Maria de la Luz Sierra

Identification of a Family of Sorting Nexin Molecules and Characterization of Their Association with Receptors

ABSTRACT Sorting nexin 1 (SNX1) is a protein that binds to the epidermal growth factor (EGF) receptor and is proposed to play a role in directing EGF receptors to lysosomes for degradation (R. C. Kurten, D. L. Cadena, and G. N. Gill, Science 272:1008–1010, 1996). We have obtained full-length cDNAs and deduced the amino acid sequences of three novel homologous proteins, which were denoted human sorting nexins (SNX2, SNX3, and SNX4). In addition, we identified a presumed splice variant isoform of SNX1 (SNX1A). These molecules contain a conserved domain of ∼100 amino acids, which was termed the phox homology (PX) domain. Human SNX1 (522 amino acids), SNX1A (457 amino acids), SNX2 (519 amino acids), SNX3 (162 amino acids), and SNX4 (450 amino acids) are part of a larger family of hydrophilic molecules including proteins identified in Caenorhabditis elegans andSaccharomyces cerevisiae. Despite their hydrophilic nature, the sorting nexins are found partially associated with cellular membranes. They are widely expressed, although the tissue distribution of each sorting nexin mRNA varies. When expressed in COS7 cells, epitope-tagged sorting nexins SNX1, SNX1A, SNX2, and SNX4 coimmunoprecipitated with receptor tyrosine kinases for EGF, platelet-derived growth factor, and insulin. These sorting nexins also associated with the long isoform of the leptin receptor but not with the short and medium isoforms. Interestingly, endogenous COS7 transferrin receptors associated exclusively with SNX1 and SNX1A, while SNX3 was not found to associate with any of the receptors studied. Our demonstration of a large conserved family of sorting nexins that interact with a variety of receptor types suggests that these proteins may be involved in several stages of intracellular trafficking in mammalian cells.

Succinate Dehydrogenase (SDH) D Subunit (SDHD) Inactivation in a Growth-Hormone-Producing Pituitary Tumor: A New Association for SDH?

BACKGROUND Mutations in the subunits B, C, and D of succinate dehydrogenase (SDH) mitochondrial complex II have been associated with the development of paragangliomas (PGL), gastrointestinal stromal tumors, papillary thyroid and renal carcinoma (SDHB), and testicular seminoma (SDHD). AIM Our aim was to examine the possible causative link between SDHD inactivation and somatotropinoma. PATIENTS AND METHODS A 37-yr-old male presented with acromegaly and hypertension. Other family members were found with PGL. Elevated plasma and urinary levels of catecholamines led to the identification of multiple PGL in the proband in the neck, thorax, and abdomen. Adrenalectomy was performed for bilateral pheochromocytomas (PHEO). A GH-secreting macroadenoma was also found and partially removed via transsphenoidal surgery (TTS). Genetic analysis revealed a novel SDHD mutation (c.298_301delACTC), leading to a frame shift and a premature stop codon at position 133 of the protein. Loss of heterozygosity for the SDHD genetic locus was shown in the GH-secreting adenoma. Down-regulation of SDHD protein in the GH-secreting adenoma by immunoblotting and immunohistochemistry was found. A literature search identified other cases of multiple PGL and/or PHEO in association with pituitary tumors. CONCLUSION We describe the first kindred with a germline SDHD pathogenic mutation, inherited PGL, and acromegaly due to a GH-producing pituitary adenoma. SDHD loss of heterozygosity, down-regulation of protein in the GH-secreting adenoma, and decreased SDH enzymatic activity supports SDHDs involvement in the pituitary tumor formation in this patient. Older cases of multiple PGL and PHEO and pituitary tumors in the literature support a possible association between SDH defects and pituitary tumorigenesis.

