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A degradable, poly (lactic-co-glycolic acid) (PLGA), gentamicin-loaded prophylactic coating for hydroxyapatite (HA)-coated cementless hip prostheses is developed with similar antibacterial efficacy as offered by gentamicin-loaded cements for fixing traditional, cemented prostheses in bone. We describe the development pathway, from in vitro investigation of antibiotic release and antibacterial properties of this PLGA-gentamicin-HA-coating in different in vitro models to an evaluation of its efficacy in preventing implant-related infection in rabbits. Bone in-growth in the absence and presence of the coating was investigated in a canine model. The PLGA-gentamicin-HA-coating showed high-burst release, with antibacterial efficacy in agar-assays completely disappearing after 4 days, minimising risk of inducing antibiotic resistance. Gentamicin-sensitive and gentamicin-resistant staphylococci were killed by the antibiotic-loaded coating, in a simulated prosthesis-related interfacial gap. PLGA-gentamicin-HA-coatings prevented growth of bioluminescent staphylococci around a miniature-stem mounted in bacterially contaminated agar, as observed using bio-optical imaging. PLGA-gentamicin-HA-coated pins inserted in bacterially contaminated medullary canals in rabbits caused a statistically significant reduction in infection rates compared to HA-coated pins without gentamicin. Bone ingrowth to PLGA-gentamicin-HA-coated pins, in condylar defects of Beagle dogs was not impaired by the presence of the degradable, gentamicin-loaded coating. In conclusion, the PLGA-gentamicin-HA-coating constitutes an effective strategy for infection prophylaxis in cementless prostheses.

Lutein is a carotenoid that is beneficial to human health. However, its low stability and bioavailability have limited its application in the pharmaceutical and food industries. Herein, lutein has been successfully modified with carbon-dots (CDs) via a simple and mild solvent-ultrasonic method at room temperature. The synthesized lutein/CDs composites (LCs) showed 3.4 times higher photostability than the pristine lutein under UV irradiation, and 3.5 times higher than that under visible light, and the retention rate of lutein in the air rose from 3.6 % to 68 % over 25 days. Meanwhile, the antioxidant capacity of lutein has been improved by 6.4 times. Based on the photoluminescence (PL) measurements, a possible mechanism for the enhanced photostability and antioxidant ability of LCs has been proposed. The work provides a simple but effective approach to enhancing the stability and antioxidant capacity of lutein, and is expected to extend its application in food and biomedical fields., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Dye degradation via photocatalysis technology has been investigated intensively to tackle environmental issues and energy crisis concerns. In this study, a newly designed ternary photocatalyst was facilely prepared by a simple one-pot hydrothermal process by directly mixing TiO 2 nanoparticles with carbon dots (C-dots) and graphitic carbon nitride (g-C 3 N 4 ). The optimized precursor treatments and heterostructure components show significantly enhanced photodegradation activity towards organic dyes Rhodamine B (RhB) and methylene blue (MB). Excellent photocatalytic activities were achieved owing to the better attachment of anatase-type TiO 2 nanoparticle-aggregations to the C-dots/g-C 3 N 4 (CC) nanocomposite, which impressively displays superhydrophilicity by employing the hydrothermal activation process. FT-IR spectra revealed that the hydrothermal treatment could remarkably increase the coupling interactions between TiO 2 nanoparticles and the CC nanosheets within the ternary catalyst, enhancing the photocatalytic activity. Thus, it was concluded that this ternary photocatalyst is highly suitable for the remediation of dye-contaminated wastewater., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Cu-chlorophyllin-bonded carbon dots (CCPh-CDs) have been synthesized at room temperature, and the energy/electron co-transfer behavior between Cu-chlorophyllin molecules (CCPh) and carbon dots (CDs) is investigated via various techniques. The mean diameters of CDs and CCPh-CDs are 2.8 nm and 3.1 nm, respectively, measured by HRTEM. The absorption spectra of CCPh-CDs show two parts: the absorptions of CDs and CCPh are in the wavelength range of 300-500 nm. The PL spectra of CCPh-CDs exhibit very weak intensities, and with the decreasing of CCPh content on CDs, the corresponding intensity increases. Luminescent decay spectra show that the PL decay times of CCPh and CCPh-CDs with the highest CCPh content are single-exponentially fitted to be 3.20 ns and 12.64 ns, respectively. Furthermore, based on the electron transfer and reducibility of CCPh-CDs, Ag/Ag 2 O nanoparticles with a mean diameter of 10 nm can be easily prepared at room temperature under ultraviolet irradiation. The PL measurement result reveals that both electron transfer and FRET behavior take place from CCPh-CDs to Ag., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

In the paper, ZnO-deposited activated carbon composite (ZnO-AC) was firstly prepared in a simple two-step preparation process, and then covered with nano-graphene oxide to give the NGO-ZnOAC composite. The successful deposition of ZnO and NGO on the AC surface was demonstrated by various experiments, and the ZnO nanoparticles showed a mean diameter size mainly within about 10 nm. The specific surface area of the NGO-AC and NGO-ZnO-AC decreased from 67.74 m²/g of the parent AC to 32.54 and 11.43 m²/g, respectively. The fabricated NGO-ZnO-AC showed excellent adsorptive behaviors towards CrO 2- ₄ and Cu 2+ ions, outperforming both ZnO-AC and AC.

