Adeno-Associated Virus Type 5 Infection via PDGFRα Is Associated With Interstitial Lung Disease in Systemic Sclerosis and Generates Composite Peptides and Epitopes Recognized by the Agonistic Immunoglobulins Present in Patients With Systemic Sclerosis.

OBJECTIVE: The etiopathogenesis of systemic sclerosis (SSc) is unknown. Platelet-derived growth factor receptors (PDGFRs) are overexpressed in patients with SSc. Because PDGFRα is targeted by the adeno-associated virus type 5 (AAV5), we investigated whether AAV5 forms a complex with PDGFRα exposing epitopes that may induce the immune responses to the virus-PDGFRα complex. METHODS: The binding of monomeric human PDGFRα to the AAV5 capsid was analyzed by in silico molecular docking, surface plasmon resonance (SPR), and genome editing of the PDGFRα locus. AAV5 was detected in SSc lungs by in situ hybridization, immunohistochemistry, confocal microscopy, and molecular analysis of bronchoalveolar lavage (BAL) fluid. Immune responses to AAV5 and PDGFRα were evaluated by SPR using SSc monoclonal anti-PDGFRα antibodies and immunoaffinity-purified anti-PDGFRα antibodies from sera of patients with SSc. RESULTS: AAV5 was detected in the BAL fluid of 41 of 66 patients with SSc with interstitial lung disease (62.1%) and in 17 of 66 controls (25.75%) (P < 0.001). In SSc lungs, AAV5 localized in type II pneumocytes and in interstitial cells. A molecular complex formed of spatially contiguous epitopes of the AAV5 capsid and of PDGFRα was identified and characterized. In silico molecular docking analysis and binding to the agonistic anti-PDGFRα antibodies identified spatially contiguous epitopes derived from PDGFRα and AAV5 that interacted with SSc agonistic antibodies to PDGFRα. These peptides were also able to bind total IgG isolated from patients with SSc, not from healthy controls. CONCLUSION: These data link AVV5 with the immune reactivity to endogenous antigens in SSc and provide a novel element in the pathogenesis of SSc.

CD38 Expression by Circulating and Skin-Infiltrating Lymphocytes from Sezary Syndrome Patients: A Flow Cytometry and Immunohistochemistry Study.

BACKGROUND: Reports on the expression of CD38 in Sézary syndrome (SS), erythrodermic primary cutaneous T cell lymphoma with leukemic involvement, are limited. The aim of the present study is the analysis of the expression of CD38 by skin-infiltrating mononuclear cells and circulating T lymphocytes in a cohort of SS patients. METHODS: SS patients diagnosed since 1985 in our clinic were retrospectively analyzed for CD38 expression in biopsy and blood samples by immunohistochemistry and flow cytometry, respectively. RESULTS: SS patients show a predominant CD38-negative phenotype on both skin and blood. A subgroup of patients was found expressing CD38 (12 cases) in either the skin (>25% cell infiltrate) or blood (CD4+CD38+ >50%), among whom 4 in the blood, 7 in the skin, and 1 in both blood and skin. CONCLUSION: The implications of these observations may be twofold: the relevance in basic science is related to a potential role in immune defense regulation, whilst in perspective CD38 may become a target for antibody therapy, considering the availability of different anti-CD38 monoclonal antibodies.

Sézary Syndrome: Different Erythroderma Morphological Features with Proposal for a Clinical Score System.

Sézary syndrome is a rare subtype of cutaneous T-cell lymphoma characterized by erythroderma, peripheral lymphadenopathies, and circulating atypical cerebriform T-cells. To date, no definite staging system has been developed for these patients. In this retrospective analysis of the archive of the Dermatological Clinic of the University of Turin, Italy, erythrodermic SS patients were classified according to clinical records and photographs into three main presentations: erythematous, infiltrated, or melanodermic. The pattern of erythroderma was found to be associated with disease outcome, as better survivals were recorded in patients with erythematous and infiltrative erythroderma. Patients in the melanodermic group, though less represented in our investigation, seemed to show a worse trend in survival. According to this preliminary evidence, a new prognostic classification, with a revised score specific for Sézary syndrome patients, can be proposed to usefully integrate the current staging system. The correlation displayed in our research will be hopefully confirmed by prospective studies with larger cohorts, with the aim of identifying significant prognostic features in this subset of cutaneous T-cell lymphoma patients.

