Wnt/ß-catenin pathway and cell adhesion deregulation in CSDE1-related intellectual disability and autism spectrum disorders.

Among the genetic factors playing a key role in the etiology of intellectual disabilities (IDs) and autism spectrum disorders (ASDs), several encode RNA-binding proteins (RBPs). In this study, we deciphered the molecular and cellular bases of ID-ASD in a patient followed from birth to the age of 21, in whom we identified a de novo CSDE1 (Cold Shock Domain-containing E1) nonsense variation. CSDE1 encodes an RBP that regulates multiple cellular pathways by monitoring the translation and abundance of target transcripts. Analyses performed on the patient's primary fibroblasts showed that the identified CSDE1 variation leads to haploinsufficiency. We identified through RNA-seq assays the Wnt/ß-catenin signaling and cellular adhesion as two major deregulated pathways. These results were further confirmed by functional studies involving Wnt-specific luciferase and substrate adhesion assays. Additional data support a disease model involving APC Down-Regulated-1 (APCDD1) and cadherin-2 (CDH2), two components of the Wnt/ß-catenin pathway, CDH2 being also pivotal for cellular adhesion. Our study, which relies on both the deep phenotyping and long-term follow-up of a patient with CSDE1 haploinsufficiency and on ex vivo studies, sheds new light on the CSDE1-dependent deregulated pathways in ID-ASD.

Mutations in LHX3 result in a new syndrome revealed by combined pituitary hormone deficiency.

Combined pituitary hormone deficiency (CPHD) has been linked with rare abnormalities in genes encoding transcription factors necessary for pituitary development. We have isolated LHX3, a gene involved in a new syndrome, using a candidate-gene approach developed on the basis of documented pituitary abnormalities of a recessive lethal mutation in mice generated by targeted disruption of Lhx3 (ref. 2). LHX3, encoding a member of the LIM class of homeodomain proteins, consists of at least six exons located at 9q34. We identified a homozygous LHX3 defect in patients of two unrelated consanguineous families displaying a complete deficit in all but one (adrenocorticotropin) anterior pituitary hormone and a rigid cervical spine leading to limited head rotation. Two of these patients also displayed a severe pituitary hypoplasia, whereas one patient presented secondarily with an enlarged anterior pituitary. These LHX3 mutations consist of a missense mutation (Y116C) in the LIM2 domain at a phylogenetically conserved residue and an intragenic deletion predicting a severely truncated protein lacking the entire homeodomain. These data are consistent with function of LHX3 in the proper development of all anterior pituitary cell types, except corticotropes, and extrapituitary structures.

Oxidative stress induces unfolding protein response and inflammation in nasal polyposis.

BACKGROUND: Nasal polyposis, a chronic inflammatory disease affecting the upper airways, is a valuable and accessible model to investigate the mechanisms underlying chronic inflammation. The main objective of this study was to investigate a potential involvement of the unfolded protein response (UPR) in the context of oxidative stress and inflammation in nasal epithelial cells from nasal polyps (NP). METHODS: Epithelial cells from NP (n = 20) and normal mucosa (Controls, n = 15) in primary culture were analyzed by global proteomic approach and cell biology techniques for the glucose-regulated protein 78 (GRP78), the spliced X-box-binding protein 1 (sXBP-1), the glucose-regulated protein 94 (GRP94), and the calreticulin (immunoblot, mass spectrometry, immunocytochemistry). RESULTS: Proteomics analysis of human nasal epithelial cells in culture revealed the activation of the unfolded protein response in NP. Systematic cell biology and biochemical analysis of two markers (GRP78, sXBP-1) in the presence and absence of oxidative stress in NP showed a susceptibility of the unfolded protein response to oxidative stress compared to controls at least partially linked to an abnormal redox state of the protein disulfide-isomerase 4. This unfolded protein response was correlated with mitochondrial depolarization and secretion of interleukin 8 (IL-8) and leukotriene B4 (LTB4) and was prevented by mitochondrial antioxidant. CONCLUSIONS: We show the existence of UPR in nasal epithelial cells that is linked to oxidative stress leading to IL-8 and LTB4 secretions. These mechanisms may participate in chronic inflammation in nasal polyposis.

Neutrophilic dermatoses.