Glutamine addiction is a major feature of glioma cells and plays an important role in its growth and proliferation. GLUL (glutamate-ammonia ligase), which catalyzes glutamate and ammonia to synthesize glutamine, plays a crucial role in tumor growth and proliferation. We attempt to determine a pathway that limits the growth of glioma by targeting GLUL and explore effective strategies blocking glutamine metabolism. We note that miRNAs mediate regulation of genes participating directly or indirectly in cancer cell metabolism. The regulatory roles of miRNAs on metabolic enzymes are widely discussed, however miRNAs regulation of glutamine metabolism by targeting GLUL in glioma has not yet been reported. Here, we examined both the expression and functions of GLUL in glioma cells. Findings indicated that the expression of GLUL was upregulated in high-grade compared to low-grade glioma cells. Knockdown of GLUL effectively inhibited proliferation, migration and invasion of glioma cells in vitro. Bioinformatics analyses, as well as dual-luciferase reporter assays, revealed that miR-140-5p bound to GLUL mRNA at the 3'-UTR location. Furthermore, the proliferation, migration and invasion of glioma cells were also repressed by miR-140-5p. Overall, these results showed that miR-140-5p exerted its inhibitory effects on proliferation, migration and invasion in glioma cells through downregulating GLUL. Thus, the miR-140-5p/GLUL axis may function as a potential target for glioma treatment.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 bp with no protein-coding capacity. Transcribed ultraconserved regions (T-UCRs) are a type of lncRNA and are conserved among human, chick, dog, mouse and rat genomes. These sequences are involved in cancer biology and tumourigenesis. Nevertheless, the clinical significance and biological mechanism of T-UCRs in breast cancer remain largely unknown. The expression of uc.38, a T-UCR, was down-regulated in both breast cancer tissues and breast cancer cell lines. However, uc.38 was expressed at significantly lower levels in larger tumours and tumours of more advanced stages. Based on the results of in vitro and in vivo experiments, up-regulation of uc.38 expression inhibited cell proliferation and induced cell apoptosis. Thus, uc.38 suppressed breast cancer. Additional experiments revealed that uc.38 negatively regulated the expression of the pre-B-cell leukaemia homeobox 1 (PBX1) protein and subsequently affected the expression of Bcl-2 family members, ultimately inducing breast cancer cell apoptosis. Describing the uc.38/PBX1 axis has improved our understanding of the molecular mechanisms involved in breast cancer apoptosis and has suggested that this axis is a potential therapeutic target for breast cancer., Competing Interests: None.

Tamoxifen resistance is a serious problem in the endocrine therapy of breast cancer. Long non-coding RNAs play important roles in tumor development. In this study, we revealed the involvement of lncRNA uc.57 and its downstream gene BCL11A in TAM resistance. Tamoxifen-resistant MCF-7R cells showed lower expression of uc.57 and higher expression of BCL11A mRNA and protein than the parental MCF-7 cells. Moreover, levels of uc.57 mRNA were lower and BCL11A mRNA were higher in breast cancer tissues than in precancerous breast tissues. Shikonin treatment reduced tamoxifen resistance in MCF-7R cells both in vitro and in vivo , targeting uc.57/BCL11A. Fluorescence in situ hybridization and RNA immunoprecipitation analyses showed that uc.57 binds to BCL11A. Uc.57 overexpression downregulated BCL11A and reduced tamoxifen resistance in MCF-7R cells both in vitro and in vivo . BCL11A knockdown also reduced tamoxifen resistance by inhibiting PI3K/AKT and MAPK signaling pathways. It thus appears shikonin reduces tamoxifen resistance of MCF-7R breast cancer cells by inducing uc.57, which downregulates BCL11A to inhibit PI3K/AKT and MAPK signaling pathways., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

NaYF4 : Yb, Er/rGO and SiO2-coated NaYF4 : Yb, Er/rGO nanocomposites can be prepared through "one-pot" and directly mixing preparation routes. Various measurement results show that the NaYF4 : Yb, Er in the nanocomposites exhibits a cubic a-type structure and nanoparticle-like morphology with a diameter range of 30-70 nm; the rGO layers are well-dispersed in the nanocomposites, and whereas the rGO obtained from "one-pot" preparation renders relatively better dispersion. Raman spectra demonstrate that there exists a surface coupling action between the two kinds of nanomaterials, and with the increase in the relative rGO content, such action becomes stronger. UC fluorescence measurement results reveal that the rGO has significantly quenching effect and optical-limiting performance on the UC fluorescence, particularly on the red-emission of the NaYFa : Yb, Er or SiO2-coated NaYF4 : Yb, Er nanoparticles. The red-emission intensity gradually decreases with an increase in the rGO content, but the green-emission shows less change. It should be stressed that, in comparison with NaYF4 : Yb, Er/rGO, with a similar rGO content, the red-emission intensity of SiO2-coated NaYF4 : Yb, Er/rGO decreases much obviously due to a stronger light-absorption caused by part rGO aggregation.

Objective: To investigate co-expression of CD99/MIC2 and anaplastic lymphoma kinase (ALK) protein in anaplastic large-cell lymphoma (ALCL) tissues and Karpas 299 cells and its significance., Methods: Clinical prognoses and ALK protein expressions of 25 cases of ALCL were reviewed retrospectively, the median duration of survival was analyzed for patients with ALK(+) ALCL and ALK(-) ALCL. Histological and immunohistochemical staining were applied to other 25 cases of ALCL and paraffin-embedded tissue from human anaplastic large-cell lymphoma Karpas 299 cells to detect the protein of CD99 and ALK., Results: Of former 25 cases of ALCL, median duration of survival for ALK(+) patients was 59 months, whereas 20 months for ALK(-) patients. The prognosis of ALK(+) group was better than that of ALK(-) group, survival curves of these two groups showed statistically significant (P < 0.05). CD99 was positive in 18 cases (72.0%) while negative in 7 cases (28.0%) of the latter 25 ALCL, ALK was positive in 19 cases (76.0%) while negative in 6 cases (24.0%); Of 19 ALK(+) ALCL, 16 (84.2%) cases co-expressed CD99-ALK; and in 6 ALK(-) ALCL, 2(33.3%) were CD99-ALK double negative, the expression of CD99 protein strongly correlated with that of ALK protein (P < 0.05). ALK and CD99 protein expressed in Karpas 299 cells with diffuse distribution., Conclusions: CD99 highly expressed in ALCL, and showed high rate of co-expression with ALK. CD99 protein expression could be considered as a helpful diagnostic and prognostic factor of ALCL, especially for ALK(+) ALCL.

Objective: To explore correlation of seven apoptosis-related proteins (Hsp90a, p53, MDM2, Bcl-2, Bax, Cytochrome C, and Cleaved caspase3) with clinical outcomes of ALK+ anaplastic large-cell lymphoma (ALCL)., Methods: Using immunohistochemistry and immunofluorescence double staining methods, the expressions of these seven apoptosis-associated proteins were studied to clarify their relationship with clinical outcomes of 36 ALK+ and 25 ALK-systemic ALCL patients enrolled between 1996 and 2006. The relationship of these apoptosis-regulating proteins with NPM-ALK status was also evaluated with the tyrosine inhibitor herbimycin A (HA) in vitro by immunocytochemistry, Western blotting and flow cytometric assays., Results: The presence of Hsp90α-, MDM2-, Bax-, Cytochrome C, and Cleaved caspase3-positive tumor cells was found significantly different in ALK+ and ALK-ALCLs, which was correlated with highly favorable clinical outcome. The Bcl-2- and p53-positive tumor cells were found in groups of patients with unfavorable prognosis. Inhibition of NPM-ALK by HA could reactivate the p53 protein and subsequent apoptosis-related proteins and therefore induced apoptosis in ALK+ ALCL cells., Conclusion: Our results suggest that these seven proteins might be involved in apoptosis regulation and associated with clinical outcome of ALK+ systemic ALCLs. We also reveal a dynamic chain relation that NPM-ALK regulates p53 expression and subsequent apoptosis cascade in ALK+ ALCLs., (Copyright © 2011 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.)