CD157 signaling promotes survival of acute myeloid leukemia cells and modulates sensitivity to cytarabine through regulation of anti-apoptotic Mcl-1.

CD157/BST-1 (a member of the ADP-ribosyl cyclase family) is expressed at variable levels in 97% of patients with acute myeloid leukemia (AML), and is currently under investigation as a target for antibody-based immunotherapy. We used peripheral blood and bone marrow samples from patients with AML to analyse the impact of CD157-directed antibodies in AML survival and in response to cytarabine (AraC) ex vivo. The study was extended to the U937, THP1 and OCI-AML3 AML cell lines of which we engineered CD157-low versions by shRNA knockdown. CD157-targeting antibodies enhanced survival, decreased apoptosis and reduced AraC toxicity in AML blasts and cell lines. CD157 signaling activated the PI3K/AKT/mTOR and MAPK/ERK pathways and increased expression of Mcl-1 and Bcl-XL anti-apoptotic proteins, while decreasing expression of Bax pro-apoptotic protein, thus preventing Caspase-3 activation. The primary CD157-mediated anti-apoptotic mechanism was Bak sequestration by Mcl-1. Indeed, the Mcl-1-specific inhibitor S63845 restored apoptosis by disrupting the interaction of Mcl-1 with Bim and Bak and significantly increased AraC toxicity in CD157-high but not in CD157-low AML cells. This study provides a new role for CD157 in AML cell survival, and indicates a potential role of CD157 as a predictive marker of response to therapies exploiting Mcl-1 pharmacological inhibition.

CD157: From Myeloid Cell Differentiation Marker to Therapeutic Target in Acute Myeloid Leukemia.

: Human CD157/BST-1 and CD38 are dual receptor-enzymes derived by gene duplication that belong to the ADP ribosyl cyclase gene family. First identified over 30 years ago as Mo5 myeloid differentiation antigen and 10 years later as Bone Marrow Stromal Cell Antigen 1 (BST-1), CD157 proved not to be restricted to the myeloid compartment and to have a diversified functional repertoire ranging from immunity to cancer and metabolism. Despite being a NAD+-metabolizing ectoenzyme anchored to the cell surface through a glycosylphosphatidylinositol moiety, the functional significance of human CD157 as an enzyme remains unclear, while its receptor role emerged from its discovery and has been clearly delineated with the identification of its high affinity binding to fibronectin. The aim of this review is to provide an overview of the immunoregulatory functions of human CD157/BST-1 in physiological and pathological conditions. We then focus on CD157 expression in hematological tumors highlighting its emerging role in the interaction between acute myeloid leukemia and extracellular matrix proteins and its potential utility for monoclonal antibody targeted therapy in this disease.

CD157: From immunoregulatory protein to potential therapeutic target.

CD157/BST1 glycosylphosphatidylinositol-anchored glycoprotein is an evolutionary conserved dual-function receptor and ß-NAD+-metabolizing ectoenzyme of the ADP-ribosyl cyclases gene family. Identified as bone marrow stromal cell and myeloid cell differentiation antigen, CD157 turned out to have a wider expression than originally assumed. The functional significance of human CD157 as an enzyme remains unclear, while it was well established in mouse models. Conversely, the receptor role of CD157 has been clearly delineated. In physiological conditions, CD157 is a key player in regulating leukocyte adhesion, migration and diapedesis. Underlying these functional roles is the ability of CD157 to bind with high affinity selected extracellular matrix components within their heparin-binding domains. CD157 binding to extracellular matrix promotes its interaction with ß1 and ß2-integrins and induces the organization of a multimolecular complex that is instrumental to the delivery of synergistic outside-in signals leading to optimal cell adhesion and migration, both in physiological and in pathological situations. CD157 also regulates cell adhesion and migration and is a marker of adverse prognosis in epithelial ovarian cancer and pleural mesothelioma. This review focuses on human CD157 expression and functions and provides an overview on its role in human pathology and its emerging potential as target for antibody-mediated immunotherapy.