Neutrophilic dermatoses (ND) are a group of inflammatory skin conditions characterized by a neutrophilic infiltrate on histopathology with no evidence of infection. ND are classified based upon the localization of neutrophils within the skin and clinical features. Recent findings suggest that ND are due to two main mechanisms: i) a polyclonal hereditary activation of the innate immune system (polygenic or monogenic); or ii) a clonal somatic activation of myeloid cells such as encountered in myelodysplastic syndrome or VEXAS syndrome. ND belong to internal medicine as a great number of patients with ND suffer from an underlying condition (such as hematological malignancy, inflammatory bowel disease, auto-immune and auto-inflammatory diseases). ND are diagnoses of exclusion and physicians should always consider differential diagnoses, particularly skin infections. Here, we review the pathophysiology and classification of the main ND (i.e., subcorneal pustular dermatosis (Sneddon-Wilkinson Disease) and Intercellular IgA dermatoses, aseptic pustulosis of the folds, Sweet syndrome, neutrophilic eccrine hidradenitis, pyoderma gangrenosum, erythema elevatum diutinum, neutrophilic urticarial dermatosis and neutrophilic panniculitis), their clinical and histopathological features, and we highlight the investigations that are useful to identify ND-associated diseases and to exclude the differential diagnoses.

Mutations in the autoinflammatory cryopyrin-associated periodic syndrome gene: epidemiological study and lessons from eight years of genetic analysis in France.

BACKGROUND: Cryopyrin-associated periodic syndromes (CAPS) consist of a continuum of autoinflammatory diseases caused by a defect in interleukin 1ß regulation. Although symptoms may vary widely, the discovery, in 2001, of the gene involved (NLRP3) has dramatically helped diagnosis. OBJECTIVES: To define the spectrum and prevalence of NLRP3 mutations in France and to delineate initial criteria before molecular analysis. METHODS: Retrospective review (2001-9) of genetic analysis data and request forms of patients living in France with an NLRP3 mutation since the set up of CAPS molecular diagnosis by the three French laboratories providing this test (GenMAI network). RESULTS: Over 800 analyses of this gene have been conducted, identifying 135 cases with an NLRP3 mutation (55 probands; 33 multiplex families); the estimated prevalence in France was equal to 1/360 000. A total of 21 different sequence variants were detected, among which four are common and nine are new mutations. CONCLUSIONS: Although the number of NLRP3 test requests has doubled over the past 5 years, genetic screening has not contributed to enhanced detection of new index cases each year. There are two possible reasons for this: (i) no clinical prerequisite for genetic diagnosis and (ii) few new large families are now identified (unlike the initial study based on a selection by linkage). A set of initial clinical criteria have been drawn up which it is recommended should be fulfilled before a patient is tested: at least three recurrent bouts, age at disease onset < 20 years and elevated levels of C-reactive protein, especially in individuals with urticaria and moderate fever.

Mutations in NALP12 cause hereditary periodic fever syndromes.

NALP proteins, also known as NLRPs, belong to the CATERPILLER protein family involved, like Toll-like receptors, in the recognition of microbial molecules and the subsequent activation of inflammatory and immune responses. Current advances in the function of NALPs support the recently proposed model of a disease continuum bridging autoimmune and autoinflammatory disorders. Among these diseases, hereditary periodic fevers (HPFs) are Mendelian disorders associated with sequence variations in very few genes; these variations are mostly missense mutations whose deleterious effect, which is particularly difficult to assess, is often questionable. The growing number of identified sporadic cases of periodic fever syndrome, together with the lack of discriminatory clinical criteria, has greatly hampered the identification of new disease-causing genes, a step that is, however, essential for appropriate management of these disorders. Using a candidate gene approach, we identified nonambiguous mutations in NALP12 (i.e., nonsense and splice site) in two families with periodic fever syndromes. As shown by means of functional studies, these two NALP12 mutations have a deleterious effect on NF-kappaB signaling. Overall, these data identify a group of HPFs defined by molecular defects in NALP12, opening up new ways to manage these disorders. The identification of these first NALP12 mutations in patients with autoinflammatory disorder also clearly demonstrates the crucial role of NALP12 in inflammatory signaling pathways, thereby assigning a precise function to this particular member of an emerging family of proteins whose putative biological properties are currently inferred essentially through in vitro means.

A 20-year experience of electron microscopy in the diagnosis of primary ciliary dyskinesia.

