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Adaptor protein 2 (AP2), a heterotetrameric complex comprising AP2α, AP2β2, AP2μ2 and AP2σ2 subunits, is ubiquitously expressed and involved in endocytosis and trafficking of membrane proteins, such as the calcium-sensing receptor (CaSR), a G-protein coupled receptor that signals via Gα11. Mutations of CaSR, Gα11 and AP2σ2, encoded by AP2S1, cause familial hypocalciuric hypercalcaemia types 1–3 (FHH1–3), respectively. FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with hypercalcaemia, which may be marked and symptomatic, and occasional hypophosphataemia and osteomalacia. To further characterize the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous (Ap2s1+/L15) mice were viable, and had marked hypercalcaemia, hypermagnesaemia, hypophosphataemia, and increases in alkaline phosphatase activity and fibroblast growth factor-23. Plasma 1,25-dihydroxyvitamin D was normal, and no alterations in bone mineral density or bone turnover were noted. Homozygous (Ap2s1L15/L15) mice invariably died perinatally. Co-immunoprecipitation studies showed that the AP2S1 p.Arg15Leu mutation impaired protein–protein interactions between AP2σ2 and the other AP2 subunits, and also with the CaSR. Cinacalcet, a CaSR positive allosteric modulator, decreased plasma calcium and parathyroid hormone concentrations in Ap2s1+/L15 mice, but had no effect on the diminished AP2σ2-CaSR interaction in vitro. Thus, our studies have established a mouse model that is representative for FHH3 in humans, and demonstrated that the AP2S1 p.Arg15Leu mutation causes a predominantly calcitropic phenotype, which can be ameliorated by treatment with cinacalcet.

Congenital diarrheal disorders (CDD) comprise > 50 monogenic entities featuring chronic diarrhea of early-onset, including defects in nutrient and electrolyte absorption, enterocyte polarization, enteroendocrine cell differentiation, and epithelial integrity. Diarrhea is also a predominant symptom in many immunodeficiencies, congenital disorders of glycosylation, and in some defects of the vesicular sorting and transporting machinery. We set out to identify the etiology of an intractable diarrhea in 2 consanguineous families by whole-exome sequencing, and identified two novel AP1S1 mutations, c.269T>C (p.Leu90Pro) and c.346G>A (p.Glu116Lys). AP1S1 encodes the small subunit of the adaptor protein 1 complex (AP-1), which plays roles in clathrin coat-assembly and trafficking between trans-Golgi network, endosomes and the plasma membrane. An AP1S1 knock-out (KO) of a CaCo2 intestinal cell line was generated to characterize intestinal AP1S1 deficiency as well as identified mutations by stable expression in KO background. Morphology and prototype transporter protein distribution were comparable between parental and KO cells. We observed altered localization of tight-junction proteins ZO-1 and claudin 3, decreased transepithelial electrical resistance and an increased dextran permeability of the CaCo2-AP1S1-KO monolayer. In addition, lumen formation in 3D cultures of these cells was abnormal. Re-expression of wild-type AP1S1 in CaCo2-AP1S1-KO cells reverted these abnormalities, while expression of AP1S1 containing either missense mutation did not. Our data indicate that loss of AP1S1 function causes an intestinal epithelial barrier defect, and that AP1S1 mutations can cause a non-syndromic form of congenital diarrhea, whereas 2 reported truncating AP1S1 mutations caused MEDNIK syndrome, characterized by mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia.

Epithelial-to-mesenchymal transition (EMT) is a transcriptionally governed process by which cancer cells establish a front-rear polarity axis that facilitates motility and invasion. Dynamic assembly of focal adhesions and other actin-based cytoskeletal structures on the leading edge of motile cells requires precise spatial and temporal control of protein trafficking. Yet, the way in which EMT-activating transcriptional programs interface with vesicular trafficking networks that effect cell polarity change remains unclear. Here, by utilizing multiple approaches to assess vesicular transport dynamics through endocytic recycling and retrograde trafficking pathways in lung adenocarcinoma cells at distinct positions on the EMT spectrum, we find that the EMT-activating transcription factor ZEB1 accelerates endocytosis and intracellular trafficking of plasma membrane-bound proteins. ZEB1 drives turnover of the MET receptor tyrosine kinase by hastening receptor endocytosis and transport to the lysosomal compartment for degradation. ZEB1 relieves a plus-end-directed microtubule-dependent kinesin motor protein (KIF13A) and a clathrin-associated adaptor protein complex subunit (AP1S2) from microRNA-dependent silencing, thereby accelerating cargo transport through the endocytic recycling and retrograde vesicular pathways, respectively. Depletion of KIF13A or AP1S2 mitigates ZEB1-dependent focal adhesion dynamics, front-rear axis polarization, and cancer cell motility. Thus, ZEB1-dependent transcriptional networks govern vesicular trafficking dynamics to effect cell polarity change., The way in which metastatic tumour cells control endocytic vesicular trafficking networks to establish a front-rear polarity axis that facilitates motility remains unclear. Here, the authors show that the EMT activator ZEB1 influences vesicular trafficking dynamics to execute cell polarity change.

OBJECTIVE: Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominantly inherited disorder with overlapping biochemistry profile with primary hyperparathyroidism (PHPT), making the correct diagnosis a challenge. The objective of the study was to evaluate the results of the clinical work-up of a large group of hypercalcemic individuals.DESIGN: Cross-sectional study.PATIENTS: Patients undergoing clinical work-up of hypercalcemia.MEASUREMENTS: Molecular genetic analysis of the CASR gene and exon 2 of the AP2S1 gene. Plasma levels of ionized calcium and PTH as well as calcium creatinine clearance ratio (CCCR).RESULTS: A rare CASR variant was identified in 38 of 624 index patients (6.1%). A total of 18 CASR variants identified in this study were novel. No variants were identified in exon 2 of the AP2S1 gene. The majority of the variants (N = 16) were classified as likely pathogenic. The level of plasma calcium, plasma PTH and the CCCR was not affected by the type of variant (ie nonsense vs missense) (all P-values >.05). The CCCR was found to be significantly lower for variants in the transmembrane domain compared with variants located in the extracellular domain (P < .05). Plasma levels of calcium and PTH were not associated with the location of the variant (P > .05).CONCLUSIONS: We expanded the spectrum of CASR variants in hypercalcemia with 18 novel variants, and suggest that the location of the CASR variant may affect calcium excretion as determined by the CCCR.