A series of CdSe nanoparticle-deposited anatase TiO2 nanobelts were prepared by hydrothermal process. Their crystal structures, morphologies, depositing content of CdSe and visible-light photocatalytic activities were characterized using various measurement techniques. The measurement results show that the CdSe nanoparticles with zinc blende face-center-cubic structure and controlled content have deposited on anatase TiO2 nanobelts; the special surface areas of the products become lower in comparison with that of TiO2 nanobelts due to the deposited CdSe, whereas their visible-light absorptions exhibit much more obviously. The visible-light photocatalytic activities in degradation of rhodamine B solution demonstrate that the products with excess depositing content of CdSe nanoparticles and existence of NH3 molecules on the surface, show much lower photodegrada tion activities. On the contrary, the products with less CdSe nanoparticles and less NH3 molecules exhibit much higher photodegradation activities.

Cubic phase MTiO3 (M = Ba or Sr) nanoparticle-supported TiO2 nanocomposites were prepared using Sr(OH)2 or Ba(OH)2 and TiO2 nanobelts as precursors by hydrothermal process. Their component, phase, morphology, structure and optical property were characterized using various XRD, SEM, TEM, HRTEM and UV-Vis techniques. The measurement results show that the more or less MTiO3 nanoparticles are affected by the amount of added Sr(OH)2 or Ba(OH)2 and the change in reaction time, and to some extent, the content of the MTiO3 is increased with the increase in the added hydroxide precursor and the prolonging of reaction time. Either the pure TiO2 nanobelts or the nanocomposites show the similar absorption and emission spectra. Their visible photodegradation activities of rhodamine B appear much higher than that of P-25.

Objective: to map out the frequency and types of K-ras gene mutations present in colorectal and lung cancer patients; to evaluate the clinical applicability of a novel real-time double-loop probe PCR using the ADx-K-ras kit, and to compare its performance with the result by using traditional Sanger DNA sequencing in detection of somatic mutations of the tumor genes., Methods: a total of 827 formalin-fixed paraffin-embedded (FFPE) blocks including 583 from the colorectal and 244 from the lung cancer patients were assayed. Genomic DNA of the sample tissues was extracted, purified and subjected to PCR amplification of K-ras gene codon 12 and 13 and DNA sequencing was carried on using both the traditional Sanger sequencing method and the ADx's K-ras mutation detection kit, respectively. The mutation rates for K-ras gene at codon 12 and 13, and the mutation frequencies detected by using both methods were analyzed., Results: 533 out of 583 (91.4%) colorectal cancer samples and 144 out of 244 lung cancer samples (59.0%) were detected using the traditional Sanger DNA sequencing technique, and 583 out of 583 (100.0%) colorectal plus 244 out of 244(100.0%) lung cancers were detected, respectively by using the ADx-K-ras kit. Of the 583 colorectal cancer samples, 192 (32.9%) showed mutations by using the ADx-K-ras kit in comparing with a result of 160 samples (27.4%) with K-ras gene mutation by using the traditional Sanger DNA sequencing technique. Of the 244 lung cancer samples, 26 (10.7%) showed K-ras gene mutations by using ADx-K-ras kit, while in 144 samples detected by using the traditional Sanger DNA sequencing technique, only 12 samples (8.3%) showed K-ras gene mutations. In colorectal cancer analyzed, GGT→GAT at codon 12 was the most common event with 35.1% (66/188) mutations, followed by GGC→GAC at codon 13 with 26.6% (50/188) and GGT→GTT at codon 12 with 18.6% (35/188), while GGT→GCT at codon12 was the most rare with only 1.6% (3/188) of the total mutation cases. In patients with lung cancer analyzed, GGT→GTT at codon 12 was the most common mutation, accounting for 40.9% (9/22), and GGT→GCT at codon 12 the most rare with only about 4.5% (1/22) of the total mutation cases., Conclusions: K-ras gene mutations were present in colorectal cases, and significantly more frequent than that in lung cancer. There were significant statistical differences between the two methods. ADx-K-ras real-time PCR showed much higher successful detection rates and mutation ratios compared to Sanger sequencing. As a result, the real-time PCR with ADx-K-ras kit proves to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is a effective and reliable tool for clinical screening of somatic gene mutations in tumors.

The two kinds of new nanocomposites BiVO4/TiO2 nanowires were synthesized by hydrothermal process. Their crystal structure, morphology and photocatalytic activities for degradation of methylene blue solution were characterized using various measurement techniques. The XRD results indicate that they are made up of monoclinic BiVO4 and anatase TiO2 phases. The SEM, TEM and HRTEM images show that the two samples include BiVO4 nanoparticles supported onto TiO2 nanowires. The UV-Vis absorption spectra reveal that the absorption edges of the samples exhibit red-shift in comparison with that of the pure TiO2 nanowires. The measurement results for the visible-light photodegradation of methylene blue show that the nanocomposite sample prepared from the layered titanate nanowires with Bi3+ has the highest photocatalytic activity.

G protein-coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one ligand/one receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/oligomerization in vivo is still missing. Using the mouse luteinizing hormone receptor (LHR) as a model GPCR, we demonstrate that transgenic mice coexpressing binding-deficient and signaling-deficient forms of LHR can reestablish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling.

Objective: To explore the role of abnormal Tbx3 expression in the pathogenesis of breast cancer., Method: The total RNA of 4 breast cancer cell lines and 5 normal breast samples was extracted by routine Trizol method. After reverse transcription of the total RNA into cDNA, Tbx3 mRNA expression was detected in these samples by real-time PCR. Immunohistochemistry was used to examine the differences in Tbx3 protein expression between 60 breast cancer samples and 34 normal breast tissue samples., Results: Compared to normal breast tissue samples, the breast cancer cell lines showed markedly increased Tbx3 mRNA expression. The results of immunohistochemistry demonstrated a significant upregulation of Tbx3 protein expression in the 60 breast cancer tissues in comparison with the normal breast tissues, as was consistent with Tbx3 mRNA expressions in these tissue samples., Conclusions: The mRNA and protein expressions of Tbx3 are markedly upregulated in breast cancer cell lines and tissue samples, suggesting that Tbx3 may serve as one of the malignant biomarkers in the pathogenesis of breast cancer.