Soluble CD157 in pleural effusions: a complementary tool for the diagnosis of malignant mesothelioma.

BACKGROUND: CD157/Bst1 glycoprotein is expressed in >85% of malignant pleural mesotheliomas and is a marker of enhanced tumor aggressiveness. RESULTS: In vitro, mesothelial cells (malignant and non-malignant) released CD157 in soluble form or as an exosomal protein. In vivo, sCD157 is released and can be measured in pleural effusions by ELISA. Significantly higher levels of effusion sCD157 were detected in patients with malignant pleural mesothelioma than in patients with non-mesothelioma tumors or with non-malignant conditions. In our patient cohort, the area under the receiver-operating characteristic curve for sCD157 that discriminated malignant pleural mesothelioma from all other causes of pleural effusion was 0.685, cut-off (determined by the Youden Index) = 23.66 ng/ml (62.3% sensitivity; 73.93% specificity). Using a cut-off that yielded 95.58% specificity, measurement of sCD157 in cytology-negative effusions increased sensitivity of malignant pleural mesothelioma diagnosis from 34.42% to 49.18%. CONCLUSIONS: Evaluation of soluble CD157 in pleural effusions provides a diagnostic aid in malignant mesothelioma. METHODS: Soluble CD157 (sCD157) was detected biochemically in culture supernatants of malignant and non-malignant mesothelial cells, and in pleural effusions from various pathological conditions. An ELISA system was established to measure the concentration of sCD157 in fluids, and extended to analyze sCD157 in pleural effusions from a cohort of 295 patients.

Human canonical CD157/Bst1 is an alternatively spliced isoform masking a previously unidentified primate-specific exon included in a novel transcript.

CD157/Bst1 is a dual-function receptor and ß-NAD+-metabolizing ectoenzyme of the ADP-ribosyl cyclase family. Expressed in human peripheral blood neutrophils and monocytes, CD157 interacts with extracellular matrix components and regulates leukocyte diapedesis via integrin-mediated signalling in inflammation. CD157 also regulates cell migration and is a marker of adverse prognosis in epithelial ovarian cancer and pleural mesothelioma. One form of CD157 is known to date: the canonical sequence of 318 aa from a 9-exon transcript encoded by BST1 on human chromosome 4. Here we describe a second BST1 transcript, consisting of 10 exons, in human neutrophils. This transcript includes an unreported exon, exon 1b, located between exons 1 and 2 of BST1. Inclusion of exon 1b in frame yields CD157-002, a novel proteoform of 333 aa: exclusion of exon 1b by alternative splicing generates canonical CD157, the dominant proteoform in neutrophils and other tissues analysed here. In comparative functional analyses, both proteoforms were indistinguishable in cell surface localization, specific mAb binding, and behaviour in cell adhesion and migration. However, NAD glycohydrolase activity was detected in canonical CD157 alone. Comparative phylogenetics indicate that exon 1b is a genomic innovation acquired during primate evolution, pointing to the importance of alternative splicing for CD157 function.

Induction of Scleroderma Fibrosis in Skin-Humanized Mice by Administration of Anti-Platelet-Derived Growth Factor Receptor Agonistic Autoantibodies.