Background & Aims: Hepatocyte polarity is essential for the development of bile canaliculi and for safely transporting bile and waste products from the liver. Functional studies of autologous mutated proteins in the context of the polarized hepatocyte have been challenging because of the lack of appropriate cell models. The aims of this study were to obtain a patient-specific hepatocyte model that recapitulated hepatocyte polarity and to employ this model to study endogenous mutant proteins in liver diseases that involve hepatocyte polarity.Methods: Urine cell-derived pluripotent stem cells, taken from a patient with a homozygous mutation in ATP7B and a patient with a heterozygous mutation, were differentiated towards hepatocyte-like cells (hiHeps). HiHeps were also derived from a patient with MEDNIK syndrome.Results: Polarized hiHeps that formed in vivo-like bile canaliculi could be generated from embryonic and patient urine cell-derived pluripotent stem cells. HiHeps recapitulated polarized protein trafficking processes, exemplified by the Cu2+-induced redistribution of the copper transporter protein ATP7B to the bile canalicular domain. We demonstrated that, in contrast to the current dogma, the most frequent yet enigmatic Wilson disease-causing ATP7B-H1069Q mutation per se did not preclude trafficking of ATP7B to the trans-Golgi Network. Instead, it prevented its Cu2+-induced polarized redistribution to the bile canalicular domain, which could not be reversed by pharmacological folding chaperones. Finally, we demonstrate that hiHeps from a patient with MEDNIK syndrome, suffering from liver copper overload of unclear etiology, showed no defect in the Cu2+-induced redistribution of ATP7B to the bile canaliculi.Conclusions: Functional cell polarity can be achieved in patient pluripotent stem cell-derived hiHeps, enabling, for the first time, the study of the endogenous mutant proteins, patient-specific pathogenesis and drug responses for diseases where hepatocyte polarity is a key factor.Lay summary: This study demonstrates that cells that are isolated from urine can be reprogrammed in a dish towards hepatocytes that display architectural characteristics similar to those seen in the intact liver. The application of this methodology to cells from patients diagnosed with inherited copper metabolism-related liver diseases (that is, Wilson disease and MEDNIK syndrome) revealed unexpected and novel insights into patient mutation-specific disease mechanisms and drug responses. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Background Familial hypocalciuric hypercalcemia (FHH) is a heterogeneous autosomal-dominant disorder of calcium hemostasis that may be difficult to distinguish clinically from mild primary hyperparathyroidism. Loss-of-function mutations mainly involving Arg15 residue of the adaptor-related protein complex 2, sigma subunit 1 (AP2S1) cause a rarer, more recently recognized form of FHH, FFH type-3. Recently, 18F-fluorocholine positron emission tomography/computed tomography (FCH-PET/CT) showed superior sensitivity to conventional imaging in localizing parathyroid adenomas. We report a new FFH type-3 patient who underwent unnecessary parathyroidectomy in association with misleading FCH-PET/CT imaging. Case presentation A 29-year old woman was initially evaluated for parathyroid hormone (PTH)-dependent hypercalcemia in 2013. Medical history was positive only for chronic constipation and malaise with no personal or family history of hypercalcemia, kidney stones, or neck surgery. Over seven years, serum calcium level was 2.51–2.89 mmol/L with concomitant PTH level of 58.7–94.8 mmol/L. Serum phosphate levels were in the low/low normal range. Serum creatinine and magnesium levels were normal. 25-hydroxy vitamin D level was 13 nmol/L. 24-hour urine calcium level was 1.92 mmol/day but increased to 6.99 mmol/day after treatment with cholecalciferol 1000 IU daily. Bone mineral density and renal ultrasound were normal. Parathyroid ultrasound showed two hypoechoic nodules inferior to the left and right thyroid lobes; however, 99mtechnitium-sestamibi scans (2013, 2016, 2018) were negative. FCH-PET/CT (2019) showed focal uptake co-localizing with the nodule inferior to the left thyroid lobe. The patient underwent left inferior parathyroidectomy and pathology was consistent with parathyroid hyperplasia. However, postoperatively, serum calcium and PTH levels remained elevated and FCH-PET/CT and ultrasound showed persistence of the uptake/nodule. Whole exome sequencing showed Arg15Cys mutation in the AP2S1 gene characteristic of FHH type-3. Conclusions In this new case of FHH type-3, FCH-PET/CT failed to localize to the hyperplastic parathyroid glands and localized instead to apparently a lymph node. This, together with increased urinary calcium after vitamin D supplementation, led to unnecessary parathyroidectomy. Given the increasingly lower cost of genetic testing and the cost of follow up and unnecessary surgery, it may prudent to include genetic testing for FHH early on in patients with mild PTH-dependent hypercalcemia.

A 45-year-old man was referred to endocrine for the evaluation of hypercalcaemia. The calcium was elevated, vitamin D was low with a normal parathyroid hormone. Dual-energy X-ray absorptiometry scan revealed osteoporosis at the lumbar spine and femoral neck. A 24-hour urine collection revealed low urinary calcium, which was believed to be secondary to vitamin D deficiency. A diagnosis of primary hyperparathyroidism was made. The patient underwent a four-gland parathyroid exploration surgery in which three of his parathyroid glands were removed. The pathology was consistent with benign parathyroid tissue. Post surgery, the patient had persistently elevated calcium levels. He was then started on bisphosphonate and cinacalcet for osteoporosis and hypercalcaemia, respectively. Genetic analysis of familial hypocalciuric hypercalcaemia (FHH) showed a p.arg15cys mutation in the AP2S1 gene, confirming the diagnosis of FHH type 3.

Context: Familial hypocalciuric hypercalcemia (FHH) causes lifelong hypercalcemia that even persists after subtotal parathyroidectomy. Symptoms are usually mild. Past recommendations have often been for monitoring and against surgical or pharmacologic treatments. Methods: Review of publications about FHH, calcium-sensing receptors (CaSRs), and calcimimetics. Results: FHH reflects heterozygous germline mutation of CASR, GNA11, or AP2S1. These mutations inactivate the CaSRs in the parathyroid cell. Thereby, they shift the serum calcium set point to higher values and cause hypercalcemia. Calcimimetic drugs enhance the effects of calcium on the CaSRs and thereby inhibit the parathyroid cell. Calcimimetic drugs are indicated in adults with primary hyperparathyroidism without a good surgical option. Calcimimetic safety and efficacy are not established in children younger than age 18 years. Recent case reports have described treatment of FHH with calcimimetics. Success was classified as combinations of subjective improvements and decreases of serum calcium levels, but not necessarily into the normal range. Treatment was successful in 14 of 16 cases (88%). Conclusion: Deductions based on these case reports have limitations. For example, failures of therapy may not have been reported. Cost of the drug might be rate limiting. Calcimimetics can be offered to adults with FHH and those in whom the serum calcium level is >0.25 mM (1 mg/dL) beyond the upper limit of normal or with possible symptoms of hypercalcemia. Calcimimetics can now be offered to more adults with FHH., Treatment with a calcimimetic was successful in 14 of 16 reported cases of FHH. Calcimimetics can be used in more cases of FHH.

Patients with familial hyperparathyroidism and low urinary calcium excretion may have familial hypocalciuric hypercalcemia (FHH) with mutations in one of three genes: the calcium-sensing receptor (CaSR) defining FHH-type 1, the adaptor-related protein complex 2 (AP2S1) related to FHH-type 3 or the G-protein subunit alpha11 (GNA11) associated with FHH-type 2. We aimed to evaluate the presence of mutations in these genes and to identify phenotypic specificities and differences in these patients. Selected patients were recruited for genetic evaluation. After informed consent was signed, blood for DNA extraction was obtained and genetic sequencing of CaSR was done. In negative cases, we further performed sequencing of AP2S1 and GNA11. A total of 10 index cases were recruited. CaSR sequencing yielded three missense heterozygous mutations (30%): c.554G > A (p.I32V) previously characterized by our team, c.1394 G > A (p.R465Q) and a novel expected disease-causing mutation c.2479 A > C (p.S827R). We identified 2 additional patients (20%) carrying the deleterious recurrent mutation c.44G > T (p.R15L) in the AP2S1 gene. No GNA11 mutation was found. Clinically, patients with AP2S1 mutations had significant cognitive and behavioral disorders, and higher blood calcium and magnesium levels than patients with FHH1. CaSR and AP2S1 sequencing is worthwhile in patients with familial hyperparathyroidism and phenotype suggesting FHH as it can diagnose up to 50% of cases. GNA11 mutations seem much rarer. Learning disabilities in these patients, associated with higher serum calcium and magnesium levels may suggest the presence of AP2S1 rather than CaSR mutation and may guide the first step in the genetic evaluation.

Objective Familial hypocalciuric hypercalcemia (FHH) type 1 is caused by mutations in the gene encoding the calcium-sensing receptor (CASR). Recently, mutations affecting codon 15 in the gene AP2S1 have been shown to cause FHH type 3 in up to 26% of CASR-negative FHH patients. Similarly, mutations in the gene GNA11 have been shown to cause FHH type 2. We hypothesized that mutations in AP2S1 and GNA11 are causative in Danish patients with suspected FHH and that these mutations are not found in patients with primary hyperparathyroidism (PHPT), which is the main differential diagnostic disorder. Design Cross-sectional study. Methods We identified patients with unexplained hyperparathyroid hypercalcemia and a control group of verified PHPT patients through review of 421 patients tested for CASR mutations in the period 2006–2014. DNA sequencing of all amino acid coding exons including intron–exon boundaries in AP2S1 and GNA11 was performed. Results In 33 CASR-negative patients with suspected FHH, we found two (~6%) with a mutation in AP2S1 (p.Arg15Leu and p.Arg15His). Family screening confirmed the genotype–phenotype correlations. We did not identify any pathogenic mutations in GNA11. No pathogenic mutations were found in the PHPT control group. Conclusions We suggest that the best diagnostic approach to hyperparathyroid hypercalcemic patients suspected to have FHH is to screen the CASR and AP2S1 codon 15 for mutations. If the results are negative and there is still suspicion of an inherited condition (i.e. family history), then GNA11 should be examined.

Familial hypocalciuric hypercalcemia is an autosomal dominant genetic disorder characterized by hypercalcemia associated with inappropriate hypocalciuria and normal parathyroid hormone levels. Acute recurrent pancreatitis (ARP) is rare in children. Predisposing factors include hypercalcemia and mutations in the serine protease inhibitor Kazal-type 1 (SPINK1) gene. The disease carries a heavy morbidity and preventive treatment options are scant. Here, we report a child with a novel genetic/metabolic form of ARP associated with compound heterozygous SPINK1/AP2S1 (adaptor protein-2 σ1-subunit) mutations, recurrence of which was completely abrogated for 6 years by cinacalcet treatment.