Objective: To explore the role of TBX3 gene in the pathogenesis of breast cancer., Methods: The total RNA of 51 fresh breast cancer tissues and the corresponding adjacent tissues were extracted and reverse transcribed into cDNA to detect the expression of TBX3 mRNA by real-time PCR. The correlation between TBX3 mRNA expression and the clinicopathologic parameters in relation to breast cancer metastasis was analyzed., Result: Compared to that in the adjacent tissues, the expression of TBX3 mRNA was markedly increased in breast cancer tissues. TBX3 mRNA expression was significantly higher in metastatic breast cancer than in non-metastatic tumors., Conclusion: Increased expression of TBX3 mRNA suggests the involvement of TBX3 in the pathogenesis and metastasis of breast cancer.

The aim of this study was to investigate the expression of anaplastic lymphoma kinase (ALK) protein resulted from chromosome translocation in anaplastic large cell lymphoma (ALCL) and its relationship with the age and prognosis of patients with ALCL. The tissue microarray including 30 cases of ALCL and 2 normal control tissues were established, the expression of anaplastic lymphoma kinase (ALK) protein was detected by immunohistochemistry, the statistical analysis of detected results was carried out by SPSS software. The results showed that the ALK protein was expressed negatively in 2 cases of primary skin ALCL, but in 20 out of 28 cases of systematic ALCL the ALK protein was expressed positively and mainly located in cytoplasm and/or nucleus (71.4%). Clinically, the patients with ALK expression were younger than those without ALK expression (p < 0.05). The prognosis of patients with ALK expression was better than those without ALK expression (p < 0.05). It is concluded that there is a high incidence of ALK expression in ALCL, especially in younger group. ALK expression may be an useful and independent marker for the differential diagnosis and prognosis evaluation of ALCL.

Objective: To compare the efficacy of nuclear microarray combined with fluorescence in situ hybridization (FISH) and immunohistochemistry in detecting ALK gene translocation and ALK fusion protein in anaplastic large cell lymphoma (ALCL)., Methods: ALK gene translocation and ALK fusion protein in 17 paraffin-embedded ALCL specimens were detected using nuclear microarray combined with FISH and immunohistochemical straining, respectively., Results: The expression of ALK fusion protein was detected immunohistochemically with ALK antibody in 8 of the 17 specimens of systemic ALCL, including 4 with both nuclear and cytoplasmic positivity and 4 with only cytoplasmic positivity. Dual-color FISH identified 6 positive specimens, including the 4 specimens with both nuclear and cytoplasmic positivity as identified immunohistochemically, and 2 with immunohistochemical cytoplasmic positivity. FISH yielded negative results for the 2 specimens with immunohistochemical cytoplasmic positivity., Conclusion: Nuclear microarray combined with FISH eliminated the cytoplasmic interference of the results of conventional FISH and provides a high-throughput platform for clinical detection with greater specificity than immunohistochemistry.

Recently, it has been reported that Kalirin gene transcripts are under-expressed in AD hippocampal specimens compared to the controls. The Kalirin gene generates a dozen Kalirin isoforms. Kalirin-7 is the predominant protein expressed in the adult brain and plays crucial roles in growth and maintenance of neurons. Yet its role in human diseases is unknown. We report that Kalirin-7 is significantly diminished both at the mRNA and protein levels in the hippocampus specimens from 19 AD patients compared to the specimens from 15 controls. Kalirin-7 associates with iNOS in the hippocampus, and therefore, Kalirin-7 is complexed with iNOS less in AD hippocampus extracts than in control hippocampus extracts. In cultured cells, Kalirin-7 associates with iNOS and down-regulates the enzyme activity. The down-regulation is attributed to the highly conserved 33 amino acid sequence, K(617) -H(649), of the 1,663 amino acids long Kalirin-7. Remarkably, the iNOS activity is considerably higher in the hippocampus specimens from AD patients than the specimens from 15 controls. These observations suggest that the under-expression of Kalirin-7 in AD hippocampus correlates to the elevated iNOS activity.

To identify genes aberrantly expressed in the brain of individuals with Alzheimer's Disease (AD), we analyzed RNA extracts from the hippocampus and cerebellum from 19 AD patients and 15 age- and sex-matched control subjects. Our analysis identified a number of genes that were over-expressed or under-expressed specifically in AD hippocampus. Among these genes, kalirin was the most consistently under-expressed in AD hippocampus, which was verified by semi-quantitative RT-PCR and real time PCR. Kalirin is predominantly expressed in the brain, particularly in the hippocampus, and plays crucial roles in neuronal stability and growth. Our observation is the first to relate kalirin to AD and a human disease. In addition to kalirin, the genes for voltage-gated Ca++ channel gamma subunit 3 and visinin-like protein 1 (a Ca++ sensor protein) were under-expressed, whereas inositol 1,4,5-triphosphate 3-kinase B was over-expressed in AD hippocampus. Collectively, these differential expressions could severely impair calcium homeostasis. Remarkably, these aberrant gene expressions in AD hippocampus were not observed in AD cerebellum. Furthermore, housekeeping genes such as ribosomal protein genes are not affected by AD. These results provide new insights into the biochemistry of AD.

It has been thought that when a hormone binds to a receptor, the liganded receptor activates itself and generates hormone signals, such as the cAMP signal and the inositol phosphate signal (cis-activation). We describe that a liganded LH receptor or FSH receptor molecule is capable of intermolecularly activating nonliganded receptors (trans-activation). Particularly, intriguing is the possibility that a pair of compound heterozygous mutants, one defective in binding and the other defective in signaling, may cooperate and rescue signaling. Furthermore, trans-activation of the binding deficient receptors examined in our studies generates either the cAMP signal or the IP signal, but not both. Trans-activation and selective signal generation have broad implications on signal generation mechanisms, and suggest new therapeutic approaches.

