performed upon 100 patients (age range: 36-77 years, mean age: 52.9 years; 98 women and 2 men) with electrodiagnostically proven CTS for 2.5 years from 2001. Preoperative clinical severity and results of electrodiagnostic studies were compared with surgical outcomes at the minimal 3-month postoperative period. Among 131 cases 125 (95.4 %) with complete or significant relief of symptoms were satisfied and 6 (4.6 %) with partial or no relief of symptoms were dissatisfied. There were 2 cases of major complications (one with ulnar nerve injury and the other with ulnar artery injury) that developed in our early experience of ECTR and 1 case of recurrence. The grade of electrodiagnostic abnormalities was associated with surgical outcome but there was no statistical significance between them. The severity of clinical findings, age at onset and symptom duration were not correlated with surgical outcome. In conclusion, ECTR surgery was effective in relieving the symptoms of CTS with a low complication rate after the learning curve period. Thus, ECTR can be an alternative to the traditional open surgery and can be the first procedure for CTS with several advantages over open methods.

Purification, cDNA cloning and expression of an insect defensin from the great wax moth, Galleria mellonella.

An insect defensin, named Galleria defensin, was purified from the larval haemolymph of Galleria mellonella immunized against E. coli. The peptide was composed of forty-three amino acid residues containing six cysteines that might be engaged in intramolecular disulphide bridges. The primary structure of Galleria defensin shared about 90.7% identity to that of heliomicin, which was an insect defensin isolated from Heliothis virescens. The full-length cDNA encoding Galleria defensin was cloned from the fat body of the immunized G. mellonella larvae. Northern blot analysis revealed that Galleria defensin was expressed not only in the fat body but also in the midgut against invading bacteria into haemocoel. This is the first report presenting cDNA and expression of an insect defensin in the lepidopteran species.

Morphological and photoelectrochemical characterization of core-shell nanoparticle films for dye-sensitized solar cells: Zn-O type shell on SnO2 and TiO2 cores.

Core-shell type nanoparticles with SnO2 and TiO2 cores and zinc oxide shells were prepared and characterized by surface sensitive techniques. The influence of the structure of the ZnO shell and the morphology ofnanoparticle films on the performance was evaluated. X-ray absorption near-edge structure and extended X-ray absorption fine structure studies show the presence of thin ZnO-like shells around the nanoparticles at low Zn levels. In the case of SnO2 cores, ZnO nanocrystals are formed at high Zn/Sn ratios (ca. 0.5). Scanning electron microscopy studies show that Zn modification of SnO2 nanoparticles changes the film morphology from a compact mesoporous structure to a less dense macroporous structure. In contrast, Zn modification of TiO2 nanoparticles has no apparent influence on film morphology. For SnO2 cores, adding ZnO improves the solar cell efficiency by increasing light scattering and dye uptake and decreasing recombination. In contrast, adding a ZnO shell to the TiO2 core decreases the cell efficiency, largely owing to a loss of photocurrent resulting from slow electron transport associated with the buildup of the ZnO surface layer.

Common and differential mechanisms of gonadotropin receptors.

The gonadotropin receptors are G-protein-coupled receptors with unique structural and functional features, consisting of two halves. The N-terminal extracellular half (exodomain) binds the hormones, whereas the C-terminal membrane-associated half (endodomain) is responsible for receptor activation. In this review, the novel ternary interactions, contact points and mutual modulations among the exodomain, endodomain and hormone for hormone binding and signal generation are described based on the latest observations. This discussion is contrary to the yiew that the exodomain and endodomain are independent, at least functionally, and provides new insights into the receptor mechanisms for the gonadotropins and other G-protein-coupled receptors.

Alterations of HLA class I and II antigen expression in preinvasive, invasive and metastatic cervical cancers.