Hepatitis C virus (HCV) infects 130 million people worldwide and is a leading cause of liver cirrhosis, end-stage liver disease and hepatocellular carcinoma. The interactions between viral elements and host factors play critical role on HCV invade, replication and release. Here, we identified adaptor protein complex 1 sigma 3 subunit (AP1S3) as a dependency factor for the efficient HCV infection in hepatoma cells. AP1S3 silencing in cultivated Huh7.5.1 cells significantly reduced the production of HCV progeny particles. Immunoprecipitation analysis revealed that AP1S3 interacted with the HCV E2 protein. With this interaction, AP1S3 could protect HCV E2 from ubiquitin-mediated proteasomal degradation. Using in vivo ubiquitylation assay, we identified that E6-Associated Protein (E6AP) was associated with HCV E2. In addition, treatment with synthetic peptide that contains the AP1S3-recognized motif inhibited HCV infection in Huh7.5.1 cells. Our data reveal AP1 as a novel host network that is required by viruses during infection and provides a potential target for developing broad-spectrum anti-virus strategies.

The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning “grapes”). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to σ2-adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the σ2-adaptin mutation. Genetic perturbation of σ2-adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38°. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor β (TGFβ) signaling was altered in these mutants and was restored by normalizing σ2-adaptin in neurons. Thus, our data suggest that (1) while σ2-adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during high-frequency nerve stimulation, and (2) σ2-adaptin regulates NMJ morphology by attenuating TGFβ signaling.

Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogeneous condition resembling primary hyperparathyroidism (PHPT) but not curable by surgery; FHH types 1, 2, and 3 are due to loss-of-function mutations of the CASR, GNA11, or AP2S1 genes, respectively.This study aimed to compare the phenotypes of patients with genetically proven FHH types 1 or 3 or PHPT.This was a mutation analysis in a large cohort, a cross-sectional comparison of 52 patients with FHH type 1, 22 patients with FHH type 3, 60 with PHPT, and 24 normal adults.There were no interventions.Abnormalities of the CASR, GNA11, and AP2S1 genes, blood calcium, phosphate, and PTH concentrations, urinary calcium excretion were measured.In 133 families, we detected 101 mutations in the CASR gene, 68 of which were previously unknown, and in 19 families, the three recurrent AP2S1 mutations. No mutation was detected in the GNA11 gene. Patients with FHH type 3 had higher plasma calcium concentrations than patients with FHH type 1, despite having similar PTH concentrations and urinary calcium excretion. Renal tubular calcium reabsorption levels were higher in patients with FHH type 3 than in those with FHH type 1. Plasma calcium concentration was higher whereas PTH concentration and urinary calcium excretion were lower in FHH patients than in PHPT patients. In patients with FHH or PHPT, all data groups partially overlapped.In our population, AP2S1 mutations affect calcium homeostasis more severely than CASR mutations. Due to overlap, the risk of confusion between FHH and PHPT is high.

In 2012 Acta Paediatrica published our paper, which reported a possible new form of cerebral folate deficiency, with microcephaly, growth delay, severe intellectual disability and compulsive self-harm behaviour (1). Our patient has now been included in the Telethon Undiagnosed Diseases Program, which aims at providing diagnoses for paediatric patients with an unnamed genetic disease. This article is protected by copyright. All rights reserved.

Ring finger protein 41 (RNF41) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of many proteins including ErbB3 receptors, BIRC6, and parkin. Next to this, RNF41 regulates the intracellular trafficking of certain JAK2-associated cytokine receptors by ubiquitinating and suppressing USP8, which, in turn, destabilizes the ESCRT-0 complex. To further elucidate the function of RNF41 we used different orthogonal approaches to reveal the RNF41 protein complex: affinity purification-mass spectrometry, BioID, and Virotrap. We combined these results with known data sets for RNF41 obtained with microarray MAPPIT and Y2H screens. This way, we establish a comprehensive high-resolution interactome network comprising 175 candidate protein partners. To remove potential methodological artifacts from this network, we distilled the data into a high-confidence interactome map by retaining a total of 19 protein hits identified in two or more of the orthogonal methods. AP2S1, a novel RNF41 interaction partner, was selected from this high-confidence interactome for further functional validation. We reveal a role for AP2S1 in leptin and LIF receptor signaling and show that RNF41 stabilizes and relocates AP2S1.

PubMedID: 30244301 MEDNIK syndrome is an autosomal recessive rare disease as one of the most recently described copper metabolism disorder characterized by intellectual disability, ichthyosis, hearing loss, peripheral neuropathy, enteropathy and keratodermia. Here in, we reported a case presented with ichthyosis and intellectual disability with MEDNIK syndrome that confirmed by mutation analysis in a Turkish child. She was finally diagnosed with MEDNIK syndrome by clinical findings, which were confirmed by molecular genetic testing. Sequencing of AP1S1 gene showed a homozygous insertion c.364dupG (NM_001283.4), which is predicted to cause a frameshift of the reading frame (p.D122Gfs*18). To our knowledge, this is the first case of MEDNIK syndrome from Turkey. Diagnosis of MEDNIK syndrome is still challenging and we hope that this case will contribute to further understanding. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.

Mutations of the sigma subunit of the heterotetrameric adaptor-related protein complex 2 (AP2σ) impair signalling of the calcium-sensing receptor (CaSR), and cause familial hypocalciuric hypercalcaemia type 3 (FHH3). To date, FHH3-associated AP2σ mutations have only been identified at one residue, Arg15. We hypothesized that additional rare AP2σ variants may also be associated with altered CaSR function and hypercalcaemia, and sought for these by analysing >111 995 exomes (>60 706 from ExAc and dbSNP, and 51 289 from the Geisinger Health System-Regeneron DiscovEHR dataset, which also contains clinical data). This identified 11 individuals to have 9 non-synonymous AP2σ variants (Arg3His, Arg15His (x3), Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) with 3 of the 4 individuals who had Arg15His and Met117Ile AP2σ variants having mild hypercalcaemia, thereby indicating a prevalence of FHH3-associated AP2σ mutations of ∼7.8 per 100 000 individuals. Structural modelling of the novel eight AP2σ variants (Arg3His, Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) predicted that the Arg3His, Thr112Met, Glu122Gly and Glu142Lys AP2σ variants would disrupt polar contacts within the AP2σ subunit or affect the interface between the AP2σ and AP2α subunits. Functional analyses of all eight AP2σ variants in CaSR-expressing cells demonstrated that the Thr112Met, Met117Ile and Glu142Lys variants, located in the AP2σ α4-α5 helical region that forms an interface with AP2α, impaired CaSR-mediated intracellular calcium (Cai2+) signalling, consistent with a loss of function, and this was rectified by treatment with the CaSR positive allosteric modulator cinacalcet. Thus, our studies demonstrate another potential class of FHH3-causing AP2σ mutations located at the AP2σ-AP2α interface.

Copper has previously been implicated in the regulation of immune responses, but the impact of this metal on mast cells is poorly understood. In this article, we address this issue and show that copper starvation of mast cells causes increased granule maturation, as indicated by higher proteoglycan content, stronger metachromatic staining, and altered ultrastructure in comparison with nontreated cells, whereas copper overload has the opposite effects. In contrast, copper status did not impact storage of histamine in mast cells, nor did alterations in copper levels affect the ability of mast cells to degranulate in response to IgER cross-linking. A striking finding was decreased tryptase content in mast cells with copper overload, whereas copper starvation increased tryptase content. These effects were associated with corresponding shifts in tryptase mRNA levels, suggesting that copper affects tryptase gene regulation. Mechanistically, we found that alterations in copper status affected the expression of microphthalmia-associated transcription factor, a transcription factor critical for driving tryptase expression. We also found evidence supporting the concept that the effects on microphthalmia-associated transcription factor are dependent on copper-mediated modulation of MAPK signaling. Finally, we show that, in MEDNIK syndrome, a condition associated with low copper levels and a hyperallergenic skin phenotype, including pruritis and dermatitis, the number of tryptase-positive mast cells is increased. Taken together, our findings reveal a hitherto unrecognized role for copper in the regulation of mast cell gene expression and maturation.