Pituitary Adenoma With Paraganglioma/Pheochromocytoma (3PAs) and Succinate Dehydrogenase Defects in Humans and Mice

CONTEXT Germline mutations in genes coding succinate dehydrogenase (SDH) subunits A, B, C, and D have been identified in familial paragangliomas (PGLs)/pheochromocytomas (PHEOs) and other tumors. We described a GH-secreting pituitary adenoma (PA) caused by SDHD mutation in a patient with familial PGLs. Additional patients with PAs and SDHx defects have since been reported. DESIGN We studied 168 patients with unselected sporadic PA and with the association of PAs, PGLs, and/or pheochromocytomas, a condition we named the 3P association (3PAs) for SDHx germline mutations. We also studied the pituitary gland and hormonal profile of Sdhb(+/-) mice and their wild-type littermates at different ages. RESULTS No SDHx mutations were detected among sporadic PA, whereas three of four familial cases were positive for a mutation (75%). Most of the SDHx-deficient PAs were either prolactinomas or somatotropinomas. Pituitaries of Sdhb(+/-) mice older than 12 months had an increased number mainly of prolactin-secreting cells and several ultrastructural abnormalities such as intranuclear inclusions, altered chromatin nuclear pattern, and abnormal mitochondria. Igf-1 levels of mutant mice tended to be higher across age groups, whereas Prl and Gh levels varied according to age and sex. CONCLUSION The present study confirms the existence of a new association that we termed 3PAs. It is due mostly to germline SDHx defects, although sporadic cases of 3PAs without SDHx defects also exist. Using Sdhb(+/-) mice, we provide evidence that pituitary hyperplasia in SDHx-deficient cells may be the initial abnormality in the cascade of events leading to PA formation.

KCNJ5 mutations in the National Institutes of Health cohort of patients with primary hyperaldosteronism: an infrequent genetic cause of Conn's syndrome.

KCNJ5 mutations were recently described in primary hyperaldosteronism (PH or Conns syndrome). The frequency of these mutations in PH and the way KCNJ5 defects cause disease remain unknown. A total of 53 patients with PH have been seen at the National Institutes of Health over the last 12 years. Their peripheral and tumor DNAs (the latter from 16 that were operated) were screened for KCNJ5 mutations; functional studies on the identified defects were performed after transient transfection. Only two mutations were identified, and both in the tumor DNA only. There were no germline sequencing defects in any of the patients except for known synonymous variants of the KCNJ5 gene. One mutation was the previously described c.G451C alteration; the other was a novel one in the same codon: c.G451A; both lead to the same amino acid substitution (G151R) in the KCNJ5 protein. Functional studies confirmed previous findings that both mutations caused loss of channel selectivity and a positive shift in the reversal potential. In conclusion, the KCNJ5 protein was strongly expressed in the zona glomerulosa of normal adrenal glands but showed variable expression in the aldosterone-producing adenomas with and without mutation. The rate of KCNJ5 mutations among patients with PH and/or their tumors is substantially lower than what was previously reported. The G151R amino acid substitution appears to be the most frequent one so far detected in PH, despite additional nucleotide changes. The mutation causes loss of this potassium channels selectivity and may assist in the design of new therapies for PH.

Serum inactivation contributes to the failure of stromal‐derived factor‐1 to block HIV‐I infection in vivo

The chemokine stromal‐derived factor‐1 (SDF‐1) can block human immunodeficiency virus type 1 (HIV‐1) infection in vitro by binding to the CXC chemokine receptor, CXCR‐4, which serves as a coreceptor for T cell tropic HIV‐1. In spite of being constitutively expressed in vivo, SDF‐1 does not appear to block HIV‐1 infection and spread in vivo. We report that SDF‐1 is consistently measured in normal serum (15.4±3.0 ng/ml; mean±sd) and in serum from AIDS patients (16.6±3.7 ng/ml). However, we find that circulating SDF‐1 is modified to an inactive form. When exposed to serum, recombinant SDF‐1 is specifically and rapidly altered to yield an apparently smaller chemokine that does not bind to SDF‐1 receptor‐expressing cells, does not have chemoattractive or pre‐B cell stimulatory activity, and does not block HIV‐1 infection. Thus, serum modification and inactivation contribute to the failure of SDF‐1 to block HIV‐1 infection and spread in man. The inactivation of circulating SDF‐1 may be critical in permitting local gradients to develop and direct cell trafficking.