The goal of this study is to determine the prevalence and age of onset of Diagnostic and Statistical Manual of Mental Disorders and latent class-derived attention deficit/hyperactivity disorder (ADHD) subtypes in a population-based twin sample of boys and girls. Missouri birth records identified families with a twin pair 7 to 18 years of age. Telephone screening interviews for ADHD symptoms were completed for 5007 families. Diagnostic assessments were administered to 564 families with at least one twin meeting screening criteria, plus 183 control families. Prevalence and age of onset for both ADHD nosologies were calculated by sex and zygosity from parent report data. The prevalence of any DSM-IV ADHD was 6.2% overall, 7.4% in boys and 3.9% in girls. The inattentive subtype was most common in boys; the combined subtype was most common in girls. The mean age of onset of symptoms in children with any DSM-IV ADHD was 3.5 years, with no significant differences between boys and girls. Prevalences of latent class defined ADHD subtypes also varied by sex with the severe inattentive and combined classes more common in boys than girls. The age of onset of symptoms did not differ between boys and girls but were higher than in the DSM-IV subtypes. Findings in this twin sample showed that clinically significant ADHD, defined by either DSM-IV or latent class criterion, has an early age of onset and is more common in boys than girls. As clinical samples are most commonly composed of male combined subtypes, the inattentive subtype of both sexes in the general population is an under-treated segment of the general population.

The major protein component of the extracellular deposits in Alzheimer's disease (AD) is a 4 kDa peptide termed amyloid-beta (Abeta). This peptide is known to bind apolipoprotein E (apoE), a key mediator of lipoprotein transport, in an isoform specific manner. Whilst these isoform specific effects on apoE are well recognized, the functional significance of this interaction is poorly understood. Here, we investigated the influence of Abeta on apoE-mediated lipoprotein binding to cells using fluorescently tagged lipoprotein-like emulsions. Using this approach, we demonstrate that Abeta enhanced the normally poor binding of apoE2 lipoprotein-like particles to fibroblasts in culture, whilst markedly reducing the binding of apoE3 and apoE4. This suggests that the action of apoE isoforms on cellular lipoprotein or cholesterol metabolism is differentially modulated by Abeta. This also suggests that Abeta may also compromise apoE function in the Alzheimer disease affected brain.

Expression of the rat LH receptor (rLHR) is characterized by a dynamic response to a variety of hormonal stimuli. In addition to activation, the pattern of rLHR expression is also modulated by repression. In this report, an upstream initiator-like element (UInr-lE), CTCACTCTAA, of which the CTC direct repeat motif (CTCACTC) is conserved in the rat, mouse, and human, was identified as a suppressor element. Disruption of the element resulted in a 2-fold enhancement of promoter activity in the LHR-expressing murine Leydig tumor cells. The sequences of the two major initiators (Inr), Inr3 and Inr4, of the rLHR core promoter are similar to UInr-lE and competed efficiently with UInr-lE in the formation of specific protein complexes, suggesting that the same proteins interact with both UInr-lE and the Inrs in vivo. The Inrs are necessary for full promoter activity because a mutant promoter lacking Inrs showed a 70% reduction in activity. UInr-lE also further suppressed the activity of a mutant promoter lacking Inrs. UInr-lE interacted with transcription factor II-I (TFII-I) and an unidentified nuclear protein. However, dominant-negative inhibition experiments using p70 indicated that TFII-I positively regulates LHR promoter activity through UInr-lE and Inrs, suggesting that TFII-I can compromise the suppression of promoter activity mediated by UInr-lE. UInr-lE also showed binding properties distinct from that of the upstream initiator-like suppressor element (upstream regulatory element: CACTCTCC) of rat and human dynorphin promoters. Transfection assays using mutated promoters indicate that the suppression of rLHR promoter activity could be regulated via specific interactions between UInr-lE and trans-acting factors.

Asp578Gly is the major mutation of luteinizing hormone (LH) receptors in humans. It is a dominant mutant, constitutively activates Galphas, and induces cAMP production in the absence of the cognate hormone, causing the familial male precocious puberty. The mechanism of the elevated basal cAMP level is unclear. Our data show strikingly different mechanisms between the elevated basal cAMP induced by the activating mutant and the cAMP induced by the wild-type receptor activated by human chorionic gonadotropin (hCG) binding. The study suggests an approach to attenuating the elevated basal cAMP of the activating mutant LH receptor, which could be useful for controlling the familial male precocious puberty. For the study, we used the C-terminal peptides of Galphas and Galphai2, which couple to the receptor.

Previously, we reported that a liganded LH receptor (LHR) is capable of activating itself (cis-activation) and other nonliganded LHRs to induce cAMP (trans-activation). Trans-activation of the LHR raises two crucial questions. Is trans-activation unique to LHR or common to other G protein-coupled receptors? Does trans-activation stimulate phospholipase Cbeta as it does adenylyl cyclase? To address these questions, two types of novel FSH receptors (FSHRs) were constructed, one defective in hormone binding and the other defective in signal generation. The FSHR, a G protein-coupled receptor, comprises two major domains, the N-terminal extracellular exodomain that binds the hormone and the membrane-associated endodomain that generates the hormone signals. For signal defective receptors, the exodomain was attached to glycosyl phosphatidylinositol (ExoGPI) or the transmembrane domain of CD8 immune receptor (ExoCD). ExoGPI and ExoCD can trans-activate another nonliganded FSH. Surprisingly, the trans-activation generates a signal to activate either adenylyl cyclase or phospholipase Cbeta, but not both. These results indicate that trans-activation in these mutant receptors is selective and limited in signal generation, thus providing new approaches to investigating the generation of different hormone signals and a novel means to selectively generate a particular hormone signal. Our data also suggest that the FSHR's exodomain could not trans-activate LHR.

Follicle-stimulating hormone (FSH) comprises an alpha subunit and a beta subunit, whereas the FSH receptor consists of two halves with distinct functions: the N-terminal extracellular exodomain and C-terminal membrane-associated endodomain. FSH initially binds to exodomain, and the resulting FSH/exodomain complex modulates the endodomain and generates signal. However, it has been difficult to determine which subunit of FSH contacts the exodomain or endodomain and in what orientation FSH interacts with them. To address these crucial issues, the receptor was Ala-scanned and the hormone subunits were probed with photoaffinity labeling with receptor peptides corresponding to the N-terminal region of the exodomain and exoloop 3 of the endodomain. Our results show that both regions of the receptors are important for hormone binding and signal generation. In addition, the FSH beta subunit is specifically labeled with the N-terminal peptide, whereas the alpha subunit is labeled with the exoloop 3 peptide. These contrasting results show that the FSH beta subunit is close to the N-terminal region and that the alpha subunit is projected toward exoloop 3 in the endodomain. The results raise the fundamental question whether the alpha subunit, common among the glycoprotein hormones, plays a major role in generating the hormone signal common to all glycoprotein hormones.