OBJECTIVE: To describe a skin-SCID mouse chimeric model of systemic sclerosis (SSc; scleroderma) fibrosis based on engraftment of ex vivo-bioengineered skin using skin cells derived either from scleroderma patients or from healthy donors. METHODS: Three-dimensional bioengineered skin containing human keratinocytes and fibroblasts isolated from skin biopsy specimens from healthy donors or SSc patients was generated ex vivo and then grafted onto the backs of SCID mice. The features of the skin grafts were analyzed by immunohistochemistry, and the functional profile of the graft fibroblasts was defined before and after treatment with IgG from healthy controls or SSc patients. Two procedures were used to investigate the involvement of platelet-derived growth factor receptor (PDGFR): 1) nilotinib, a tyrosine kinase inhibitor, was administered to mice before injection of IgG from SSc patient sera (SSc IgG) into the grafts, and 2) human anti-PDGFR monoclonal antibodies were injected into the grafts. RESULTS: Depending on the type of bioengineered skin grafted, the regenerated human skin exhibited either the typical scleroderma phenotype or the healthy human skin architecture. Treatment of animals carrying healthy donor skin grafts with SSc IgG resulted in the appearance of a bona fide scleroderma phenotype, as confirmed by increased collagen deposition and fibroblast activation markers. Results of the experiments involving administration of nilotinib or monoclonal antibodies confirmed the involvement of PDGFR. CONCLUSION: Our results provide the first in vivo demonstration of the fibrotic properties of anti-PDGFR agonistic antibodies. This bioengineered skin-humanized mouse model can be used to test in vivo the progression of the disease and to monitor response to antifibrotic drugs.

CD157 enhances malignant pleural mesothelioma aggressiveness and predicts poor clinical outcome.

Malignant mesothelioma is a deadly tumor whose diagnosis and treatment remain very challenging. There is an urgent need to advance our understanding of mesothelioma biology and to identify new molecular markers for improving management of patients. CD157 is a membrane glycoprotein linked to ovarian cancer progression and mesenchymal differentiation. The common embryonic origin of ovarian epithelial cells and mesothelial cells and the evident similarities between ovarian and mesothelial cancer prompted us to investigate the biological role and clinical significance of CD157 in malignant pleural mesothelioma (MPM). CD157 mRNA and protein were detected in four of nine MPM cell lines of diverse histotype and in 85.2% of MPM surgical tissue samples (32/37 epithelioid; 37/44 biphasic). CD157 expression correlated with clinical aggressiveness in biphasic MPM. Indeed, high CD157 was a negative prognostic factor and an independent predictor of poor survival for patients with biphasic MPM by multivariate survival analysis (HR = 2.433, 95% CI 1.120-5.284; p = 0.025). In mesothelioma cell lines, CD157 gain (in CD157-negative cells) or knockdown (in CD157-positive cells) affected cell growth, migration, invasion and tumorigenicity, most notably in biphasic MPM cell lines. In these cells, CD157 expression was associated with increased activation of the mTOR signaling pathway, resulting in decreased platinum sensitivity. Moreover, a trend towards reduced survival was observed in patients with biphasic MPM receiving postoperative platinum-based chemotherapy. These findings indicate that CD157 is implicated in multiple aspects of MPM progression and suggest that CD157 expression could be used to stratify patients into different prognostic groups or to select patients that might benefit from particular chemotherapeutic approach.

The ADP-ribosyl cyclases--the current evolutionary state of the ARCs.

The major ADP-ribosylating enzyme families are the focus of this special issue of Frontiers in Bioscience . However, there is room for another family of enzymes with the capacity to utilize nicotinamide adenine dinucleotide (NAD): the ADP-ribosyl cyclases (ARCs). These unique enzymes catalyse the cyclization of NAD to cyclic ADP ribose (cADPR), a widely distributed second messenger. However, the ARCs are versatile enzymes that can manipulate NAD, NAD phosphate (NADP) and other substrates to generate various bioactive molecules including nicotinic acid adenine dinucleotide diphosphate (NAADP) and ADP ribose (ADPR). This review will focus on the group of well-characterized invertebrate and vertebrate ARCs whose common gene structure allows us to trace their origin to the ancestor of bilaterian animals. Behind a facade of gene and protein homology lies a family with a disparate functional repertoire dictated by the animal model and the physical trait under investigation. Here we present a phylogenetic view of the ARCs to better understand the evolution of function in this family.

Binding of CD157 protein to fibronectin regulates cell adhesion and spreading.

