Evolutionary divergence of thyrotropin receptor structure

The availability of 18 thyrotropin receptor (TSHR) sequences, including two recent entries for primates and seven from fish, have allowed us to investigate diversification of residues or domains during evolution. We used a likelihood ratio test for evolutionary rate shifts [Proc. Natl. Acad. Sci. 98 (2001) 14512] using LH/CGR sequences as an out-group. At each residue in the alignment, a statistical test was performed for a rate shift at the divergence between mammals and fish. Eighty-two rate shift sites were found, significantly more than was expected (p < 0.0001). The occurrence of rate shifts was highest in the intracellular tail, lowest in the transmembrane serpentine and intermediate in the ectodomain. In 52 mammalian sites, the rates were significantly faster than for the corresponding sites in fish. We have identified rate shift in sites important to TSHR function or in intimate proximity to such regions. The former category includes residues 53 and 55 (of LLR1 beta strand) and 253 and 255 (of LLR9 beta strand), crucial to TSH thyrotropic activity, residue 113, the site of N-linked glycosylation limited to humans, residue 310, an important switch in the hinge region for receptor binding and constitutive activity and residue 382 which centres a motif important for TSH-mediated receptor activation. The rate shifts positions close to functional region include a site proximal to a TSHR-specific motif on LLR3 beta strand, sites important in TM helix structure and homodimerization as well as, in the case of the third intracellular loop, to TSHR/G protein coupling. Rate shift analyses have identified residues whose manipulation in the human TSHR may lead to better understanding of receptor functions and help in the creation of designer analogues.     

大鼠脂肪细胞TSH受体机能研究

目的：研究TSH及Graves'TgC对大鼠脂肪细胞的作用。方法：获取大鼠游离的脂肪细胞。溶脱TSH受体。采用A蛋白尾析柱纯化Graves患者的IgG以脂肪细胞内cAMP浓度及释放到孵化液中甘油浓度作为脂肪分解指标,用cAMP及甘油试剂盒测定cAMP浓度及甘油浓度。结果：1mU／ml的TSll能增加大鼠雅高脂肪细胞的cAMP浓度及甘油的释放。脓苷引起cAMP及甘油的TSH剂量依赖曲线向右移。Gare’TgG抑制[125I]-TSH结合于大鼠脂肪细胞膜上ISH受体,并能刺激雅高脂肪细胞内cAMP的形成。结论：ISH受体功能性表达于脂肪细胞,其生物学效应是通过活化腺耷环化酶而实现的。     

Regulation of dihydropyridine receptor and ryanodine receptor gene expression in regenerating skeletal muscle

 One of the the major properties of mature skeletal muscle is its ability to regenerate after injury. The purpose of the present study was to determine whether the expression of genes encoding the dihydropyridine receptor calcium channel (DHPR) and the ryanodine receptor (RyR), which play a critical role in excitation–contraction coupling, is regulated by skeletal muscle regeneration. The process of regeneration was induced by bupivacaine injection in surgically exposed rat extensor digitorum longus (EDL) muscle. After total RNA isolation from the injected and the contralateral control EDL muscles performed 3, 7, 15 and 30 days following injection, Northern blot and RNase protection assays were carried out with four cDNA probes specific for the skeletal and cardiac muscle isoforms of both the DHPR α1-subunit and the RyR. After 3 days, an initial precipitous decrease in the expression of the genes encoding the skeletal muscle isoforms of the DHPR and RyR was observed, followed by an increase. Moreover, regenerating skeletal muscle transiently expressed mRNA for the DHPR cardiac isoform, mainly at the beginning of regeneration. No expression of mRNA for the cardiac RyR was observed. Contraction experiments, performed using EDL muscle at the same times after bupivacaine injection, showed that twitch amplitude was markedly decreased in the absence of external calcium, but only during the early stages of regeneration. Similar findings in relation to expression of skeletal and cardiac muscle DHPR message were previously reported from experiments conducted during early developmental stages using fetal skeletal muscle and muscle cell cultures [Chaudhari N, Beam KG (1993) Dev Biol 155:507–515]. These results suggest that expression of the DHPR cardiac isoform in skeletal muscle could explain certain cardiac-like aspects of excitation–contraction coupling of regenerating skeletal muscle and developing skeletal muscle as well. Received: 2 July 1996 / Received after revision: 18 September 1996 / Accepted: 20 September 1996     