γ2 adaptin is homologous to γ1, but is only expressed in vertebrates while γ1 is found in all eukaryotes. We know little about γ2 functions and their relation to γ1. γ1 is an adaptin of the heterotetrameric AP-1 complexes, which sort proteins in and do form clathrin-coated transport vesicles and they also regulate maturation of early endosomes. γ1 knockout mice develop only to blastocysts and thus γ2 does not compensate γ1-deficiency in development. γ2 has not been classified as a clathrin-coated vesicle adaptor protein in proteome analyses and functions for monomeric γ2 in endosomal protein sorting have been proposed, but adaptin interaction studies suggested formation of heterotetrameric AP-1/γ2 complexes. We detected γ2 at the trans-Golgi network, on peripheral vesicles and identified γ2 clathrin-coated vesicles in mice. Ubiquitous σ1A and tissue-specific σ1B adaptins bind γ2 and γ1. σ1B knockout in mice does not effect γ1/σ1A AP-1 levels, but γ2/σ1A AP-1 levels are increased in brain and adipocytes. Also γ2 is essential in development. In zebrafish AP-1/γ2 and AP-1/γ1 fulfill different, essential functions in brain and the vascular system.

// Marianna Vitiello 1, 2 , Andrea Tuccoli 1 , Romina D’Aurizio 3 , Samanta Sarti 1, 4 , Laura Giannecchini 1 , Simone Lubrano 1, 4 , Andrea Marranci 1, 4 , Monica Evangelista 2 , Silvia Peppicelli 5 , Chiara Ippolito 6 , Ivana Barravecchia 7 , Elena Guzzolino 7 , Valentina Montagnani 8 , Michael Gowen 9 , Elisa Mercoledi 1 , Alberto Mercatanti 2 , Laura Comelli 2 , Salvatore Gurrieri 1 , Lawrence W. Wu 10 , Omotayo Ope 10 , Keith Flaherty 11 , Genevieve M. Boland 11 , Marc R. Hammond 11 , Lawrence Kwong 12 , Mario Chiariello 2, 13 , Barbara Stecca 8 , Gao Zhang 10 , Alessandra Salvetti 14 , Debora Angeloni 7 , Letizia Pitto 2 , Lido Calorini 5 , Giovanna Chiorino 15 , Marco Pellegrini 3 , Meenhard Herlyn 10 , Iman Osman 9 , Laura Poliseno 1, 2 1 Oncogenomics Unit, Core Research Laboratory, Istituto Toscano Tumori (ITT), AOUP, Pisa, Italy 2 Institute of Clinical Physiology (IFC), CNR, Pisa, Italy 3 Laboratory of Integrative Systems Medicine (LISM), Institute of Informatics and Telematics (IIT), CNR, Pisa, Italy 4 University of Siena, Italy 5 Section of Experimental Pathology and Oncology, Department of Experimental and Clinical Biomedical Sciences, University of Firenze, Italy 6 Unit of Histology, Department of Clinical and Experimental Medicine, University of Pisa, Italy 7 Scuola Superiore Sant’Anna, Pisa, Italy 8 Tumor Cell Biology Unit, Core Research Laboratory, Istituto Toscano Tumori (ITT), AOUC, Firenze, Italy 9 New York University, New York, NY, USA 10 The Wistar Institute, Philadelphia, PA, USA 11 Massachusetts General Hospital, Boston, MA, USA 12 MD Anderson Cancer Center, Houston, TX, USA 13 Signal Transduction Unit, Core Research Laboratory, Istituto Toscano Tumori (ITT), AOUS, Siena, Italy 14 Unit of Experimental Biology and Genetics, Department of Clinical and Experimental Medicine, University of Pisa, Italy 15 Fondazione Edo and Elvo Tempia, Biella, Italy Correspondence to: Laura Poliseno, email: laura.poliseno@gmail.com Keywords: melanoma, BRAFV600E, ERK pathway, miR-204 family, context-dependency Received: March 11, 2016 Accepted: February 06, 2017 Published: March 06, 2017 ABSTRACT Despite increasing amounts of experimental evidence depicting the involvement of non-coding RNAs in cancer, the study of BRAFV600E-regulated genes has thus far focused mainly on protein-coding ones. Here, we identify and study the microRNAs that BRAFV600E regulates through the ERK pathway. By performing small RNA sequencing on A375 melanoma cells and a vemurafenib-resistant clone that was taken as negative control, we discover miR-204 and miR-211 as the miRNAs most induced by vemurafenib. We also demonstrate that, although belonging to the same family, these two miRNAs have distinctive features. miR-204 is under the control of STAT3 and its expression is induced in amelanotic melanoma cells, where it acts as an effector of vemurafenib’s anti-motility activity by targeting AP1S2. Conversely, miR-211, a known transcriptional target of MITF, is induced in melanotic melanoma cells, where it targets EDEM1 and consequently impairs the degradation of TYROSINASE (TYR) through the ER-associated degradation (ERAD) pathway. In doing so, miR-211 serves as an effector of vemurafenib’s pro-pigmentation activity. We also show that such an increase in pigmentation in turn represents an adaptive response that needs to be overcome using appropriate inhibitors in order to increase the efficacy of vemurafenib. In summary, we unveil the distinct and context-dependent activities exerted by miR-204 family members in melanoma cells. Our work challenges the widely accepted “same miRNA family = same function” rule and provides a rationale for a novel treatment strategy for melanotic melanomas that is based on the combination of ERK pathway inhibitors with pigmentation inhibitors.

CONTEXT: Autosomal dominant hypocalcemia (ADH) types 1 and 2 are due to calcium-sensing receptor (CASR) and G-protein subunit-α11 (GNA11) gain-of-function mutations, respectively, whereas CASR and GNA11 loss-of-function mutations result in familial hypocalciuric hypercalcemia (FHH) types 1 and 2, respectively. Loss-of-function mutations of adaptor protein-2 sigma subunit (AP2σ 2), encoded by AP2S1, cause FHH3, and we therefore sought for gain-of-function AP2S1 mutations that may cause an additional form of ADH, which we designated ADH3. OBJECTIVE: The objective of the study was to investigate the hypothesis that gain-of-function AP2S1 mutations may cause ADH3. DESIGN: The sample size required for the detection of at least one mutation with a greater than 95% likelihood was determined by binomial probability analysis. Nineteen patients (including six familial cases) with hypocalcemia in association with low or normal serum PTH concentrations, consistent with ADH, but who did not have CASR or GNA11 mutations, were ascertained. Leukocyte DNA was used for sequence and copy number variation analysis of AP2S1. RESULTS: Binomial probability analysis, using the assumption that AP2S1 mutations would occur in hypocalcemic patients at a prevalence of 20%, which is observed in FHH patients without CASR or GNA11 mutations, indicated that the likelihood of detecting at least one AP2S1 mutation was greater than 95% and greater than 98% in sample sizes of 14 and 19 hypocalcemic patients, respectively. AP2S1 mutations and copy number variations were not detected in the 19 hypocalcemic patients. CONCLUSION: The absence of AP2S1 abnormalities in hypocalcemic patients, suggests that ADH3 may not occur or otherwise represents a rare hypocalcemic disorder.

Background Pettigrew syndrome (PGS) is a rare X‐linked mental retardation that caused by AP1S2 mutation. The pathogenesis of AP1S2 deficiency has remained elusive. The purpose of this study is to give a comprehensive overview of the phenotypic and genetic spectrum of AP1S2 mutations. Methods This study systematically analyzed clinical features and genetic information of a Chinese family with AP1S2 variation, and reviewed previously reported literatures with the same gene variation. Results We identified a new c.1‐1 G>C mutation in AP1S2 gene from a four generation family with seven affected individuals and found the elevated neuron‐specific enolase (NSE) in a patient. We summarized the clinical manifestation of 59 patients with AP1S2 mutation. We found that pathogenic point mutations affecting AP1S2 are associated with dysmorphic features and neurodevelopmental problems, which included highly variable mental retardation (MR), delayed in walking, abnormal speech, hypotonia, abnormal brain, abnormal behavior including aggressive behavior, ASD, self‐abusive, and abnormal gait. Patients with splice site mutation were more likely to lead to seizures. By contrast, patients with nonsense mutations are more susceptible to microcephaly. Conclusion Our findings suggest AP1S2 mutations contribute to a broad spectrum of neurodevelopmental disorders and are important in the etiological spectrum of PGS.