Transmission electron microscopy (TEM) analysis of ciliary ultrastructure is classically used for the diagnosis of primary ciliary dyskinesia (PCD). We report our extensive experience of TEM analysis in a large series of patients in order to evaluate its feasibility and results. TEM analysis performed in 1,149 patients with suspected PCD was retrospectively reviewed. Biopsies (1,450) were obtained from nasal (44%) or bronchial (56%) mucosa in children (66.5%) and adults (33.5%). TEM analysis was feasible in 71.4% of patients and showed a main defect suggestive of PCD in 29.9%. TEM was more feasible in adults than in children, regardless of the biopsy site. Main defects suggestive of PCD were found in 76.9% of patients with sinopulmonary symptoms and in only 0.4% of patients with isolated upper and 0.4% with isolated lower respiratory tract infections. The defect pattern was similar in children and adults, involving dynein arms (81.2%) or central complex (CC) (18.8%). Situs inversus was never observed in PCD patients with CC defect. Kartagener syndrome with normal ciliary ultrastructure was not an exceptional condition (10.2% of PCD). In conclusion, TEM analysis is feasible in most patients and is particularly useful for PCD diagnosis in cases of sinopulmonary syndrome of unknown origin.

Muco-ciliary differentiation of nasal epithelial cells is decreased after wound healing in vitro.

BACKGROUND: Epithelial damage and modifications of cell differentiation are frequent in airway diseases with chronic inflammation, in which transforming growth factor-beta1 (TGF-beta1) plays an important role. The aim of this study was to evaluate the differentiation of human nasal epithelial cells (HNEC) after wound healing and the potential effects of TGF-beta1. METHODS: Basal, mucus, and ciliated cells were characterized by cytokeratin-14, MUC5AC, and betaIV tubulin immunodetection, respectively. Their expression was evaluated in situ in nasal polyps and in an in vitro model of wound healing in primary cultures of HNEC after wound closure, under basal conditions and after TGF-beta1 supplementation. Using RT-PCR, the effects of TGF-beta1 on MUC5AC and DNAI1 genes, specifically transcribed in mucus and ciliated cells, were evaluated. RESULTS: In situ, high TGF-beta1 expression was associated with low MUC5AC and betaIV tubulin expression. In vitro, under basal conditions, MUC5AC expression remained stable, cytokeratin-14 expression was strong and decreased with time, while betaIV tubulin expression increased. Transforming growth factor-beta1 supplementation downregulated MUC5AC and betaIV tubulin expression as well as MUC5AC and DNAI1 transcripts. CONCLUSION: After a wound, differentiation into mucus and ciliated cells was possible and partially inhibited in vitro by TGF-beta1, a cytokine that may be involved in epithelial remodeling observed in chronic airway diseases.

Exclusion of linkage between the collagenase gene and generalized recessive dystrophic epidermolysis bullosa phenotype.

Generalized recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited autosomal disease characterized by dermolytic blister formation. Enhanced collagenase and/or abnormal collagenase have been reported in skin from affected patients, suggesting that collagenase could be responsible for the absence of anchoring fibrils in this disorder. We used a genetic linkage approach to test the hypothesis that this disease is due to a defect in the collagenase gene in nine affected families. Analysis of amplified genomic DNA fragments of the collagenase gene by means of denaturing gradient gel electrophoresis (DGGE) allowed us to detect intragenic polymorphisms, which were subsequently characterized by direct genomic sequencing. Segregation analysis of these polymorphic sites showed exclusion of linkage between the collagenase gene and generalized RDEB phenotype in a family with consanguineous parents and three affected children. However, the possibility of linkage with the collagenase gene in the other eight families tested could not be excluded. The genetic markers described here provide a tool for investigating genetic linkage in other affected families. Overall, our results show that generalized RDEB can be caused by a defect in a gene other than the collagenase gene, and support the hypothesis that the genetic defect lies in abnormal anchoring fibril formation.

Recurrent nonsense mutations in the growth hormone receptor from patients with Laron dwarfism.