CD157/BST-1 behaves both as an ectoenzyme and signaling receptor and is an important regulator of leukocyte trafficking and ovarian cancer progression. However, the molecular interactions underpinning the role of CD157 in these processes remain obscure. The biological functions of CD157 and its partnership with members of the integrin family prompted us to assume the existence of a direct interaction between CD157 and an unknown component of the extracellular matrix. Using solid-phase binding assays and surface plasmon resonance analysis, we demonstrated that CD157 binds fibronectin with high affinity within its heparin-binding domains 1 and 2. Furthermore, we found that CD157 binds to other extracellular matrix proteins containing heparin-binding domains. Finally, we proved that the CD157-fibronectin interaction occurs with living cells, where it elicits CD157-mediated cell responses. Indeed, knockdown of CD157 in Met-5A mesothelial cells changed their morphology and cytoskeleton organization and attenuated the activation of intracellular signaling pathways triggered by fibronectin. This led to impaired cell spreading and adhesion to selected extracellular matrix proteins. Collectively, these findings indicate a central role of CD157 in cell-extracellular matrix interactions and make CD157 an attractive therapeutic target in inflammation and cancer.

CD157 at the intersection between leukocyte trafficking and epithelial ovarian cancer invasion.

CD157 is a member of the ADP-ribosyl cyclase gene family that is involved in the metabolism of NAD. CD157 behaves both as an ectoenzyme and as a receptor. Though CD157 is anchored to the membrane by a glycosylphosphatidylinositol moiety, which makes it unsuitable to transduce signals on its own, it exploits its localization in selected membrane microdomains and its proclivity to interact with integrins to accomplish receptor functions. Initially characterized as a stromal and myeloid antigen involved in the control of leukocyte adhesion, migration and diapedesis, CD157 was subsequently found to have a far wider distribution. In particular, CD157 was found to be expressed by epithelial ovarian cancer cells where it is involved in interactions among tumor cells, extracellular matrix proteins and mesothelium. The overall picture inferred from experimental and clinical observations is that CD157 is a critical player both in leukocyte trafficking and in ovarian cancer invasion and metastasis formation. In this review, we will discuss the biological mechanisms underpinning the role of CD157 in the control of leukocyte migration and ovarian cancer dissemination.

Genome-wide expression profiling and functional characterization of SCA28 lymphoblastoid cell lines reveal impairment in cell growth and activation of apoptotic pathways.

BACKGROUND: SCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We performed a whole genome expression profiling using lymphoblastoid cell lines (LCLs) from four SCA28 patients and six unrelated healthy controls matched for sex and age. METHODS: Gene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR. RESULTS: We found 66 genes whose expression was statistically different in SCA28 LCLs, 35 of which were up-regulated and 31 down-regulated. The differentially expressed genes were clustered in five functional categories: (1) regulation of cell proliferation; (2) regulation of programmed cell death; (3) response to oxidative stress; (4) cell adhesion, and (5) chemical homeostasis. To validate these data, we performed functional experiments that proved an impaired SCA28 LCLs growth compared to controls (p < 0.005), an increased number of cells in the G0/G1 phase (p < 0.001), and an increased mortality because of apoptosis (p < 0.05). We also showed that respiratory chain activity and reactive oxygen species levels was not altered, although lipid peroxidation in SCA28 LCLs was increased in basal conditions (p < 0.05). We did not detect mitochondrial DNA large deletions. An increase of TFAM, a crucial protein for mtDNA maintenance, and of DRP1, a key regulator of mitochondrial dynamic mechanism, suggested an alteration of fission/fusion pathways. CONCLUSIONS: Whole genome expression profiling, performed on SCA28 LCLs, allowed us to identify five altered functional categories that characterize the SCA28 LCLs phenotype, the first reported in human cells to our knowledge.

CD38 and CD157: a long journey from activation markers to multifunctional molecules.