MEDNIK syndrome-acronym for mental retardation, enteropathy, deafness, neuropathy, ichthyosis, keratodermia-is caused by AP1S1 gene mutations, encoding σ1A, the small subunit of the adaptor protein 1 complex, which plays a crucial role in clathrin coat assembly and mediates trafficking between trans-Golgi network, endosomes and the plasma membrane. MEDNIK syndrome was first reported in a few French-Canadian families sharing common ancestors, presenting a complex neurocutaneous phenotype, but its pathogenesis is not completely understood. A Sephardic-Jewish patient, carrying a new AP1S1 homozygous mutation, showed severe perturbations of copper metabolism with hypocupremia, hypoceruloplasminemia and liver copper accumulation, along with intrahepatic cholestasis. Zinc acetate treatment strikingly improved clinical conditions, as well as liver copper and bile-acid overload. We evaluated copper-related metabolites and liver function retrospectively in the original French-Canadian patient series. Intracellular copper metabolism and subcellular localization and function of copper pump ATP7A were investigated in patient fibroblasts. Copper metabolism perturbation and hepatopathy were confirmed in all patients. Studies in mutant fibroblasts showed abnormal copper incorporation and retention, reduced expression of copper-dependent enzymes cytochrome-c-oxidase and Cu/Zn superoxide dismutase, and aberrant intracellular trafficking of Menkes protein ATP7A, which normalized after rescue experiments expressing wild-type AP1S1 gene. We solved the pathogenetic mechanism of MEDNIK syndrome, demonstrating that AP1S1 regulates intracellular copper machinery mediated by copper-pump proteins. This multisystem disease is characterized by a unique picture, combining clinical and biochemical signs of both Menkes and Wilson's diseases, in which liver copper overload is treatable by zinc acetate therapy, and can now be listed as a copper metabolism defect in humans. Our results may also contribute to understand the mechanism(s) of intracellular trafficking of copper pumps.

The adaptor protein-2 sigma subunit (AP2sigma;2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2sigma;2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Cao2+) homeostasis. To elucidate the role of AP2sigma;2 in Cao2+ regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2sigma;2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2sigma;2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2sigma;2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2sigma;2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2sigma;2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Adaptor protein-2 (AP2), a central component of clathrin-coated vesicles (CCVs), is pivotal in clathrin-mediated endocytosis which internalises plasma membrane constituents such as G protein-coupled receptors (GPCRs)1-3 . AP2, a heterotetramer of alpha, beta, mu and sigma subunits, links clathrin to vesicle membranes and binds to tyrosine-based and dileucine-based motifs of membrane-associated cargo proteins1,4. Here, we show that AP2 sigma subunit (AP2S1) missense mutations, which all involved the Arg15 residue (Arg15Cys, Arg15His and Arg15Leu) that forms key contacts with dileucine-based motifs of CCV cargo proteins4, result in familial hypocalciuric hypercalcemia type 3 (FHH3), an extracellular-calcium homeostasis disorder affecting parathyroids, kidneys and bone5-7 These AP2S1 mutations occurred in >20% of FHH patients without calcium-sensing GPCR (CaSR) mutations which cause FHH18-12. AP2S1 mutations decreased the sensitivity of CaSR-expressing cells to extracellular-calcium and reduced CaSR endocytosis, likely through a loss of interaction with a C-terminus CaSR dileucine-based motif whose disruption also decreased intracellular signalling. Thus, our results reveal a new role for AP2 in extracellular-calcium homeostasis.

The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1 and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5GTP-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation.

The assembly of clathrin-coated vesicles is important for numerous cellular processes, including nutrient uptake and membrane organization. Important contributors to clathrin assembly are four tetrameric assembly proteins, also called adaptor proteins (APs), each of which contains a β subunit. We identified a single β subunit, named β1/2, that contributes to both the AP1 and AP2 complexes of Dictyostelium. Disruption of the gene encoding β1/2 resulted in severe defects in growth, cytokinesis and development. Additionally, cells lacking β1/2 displayed profound osmoregulatory defects including the absence of contractile vacuoles and mislocalization of contractile vacuole markers. The phenotypes of β1/2 null cells were most similar to previously described phenotypes of clathrin and AP1 mutants, supporting a particularly important contribution of AP1 to clathrin pathways in Dictyostelium cells. The absence of β1/2 in cells led to significant reductions in the protein amounts of the medium-sized subunits of the AP1 and AP2 complexes, establishing a role for the β subunit in the stability of the medium subunits. Dictyostelium β1/2 could resemble a common ancestor of the more specialized β1 and β2 subunits of the vertebrate AP complexes. Our results support the essential contribution of a single β subunit to the stability and function of AP1 and AP2 in a simple eukaryote.

The relationship between cargo accumulation and clathrin-coated pit initiation and maturation is examined by direct visualization of receptor-engaged clathrin-coated pits., Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor–ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.

Adaptins are subunits of the heterotetrameric (beta/mu/gamma/sigma) adaptor protein (AP) complexes that are involved in clathrin-mediated membrane trafficking. Here, we show that in Schizosaccharomyces pombe the deletion strains of each individual subunit of the AP-1 complex [Apl2 (beta), Apl4 (gamma), Apm1 (mu) and Aps1 (sigma)] caused distinct phenotypes on growth sensitivity to temperature or drugs. We also show that the Deltaapm1 and Deltaapl2 mutants displayed similar but more severe phenotypes than those of Deltaaps1 or Deltaapl4 mutants. Furthermore, the Deltaapl2Deltaaps1 and Deltaapl2Deltaapl4 double mutants displayed synthetic growth defects, whereas the Deltaaps1Deltaapl4 and Deltaapl2Deltaapm1 double mutants did not. In pull-down assay, Apm1 binds Apl2 even in the absence of Aps1 and Apl4, and Apl4 binds Aps1 even in the absence of Apm1 and Apl2. Consistently, the deletion of any subunit generally caused the disassociation of the heterotetrameric complex from endosomes, although some subunits weakly localized to endosomes. In addition, the deletion of individual subunits caused similar endosomal accumulation of v-SNARE synaptobrevin Syb1. Altogether, results suggest that the four subunits are all essential for the heterotetrameric complex formation and for the AP-1 function in exit transport from endosomes.

The calcium-sensing receptor (CASR) is the main calcium sensor in the maintenance of calcium metabolism. Mutations of theCASR, the G protein alpha 11 (GNA11) and the adaptor-related protein complex 2 sigma 1 subunit (AP2S1) genes can shift the set point for calcium sensing causing hyper- or hypo-calcemic disorders. Therapeutic concepts for these rare diseases range from general therapies of hyper- and hypo-calcemic conditions to more pathophysiology oriented approaches such as parathyroid hormone (PTH) substitution and allosteric CASR modulators. Cinacalcet is a calcimimetic that enhances receptor function and has gained approval for the treatment of hyperparathyroidism. Calcilytics in turn attenuate CASR activity and are currently under investigation for the treatment of various diseases. We conducted a literature search for reports about treatment of patients harboring inactivating or activating CASR, GNA11 or AP2S1 mutants and aboutin vitroeffects of allosteric CASR modulators on mutated CASR. The therapeutic concepts for patients with familial hypocalciuric hypercalcemia (FHH), neonatal hyperparathyroidism (NHPT), neonatal severe hyperparathyroidism (NSHPT) and autosomal dominant hypocalcemia (ADH) are reviewed. FHH is usually benign, but symptomatic patients benefit from cinacalcet. In NSHPT patients pamidronate effectively lowers serum calcium, but most patients require parathyroidectomy. In some patients cinacalcet can obviate the need for surgery, particularly in heterozygous NHPT. Symptomatic ADH patients respond to vitamin D and calcium supplementation but this may increase calciuria and renal complications. PTH treatment can reduce relative hypercalciuria. None of the currently available therapies for ADH, however, prevent tissue calcifications and complications, which may become possible with calcilytics that correct the underlying pathophysiologic defect.

The sorting of membrane-bound proteins from the trans-Golgi network to lysosomal/endosomal compartments is achieved by preferential inclusion into clathrin-coated vesicles. Contained within the cytoplasmic domains of such proteins, specific sequence motifs have been identified (tyrosine-based and/or di-leucine-based) that are essential for targeting and are recognized by a family of heterotetrameric adaptor complexes, which then recruit clathrin. These cytosolic protein complexes, which have been found in a wide variety of higher eukaryotic organisms, are essential for the development of multicellular organisms. In trypanosomatids, the adaptin-mediated sorting of proteins is largely uncharacterized. In order to identify components of the adaptor-complex machinery, this study reports the cloning and characterization of sigma 1- and mu 1-adaptin gene homologues from the eukaryotic protozoan parasite, Leishmania mexicana. Generation of sigma 1- and mu 1-adaptin gene deletion mutants shows that these promastigote parasites are viable in culture, but are unable to establish infection of macrophages or mice, indicating that adaptin function is crucial for pathogenesis in these unicellular organisms.