FXR通过调节促甲状腺素胚胎因子减轻Con A诱导的自身免疫性肝炎病变

 目的：观察法尼酯衍生物X受体（farnesoid X receptor,FXR)-促甲状腺素胚胎因子（thyrotropin embryonic factor,TEF）通路在自身免疫性肝炎模型小鼠肝损害中的作用,探讨FXR-TEF通路改善自身免疫性肝炎的部分可能机制。方法：检测FXR在伴刀豆球蛋白A (concanavalin A, Con A) 诱导的肝炎（Con A-induced hepatitis, CIH）小鼠肝脏的表达;检测FXR激活对TEF表达的影响;观察C57BL/6小鼠和鹅去氧胆酸（chenodeoxycholic acid,CDCA）激活FXR的CIH小鼠肝脏病理、肝脏酶学及炎症因子变化。结果：FXR在CIH小鼠中低表达;CDCA激活FXR的C57BL/6小鼠TEF表达上调;FXR被激活的CIH小鼠的肝损害较轻,FXR激活可减轻肝脏炎症因子释放。结论：CDCA激活FXR能减轻CIH引起的肝功能损害和炎症反应。FXR激活使TEF上调。FXR可能是自身免疫性肝炎的保护因素,其保护作用可能是通过TEF来实现的。激活FXR可能成为治疗自身免疫性肝炎的一个途径。     

Androgen receptor gene and male infertility

Androgens are critical steroid hormones that determine the expression of the male phenotype. Their actions are mediated by a single androgen receptor (AR) which, upon ligand binding, translocates to the nucleus to regulate the expression of androgen-responsive genes. Mutations that disrupt AR function totally result in the complete feminization of 46 XY individuals and the complete androgen insensitivity syndrome. Studies have revealed that AR mutations that do not lead to complete abrogation of its activity can cause a wide spectrum of milder androgen insensitivity syndromes, from ambiguous genitalia in newborn infants to 'idiopathic' male infertility. Recent studies indicate that missense amino-acid substitutions in the ligand-binding domain of the AR result in infertility through a novel mechanism that involves defective protein-protein interactions between receptor domains and coactivator proteins. Independent of missense mutations, studies involving Singaporean, Australian, North American and Japanese subjects indicate that increases in length of a trinucleotide repeat (CAG) tract, encoding a polyglutamine stretch in the transactivation domain of the AR, are associated with increased risk of defective spermatogenesis and undermasculinization. This association was however not observed in European populations, suggesting that the genetic background may play a significant role in the expression of the AR defects.     

Clinical and molecular genetics of the human GnRH receptor

A functional GnRH receptor (GnRH-R) in the anterior pituitary is critical for normal LH/FSH secretion, pubertal development and reproduction. Inactivating mutations of the GnRH-R have been identified in patients with idiopathic hypogonadotrophic hypogonadism. In this article we summarize phenotypic characteristics of these patients and focus on specific functional alterations of the human GnRH-R. In-vitro studies using recombinant receptor constructs demonstrate that GnRH-R missense mutations result in impaired ligand binding and reduced signal transduction, causing gonadotrophin deficiency. A detailed molecular understanding of receptor inactivation may help to design new GnRH agonists to therapeutically modulate GnRH-R function.     