HLA expression is altered in a large variety of human cancers. We performed immunohistochemical staining on tissues from normal, preinvasive, invasive and metastatic cervical cancer tissues using anti-HLA class I or class II antibody. In tissues from normal squamous epithelium, carcinoma in situ (CIS) and microinvasive carcinoma (MIC), the expressions of HLA-B, C heavy chains and class II heavy chain were significantly decreased as disease progressed. When the expression patterns were compared between primary and metastatic squamous cell carcinoma (SCC) lesions, statistically significant down-regulation of HLA class I and class II antigen in metastatic lesions was observed. The rates of HLA-B, C heavy chains and class II heavy chain expressions were all significantly down-regulated compared to the down-regulation rate of class I beta2-microglobulin (beta2m) in invasive squamous lesions, and the expressions of class II heavy chain in metastatic lesions was decreased further than that in primary lesions. Unlike SCC, the degree of HLA class I and class II loss was not evident as disease progressed in early stage of adenocarcinoma. In invasive adenocarcinoma lesions, only the expression of HLA-B, C heavy chains was decreased and no differences were seen in HLA-B, C heavy chain expression patterns between primary and metastatic lesions. These results suggest that alterations of HLA class I and II expressions seem to occur at a particular step in cervical cancer development and depend on tissue types: when the tumor becomes invasive and starts to metastasize.

Polyhydroxylated alkaloids isolated from mulberry trees (Morusalba L.) and silkworms (Bombyx mori L.).

New polyhydroxylated alkaloids, (2R,3R,4R)-2-hydroxymethyl-3,4-dihydroxypyrrolidine-N-propionamide from the root bark of Morus alba L., and 4-O-alpha-D-galactopyranosyl-calystegine B(2) and 3 beta,6 beta-dihydroxynortropane from the fruits, were isolated by column chromatography using a variety of ion-exchange resins. Fifteen other polyhydroxylated alkaloids were also isolated. 1-Deoxynojirimycin, a potent alpha-glucosidase inhibitor, was concentrated 2.7-fold by silkworms feeding on mulberry leaves. Some alkaloids contained in mulberry leaves were potent inhibitors of mammalian digestive glycosidases but not inhibitors of silkworm midgut glycosidases, suggesting that the silkworm has enzymes specially adapted to enable it to feed on mulberry leaves. The possibility of preventing the onset of diabetes and obesity using natural dietary supplements containing 1-deoxynojirimycin and other alpha-glucosidase inhibitors in high concentration is of great potential interest.

Cytotoxicity and L-amino acid oxidase activity of crude insect drugs.

The cytotoxicity of crude insect drugs was measured using HeLa cells originating from human cervix and uterine cancer, using the dye uptake assay in order to find potential anticancer agents. Three kinds of extracts (buffer, methanol and ethylacetate) were prepared from 26 insects and used as raw materials for the activity assay. Among these, the buffer extracts from Tabanus, Mylabris and Huechys showed a potent anticancer activity, and those from Catharsius, Red ant, Scorpion, Tabanus and Vespae Nidus showed a strong L-amino acid oxidase (AAO) activity as well as cytotoxicity. In contrast, buffer extracts from Gryllotalpa orientalis and Apriona germari larvae showed greater/more rapid Hela cell growth than that of other insects.

Structures of ovine corticotropin-releasing factor and its Ala32 mutant as studied by CD and NMR techniques.

The corticotropin-releasing factor (CRF) is a 41-amino acid peptide-amide hormone, which mediates a general stress-response. It has been reported that the substitution of His-32 in the ovine CRF (oCRF) with Ala brings about a 4.5-fold increase in activity [Kornreich et al. (1992) J. Med. Chem. 35, 1870-76]. Here, we have determined the secondary structure of this Ala-substituted ovine CRF ([Ala32]oCRF) and compare it with that of oCRF using circular dichroism (CD) and NMR techniques in trifluoroethanol (TFE) solution, which is known to stabilize the alpha-helix formation. In contrast to an earlier report, it was observed the alpha-helical structure extends to the C-terminus of oCRF. By analyzing the CalphaH and NH chemical shifts, the properties of local structures of oCRF were elucidated. The oCRF and [Ala32]oCRF have stable alpha-helical structures in the middle region, regardless of pH and temperature, and the alpha-helix initiation regions of these peptides are stabilized as the pH is decreased. However, the [Ala32]oCRF has a more stable alpha-helical structure than oCRF in the vicinity of the substitution region, and it is thought that this is the cause of the increased activity of [Ala32]oCRF.