Recognition of dileucine signals by AP-1 mediates somatodendritic sorting of the copper transporter ATP7B and the SNARE VAMP4 in hippocampal neurons, establishing AP-1 as a global regulator of polarized sorting and contributing to the understanding of neuronal copper metabolism under physiological and pathological conditions., Neurons are highly polarized cells having distinct somatodendritic and axonal domains. Here we report that polarized sorting of the Cu2+ transporter ATP7B and the vesicle-SNARE VAMP4 to the somatodendritic domain of rat hippocampal neurons is mediated by recognition of dileucine-based signals in the cytosolic domains of the proteins by the σ1 subunit of the clathrin adaptor AP-1. Under basal Cu2+ conditions, ATP7B was localized to the trans-Golgi network (TGN) and the plasma membrane of the soma and dendrites but not the axon. Mutation of a dileucine-based signal in ATP7B or overexpression of a dominant-negative σ1 mutant resulted in nonpolarized distribution of ATP7B between the somatodendritic and axonal domains. Furthermore, addition of high Cu2+ concentrations, previously shown to reduce ATP7B incorporation into AP-1–containing clathrin-coated vesicles, caused loss of TGN localization and somatodendritic polarity of ATP7B. These findings support the notion of AP-1 as an effector of polarized sorting in neurons and suggest that altered polarity of ATP7B in polarized cell types might contribute to abnormal copper metabolism in the MEDNIK syndrome, a neurocutaneous disorder caused by mutations in the σ1A subunit isoform of AP-1.

BACKGROUND: Inactivating mutations of the calcium-sensing receptor (CaSR), of the G-protein subunit α11 (GNA11) and of the adaptor-related protein complex 2, sigma 1 subunit (AP2S1) genes are responsible for familial hypocalciuric hypercalcaemia (FHH). The aim of this study was to analyse prevalence and pathogenicity of CaSR, GNA11 and AP2S1 mutations in patients with an FHH phenotype and to compare them with a sample of patients with primary hyperparathyroidism (PHPT) in order to identify the most useful laboratory parameter for a differential diagnosis. METHODS: Patients with an FHH phenotype were studied with polymerase chain reaction amplification and direct sequencing of the entire CaSR, GNA11 and AP2S1 coding sequences. Novel mutations were introduced in a Myc-tagged human wild-type (WT) CaSR cDNA-expressing vector, and functional assay was performed on human embryonic kidney cells evaluating expression and function of mutated proteins. RESULTS: Among 16 FHH patients, none had an inactivating GNA11 or AP2S1 mutation while 3 (18.8%) carried a CaSR mutation and 10 (62.5%) at least one CaSR polymorphism. Within the latter group, 7 of 10 patients had more than one polymorphism (4.1 ± 2.1 per patient). Two novel CaSR mutations [c.2120A>T (E707V) and c.2320G>A (G774S)] were identified: the E707V mutation prevented CaSR expression (western blot), whereas the G774S mutation determined a reduced receptor sensitivity to calcium (IP3 assay). PHPT patients showed significantly (P < 0.001) higher serum calcium, parathyroid hormone, urinary calcium and calcium-creatinine clearance ratio (CCCR) and significantly lower serum phosphate than FHH ones. CONCLUSIONS: FHH should be clearly differentiated by PHPT to avoid unnecessary surgery: CCCR could be a useful screening tool while genetic analysis should include the two novel CaSR mutations herein described. The role of multiple polymorphisms deserves further investigation in patients with an FHH phenotype.

MEDNIK (mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia) syndrome has been recently described as a new disorder of copper metabolism. This multisystem disease combines clinical and biochemical signs of both Menkes and Wilson's diseases, in which liver copper overload is treatable using zinc acetate therapy. MEDNIK syndrome is caused by mutation of the AP1S1 gene, which codes for the σ1A subunit of adaptor protein complex 1, and directs intracellular trafficking of copper pumps ATP7A and ATP7B. Adaptor protein complexes regulate clathrin-coated vesicle assembly, protein cargo sorting, and vesicular trafficking between organelles in eukaryotic cells. A growing number of diseases have been associated with mutations in genes coding for adaptor protein complexes subunits and we propose for them the term adaptinopathies, as a new organic category of disorders of intracellular trafficking, which offers the opportunity to dissect the mechanisms involved in the crosstalk between the Golgi apparatus and the other organelles.

Background: Except after neck surgery, hypoparathyroidism is a rare disease caused by defects in genes involved in parathyroid gland development (TBX1/22q11.2 del, GCMB, GATA3, TBCE) or function [calcium sensing receptor (CASR), GNA11, PTH], or the autoimmune polyglandular syndrome type 1 (AIRE). Approximately 90% of sporadic cases and 30% of familial cases of isolated hypoparathyroidism remain unexplained. Recurrent missense mutations in AP2S1, a calcium-sensing receptor regulator, have been recently identified in familial hyperparathyroidism. Aim: The aim of the study was to investigate AP2S1 as a putative hypoparathyroidism-causing gene. Methods: Sequencing analysis and quantitative genomic PCR of the AP2S1 gene in a large cohort of 10 index cases (from nine families) and 50 sporadic cases affected with isolated hypoparathyroidism were investigated. Results and Conclusions: None of the 60 patients presented with nucleotidic changes or copy number variation in the AP2S1 gene, thereby excluding AP2S1 def...

Rearrangements of the short arm of chromosome 12 are frequently observed in hematological disorders. Previous studies of loss of heterozygosity identified a small genetic interval on chromosome 12p12.3 that is frequently deleted in childhood acute lymphoblastic leukemia (ALL). Two genes, ETV6/TEL and p27/KIP1, are located in this interval. Evidence has accumulated that an as-yet unidentified tumor suppressor gene is closely linked to these. To facilitate the identification of candidate genes, a long-range high-resolution restriction map of the ALL locus was constructed using a contig of YAC clones. Several marker loci, including 11 STS, three newly developed YAC end-based STS, six EST, and seven genes were unambiguously positioned in the new map. The map covers 1.8Mb and extends from the distal salivary proline-rich protein gene cluster to the proximal p27/KIP1 gene. The data confirmed the order tel-D12S358-p27/KIP1-cen and excluded p27/KIP1 as a candidate tumor suppressor gene. The critical region delimited by D12S89 and D12S358 is a 750kb CpG-island rich region that includes the 240kb TEL/ETV6 gene as well as CLAPS3 (clathrin-adaptor small chain 3). The new map provides a molecular framework for the identification of novel genes and transcriptional units in the ALL interval.

Signaling through the insulin receptor tyrosine kinase involves its autophosphorylation in response to insulin and the subsequent tyrosine phosphorylation of substrate proteins such as insulin receptor substrate-1 (IRS-1). In basal 3T3-L1 adipocytes, IRS-1 is predominantly membrane-bound, and this localization may be important in targeting downstream signaling elements that mediate insulin action. Since IRS-1 localization to membranes may occur through its association with specific membrane proteins, a 3T3-F442A adipocyte cDNA expression library was screened with non-tyrosine-phosphorylated, baculovirus-expressed IRS-1 in order to identify potential IRS-1 receptors. A cDNA clone that encodes sigma3A, a small subunit of the AP-3 adaptor protein complex, was demonstrated to bind IRS-1 utilizing this cloning strategy. The specific interaction between IRS-1 and sigma3A was further verified by in vitro binding studies employing baculovirus-expressed IRS-1 and a glutathione S-transferase (GST)-sigma3A fusion protein. IRS-1 and sigma3A were found to co-fractionate in a detergent-resistant population of low density membranes isolated from basal 3T3-L1 adipocytes. Importantly, the addition of exogenous purified GST-sigma3A to low density membranes caused the release of virtually all of the IRS-1 bound to these membranes, while GST alone had no effect. These results are consistent with the hypothesis that sigma3A serves as an IRS-1 receptor that may dictate the subcellular localization and the signaling functions of IRS-1.