Loss of function by 11 of 13 naturally occurring mutations in the human GnRH receptor (hGnRHR) was thought to result from impaired ligand binding or effector coupling, but actually results from receptor misrouting. Homo- or heterodimerization of mutant receptors with wild-type (WT) receptors occurs for other G protein-coupled receptors and may result in dominant-negative or -positive effects on the WT receptor. We tested the hypothesis that WT hGnRHR function was affected by misfolded hGnRHR mutants. hGnRHR mutants were found to inhibit the function of WT GnRHR (measured by activation of effector and ligand binding). Inhibition varied depending on the particular hGnRHR mutant coexpressed and the ratio of hGnRHR mutant to WT hGnRHR cDNA cotransfected. The hGnRHR mutants did not interfere with the function of genetically modified hGnRHRs bearing either a deletion of primate-specific Lys(191) or the carboxyl-terminal tail of the catfish GnRHR; these show intrinsically enhanced expression. Moreover, a peptidomimetic antagonist of GnRH enhanced the expression of WT hGnRHR, but not of genetically modified hGnRHR species. The dominant-negative effect of the naturally occurring receptor mutants occurred only for the WT hGnRHR, which has intrinsic low maturation efficiency. The data suggest that this dominant negative effect accompanies the diminished plasma membrane expression as a recent evolutionary event.

FSH plays crucial roles in differentiation of granulosa cells and development of follicles. Considering the broad scope of FSH effects, a large number of genes are likely responsive to the hormone. However, only a limited number of genes have been identified as FSH-regulated genes, particularly during the preantral stage. In an attempt to better define genes involved in follicular development, we examined primary granulosa cell cultures, an undifferentiated rat ovarian granulosa cell line and rat ovaries, using differential display, quantitative RT-PCR, Northern blot analysis, and in situ hybridization. We report, for the first time, that nicotinamide adenine dinucleotide phosphate-dependent cytosolic T(3)-binding protein mRNA is expressed in the ovary, particularly in the granulosa cell layer of preantral and early antral follicles, but not in large preovulatory follicles. Its expression markedly declines in response to FSH, which is dependent on the period of the exposure. This FSH-responsive down-regulation is dependent on granulosa cell differentiation and follicular development. FSH down-regulates the mRNA via the adenylyl cyclase/cAMP pathway, and the down-regulation requires de novo synthesis of a regulatory protein(s). The cytosolic T(3)-binding protein may play a significant role in the regulation of steroidogenesis and follicular development in the mammalian ovary.

FSH regulates gene expression for granulosa cell differentiation and follicular development. Therefore, FSH-responsive genes are crucial, but only a few genes have been identified for the early stage of follicular development. In particular, little is known about cytoskeletal genes, which likely play essential roles in the morphological changes such as the antrum formation, a major landmark. FSH is also known to induce the differentiation of an immature, undifferentiated rat ovary granulosa (ROG) cell line. Our data show that FSH induced massive yet distinct reorganization of microtubules and the actin cytoskeletons as well as morphological changes. To identify those genes responding to FSH during the differentiation, differential display was performed on ROG cells. Of the 80 FSH-responsive genes identified, there were three cytoskeleton-related genes (class I beta-tubulin, tropomyosin 4, and kinesin heavy chain), which are crucial for intracellular morphogenesis, transport, and differentiation. Northern blots show that the level of these gene transcripts reached a peak at 6 h after FSH treatment and subsided at 24 h. FSH induced the similar temporal expression not only in granulosa cells isolated from immature rats, but also in vivo. For instance, in situ hybridization showed that beta-tubulin mRNA was transiently expressed in the granulosa cells of large preantral and early antral follicles. Despite the same temporal expression, the regulatory mechanisms of the three genes were strikingly different. As an example, cycloheximide blocked the beta-tubulin mRNA expression, whereas it increased tropomyosin-4 (TM4) mRNA. Yet, it did not impact kinesin heavy chain (Khc) mRNA. In conclusion, FSH induces the massive reorganization of the cytoskeletons and morphological changes by the selective regulation of the gene expression, protein synthesis, and rearrangement of the cytoskeletal proteins in the ROG cells and probably, specific follicles and granulosa cells.

The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of approximately 330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of (580)KVPLITVSKAK(590) was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH alpha but not FSH beta. This is in contrast to photoaffinity labeling of FSH beta by the peptide mimic of the N-terminal region of the receptor. Leu(583) and Ile(584) are crucial for the interaction of FSH and exoloop 3. Substitutions of these two residues enhanced the hormone binding affinity. This is due to the loss of the original side chains but not the introduction of new side chains. The Leu(583) and Ile(584) side chains appear to project in opposite directions. Ile(584) appears to be so specific and to require flexibility and stereo specificity so that no other amino acids can fit into its place. Leu(583) is less specific. The improvement in hormone binding by substitutions was offset by the severe impairment of signal generation of cAMP and/or inositol phosphate. For example, the Phe or Tyr substitution of Leu(583) improved the hormone binding and cAMP induction but impaired IP induction. On the other hand, the substitutions for Ile(584) and Lys(590) abolished the cAMP and IP induction. Our results open a logical question whether Leu(583), Ile(584), and Lys(590) interact with the exodomain and/or the hormone. The answers will provide new insights into the mechanisms of hormone binding and signal generation.

This article describes a novel method to access functional interactions of two defective mutant receptors. As a model, luteinizing hormone receptor, a G-protein-coupled receptor, was used by coexpressing two different mutants, one defective in hormone binding and the other defective in signal generation. When these two mutants were coexpressed in a cell, the cell responded to the hormone and induced the hormone action, indicating the interaction of the two receptors and rescue of the activity. The luteinizing hormone receptor consists of a 350-amino-acid extracellular N-terminal domain (exodomain), followed by seven transmembrane domains and connecting loops (endodomain). Hormone binds to the exodomain, whereas hormone signals are generated in the endodomain. Here, we show that binding of hormone to one receptor can activate adenylyl cyclase through its transmembrane bundle, intramolecular activation (cis-activation), as well as intermolecular activation (trans-activation) through the transmembrane bundle of an adjacent receptor, without forming a stable receptor dimer. Our observations provide new insights into the mechanism of receptor activation mechanisms, and have implications for the treatment of inherited disorders of glycoprotein hormone receptors.