In addition to its classical effects on growth, growth hormone (GH) has been shown to have a number of other actions, all of which are initiated by an interaction with specific high affinity receptors present in a variety of tissues. Purification of a rabbit liver protein via its ability to bind GH has allowed the isolation of a cDNA encoding a putative human growth hormone receptor that belongs to a new class of transmembrane receptors. We have previously shown that this putative growth hormone receptor gene is genetically linked to Laron dwarfism, a rare autosomal recessive syndrome caused by target resistance to GH. Nevertheless, the inability to express the corresponding full-length coding sequence and the lack of a test for growth-promoting function have hampered a direct confirmation of its role in growth. We have now identified three nonsense mutations within this growth hormone receptor gene, lying at positions corresponding to the amino terminal extremity and causing a truncation of the molecule, thereby deleting a large portion of both the GH binding domain and the full transmembrane and intracellular domains. Three independent patients with Laron dwarfism born of consanguineous parents were homozygous for these defects. Two defects were identical and consisted of a CG to TG transition. Not only do these results confirm the growth-promoting activity of this receptor but they also suggest that CpG doublets may represent hot spots for mutations in the growth hormone receptor gene that are responsible for hereditary dwarfism.

Genetic linkage of recessive dystrophic epidermolysis bullosa to the type VII collagen gene.

Generalized mutilating recessive dystrophic epidermolysis bullosa (RDEB) is characterized by extreme skin fragility owing to loss of dermal-epidermal adherence. Immunohistochemical studies have implicated type VII collagen, the major component of anchoring fibrils, in the etiology of RDEB. In this study, we demonstrate genetic linkage of the type VII collagen gene and the generalized mutilating RDEB phenotype. We first identified a Pvull polymorphic site by digestion of an amplified product of the type VII collagen gene, which was shown to reside within the coding region. Genetic linkage analysis between this marker and the RDEB phenotype in 19 affected families which were informative for this polymorphism showed no recombination events, and gave a maximum lod score of 3.97 at a recombination fraction (theta) of 0, demonstrating that this DNA region is involved in this form of RDEB. These data provide strong evidence that the type VII collagen gene, which has also been linked with the dominant form of the disease, harbors the mutation(s) causing the generalized mutilating form of RDEB in these families, thus underscoring the major functional importance of type VII collagen in basement membrane zone stability.

RPGR is mutated in patients with a complex X linked phenotype combining primary ciliary dyskinesia and retinitis pigmentosa.

INTRODUCTION: Primary ciliary dyskinesia (PCD) is a rare disease classically transmitted as an autosomal recessive trait and characterised by recurrent airway infections due to abnormal ciliary structure and function. To date, only two autosomal genes, DNAI1 and DNAH5 encoding axonemal dynein chains, have been shown to cause PCD with defective outer dynein arms. Here, we investigated one non-consanguineous family in which a woman with retinitis pigmentosa (RP) gave birth to two boys with a complex phenotype combining PCD, discovered in early childhood and characterised by partial dynein arm defects, and RP that occurred secondarily. The family history prompted us to search for an X linked gene that could account for both conditions. RESULTS: We found perfect segregation of the disease phenotype with RP3 associated markers (Xp21.1). Analysis of the retinitis pigmentosa GTPase regulator gene (RPGR) located at this locus revealed a mutation (631_IVS6+9del) in the two boys and their mother. As shown by study of RPGR transcripts expressed in nasal epithelial cells, this intragenic deletion, which leads to activation of a cryptic donor splice site, predicts a severely truncated protein. CONCLUSION: These data provide the first clear demonstration of X linked transmission of PCD. This unusual mode of inheritance of PCD in patients with particular phenotypic features (that is, partial dynein arm defects and association with RP), which should modify the current management of families affected by PCD or RP, unveils the importance of RPGR in the proper development of both respiratory ciliary structures and connecting cilia of photoreceptors.

The interaction of Sendai virus with negatively charged liposomes: virus-induced lysis of carboxyfluorescein-loaded small unilamellar vesicles.

The interaction of Sendai virus with small, unilamellar vesicles, lacking virus receptors and loaded with self-quenched 6-carboxyfluorescein, was studied. Sendai virions induced release of carboxyfluorescein from vesicles composed of negative charged phospholipids, despite the fact that they did not contain virus receptors. Preliminary experiments indicate that the carboxyfluorescein release is accompanied by mixing of the virus and liposome lipids and their entrapped contents, suggesting liposome-virus fusion. No release of carboxyfluorescein was observed with vesicles containing only phosphatidylcholine. The rate of virus-induced carboxyfluorescein release was temperature dependent; the lytic activity of the virus was greatly enhanced above 25 degrees C. This effect was not due to a thermal phase transition of the lipids in either the lipid vesicles or the virions. Virus-induced carboxyfluorescein release was inhibited by the presence of calcium ions in the medium and of cholesterol in the lipid vesicles. It increased with increasing concentrations of either the lipid vesicles or the virions. pretreatment of virions with increasing concentrations of three different proteolytic enzymes (trypsin, chymotrypsin and proteinase) inhibited the virus' ability to cause release of carboxyfluorescein from negatively charged liposomes. Inhibition of the viral lytic activity was also observed after virions were incubated above 56 degrees C.