Calcium homeostasis is regulated by PTH, and PTH secretion from parathyroid glands is inhibited by increases in extracellular calcium ([Ca2+]o) levels that are sensed by the calcium-sensing receptor (CaSR) (1). Primary hyperparathyroidism (PHPT) is a common endocrine disorder in which increased PTH secretion occurs despite increased [Ca2+]o, related in part to reduced CaSR expression. In familial hypocalciuric hypercalcemia (FHH), the hypercalcemia is accompanied by normal or elevated serum PTH levels, as in mild PHPT (2). However, renal and skeletal manifestations of PHPT are generally absent. A family history and calcium-to-creatinine clearance ratios that are low relative to the filtered load of calcium (T transition in exon 2 of the AP2S1 gene—was identified in affected individuals of both kindreds. The nucleotide substitution predicts a p.Arg15Cys missense alteration. Nesbit et al (7) next determined the frequency of AP2S1 mutations in 50 unrelated FHH cases in which CASR mutations had been excluded. Eleven missense heterozygous alterations, all affecting Arg15 (p.Arg15Cys, p.Arg15His, or p.Arg15Leu), were identified in patients whose ages ranged from 8 days to 78 years. The authors noted that all had similar elevations in serum calcium and PTH concentrations as well as reductions in urine calcium excretion. The AP2S1 gene encodes the σ-subunit of adaptor protein complex 2 (designated as the AP2σ2 protein; see Ref. 7) critical for clathrin-mediated endocytosis of a variety of cell-surface proteins including GPCRs. AP2 is a heterotetramer of α-, β-, μ-, and σ-subunits and links clathrin to vesicle membranes by binding to tyrosine and dileucine-based motifs of membrane-associated cargo proteins. An analysis of the crystal structure of AP2 shows that the Arg15 residue of AP2σ2 and the Arg21 residue of AP2α1 are involved in forming key contacts with a glutamine residue at position –4 relative to the first leucine of the dileucine motif of cargo proteins (7). Replacing the positively charged Arg15 residue with either the polar but uncharged Cys15 residue or the nonpolar Leu15 residue is predicted to lead to a reduced affinity for the dileucine motif and, hence, compromise receptor function. The effects of the His15 substitution are more difficult to predict. AP2 is known to have an important role in GPCR endocytosis and recycling, thereby potentially influencing GPCR sensitivity. CaSR has within its cytoplasmic C-terminal domain a variant dileucine motif (RHQPLL, residues 1009–1014). Nesbit et al (7) examined the effect of removing the CaSR dileucine motif by transiently transfecting HEK293 cells with either wild-type CaSR (Leu1013Leu1014) or mutant CaSR (Ala1013Ala1014) and measuring the intracellular calcium responses to changes in [Ca2+]o. Expression of the mutant CaSR led to a rightward shift in the concentration-response curve, with a higher EC50 value compared to wild-type, supporting the notion that the CaSR dileucine motif is functionally important. Expression of transiently expressed mutant (Cys15, Leu15, His15) AP2σ2 proteins in HEK293 cells stably expressing the CaSR led to a rightward shift in the concentration-response curves relative to those expressing the wild-type Arg15 AP2σ2 protein. This indicated a decrease in the sensitivity of cells expressing the FHH3-associated AP2S1 mutants relative to wild-type AP2S1. The effect of AP2S1 Arg15 alterations on CaSR cell-surface expression was evaluated under basal (0.5 mm [Ca2+]o) and stimulated (5.0 mm [Ca2+]o) conditions in HEK293 cells stably transfected with CaSR (7). Under short-term conditions (15 min) cell-surface expression of the CaSR was decreased in cells transfected with empty vector, and the AP2S1 wild-type decreased this level further, whereas the AP2S1 mutants were without any effect greater than that of empty vector. Hence, the AP2S1 mutants lacked the wild-type AP2S1 effect on CaSR endocytosis. Although the genetic evidence linking AP2S1 mutations to FHH3 is clear-cut, elucidation of the precise mechanism whereby AP2S1 Arg15 mutations cause impaired CaSR function will require further study. The work of Nesbit et al (7) supports the notion that endocytosis of the CaSR is important for activity. In apparent discord with this idea, however, CaSR mutations have been found in ADH cases that involve loss of a significant part of the C-terminal tail (eg, S895-V1075del), including the dileucine 1013,1014 motif (8). The higher cell-surface expression of the S895-V1075del mutant CaSR relative to wild-type had been postulated to be the basis for the greater activity and leftward shift in concentration-response assays relative to wild-type CaSR. In the present issue of the JCEM, Fujisawa et al (9) report on a female infant who was found to have idiopathic infantile hypercalcemia (IIH) after referral for poor weight gain on breast feeding. At 49 days of age, her corrected serum calcium, intact PTH, and urinary calcium-to- creatinine ratio were 13.1 mg/dL, 27 pg/mL, and 1.29, respectively. There was no evidence of hyperparathyroidism, PTHrP-producing neoplasm, or vitamin D excess. Except for hypercalciuria, the data were consistent with impaired CaSR signaling. With low calcium formula (2.6 mg/dL), the infant showed catch-up growth, and serum calcium and urinary calcium-to-creatinine ratio were decreased. However, serum PTH rose. Low-calcium and standard formula at a 2:1 ratio maintained serum calcium around 12 mg/dL without markedly increasing serum PTH. No pathological mutation was detected in the CASR gene, but a heterozygous p.Arg15Leu mutation was identified in the AP2S1 gene, the previously reported causative mutation for FHH3. Fujisawa et al (9) suggest that a lack of hypocalciuria does not necessarily argue against an AP2S1 mutation. Similarly, in a study of 32 undifferentiated IIH cases, we reported two infants with maternally inherited CASR-inactivating mutations but without hypocalciuria (10). As Fujisawa et al (9) suggest, the young age of the infant is likely playing a significant role in the variable occurrence, and possibly degree, of hypercalciuria. Important in this study is the evidence for efficacy of a low-calcium formula. The authors suggest that it may be of benefit in reducing the serum calcium level and, if used judiciously, helpful in restoring normal growth and development. Of further interest, Nesbit et al (11) have identified heterozygous germline mutations in GNA11, the gene encoding the α-subunit of G11, in affected members of a kindred with FHH2, and one unrelated FHH patient. GNA11 was selected using a candidate gene approach because it is located at 19p13.3, the locus of the FHH2 trait. In addition, Nesbit et al (11) and Mannstadt et al (12) identified four different heterozygous missense mutations in GNA11 in patients with ADH in whom CASR-activating mutations had not been detected. Gα11 mediates signaling by the CaSR; hence, GNA11-inactivating mutations would reduce and activating mutations would increase the sensitivity of the parathyroid gland and renal tubule to [Ca2+]o. Autosomal dominant hypocalcemia/hypoparathyroidism due to CASR mutations and GNAS11 are now designated as ADH type1 and type 2, respectively. Finally, it is now recognized that mutations of the 25-hydroxyvitamin D 24-hydroxylase gene (CYP24A1) also manifest in early infancy with a more severe symptomatic IIH phenotype that can include marked hypercalciuria and nephrocalcinosis (13–15). In conclusion, these recent studies give geneticists and clinical endocrinologists additional options in providing for molecular diagnostics in IIH and related PTH-independent disorders of calcium homeostasis (Table 1) in which CASR mutations have not been identified. Table 1. Disorder, Chromosomal Locus, Gene Name, and Phenotype

Although AP2S1 has recently been shown to be a causative gene for familial hypocalciuric hypercalcemia type 3 (FHH3), knowledge about FHH3 remains poor.Our objective was to report AP2S1 mutation and effects of low calcium formula in a patient with hypercalcemia and hypercalciuria.This Japanese female infant was found to have hypercalcemia by a routine laboratory test for poor weight gain on breast feeding. At 49 days of age, serum calcium (adjusted by Payne's formula) was 13.1 mg/dL, intact PTH 27 pg/mL, and urinary calcium-to-creatinine ratio 1.29 mg/mg. There was no evidence for hyperparathyroidism, PTHrP-producing neoplasm, and vitamin D excess. These data, except for hypercalciuria, appeared to be consistent with defective calcium-sensing receptor-mediated signaling. With use of low calcium formula containing 2.6 mg/dL of calcium, she showed catch-up growth, and serum calcium was decreased, as was urinary calcium-to-creatinine ratio. Furthermore, feeding with a mixture of low calcium formula and standard formula with a 2:1 ratio maintained serum calcium ∼12 mg/dL without markedly increasing serum PTH.Although no pathologic mutation was detected in CASR or GNA11, a presumably de novo heterozygous mutation (p.Arg15Leu), a previously reported causative mutation for FHH3, was identified in AP2S1 of this patient.The results imply that lack of hypocalciuria does not necessarily argue against the presence of AP2S1 mutations. The early infantile age of this patient would have played a certain role in the occurrence of hypercalciuria, and low calcium formula is worth attempting in infants with FHH.