The transcription factor Gfi1 regulates G-CSF signaling and neutrophil development through the Ras activator RasGRP1

The transcription factor growth factor independence 1 (Gfi1) and the growth factor granulocyte colony-stimulating factor (G-CSF) are individually essential for neutrophil differentiation from myeloid progenitors. Here, we provide evidence that the functions of Gfi1 and G-CSF are linked in the regulation of granulopoiesis. We report that Gfi1 promotes the expression of Ras guanine nucleotide releasing protein 1 (RasGRP1), an exchange factor that activates Ras, and that RasGRP1 is required for G-CSF signaling through the Ras/mitogen-activated protein/extracellular signal-regulated kinase (MEK/Erk) pathway. Gfi1-null mice have reduced levels of RasGRP1 mRNA and protein in thymus, spleen, and bone marrow, and Gfi1 transduction in myeloid cells promotes RasGRP1 expression. When stimulated with G-CSF, Gfi1-null myeloid cells are selectively defective at activating Erk1/2, but not signal transducer and activator of transcription 1 (STAT1) or STAT3, and fail to differentiate into neutrophils. Expression of RasGRP1 in Gfi1-deficient cells rescues Erk1/2 activation by G-CSF and allows neutrophil maturation by G-CSF. These results uncover a previously unknown function of Gfi1 as a regulator of RasGRP1 and link Gfi1 transcriptional control to G-CSF signaling and regulation of granulopoiesis.

Two mutant alleles of the insulin receptor gene in a family with a genetic form of insulin resistance: a 10 base pair deletion in exon 1 and a mutation substituting serine for asparagine-462

Mutations in the insulin receptor gene cause several genetic syndromes associated with extreme insulin resistance. We have studied three insulin resistant siblings with acanthosis nigricans, dental abnormalities, and acral hypertrophy. The female patient also had primary amenorrhea due to hyperandrogenism. All three patients were compound heterozygotes with two mutant alleles of the insulin receptor gene. One allele had a 10-bp deletion in the region of exon 1 encoding the hydrophobic signal peptide; this leads to a frameshift and premature chain termination at codon 61. The deletion occurs at the site of a direct repeat of a hexanucleotide sequence interrupted by a tetranucleotide sequence; the deletion may have resulted from recombination between the upstream and downstream hexanucleotide repeats. In the other mutant allele, there is a missense mutation substituting serine for Asn462 — a mutation identified previously in one allele of the insulin receptor gene in a patient with type-A insulin resistance. The Ser462 mutation impaired the ability of acidic pH to dissociate insulin from the receptor. Thus, like the previously described Glu460 mutation, the Ser462 mutation may retard dissociation of insulin from the receptor in the acidic compartment of the endosome and may, as a result, accelerate the rate of receptor degradation.

