Loss Of Receptor Function Research Articles

RNA Studies Demonstrate that the LDLR c.2389+4A>G Intronic Variant Causes Familial Hypercholesterolemia through an RNA Splicing Impact

Lead Author's Financial Disclosures H.Z. is an employee of Ambry Genetics. Study Funding None. Background/Synopsis Familial hypercholesterolemia (FH) is an inherited disorder characterized by high plasma levels of low-density lipoprotein cholesterol (LDL-C). Mutations in the low-density lipoprotein receptor (LDLR) gene are the most common cause of FH. However, not all alterations in LDLR are pathogenic; some represent benign genetic variation, and the clinical significance of any given variant is not always clear. Variants of uncertain significance (VUS) require additional studies to elucidate their pathogenicity. Objective/Purpose We sought to further characterize an intronic LDLR alteration originally classified as a VUS in a large family with clinical FH. Methods We performed multigene panel testing for four genes associated with FH (APOB, LDLR, LDLRAP1, and PCSK9) on the proband of a large family with a strong family history of high cholesterol and premature death; additional affected and unaffected family members were genotyped by Sanger sequencing for an identified intronic LDLR variant. The splicing impact of the LDLR variant was ascertained by RT-PCRseq performed on three affected individuals. Results Panel testing detected LDLR c.2389+4A>G in the proband. No other pathogenic variants, likely pathogenic variants, or VUSs were identified. Subsequent familial testing indicated that LDLR c.2389+4A>G cosegregates with disease. RNA studies demonstrated that this alteration causes skipping of exon 16, which is predicted to result in the in-frame deletion of 26 amino acids. This deletion removes part of the transmembrane domain and is expected to disrupt the ability of LDLR to insert into the membrane, resulting in loss of receptor function. Other pathogenic alterations impacting the same donor splice site have been shown to have a similar impact on splicing (LDLR c.2389G>T and LDLR c.2389+1G>T; Bourbon M et al. J. Med. Genet., 2009 May;46:352-7; Holla ØL et al. Mol. Genet. Metab., 2009 Apr;96:245-52). Conclusions The LDLR c.2389+4A>G intronic variant is a pathogenic mutation that results in a splice defect and cosegregates with disease. In this case, RNA and family studies resulted in a reclassification from a VUS to a pathogenic mutation and confirmed the molecular cause of the multigenerational FH. Clinical management recommendations exist for individuals with a confirmed FH diagnosis, and an accurate genetic diagnosis can improve patient outcomes through tailored treatments (Brown E et al. J. Clin. Lipidol.,2020 Jul-Aug;14:398-413). Our findings highlight the utility of familial testing and RNA studies in clarifying the classification of DNA variants. H.Z. is an employee of Ambry Genetics.

Mutation-Induced Functional Alterations of CCR6

The Cys-Cys chemokine receptor 6 (CCR6) is a well-established modulator of inflammation. Although several genetic associations have been identified between CCR6 polymorphisms and immune system disorders (e.g., rheumatoid arthritis and Crohn's disease), the pharmacological effects of naturally occurring missense mutations in this receptor have yet to be characterized. In this study, we initially assessed G protein-mediated signaling and observed that wild-type (WT) CCR6 exhibited ligand-independent activity. In addition, we found that the five most frequent CCR6 missense variants (A89T, A150V, R155W, G345S, and A369V) exhibited decreased basal and/or ligand induced Gαi protein signaling. To complement the study of these loss-of-function variants, we engineered a set of constitutively active CCR6 receptors. Selected mutations enhanced basal G protein-mediated signaling up to 3-fold relative to the WT value. Using a bioluminescence resonance energy transfer assay we investigated the ability of each naturally occurring and engineered CCR6 receptor mutant to recruit β-arrestin. In contrast to G protein-mediated signaling, β-arrestin mobilization was largely unperturbed by the naturally occurring loss-of-function CCR6 variants. Elevated recruitment of β-arrestin was observed in one of the engineered constitutively active mutants (T98P). Our results demonstrate that point mutations in CCR6 can result in either a gain or loss of receptor function. These observations underscore the need to explore how CCR6 natural variants may influence immune cell physiology and human disease.

