The microsomal enzyme cytochrome P450c17 is an important regulator of steroidogenesis. The enzyme has two functions: 17alpha-hydroxylase and 17,20-lyase activities. These functions determine the ability of adrenal glands and gonads to synthesize 17alpha-hydroxylated glucocorticoids (17alpha-hydroxylase activity) and/or sex steroids (17,20-lyase activity). Both enzyme functions depend on correct steroid binding, but it was recently shown that isolated lyase deficiency can also be caused by mutations located in the redox partner interaction domain. In this article we present the clinical history and molecular analysis of two patients with combined 17alpha-hydroxylase/17,20-lyase deficiency and four patients with isolated 17,20-lyase deficiency. In these six patients, four missense CYP17 mutations were identified. Two mutations were located in the steroid-binding domain (F114V and D116V), and the other two mutations were found in the redox partner interaction domain (R347C and R347H). We investigated the activity of these mutated proteins by transfection experiments in COS-1 cells using pregnenolone, progesterone, or their hydroxylated products as a substrate and measuring 17alpha-hydroxylase- and 17,20-lyase-dependent metabolites in the medium. The mutations in the steroid-binding domain (F114V and D116V) of P450c17 caused combined, complete (F114V), or partial (D116V) 17alpha-hydroxylase and 17,20-lyase deficiencies, whereas mutations in the redox partner interaction domain (R347C and R347H) displayed less severe 17alpha-hydroxylase deficiency, but complete 17,20-lyase deficiency. These findings are consistent with the clinical data and support the observation that the redox partner interaction domain is essential for normal 17,20-lyase function of P450c17.

In this study, molecular simulations have been combined with site-directed mutagenesis experiments to explore M398(2.43), a LH (lutropin) receptor (LHR) site in helix 2 susceptible to spontaneous activating mutations, and to develop a computational tool for predicting the functionality (i.e. active or nonactive) of LHR mutants.Site-directed mutagenesis experiments engineered 15 different substitutions for M389(2.43), which resulted in variable levels of constitutive activity, inversely correlated with the size of the replacing amino acid. This inverse correlation is suggested to be mediated by I460(3.46), M571(6.37), and Y623(7.53), the tyrosine of the NPxxY motif. In fact, size reduction at position 398(2.43), which is concurrent with constitutive receptor activity, releases the van der Waals interactions found in the wild-type LHR between M398(2.43) and these three amino acids, resulting in structural modifications in the proximity to the E/DRY/W motif. An increment, above a threshold value, in the solvent accessibility of the cytosolic ends of helices 3 and 6 is the main structural feature shared by the active mutants of the LHR. This feature has been successfully used for predicting the functionality of the engineered mutants at M398(2.43), proving that molecular simulations can be useful for in silico screening of LHR mutants.

In a substantial part of adrenal adenomas and hyperplasias from patients with Cushing's syndrome, cortisol production is controlled by the expression of aberrant hormone receptors on adrenocortical cells. We present in vivo and in vitro data of two patients with a LH-responsive Cushing's syndrome based on ACTH-independent bilateral adrenal hyperplasia. Patients 1 and 2 are women who presented with Cushing's syndrome and bilateral adrenal hyperplasia. Endocrine testing demonstrated absence of cortisol diurnal rhythm, insufficient cortisol suppression after 1 mg dexamethasone orally, and undetectable ACTH levels in both patients. Both patients were treated by laparoscopic biadrenalectomy. In in vivo testing, in patients 1 and 2, a profound cortisol rise was found after administration of GnRH [change in cortisol (Delta F), 118 and 106%, respectively], human CG (Delta F, 133 and 44%), LH (Delta F, 73 and 43%), ACTH (Delta F, 89 and 181%), and the 5-hydroxy-tryptamine receptor type 4 (5-HT(4)) agonists cisapride (Delta F, 141 and 148%) and metoclopramide (Delta F, 189 and 95%). In in vitro testing, adrenal cells from patient 2 responded, in a dose-dependent fashion, with cortisol production after exposure to human CG (Delta F, 45%), cisapride (Delta F, 68%), and metoclopramide (Delta F, 81%). ACTH induced cortisol production by cells from both patients (Delta F, 135 and 159%). In receptor studies, LH receptor mRNA was demonstrated in adrenal tissue of both patients but also in control adrenal tissue of two patients with persisting pituitary-dependent Cushing's syndrome treated by biadrenalectomy. In neither patient were mutations found in the ACTH receptor gene. LH-responsive Cushing's syndrome associated with bilateral adrenal hyperplasia may result from aberrant (or possibly increased) adrenal LH receptor expression. This variant is further characterized by adrenal responsiveness to 5-HT4 receptor agonists, possibly pointing to an interaction between LH and serotonin in the regulation of cortisol secretion. Despite the regulatory potential of LH and 5-HT4 receptor agonists on cortisol production in our patients, their adrenals seemed to be still sensitive to ACTH, both in vivo and in vitro.