Accentuated antagonism by angiotensin II on guinea-pig cardiac L-type Ca-currents enhanced by β-adrenergic stimulation

 To examine mechanism(s) underlying the accentuated antagonism by angiotensin II (A-II) on twitch tension, we recorded L-type Ca²⁺ currents (I _Ca,L) using conventional patch-clamp techniques in single, guinea-pig, ventricular myocytes. I _Ca,L was recorded by a step-pulse protocol after eliminating K⁺ conductances (internal Cs⁺ plus tetraethylammonium chloride and K⁺-free extracellular solution). A-II (100 nM) did not affect basal I _Ca,L, but inhibited I _Ca,L that had been enhanced (approximately 200% of control) by (ISO, isoproterenol 100 nM). The inhibitory action of A-II was concentration dependent (concentration eliciting 50% inhibition 88±9 pM, n=41) and the ISO-enhanced component of I _Ca,L was completely blocked by A-II at concentrations above 10 nM. CV-11974 (500 nM), an A-II type-1 receptor (AT₁) antagonist, prevented the inhibitory action of A-II. Pre-incubation with pertussis toxin (PTX) abolished the inhibitory effect of A-II. A-II also inhibited the I _Ca,L enhanced by histamine (500 nM) and forskolin (1 μM), but failed to affect I _Ca,L enhanced by intracellular cyclic adenosine monophosphate (1 mM). The inhibitory action of A-II may therefore involve AT₁ receptors/PTX-sensitive, guanine nucleotide-binding (G) proteins (Gi)/adenylate cyclase and partially explains the A-II-dependent accentuated antagonism of inotropy.     

Different alleles of the fcrA/mrp gene of Streptococcus pyogenes encode M-related proteins exhibiting an identical immunoglobulin-binding pattern

  The majority of group A streptococci (GAS, Streptococcus pyogenes) express immunoglobulin (Ig)-binding proteins. The genes encoding these proteins belong either to the emm or the emm-related (fcrA/mrp and enn) gene family and are located in close proximity on the GAS genome, where they form part of the vir regulon. In the present study analysis of sequence data of the 5′ terminal portions of the fcrA/mrp genes from GAS isolates representing 37 different M serotypes led to a classification of six different types. Thus, although fcrA/mrp genes exhibit an allelic polymorphism, they do not display the high degree of N-terminal sequence diversity found among emm genes. The nucleotide sequences of the fcrA/mrp genes from 3 GAS isolates, belonging to serotypes M8, M9, and M13 and representing newly characterized fcrA/mrp gene types, are reported. Analysis of the Ig-binding properties of recombinant FcrA/Mrp8, 9, and 13 proteins, demonstrated a similar Ig-binding profile being reactive with human IgG subclasses 1, 2, and 4. This pattern is identical to that previously described for other recombinant fcrA/mrp4, 49, 64/14 and 76 gene products, indicating that this property is not affected by the N-terminal variability. Evidence for recombination between an fcrA/mrp and an mga gene was observed in an M-type 33 strain isolate providing further support for the concept of gene rearrangement contributing to the diversity of vir regulon gene products. Received: 31 January 1996     

Immunohistochemical study of hormone receptor and hormone-regulated protein expression in phyllodes tumour: comparison with fibroadenoma

 The histogenesis of phyllodes tumour (PT) and that of fibroadenoma (FA) of the breast appear to be closely related. FA is thought to be hormonally responsive, while the hormone-responsiveness of PT is uncertain. To gain insight into hormone-responsiveness of PT, we performed immunohistochemical analysis of oestrogen-regulated pS2 and androgen-regulated prostate-specific antigen (PSA) protein expression and also of oestrogen receptor (ER), progesterone receptor (PgR) and androgen receptor (AR) expression in paraffin sections obtained from 50 female PT patients. Paraffin sections taken from 50 female fibroadenoma (FA) patients were analysed for comparison. ER, PgR, pS2, AR and PSA expression were detected in 32%, 96%, 20% 98% and 4.0% of PT sections and in 28%, 96%, 42% 80% and 10% of FA sections, respectively. No correlations were detected among ER, PgR and pS2 expression or between AR and PSA expression in PT or FA sections. PgR expression was significantly associated with AR expression in PT (P<0.0001). The present investigations indicate that PT and FA have almost similar hormone receptor status. However, different positivities of pS2 expression suggest that oestrogen-responsiveness may differ between PT and FA. In addition, a wide-ranging co-expression of AR and PgR in PT sections suggests that these receptors may play an important part in the proliferation, although the functional significance of these receptors should be elucidated. Received: 26 January 1998 / Accepted: 5 April 1998     