CD38 (also known as T10) was identified in the late 1970s in the course of pioneering work carried out at the Dana-Farber Cancer Center (Boston, MA) that focused on the identification of surface molecules involved in antigen recognition. CD38 was initially found on thymocytes and T lymphocytes, but today we know that the molecule is found throughout the immune system, although its expression levels vary. Because of this, CD38 was considered an "activation marker," a term still popular in routine flow cytometry. This review summarizes the findings obtained from different approaches, which led to CD38 being re-defined as a multifunctional molecule. CD38 and its homologue CD157 (BST-1), contiguous gene duplicates on human chromosome 4 (4p15), are part of a gene family encoding products that modulate the social life of cells by means of bidirectional signals. Both CD38 and CD157 play dual roles as receptors and ectoenzymes, endowed with complex activities related to signaling and cell homeostasis. The structure-function analysis presented here is intended to give clinical scientists and flow cytometrists a background knowledge of these molecules. The link between CD38/CD157 and human diseases will be explored here in the context of chronic lymphocytic leukemia, myeloma and ovarian carcinoma, although other disease associations are also known. Thus CD38 and CD157 have evolved from simple leukocyte activation markers to multifunctional molecules involved in health and disease. Future tasks will be to explore their potential as targets for in vivo therapeutic interventions and as regulators of the immune response.

Overexpression of CD157 contributes to epithelial ovarian cancer progression by promoting mesenchymal differentiation.

Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little or no prospect of cure. Despite advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, unraveling the biological mechanisms underpinning EOC progression is critical for improving patients' survival. Recently, we reported that CD157 (an ectoenzyme regulating leukocyte diapedesis) is expressed in EOC and that high expression of the molecule is negatively correlated with the disease outcome in patients. Here, we demonstrate that forced overexpression of CD157 in OVCAR-3, TOV-21G, A2780 and OV-90 ovarian cancer cell lines promotes morphological and phenotypic changes characterized by disruption of intercellular junctions, downregulation of epithelial markers and upregulation of mesenchymal ones. These changes in cell shape and phenotype bring to reduced sensitivity to anoikis, increased anchorage-independent growth, cell motility and mesothelial invasion. Conversely, knockdown of CD157 in OV-90 and OC314 cells reverts the mesenchymal phenotype and reduces the cells' migratory potential. Transcriptome profiling analysis highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing OVCAR-3 and OV-90 cells. The modulation of selected genes translates into alteration of protein expression that give cells a highly malignant phenotype. The overall picture deduced from the analysis of the modulated transcripts is that high expression of CD157 strengthens a number of biological processes favoring tumor progression (including development and cell motility), and weakens several biological processes hindering tumor progression (such as apoptosis, cell death and response to stress). Together, these findings implicate CD157 in the progression of EOC to metastatic disease and suggest that CD157 may represent a valuable therapeutic target.

The CD157-integrin partnership controls transendothelial migration and adhesion of human monocytes.

CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of ß(1) and ß(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.

Mutation analysis of the LH receptor gene in Leydig cell adenoma and hyperplasia and functional and biochemical studies of activating mutations of the LH receptor gene.

CONTEXT: Germline and somatic activating mutations in the LH receptor (LHR) gene have been reported. OBJECTIVE: Our objective was to perform mutation analysis of the LHR gene of patients with Leydig cell adenoma or hyperplasia. Functional studies were conducted to compare the D578H-LHR mutant with the wild-type (WT)-LHR and the D578G-LHR mutant, a classic cause of testotoxicosis. The three main signal transduction pathways in which LHR is involved were studied. PATIENTS: We describe eight male patients with gonadotropin-independent precocious puberty due to Leydig cell adenoma or hyperplasia. RESULTS: The D578H-LHR mutation was found in the adenoma or nodule with hyperplasia in all but two patients. D578H-LHR displayed a constitutively increased but noninducible production of cAMP, led to a very high production of inositol phosphates, and induced a slight phosphorylation of p44/42 MAPK in the absence of human chorionic gonadotropin. The D578G-LHR showed a response intermediate between WT-LHR and the D578H-LHR. Subcellular localization studies showed that the WT-LHR was almost exclusively located at the cell membrane, whereas the D578H-LHR showed signs of internalization. D578H-LHR was the only receptor to colocalize with early endosomes in the absence of human chorionic gonadotropin. CONCLUSIONS: Although several LHR mutations have been reported in testotoxicosis, the D578H-LHR mutation, which has been found only as a somatic mutation, appears up until now to be specifically responsible for Leydig cell adenomas. This is reflected by the different activation of the signal transduction pathways, when compared with the WT-LHR or D578G-LHR, which may explain the tumorigenesis in the D578H mutant.