Objective: Despite the general use of DSM-IV attention-deficit/hyperactivity disorder (ADHD) subtypes, there is controversy over the optimal phenotyping strategy for this disorder.This report contrasts two ADHD subtyping approaches on the prediction of cognitive function and educational achievement., Method: ADHD subtypes were determined using DSM-IV and latent class approaches for a population sample of 1,154 child and adolescent twins using parent report data. Twins completed cognitive and achievement testing and parents reported on school grades, special education placement, and history of being held back in school., Results: The DSM-IV primarily inattentive and combined subtype ADHD groups showed significant deficits in cognitive and achievement testing, worse grades, and increased use of special education resources compared with the primarily hyperactive/impulsive subtype and no-ADHD groups. Clinically relevant and less severe latent class ADHD subtypes were also associated with deficits in cognitive and achievement testing, grades, and special education use., Conclusions: DSM-IV primarily inattentive and combined subtypes of ADHD have similar significant patterns of cognitive and academic dysfunction in the general population. Latent class-defined ADHD subtypes also have patterns of serious cognitive and achievement deficits.

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.

G protein-coupled receptors (GPCRs) accommodate a wide spectrum of activators from ions to glycoprotein hormones. The mechanism of activation for this large and clinically important family of receptors is poorly understood. Although initially thought to function as monomers, there is a growing body of evidence that GPCR dimers form, and in some cases that these dimers are essential for signal transduction. Here we describe a novel mechanism of intermolecular GPCR activation, which we refer to as trans-activation, in the LH receptor, a GPCR that does not form stable dimers. The LH receptor consists of a 350-amino acid amino-terminal domain, which is responsible for high-affinity binding to human CG, followed by seven-transmembrane domains and connecting loops. This seven-transmembrane domain bundle transmits the signal from the extracellular amino terminus to intracellular G proteins and adenylyl cyclase. Here, we show that binding of hormone to one receptor can activate adenylyl cyclase through its transmembrane bundle, intramolecular activation (cis-activation), as well as trans-activation through the transmembrane bundle of an adjacent receptor, without forming a stable receptor dimer. Coexpression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescues hormone-activated cAMP production. Our observations provide new insights into the mechanism of receptor activation mechanisms and have implications for the treatment of inherited disorders of glycoprotein hormone receptors.

Luteinizing hormone receptor is a G protein-coupled receptor and consists of two halves: the N-terminal extracellular half (exodomain) and C-terminal membrane-associated half (endodomain). Hormone binds to the exodomain, and the resulting hormone-exodomain complex modulates the endodomain to generate signals. There are mutations that impair either hormone binding or signal generation. We report that the coexpression of a binding defective mutant and a signal-defective mutant rescues signal generation to produce cAMP. This rescue requires both types of mutant receptors and is dependent on the human chorionic gonadotropin dose, the surface concentration of mutant receptors, and the amino acid position of mutations. Furthermore, random collisions among mutant receptors are not involved in the rescue. Our observations provide new insights into the mechanisms of the functional and structural relationship of the exo- and endodomain, signal transduction, and receptor genetics, in particular for defective heterozygotes.

Regulators of G protein signaling (RGS) play a pivotal role in cellular signal transduction. RGS3 or RGS10 were overexpressed in GGH(3) cells [GH(3) cells stably expressing the GnRH receptor (GnRHR)]. Responsiveness to a GnRH agonist was assessed because RGS proteins attenuate production of inositol phosphates (IP) and/or cAMP, molecules believed to be involved in GnRH signaling. In addition, site-directed mutagenesis of a potentially palmitoylated Cys(60) residue of RGS10 was used to assess the significance of this site. We observed maximum inhibition of GnRH-stimulated IP responses by RGS3 and by the conserved domain of RGS10 at both 48 and 72 h after transfection, indicating their involvement in G(q)alpha mediated signaling. Significantly diminished cAMP production was observed at all times when cells overexpressed the conserved domain of RGS10; no effect was observed with RGS3 on G(s)alpha-mediated signaling. Palmitic acid incorporation into RGS3 was dependent on agonist occupancy of GnRHR, whereas palmitoylation of RGS10 was constitutive. Mutation of the conserved Cys(60) residue of RGS10 obviated its negative regulatory action on GnRH-stimulated responses, indicating that this site is crucial for its activity on this system. This study is the first demonstration of a role for palmitoylation of this conserved Cys(60) in mammalian G protein signaling.

Human chorionic gonadotropin (hCG) binds to the extracellular N-terminal domain, exodomain, of its receptor, and the resulting hCG-exodomain complex is thought to modulate the membrane associated domain, endodomain, of the receptor to generate hormone signal. The bulk of the exodomain is speculated to assume a crescent structure consisting of eight to nine Leu-rich repeats (LRRs), which may provide the hormone contact sites. Unfortunately, little experimental evidence is available for the precise hormone contact points in the exodomain and the endodomain. The two preceding articles (Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3426-3435; Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3436-3442) show that putative LRR2 and LRR4 are crucial for hormone binding. In particular, the N-terminal region of LRR4 assumes the hydrophobic core of the LRR4 loop, whereas the C-terminal region is crucial for signal generation. However, it is unclear whether LRR4 interacts hCG and the endodomain and how it might be involved in signal generation. In this article, our affinity labeling results present the first evidence that the N-terminal region of LRR4 interacts with hCG, preferentially the hCGalpha subunit and that the hCG/LRR4 complex interacts with exoloop 2 of the endodomain. This interaction offers a mechanism to generate hormone signal.

Luteinizing hormone receptor, a G protein-coupled receptor, consists of two halves, the N-terminal extracellular hormone binding domain (exodomain) and the C-terminal membrane-associated, signal-generating domain (endodomain). The exodomain has seven to nine Leu-rich repeats, which are generally thought to form a 1/3 donut-like structure and interact with human choriogonadotropin (hCG). The resulting hCG-exodomain complex adjusts the structure and its association with the endodomain, which results in signal generation in the endodomain. It is unclear whether the rigid 1/3 donut structure could provide the agility and versatility of this dynamic action. In addition, there is no clue as to where the endodomain contact point (the signal modulator) in the exodomain is. To address these issues, the exodomain was examined by Ala scan and multiple substitutions, while receptor peptides were used for photoaffinity labeling and affinity cross-linking. Our results show that the C-flanking sequence (hinge region), Thr(250)-Gln(268), of the Leu-rich repeats (LRRs) specifically interacts with hCG, preferentially hCGalpha. This interaction is inhibited by exoloop 2 of the endodomain but not by exoloops 1 and 3, suggesting an intimate relationship between Thr(250)-Gln(268), exoloop 2, and hCG. Taken together, our observations in this article suggest a new paradigm that the LRRs contact the front of hCG, while both flanking regions of the LRRs interact with the sides of hCG. This would trap hCG in the 1/3 donut structure of the LRRs and enhance the binding affinity. In addition, mutations of conserved Ser(255) in the sequence can constitutively activate the receptor. This provides a clue for the signal modulator in the exodomain. In contrast, a phenyl or phenolic group is necessary at conserved Tyr(253) for targeting the receptor to the surface.