Antioxidative flavonoids from the leaves of Morus alba.

Nine flavonoids (1-9) were isolated from the leaves of Morus alba (Moraceae). The structures of compounds were determined to be kaempferol-3-O-beta-D-glucopyranoside (astragalin, 1) kaempferol-3-O-(6"-O-acetyl)-beta-D-glucopyranoside (2), quercetin-3-O-(6"-O-acetyl)-beta-D-glucopyranoside (3), quercetin-3-O-beta-D-glucopyranoside (4), kaempferol-3-O-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (5), quercetin-3-O-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (rutin, 6), quercetin-3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (7), quercetin-3,7-di-O-beta-D-glucopyranoside (8) and quercetin (9) on the basis of spectroscopic and chemical studies. Compounds 7 and 9 exhibited significant radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radical.

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

Trophoblastic neoplasms and choriocarcinoma cells express high levels of the hCG receptor. The hCG receptor is encoded by a single gene in chromosome 2p21-p16, spanning over -70 kb with 11 exons and 10 introns. Multiple mRNA species are produced from the gene utilizing two proximal promoters and several Sp-1 elements as well as proximal and distal suppressors. In fact, regulatory proteins which bind to one of these suppressors are expressed less in choriocarcinoma cell lines than in placenta. 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 indicate that hCG and the receptor undergo multiple stages of interactions leading to receptor activation. Initial high affinity binding of hCG to the exodomain results into 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 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 are 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 of the 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 reduces 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. Interesting, the alpha C-terminal residues play distinct roles in all three hormones, despite its common nature.

Interaction of glucagon with dimyristoylphosphatidylcholine in vesicular and discoidal complexes.

Glucagon fragments dimyristoylphosphatidylcholine liposomes into discoidal complex under appropriate conditions. The concentration of glucagon required to fragment the vesicles increases with increasing pH, which appears to be related to the glucagon binding. It was also observed that the fragmentation is facilitated by NaCl, which is also due to increased glucagon binding. From the quenching of Trp fluorescence by doxyl group located at various positions of the acyl chain of the lipid, Trp of glucagon was found to be located close to the bilayer surface in the vesicular complex. However, the Trp fluorescence was quenched by the doxyl group in the discoidal complex to an equal extent regardless of the position of this spin label in the acyl chain. This and the results of second derivative UV spectroscopy of Tyr suggested that segments including Tyr-13 and Trp-25 are involved in the discoidal complex formation and that the orientation of glucagon is not normal to the bilayer surface.

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

Trophoblastic neoplasms and choriocarcinoma cells express high levels of the hCG receptor. The hCG receptor is encoded by a single gene in chromosome 2p21-p16, spanning over -70 kb with 11 exons and 10 introns. Multiple mRNA species are produced from the gene utilizing two proximal promoters and several Sp-1 elements as well as proximal and distal suppressors. In fact, regulatory proteins which bind to one of these suppressors are expressed less in choriocarcinoma cell lines than in placenta. 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 envidence indicate that hCG and the receptor undergo multiple stages of interactions leading to receptor activation. Initial high affinity binding of hCG to the exodomain results into 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 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 are 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 of the 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 reduces of the α subunit play a crucial role in receptor activation. This α subunit is shared with other glycoprotein hormones, follicle stimulating hormone and thyroid stimulating hormone. Interesting, the α C-terminal residues play distinct roles in all three hormones, despite its common nature.

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.

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.

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.

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.

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.