MRXS5 or Pettigrew syndrome was described 20 years ago in a four generation family including nine affected individuals presenting with facial dysmorphism, intellectual disability, Dandy–Walker malformation and inconstant choreoathetosis. Four individuals had iron deposition in the basal ganglia seen on MRI or at autopsy. The mutation causing Pettigrew has remained elusive since the initial description of the condition. We report the identification of a mutation in the X-linked AP1S2 gene in the original Pettigrew syndrome family using X-chromosome exome sequencing. We report additional phenotype details for several of the affected individuals, allowing us to further refine the phenotype corresponding to this X-linked intellectual disability syndrome. The AP1S2 c.426+1 G>T mutation segregates with the disease in the Pettigrew syndrome family and results in loss of 46 amino acids in the clathrin adaptor complex small chain domain that spans most of the AP1S2 protein sequence. The mutation reported here in AP1S2 is the first mutation that is not predicted to cause a premature termination of the coding sequence or absence of the AP1S2 protein. Although most of the families affected by a mutation in AP1S2 were initially described as having different disorders assigned to at least three different OMIM numbers (MIM 300629, 300630 and 304340), our analysis of the phenotype shows that they are all the same syndrome with recognition complicated by highly variable expressivity that is seen within as well as between families and is probably not explained by differences in mutation severity.

There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN. The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19. To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains. We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting. Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19. These observations suggest that these other subunits may play an important role in targeting. In contrast, beta- and beta'-adaptins are clearly not involved in this event. Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal. We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system. Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma-adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19. These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane.

The assembly of clathrin-coated vesicles is important for numerous cellular processes, including nutrient uptake and membrane organization. Important contributors to clathrin assembly are four tetrameric assembly proteins, also called adaptor proteins (APs), each of which contains a β subunit. We identified a single β subunit, named β1/2, that contributes to both the AP1 and AP2 complexes of Dictyostelium. Disruption of the gene encoding β1/2 resulted in severe defects in growth, cytokinesis and development. Additionally, cells lacking β1/2 displayed profound osmoregulatory defects including the absence of contractile vacuoles and mislocalization of contractile vacuole markers. The phenotypes of β1/2 null cells were most similar to previously described phenotypes of clathrin and AP1 mutants, supporting a particularly important contribution of AP1 to clathrin pathways in Dictyostelium cells. The absence of β1/2 in cells led to significant reductions in the protein amounts of the medium-sized subunits of the AP1 and AP2 complexes, establishing a role for the β subunit in the stability of the medium subunits. Dictyostelium β1/2 could resemble a common ancestor of the more specialized β1 and β2 subunits of the vertebrate AP complexes. Our results support the essential contribution of a single β subunit to the stability and function of AP1 and AP2 in a simple eukaryote.

We identified a 495 Kb interstitial deletion of chromosome Xp22.2, centered on the AP1S2 gene, by means of oligonucleotide array comparative genomic hybridisation (array-CGH) in a child with marked hypotonia in the first months of life, psychomotor retardation, severely delayed walking and speech development, and unspecific dysmorphic facial features. The deletion was inherited from the healthy mother. Point mutations of the AP1S2 gene have been identified in patients with X-linked mental retardation (XLMR). The clinical features of our patient are quite similar to those reported in male patients carrying point mutations, thus suggesting that point mutations and deletions of the AP1S2 gene lead to a recognisable XLMR phenotype in males.

Suicide completion rates are significantly higher in males than females in most societies. Although gender differences in suicide rates have been partially explained by environmental and behavioral factors, it is possible that genetic factors, through differential expression between genders, may also help explain gender moderation of suicide risk. This study investigated X-linked genes in suicide completers using a two-step strategy. We first took advantage of the genetic structure of the French-Canadian population and genotyped 722 unrelated French-Canadian male subjects, of whom 333 were suicide completers and 389 were non-suicide controls, using a panel of 37 microsatellite markers spanning the entire X chromosome. Nine haplotype windows and several individual markers were associated with suicide. Significant results aggregated primarily in two regions, one in the long arm and another in the short arm of chromosome X, limited by markers DXS8051 and DXS8102, and DXS1001 and DXS8106, respectively. The second stage of the study investigated differential brain expression of genes mapping to associated regions in Brodmann areas 8/9, 11, 44 and 46, in an independent sample of suicide completers and controls. Six genes within these regions, Rho GTPase-activating protein 6, adaptor-related protein complex 1 sigma 2 subunit, glycoprotein M6B, ribosomal protein S6 kinase 90 kDa polypeptide 3, spermidine/spermine N(1)-acetyltransferase 1 and THO complex 2, were found to be differentially expressed in suicide completers.

International audience; Communicated by Andreas Gal Mutations in the AP1S2 gene, encoding the r1B subunit of the clathrin-associated adaptor protein complex (AP)-1, have been recently identified in five X-linked mental retardation (XLMR) families, including the original family with Fried syndrome. Studying four patients in two unrelated families in which AP1S2 nonsense and splice-site mutations segregated, we found that affected individuals presented, in addition to previously described features, with elevated protein levels in cerebrospinal fluid (CSF). Moreover, computed tomography scans demonstrated that the basal ganglia calcifications associated with AP1S2 mutations appeared during childhood and might be progressive. Based on these observations, we propose that AP1S2 mutations are responsible for a clinically recognizable XLMR and autism syndrome associating hypotonia, delayed walking, speech delay, aggressive behavior, brain calcifications, and elevated CSF protein levels. Using the AP-2 complex, in which the r subunit is encoded by one single gene, as a model system, we demonstrated that r subunits are essential for the stability of human AP complexes. By contrast, no major alteration of the stability, subcellular localization, and function of the AP-1 complex was observed in fibroblasts derived from a patient carrying an AP1S2 mutation. Similarly, neither macro-nor microscopic defects were observed in the brain of an affected fetus. Altogether, these data suggest that the absence of an AP-1 defect in peripheral tissues is due to functional redundancy among AP-1 r subunits (r1A, r1B, and r1C) and that the phenotype observed in our patients results from a subtle and brain-specific defect of the AP-1-dependent intracellular protein traffic. Hum Mutat 29(7), 966-974, 2008. r r 2008 Wiley-Liss, Inc.

The adaptor protein (AP) complexes are involved in membrane transport of many proteins. There are 3 AP complexes in C. elegans unlike mammals that have four. To study the biological functions of the AP-3 complexes of C. elegans, we sought homologues of the mouse and human genes that encode subunits of the AP-3 complexes by screening C. elegans genomic and EST sequences. We identified single copies of homologues of the m3, s3, b3 and d genes. The medium chain of AP-3 is encoded by a single gene in C. elegans but two different genes in mammals. Since there are no known mutations in these genes in C. elegans, we performed RNAi to assess their functions in development. RNAi of each of the genes caused embryonic and larval lethal phenotypes. APM-3 is expressed in most cells, particularly strongly in spermatheca and vulva. We conclude that the products of the C. elegans m3, s3, b3 and d genes are essential for embryogenesis and larval development.

Mice and rats are naturally deficient in cholesteryl ester transfer protein (CETP) activity, although the reason behind the deficiency in activity is unknown. A search of mouse genome databases revealed sequences resembling 7 of the 16 human exons. However, these sequences could not code for a functional CETP. Analysis of the rat genome using Southern blotting revealed sequences complementary to human CETP cDNA, but RNase protection assays were unable to detect any Cetp gene expression in liver, adipose, or muscle. A search of rat whole-genome shotgun databases revealed exon-like sequences that would be unable to code for a functional CETP. An Ap3s1 pseudogene lay immediately upstream of the CETP-like sequences in mouse, but was nearly identical to the functional gene and unlikely to have been inserted prior to mouse-rat divergence. In contrast, a deletion leading to a nonsense codon was found in the exon 11-like sequences of both rat and mouse and not in any other species. Thus, the lack of CETP activity in both the mouse and the rat is most likely due to an evolutionary event that occurred before these species diverged and not to altered regulation of the gene or function of the gene product.