Isoforms and single nucleotide polymorphisms of the FSH receptor gene: implications for human reproduction

The FSH receptor shows three single nucleotide polymorphisms (SNPs), one in the promoter and two in exon 10. In addition, the FSH receptor mRNA undergoes extensive alternative splicing. While no physiological role for the SNP in the promoter and for alternative spliced isoforms has been demonstrated so far, the SNPs in exon 10 result in four discrete allelic variants characterized by the amino acid combinations Thr307-Asn680, Ala307-Ser680, Ala307-Asn680 and Thr307-Ser680. Several studies have demonstrated that the first two allelic variants are very frequent (approximately 60 and 40% respectively) in the Caucasian population. The rarer Ala307-Asn680 and Thr307-Ser680 variants are much less frequent (<5%) in the Chinese. In males the FSH receptor variants are not related to testicular volume, serum FSH or serum inhibin B levels. The two most common receptor variants transiently transfected in COS-7 cells displayed similar functional characteristics. Frequency distribution of the two polymorphisms in normal women and patients with polycystic ovarian syndrome or premature ovarian failure is still under investigation. The homozygous Ala307-Ser680 variant seems to be associated with significantly higher basal serum FSH levels and with a higher amount of FSH required for ovarian stimulation in women undergoing assisted reproduction. This suggests that the FSH receptor genotype can influence the ovarian response to FSH stimulation. The presence of SNPs in the FSH receptor gene capable of modifying FSH action paves the way for future patient-tailored, genotype-based hormone therapies.     

Positive feedback regulation of angiotensin II-AT1B receptor gene expression in rat adrenal glands

 This study aimed to investigate the role of endogenous angiotensin II (ANGII) in the upregulation of ANG-II AT₁ receptors in adrenal glands during a low-salt intake. To this end male Sprague-Dawley rats were fed a low-salt diet (0.2 mg/g) for 10 days and were treated with the ANGII-AT₁ receptor antagonist losartan (40 mg/kg per day) for 2 days, and adrenal mRNA levels for ANGII AT_1A and AT_1B receptors were determined by RNase protection. The low-salt diet increased AT_1A and AT_1B receptor mRNA levels by 90% and 220%, respectively. Losartan treatment did not change the basal AT_1A mRNA level, but decreased AT_1B mRNA by 50%. Treatment of rats on a low-salt diet with losartan did not change the increase of AT_1A mRNA but significantly attenuated the increase of AT_1B mRNA to 90% of the control value. Stimulation of endogenous ANGII levels by unilateral renal artery clipping for 2 days lowered AT_1A mRNA by 25% and increased AT_1B mRNA by 30%. Additional treatment with losartan did not affect the decreased AT_1A mRNA levels in rats with a unilateral renal artery clip, but significantly attenuated the increase of AT_1B mRNA. These findings suggest that sodium deficiency stimulates adrenal AT_1A and AT_1B receptor mRNA levels primarily via an ANGII-AT₁-independent mechanism. The preferential increase of adrenal AT_1B mRNA during a low-salt intake could be explained by the elevation of endogenous ANGII levels during sodium deficiency, suggesting that endogenous ANGII acts as an enhancer for adrenal AT_1B but not for AT_1A receptor gene expression via ANGII-AT₁ receptors. Received: 23 May 1997 / Received after revision: 23 February 1998 / Accepted: 11 March 1998     

Elevated levels of p27, p21 and cyclin D1 correlate with positive oestrogen and progesterone receptor status in node-negative breast carcinoma patients