Familial pituitary apoplexy as the only presentation of a novel AIP mutation

Growth hormone (GH)-producing tumors in AIP mutation-positive patients are typically more aggressive than their sporadic counterparts. They are also frequently associated with onset in childhood and adolescence and appear to be less responsive to medical therapy (Beckers et al. 2013). Amale predominance has been observed (Daly et al. 2010). We report on a unique clinical presentation of a kindred with a novel AIPmutation: only one of the three affected members had all the typical signs and symptoms of patients with an AIP mutation. On the contrary, the carrier father and brother had both partially empty sella turcica and, in fact, the latter ended being treated for anterior pituitary lobe hormonal deficiencies, including GH. This was presumably the result of pituitary apoplexy due to a rapidly growing AIP-induced adenoma. Briefly, a 12 years 2 month-old boy was seen for tall stature and accelerated growth velocity that was first noticed at the age of 4 years. At initial presentation, his height was 186.6 cm (z-score C4.72), over the calculated mid-parental height (180.5G4.5 cm), and his weight was 78.7 kg (z-score C2.59). He was also macrocephalic and he had several facial characteristics typical of gigantism. Hormonal investigation showed increased IGF1: 906 ng/ml (nv: 183–850) and GH: 18.3 ng/ml that did not suppress to oral glucose tolerance test (OGTT). The rest of pituitary hormones were in the lower normal range (Supplementary Table 1, see section on supplementary data given at the end of this article). Pituitary MRI showed the presence of a 1.3!1.2!1.1 cm pituitary adenoma with suprasellar extension (Fig. 1A). He underwent partial resection of the pituitary adenoma. The surgical pathology evaluation confirmed the presence of a GH-positive, sparsely granulated tumor with globules of keratin (fibrous bodies) and lack of normal reticulin framework (Fig. 2A, B, C, and D). Areas of expanded acini clearly distinctive from the adenoma were detected suggestive of hyperplasia (Fig. 2E and F). Both parents had normal heights and no history of acromegaly.

Differences in Adiposity in Cushing Syndrome Caused by PRKAR1A Mutations: Clues for the Role of Cyclic AMP Signaling in Obesity and Diagnostic Implications

CONTEXT The cAMP signaling pathway is implicated in bilateral adrenocortical hyperplasias. Bilateral adrenocortical hyperplasia is often associated with ACTH-independent Cushing syndrome (CS) and may be caused by mutations in genes such as PRKAR1A, which is responsible for primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A regulates cAMP-dependent protein kinase (PKA), an essential enzyme in the regulation of adiposity. Although CS is invariably associated with obesity, its different forms, including those associated with PKA defects, have not been compared. OBJECTIVE The purpose of this study was to characterize the phenotypic and molecular differences in periadrenal adipose tissue (PAT) between patients with CS with and without PRKAR1A mutations. DESIGN AND SETTING Samples from adrenalectomies of 51 patients were studied: patients with CS with (n = 13) and without (n = 32) PRKAR1A mutations and a comparison group with aldosterone-producing adenomas (APAs) (n = 6). In addition, clinical data from a larger group of patients with Cushing disease (n = 89) and hyperaldosteronism (n = 26) were used for comparison. METHODS Body mass index (BMI), abdominal computed tomography scans, and cortisol data were collected preoperatively. PAT was assayed for PKA activity, cAMP levels, and PKA subunit expression. RESULTS BMI was lower in adult patients with CS with PRKAR1A mutations. cAMP and active PKA levels in PAT were elevated in patients with CS with PRKAR1A mutations. CONCLUSIONS Increased PKA signaling in PAT was associated with lower BMI in CS. Differences in fat distribution may contribute to phenotypic differences between patients with CS with and without PRKAR1A mutations. The observed differences are in agreement with the known roles of cAMP signaling in regulating adiposity, but this is the first time that germline defects of PKA are linked to variable obesity phenotypes in humans.

Mutations in the insulin receptor gene in patients with genetic syndromes of insulin resistance.

Insulin resistance contributes importantly to the pathogenesis of noninsulin-depedendent diabetes mellitus (NIDDM) (1, 2). Furthermore, in longitudinal studies, insulin resistance has been shown to be among the best predictors of future development of NIDDM (3,4). Therefore, we have focused upon genetic causes of insulin resistance in the hope that this will help us to identify genetic factors that predispose to the development of NIDDM. Because of the central role of the insulin receptor in mediating the first step in insulin action (5), we have begun by examining the insulin receptor gene in insulin resistant patients. We have selected patients who manifest an extreme degree of insulin resistance in the hope that the severe insulin resistance would be associated with major biochemical defects, thereby simplifying the task of identifying the molecular defect. In this review, we will summarize the mutations that have been identified in the insulin receptor genes of patients with extreme insulin resistance. Interestingly, multiple different types of mutations have been identified (5). In addition to elucidating the molecular genetics of human insulin resistance, these studies have begun to give new insights into structure-function relationships of the insulin receptor protein.