The luteinizing hormone receptor (LHR) consists of an approximately 350-amino acid-long N-terminal extracellular exodomain and a membrane-associated endodomain of similar size. Human chorionic gonadotropin (hCG) binds to the exodomain, and then hCG/exodomain complex is thought to make a secondary contact with the endodomain and generate hormone signals. The sequence alignment of the exodomain shows imperfectly matching eight to nine Leu-rich repeats (LRRs). In the preceding article (Song, Y., Ji, I., Beauchamp, J., Isaacs, N., and Ji, T. (2001) J. Biol. Chem. 276, 3426-3435), we have shown that LRR2 and LRR4 are crucial for hormone binding. In this work, we have examined the residues of LRR4, in particular Leu(103) and Ile(105) in the putative beta strand. Our data show that Leu(103) and Ile(105) are involved in the specific, hydrophobic interaction of the LRR4 loop, likely to form the hydrophobic core. This loop is crucial for the structural integrity of all of the LRRs. In contrast, the downstream sequence consisting of Asn(107), Thr(108), Gly(109), and Ile(110) of LRR4 is crucial for cAMP induction but not for hormone binding, folding, and surface expression. This implicates, for the first time, its involvement in the interaction with the endodomain and signal generation. The evidence for the interaction is presented in the following article.

The luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) have an approximately 350-amino acid-long, N-terminal extracellular exodomain. This exodomain binds hormone with high affinity and specificity and contains eight to nine putative Leu-rich repeat (LRR) sequences. LRRs are known to assume the horseshoe structure in ribonuclease inhibitors, and the inner lining of the horseshoe consists of the beta-stranded Leu/Ile-X-Leu/Ile motif. In the case of ribonuclease inhibitors, these beta strands interact with ribonuclease. However, it is unclear whether the putative LRRs of LHR and FSHR play any role in the structure and function. In this work, the beta-stranded Leu/Ile residues in all LRRs of the human LHR and FSHR were Ala-scanned and characterized. In addition, the 23 residues around LRR2 of LHR were Ala-scanned. The results show that beta-stranded Leu and Ile residues in all LRRs are important but not equally. These Leu/Ile-X-Leu/Ile motifs appear to form the hydrophobic core of the LRR loop, crucial for the LRR structure. Interestingly, the hot spots are primarily in the upstream and downstream LRRs of the LHR exodomain, whereas important LRRs spread throughout the FSHR exodomain. This may explain the distinct hormone specificity despite the structural similarity of the two receptors.

FSH plays a crucial role in granulosa cell differentiation and follicular development during the ovulation cycle. The early events of granulosa cell differentiation in cell culture involve changes in the cell morphology and cell-to-cell interactions. To determine the cause and signaling mechanism for these changes, we examined an undifferentiated rat ovarian granulosa cell line that grows in a defined serum-free medium, expresses the FSH receptor, terminally differentiates when exposed to FSH, and undergoes apoptosis upon FSH withdrawal. FSH bound the FSH receptor on rat ovarian granulosa cells, and the liganded receptor activated adenylyl cyclase (AC) to produce cAMP but did not mobilize Ca2+. In addition, we observed massive reorganization of the actin cytoskeleton within 3 h of FSH treatment. This involves formation of lamellipodia and filopodia and spreading of multilayer cell aggregates to monolayers. This actin reorganization and cell transformation could also be induced by the AC activator, forskolin, in the absence of FSH. Furthermore, AC inhibitors blocked the FSH-dependent actin reorganization and transformation. On the other hand, phospholipase C inhibitors did not block the FSH-induced changes. Taken together, our observations indicate that the AC/cAMP signal is necessary and sufficient for FSH-dependent granulosa cell differentiation, including massive reorganization of the actin cytoskeleton and changes in the cell morphology and cell-to-cell interactions. There is no evidence that the phospholipase C signal and Ca2+ mobilization are involved in this process.

Human CG (hCG) consists of a common alpha-subunit and a hormone-specific beta-subunit. Similarly, its receptor is also composed of two domains, an extracellular N-terminal half (exodomain) and a membrane-associated C-terminal half (endodomain). hCG initially binds the exodomain of the receptor after which the resulting hCG/exodomain complex is thought to interact with the endodomain. This secondary interaction is considered responsible for signal generation. Despite the importance, it is unclear which hormone subunit interacts with the exodomain or the endodomain. As a step to determine the mechanisms of the initial and secondary interactions and signal generation, we investigated the interaction of the hormone-specific beta-subunit in hCG with the receptor's exodomain. A photoactivable hCG derivative consisting of the wild-type alpha-subunit and a photoactivable beta-subunit derivative was prepared and used to label the exodomain. The analysis and immunoprecipitation of photoaffinity labeled exodomain demonstrate that the beta-subunit in hCG makes the direct contact with the exodomain.

The LH/CG receptor, a G protein-coupled receptor, consists of two parts, the N-terminal extracellular segment (exodomain) and the membrane-associated C-terminal segment (endodomain). hCG initially binds the exodomain of the receptor and then, the hormone/exodomain complex is thought to make the secondary contact with the endodomain of the receptor and generate a hormone signal. However, little direct evidence is available about which hormone subunits (alpha or beta) interact with which domains of the receptor. To determine whether the alpha-subunit contacts the exodomain of its receptor, hCG containing [125I]alpha and truncated exodomain lacking the endodomain were prepared. They were chemically cross-linked, and the resulting cross-linked complexes were solubilized and electrophoresed. The results indicate that the alpha-subunit of hCG was directly and specifically cross-linked to the exodomain. To verify the cross-linked exodomain by the independent method, the Flag epitope was inserted between the signal sequence and the mature exodomain. hCG containing [125I]alpha was cross-linked to the Flag exodomain, and the resulting cross-linked hCG/Flag exodomain complexes were immunoprecipitated with anti-Flag antibody. The results show that the material cross-linked to hCG containing [125I]alpha is indeed the exodomain. In conclusion, our results show the direct interaction of the alpha-subunit with the exodomain and, therefore, its crucial role in the hormone-receptor interaction in addition to its involvement in signal generation.