The search for better prognostic indicators and new treatment modalities in node-negative breast carcinoma patients is important. The aim of this study was to determine the immunohistochemical expression of central cell regulator proteins in relation to hormone receptor status, tumour-cell differentiation and prognosis. We investigated the immunoreactivity of p27, p21, cdk4, cyclin D1 and p53 in 77 node-negative breast carcinomas, with long-term follow-up (mean 163 months; range 20−227). Nuclear staining for p27 was seen in 87% of the carcinomas, for cdk4 in 92%, for p21 in 68%, for cyclin D1 in 58% and for p53 in 18%. Oestrogen receptor (ER) and progesterone receptor (PgR) nuclear staining was seen in 69% and 65% of the tumours, respectively. No correlation between the levels of p21 and p53 was observed. P21 overexpression was, however, associated with positive ER status. Elevated levels of p27 and cyclin D1 correlated with positive hormone status (both ER and PgR). We did find a significant correlation between p27 and cyclin D1 and histological grade of the tumours, with extensive positive immunostaining of p27 and cyclin D1 in well-differentiated carcinomas. The only significant prognostic factor in our series was histological grading. Ten-year relapse-free survival was significantly prolonged in patients with histological grade I tumours versus histological grade II and III tumours. Our results suggest that the expression of p27 and cyclin D1 is closely linked to hormone receptor status in breast carcinomas and to tumour differentiation, a finding that may be of importance in the treatment of hormone-dependent tumours. Received: 26 March 1998 / Accepted: 29 April 1999     

Keratin subsets in papillary and follicular thyroid lesions

 Previous studies indicate that keratins 7, 8 and 18 are present in all thyroid papillary and follicular lesions, but the distribution of other keratins has been incompletely characterized. The profile of individual keratin (K) polypeptides was evaluated immunohistochemically in over 200 non-neoplastic and neoplastic thyroid papillary and follicular lesions. Monoclonal antibodies to K19, K17, K16, K5/6 and K10 were applied in paraffin sections of formaldehyde-fixed tissue. K19 was present variably, often only focally in goitres, and was present only sporadically in papillary hyperplasia. However, K19 was strongly and uniformly expressed in virtually all papillary carcinomas, indicating differential diagnostic usefulness in differentiating papillary hyperplasia and papillary carcinoma. About half of the follicular carcinomas (defined as tumours strictly excluding the follicular variant of papillary carcinoma) were also strongly K19-positive, suggesting that K19 patterns are not reliable in differentiating papillary and follicular carcinoma. K17 and K5/6 were present in cysts and squamous metaplasia of goitres, and focally in papillary but only exceptionally in follicular carcinoma in areas of squamous differentiation and tumour cells in desmoplastic stroma. K16 in turn was present only focally in well-developed squamous metaplasia in goitres but was not found in differentiated thyroid carcinomas. K10, a high-molecular-weight keratin typical of epidermal differentiation, was identified neither in non-neoplastic nor in neoplastic differentiated thyroid lesions, including squamous metaplasia. These results indicate that papillary carcinomas differ from other differentiated thyroid tumours in their varying, usually focal, expression of stratified epithelial keratins that are partly but not exclusively related to squamous differentiation in such lesions. However, papillary carcinomas do not express truly epidermally restricted keratins; their previously described reactivity with polyclonal ”epidermal keratin” antibodies most probably results from the reactivity of such antibodies with K19. Received: 14 April 1997 / Accepted: 28 May 1997     

Inhibition of the L-type calcium channel by the five muscarinic receptors (m1–m5) expressed in NIH 3T3 cells

 Modulation of L-type calcium channels by the five cloned muscarinic receptors was studied by expression of the receptors in NIH 3T3 cells. Application of acetylcholine (ACh) to cells transfected with m1–m5 resulted in a reduction in the L-type calcium current amplitude. Elevations in intracellular cAMP concentrations induced by 8-bromo-cAMP or forskolin resulted in no discernible change in the L-type calcium current. In addition, treatment with Rp-adenosine 3′,5′-cyclic monophosphothioate triethylamine (Rp-cAMPS), a protein kinase A (PKA) inhibitor, had no effect on the L-type currents. Conversely, application of phorbol dibutyrate, an activator of protein kinase C (PKC) or 8-bromo-cGMP, an activator of cGMP-dependent protein kinase (PKG), reduced the calcium currents. Incubation of the cells with KT5823, an inhibitor of PKG, resulted in a reduction of the response to 8-bromo-cGMP. The ACh-induced depression of L-type calcium current amplitude was sensitive to pertussis toxin (PTX) in cells transfected with the m2 or m4 receptor subtype. The m2-muscarinic-receptor-induced inhibition of the L-type calcium current was attenuated by preincubation of the cells with 8-bromo-cAMP and was unaffected by KT5823 or by calphostin C. The m1-muscarinic-receptor-induced inhibition of the L-type calcium conductance was insensitive to PTX treatment. However, the m1-induced response was blocked by preincubation of the cells with calphostin C. The present data indicate that the m2 (and possibly also the m4) muscarinic receptors inhibit the L-type calcium conductance by a reduction in cAMP concentration and that the m1 (and possibly also the m3 and m5) muscarinic receptors inhibit the L-type calcium channel via activation of PKC. Received: 2 September 1996 / Received after revision and accepted: 15 October 1996     