Müllerian inhibiting substance (MIS or anti-Müllerian hormone) is a member of the transforming growth factor-beta family and plays a pivotal role in proper male sexual differentiation. Members of this family signal by the assembly of two related serine/threonine kinase receptors, referred to as type I or type II receptors, and downstream cytoplasmic Smad effector proteins. Although the MIS type II receptor (MISRII) has been identified, the identity of the type I receptor is unclear. Here we report that MIS activates a bone morphogenetic protein-like signaling pathway, which is solely dependent on the presence of the MISRII and bioactive MIS ligand. Among the multiple type I candidates tested, only ALK2 resulted in significant enhancement of the MIS signaling response. Furthermore, dominant-negative and antisense strategies showed that ALK2 is essential for MIS-induced signaling in two independent assays, the cellular Tlx-2 reporter gene assay and the Müllerian duct regression organ culture assay. In contrast, ALK6, the other candidate MIS type I receptor, was not required. Expression analyses revealed that ALK2 is present in all MIS target tissues including the mesenchyme surrounding the epithelial Müllerian duct. Collectively, we conclude that MIS employs a bone morphogenetic protein-like signaling pathway and uses ALK2 as its type I receptor. The use of this ubiquitously expressed type I receptor underscores the role of the MIS ligand and the MIS type II receptor in establishing the specificity of the MIS signaling cascade.

Context: Epidemiological and animal studies indicate a carcinogenic role of estrogens in breast tissue. The pituitary gonadotropin LH is an important regulator of estrogen production in premenopausal women, whereas even in women after menopause, 10 -25% of ovarian steroid hormone production is LH dependent.Objective: We hypothesized that an LH receptor (LHR) gene variant may affect LHR function and thereby influence disease outcome in breast cancer patients. Design:The association of a polymorphic CTCCAG (Leu-Gln) insertion (insLQ), in the signal peptide encoded by exon 1 of the LHR gene with breast cancer risk, (disease-free) survival, and clinicopathological features was studied in a large cohort of 751 breast cancer patients with complete follow-up. Functional analysis of the insLQ-LHR and non-LQ-LHR (no LQ insertion) was carried out using transfection studies. Results:We found a significant association between the insLQ-LHR and a shorter disease-free survival (hazard ratio, 1.34; confidence interval, 1.11-1.63; P ϭ 0.003). The mechanism of the effect of insLQ on LHR function involves increased receptor sensitivity (insLQ-LHR has a 1.9 times lower EC 50 than non-LQ-LHR; P ϭ 0.02) and plasma membrane expression (insLQ-LHR has 1.4 times higher B max ; P ϭ 0.0006) rendering the insLQ-LHR allele more active. Conclusions:The insLQ polymorphism increases LHR activity, thereby shortening breast cancer disease-free survival, probably by increasing estrogen exposure in female carriers.

Three mutations (33 bp insertion in exon 1; W491* and I625K) were identified that explain the phenotype in two patients. In addition, most of the patients with the clinical phenotype of LCH did not have causative mutations, suggesting that changes in other regions of the LH receptor gene, such as the large introns or the promoter region, may be responsible for the majority of cases. Alternatively, the displayed phenotype may be the result of other genetic defects. Our work further underscores the importance of thorough clinical analysis of patients before molecular analysis of a particular gene is performed.