Fusion of Sendai virus with negatively charged liposomes as studied by pyrene-labelled phospholipid liposomes.

Sendai virus particles fuse with negatively charged liposomes but not with vesicles made of zwitterionic phospholipids. The liposome-virus fusion process was studied by dilution of the concentration-dependent excimer-forming fluorophore 2-pyrenyldodecanoylphosphatidylcholine contained in the liposomes by the viral lipids. The data were analyzed in the framework of a mass action kinetic model. This provided analytical solutions for the final levels of probe dilution and numerical solutions for the kinetics of the overall fusion process, in terms of rate constants for the liposome-virus adhesion, deadhesion and fusion. This analysis led to the following conclusions: At neutral pH and 37 degrees C, only 15% of the virus particles can fuse with the phospholipid vesicles, although all the virions may aggregate with the liposomes. The rate constants for aggregation, fusion and deadhesion are of the orders of magnitude of 10(7) M-1 X s-1, 10(-3) s-1 and 10(-2), s-1, respectively. The fraction of active virus increases with temperature. At acidic pH, both the fraction of 'fusable' virus and the rate of fusion increase markedly. The optimal pH for fusion is 3-4, where most of the virus particles are active. At higher pH values, an increasing fraction of the virus particles become inactive, probably due to ionization of viral glycoproteins, whereas at pH values below 3.0 the fusion is markedly reduced, most likely due to protonation of the negatively charged vesicles. While only 15% of the virions fuse with the liposomes at pH 7.4 and 37 degrees C, all the liposomes lose their content (Amselem, S., Loyter, A. Lichtenberg, D. and Barenholz, Y. (1985) Biochim. Biophys. Acta 820, 1-10). We therefore propose that release of entrapped solutes is due to liposome-virus aggregation, and not to fusion. Both trypsinization and heat inactivation of the virus particles inhibit not only the fusion process but also the release of carboxyfluorescein. This demonstrates the obligatory role of viral membrane proteins in liposome-virus aggregation. Reconstituted vesicles made of the viral lipid and the hemagglutinin/neuraminidase (HN) glycoprotein fuse with negatively charged liposomes similar to the intact virions. This suggests that the fusion of virions with negatively charged vesicles, unlike the fusion of the virus with biological membranes, requires only the HN and not the fusion glycoprotein.

Molecular basis of Laron dwarfism.

An autosomal recessive disorder, Laron-type dwarfism, results from peripheral unresponsiveness to growth hormone. Mutations in the growth hormone receptor have recently been identified in this syndrome. Analysis of patients with Laron-type dwarfism should provide insight into the mechanisms of hormone receptor binding and signal transduction pathways of this receptor, which belongs to a new class of transmembrane receptors.

Frequent detection of minimal residual disease by use of the polymerase chain reaction in long-term survivors after bone marrow transplantation for chronic myeloid leukemia.

The polymerase chain reaction (PCR) allows the detection of minimal amounts of nucleic sequences and has been successfully used to test for the chronic myeloid leukemia-specific bcr/abl transcripts. We studied blood samples from 17 patients who had undergone allogeneic bone marrow transplantation for CML, using a modified polymerase chain reaction-based assay for the detection of leukemic mRNA. This nested PCR technique was found to be highly sensitive, detecting the chimeric bcr/abl transcript in 16 of 17 patients including several long-term survivors. Cytogenetic techniques failed to detect Ph mitoses. The clinical significance of the persisting bcr/abl transcript for long periods following BMT is poorly understood and remains to be elucidated by further studies.

A naturally occurring growth hormone receptor mutation: in vivo and in vitro evidence for the functional importance of the WS motif common to all members of the cytokine receptor superfamily.