Detection of gonadotropin-releasing hormone receptor in normal human pituitary cells and pituitary adenomas using immunohistochemistry

Gonadotropin-releasing hormone (GnRH), which is a well-known regulator of gonadotroph function, has recently been considered to be a paracrine factor involved in the control of somatotroph, lactotroph, and corticotroph cells. GnRH action is initiated by binding to a specific cell surface receptor, the gonadotropin-releasing hormone receptor (GnRHR), which is expressed by follicle-stimulating hormone/luteinizing hormone (FSH/LH) cells. Using in situ hybridization techniques, GnRHR messenger ribonucleic acid (mRNA) has recently been detected in normal human anterior pituitary gland and in various pituitary adenomas, including FSH/LH-cell, growth hormone (GH)-cell, adrenocorticotropic hormone (ACTH)-cell, and null-cell adenomas. However, immunohistochemical studies indicating the specific cell distribution of GnRHR in normal pituitary cells have never been reported. The aim of the present investigation was to evaluate the immunohistochemical expression of GnRHR in different types of normal pituitary cells and related tumors. Using double-label immunohistochemical techniques on formalin-fixed and paraffin-embedded tissues and specific antibodies directed against pituitary hormones and GnRHR, we found GnRHR immunoreactivity not only in FSH/LH cells, but also in GH- and thyroid-stimulating hormone (TSH) cells. GnRHR was detected in FSH/LH-cell, GH-cell, mixed GH- and prolactin (PRL)-cell, and α-subunit (α-SU)/null-cell adenomas. The findings of this study suggest that the interaction between GnRH and GnRHR may play a role in paracrine/autocrine regulation of different types of normal pituitary cells and pituitary adenomas. Received: 24 January 2000 / Accepted: 12 April 2000     

Dual modulation of calcium channel current via recombinant α2-adrenoceptors in pheochromocytoma (PC-12) cells

 The ability of recombinant rat α_2D-and α_2B-adrenoceptors expressed in nerve-growth-factor-differentiated pheochromocytoma PC-12 cells to modulate Ca²⁺ currents, recorded by the whole-cell patch-clamp technique, has been studied. Ca²⁺ currents in different cells were either reversibly reduced or increased by dexmedetomidine, an α₂-adrenergic agonist, in a concentration-dependent manner. Pertussis toxin pretreatment reduced the number of cells that showed an inhibitory response and reduced the magnitude of inhibition. In cells expressing the α_2B-adrenoceptor, pertussis toxin increased the proportion of cells from which a stimulatory effect on Ca²⁺ currents could be recorded. The magnitude of the inhibitory responses was unaffected but the stimulatory responses were considerably reduced by the dihydropyridine Ca²⁺ channel blocker nifedipine (5 μM). All effects of dexmedetomidine were reversible upon wash-out and inhibited by the antagonist rauwolscine. The results support the idea that modulation of voltage-dependent Ca²⁺ channels in transfected PC-12 cells is mediated by activation of recombinant α_2D- and α_2B-adrenoceptors. This receptor activation predominantly causes inhibition of dihydropyridine-insensitive Ca²⁺ channels via pertussis-toxin-sensitive G proteins. Additionally receptor activation can also lead to stimulation of dihydropyridine-sensitive Ca²⁺ channels via pertussis-toxin-insensitive mechanisms. Received: 25 March 1997 / Received after revision: 27 August 1997 / Accepted: 12 September 1997