Polymorphic variation of the LHR gene may affect receptor function and accordingly may influence ovarian steroid hormone action, including steroid hormone-dependent clinical outcome. The functional effects of two single nucleotide polymorphisms (SNPs), i.e. LHR 291Asn/Ser (rs12470652) and 312Ser/Asn (rs2293275) in the biologically interesting exon 10 of the LHR gene are described. Furthermore, ethnic diversity in allele frequencies and genotype distributions of both SNPs was determined. In addition associations with breast cancer were studied in 751 breast cancer patients. In vitro transfection studies revealed altered glycosylation status and increased receptor sensitivity for the 291Ser LHR variant. No functional consequences were observed for the 312SerAsn LHR SNP. The LHR 312Asn allele was slightly more often present in two independent breast cancer patient cohorts as compared to controls (OR=1.15; p=0.03 and 1.26; p=0.001, respectively). In conclusion, although functional changes of the LHR 291Ser candidate allele were observed, no associations with breast cancer were found, while the LHR 312Asn allele can be regarded as a weak breast cancer risk allele.

The luteinizing hormone (LH) receptor plays an essential role in male and female gonadal function. Together with the follicle-stimulating hormone (FSH) and thyroid stimulating hormone (TSH) receptors, the LH receptor forms the family of glycoprotein hormone receptors. All glycoprotein hormone receptors share a common modular topography, with an N-terminal extracellular ligand binding domain and a C-terminal seven-transmembrane transduction domain. The ligand binding domain consists of 9 leucine-rich repeats, flanked by N-and C-terminal cysteine-rich regions. Recently, crystal structures have been published of the extracellular domains of the FSH and TSH receptors. However, the C-terminal cysteine-rich region (CCR), also referred to as the "hinge region," was not included in these structures. Both structure and function of the CCR therefore remain unknown. In this study we set out to characterize important domains within the CCR of the LH receptor. First, we mutated all cysteines and combinations of cysteines in the CCR to identify the most probable disulfide bridges. Second, we exchanged large parts of the LH receptor CCR by its FSH receptor counterparts, and characterized the mutant receptors in transiently transfected HEK 293 cells. We zoomed in on important regions by focused exchange and deletion mutagenesis followed by alanine scanning. Mutations in the CCR specifically decreased the potencies of LH and hCG, because the potency of the low molecular weight agonist Org 41841 was unaffected. Using this unbiased approach, we identified Asp 330 and Tyr 331 as key amino acids in LH/hCG mediated signaling.The glycoprotein hormone luteinizing hormone (LH) 3 has an essential role in reproduction. In both sexes, LH regulates the production of gonadal androgens, which in women are almost completely converted to estrogens. Furthermore, in women the mid-cycle LH peak triggers ovulation of the mature oocyte ready to be fertilized, whereas the closely related pregnancy hormone, chorionic gonadotropin (hCG), supports the corpus luteum of pregnancy. Both LH and hCG act by binding and activating the LH receptor. The LH receptor has a modular architecture consisting of an ectodomain or extracellular hormone binding domain (ECD), linked to a seven-transmembrane signal transduction domain (7TMD). Together with the receptors for thyroid stimulating hormone (TSH) and follicle stimulating hormone (FSH), the LH receptor belongs to the glycoprotein hormone receptor family. The extracellular ligand binding domain of this family consists of a stretch of nine leucine-rich repeats (LRRs), flanked by N-terminal and C-terminal cysteine-rich clusters (NCR and CCR, respectively). The crystal structures that have been determined for major parts of FSHR and TSHR ECDs show that the LRRs are organized as ␤ sheets, and give rise to a curved helical tube of which the concave surface forms the hormone-binding surface (1, 2). The NCR provides an additional ␤ strand to the binding surface. Because the CCR was not included in the protein expression...