To obtain an animal model for studying the role of the GH receptor (GHR) in growth and development, we analyzed a sex-linked dwarf (SLD) chicken strain (Leghorn) which exhibits phenotype similarities with a human genetic growth disorder, an autosomal recessive GH resistance condition (Laron dwarfism). Having previously demonstrated the responsibility of the human GHR gene in the Laron phenotype, we focused our analysis on the corresponding gene in SLD chickens. Sequencing of the whole coding region of the chicken GHR cDNA identified a G-to-T transversion segregating with the SLD phenotype and generating an isoleucine instead of a serine at position 199 within a highly conserved region close to the junction between the extracellular and transmembrane domains. This defect involves the last invariant amino acid of the WS-like motif (amino acid sequence WSXWS) common to all members of the cytokine receptor superfamily. Transfection of a mutated GHR cDNA containing this mutation into eukaryotic cells led to the synthesis of a receptor protein that displayed impaired plasma membrane expression and binding activity. These data define the molecular basis for the SLD phenotype and identify this strain as an interesting model for studying Laron dwarfism in humans; this animal model may also represent a system in which therapeutic strategies to promote growth can be evaluated. Finally, the nature of the molecular defect identified provides direct evidence for the functional importance of the WS motif in GHRs and related receptors.

NF1 gene analysis focused on CpG-rich exons in a cohort of 93 patients with neurofibromatosis type 1.

We studied the NF1 gene in 93 unrelated patients with neurofibromatosis type1, focusing the analysis on four exons that contain the highest number of possible mutations occurring at CpG sites. We used denaturing gradient gel electrophoresis to analyse exons 16, 28, 29 and 49, which contain 45 (25%) of the 183 possible mutations that could occur at the 120 CpG dinucleotides of the coding sequence. Six different mutations were identified, five of which are novel: two truncating mutations, W1810X and 5448insG, located in exon29; two splice defects leading to exon29 skipping, 5206-2A>G and 5546G>A; and one missense mutation, L844F, located in exon16. The already described R1748X mutation located in exon29 was found in two unrelated patients. The 5546G>A and R1748X mutations are located at CpG sites, whereas the W1810X involves a CpNpG site. Four novel polymorphisms, which may be helpful for family studies, were also identified. Overall, all but one mutations were found in exon29, a result which suggests that all the CpG sites of the NF1 coding sequence do not have the same mutability, and that exon29, the most CpG-rich exon, contains mutational hotspots associated with NF1.

The human growth hormone (GH) receptor and its truncated isoform: sulfhydryl group inactivation in the study of receptor internalization and GH-binding protein generation.

The human GH receptor (hGHR) contains nine intracellular and seven extracellular cysteines, of which six are linked by disulfide bonds and one, at position 241 proximal to the membrane, is free. Recently, an alternatively spliced GHR isoform has been isolated; it encodes a truncated receptor lacking most of the cytoplasmic domain (hGHRtr). In the present study, we have examined the effect of sulfhydryl group(s) inactivation on receptor internalization and GH binding-protein (GHBP) generation from the human (h) and rabbit (rb) full-length GHR, as well as from hGHRtr and a mutant of the free extracellular cysteine (hGHRtr-C241A), expressed in Chinese hamster ovary (CHO) cells. In CHO/rbGHR and CHO/hGHR cells, permeable sulfhydryl-reactive agents, like N-ethylmaleimide (NEM) and iodacetamide (IA), inhibited GHR internalization and induced an immediate dose-dependent loss of cellular GHR, associated with a concomitant marked increase in released GHBP. In contrast, the membrane impermeable IA derivative A-484 had no effect on either GHBP release or on GHR internalization. NEM exposure of CHO cells, expressing hGHRtr, resulted in a dose-dependent increase in GHBP generation, but only a moderate decrease in cellular hGHRtr. The importance of the only unpaired cysteine in these processes was evaluated in CHO/hGHRtr-C241A cells. hGHRtr-C241A was similar to hGHRtr in its impaired internalization and enhanced GHBP release by NEM. Taken together, these data suggest that intracellular sulfhydryl groups, within membranal endocytic vesicles, that do not belong to the GHR molecule, are involved in receptor internalization and GHBP generation. In addition, the present study demonstrates that despite impaired hGHR internalization/down-regulation, the inducible release of GHBP was not affected, further suggesting that GHR endocytosis is not a prerequisite for GHBP generation.