Lack of IL7Rα expression in T cells is a hallmark of T-cell immunodeficiency in Schimke immuno-osseous dysplasia (SIOD)

Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, fatal childhood disorder associated with skeletal dysplasia, renal dysfunction, and T-cell immunodeficiency. This disease is linked to biallelic loss-of-function mutations of the SMARCAL1 gene. Although recurrent infection, due to T-cell deficiency, is a leading cause of morbidity and mortality, the etiology of the T-cell immunodeficiency is unclear. Here, we demonstrate that the T cells of SIOD patients have undetectable levels of protein and mRNA for the IL-7 receptor alpha chain (IL7Rα) and are unresponsive to stimulation with IL-7, indicating a loss of functional receptor. No pathogenic mutations were detected in the exons of IL7R in these patients; however, CpG sites in the IL7R promoter were hypermethylated in SIOD T cells. We propose therefore that the lack of IL7Rα expression, associated with hypermethylation of the IL7R promoter, in T cells and possibly their earlier progenitors, restricts T-cell development in SIOD patients.

Dysfunctional Crohn's Disease-Associated NOD2 Polymorphisms Cannot be Reliably Predicted on the Basis of RIPK2 Binding or Membrane Association.

Polymorphisms in NOD2 represent the single greatest genetic risk factor for the development of Crohn’s disease. Three different non-synonomous NOD2 polymorphisms – R702W, G908R, and L1007fsincC – account for roughly 80% of all NOD2-associated cases of Crohn’s disease and are reported to result in a loss of receptor function in response to muramyl dipeptide (MDP) stimulation. Loss of NOD2 signaling can result from a failure to detect ligand; alterations in cellular localization; and changes in protein interactions, such as an inability to interact with the downstream adaptor protein RIPK2. Using an overexpression system, we analyzed ~50 NOD2 polymorphisms reportedly connected to Crohn’s disease to determine if they also displayed loss of function and if this could be related to alterations in protein localization and/or association with RIPK2. Just under half the polymorphisms displayed a significant reduction in signaling capacity following ligand stimulation, with nine of them showing near complete ablation. Only two polymorphisms, R38M and R138Q, lost the ability to interact with RIPK2. However, both these polymorphisms still associated with cellular membranes. In contrast, L248R, W355stop, L550V, N825K, L1007fsinC, L1007P, and R1019stop still bound RIPK2, but showed impaired membrane association and were unable to signal in response to MDP. This highlights the complex contributions of NOD2 polymorphisms to Crohn’s disease and reiterates the importance of both RIPK2 binding and membrane association in NOD2 signaling. Simply ascertaining whether or not NOD2 polymorphisms bind RIPK2 or associate with cellular membranes is not sufficient for determining their signaling competency.

Mouse model of OPRM1 (A118G) polymorphism has altered hippocampal function

A single nucleotide polymorphism (SNP) in the human μ-opioid receptor gene (OPRM1 A118G) has been widely studied for its association in a variety of drug addiction and pain sensitivity phenotypes; however, the extent of these adaptations and the mechanisms underlying these associations remain elusive. To clarify the functional mechanisms linking the OPRM1 A118G SNP to altered phenotypes, we used a mouse model possessing the equivalent nucleotide/amino acid substitution in the Oprm1 gene. In order to investigate the impact of this SNP on circuit function, we used voltage-sensitive dye imaging in hippocampal slices and in vivo electroencephalogram recordings of the hippocampus following MOPR activation. As the hippocampus contains excitatory pyramidal cells whose activity is highly regulated by a dense network of inhibitory neurons, it serves as an ideal structure to evaluate how putative receptor function abnormalities may influence circuit activity. We found that MOPR activation increased excitatory responses in wild-type animals, an effect that was significantly reduced in animals possessing the Oprm1 SNP. Furthermore, in order to assess the in vivo effects of this SNP during MOPR activation, EEG recordings of hippocampal activity following morphine administration corroborated a loss-of-function phenotype. In conclusion, as these mice have been shown to have similar MOPR expression in the hippocampus between genotypes, these data suggest that the MOPR A118G SNP results in a loss of receptor function.

A Novel F2RL3 Variant Predicting a Tyr157Cys Substitution in Protease-Activated Receptor 4 Is Associated with Reduced Receptor Function in Platelets

IntroductionProtease-activated receptor 4 (PAR4) is expressed widely in haemopoietic and vascular tissues and mediates thrombin-induced platelet activation primarily through Gq, protein kinase C and Ca2+ dependent pathways. Platelet PAR4 function may be modified by common non-coding variants near F2RL3 which encodes PAR4, and by epigenetic regulators which affect F2RL3 expression. However, coding sequence variants which affect PAR4 expression or function have not been reported previously. Here we characterise a novel missense variant in F2RL3associated with loss of PAR4 function in platelets from heterozygous subjects and reduced surface expression in platelets and a cell model.MethodsThe index case (P1) was identified by re-sequencing platelet G-protein coupled receptor genes in sub-groups of a collection of 2,400 adult cardiac surgery patients (GRAPHICS study; UK REC 12/SW/0286). Platelets were studied by light transmission aggregation (LTA) and by measuring P-selectin exposure, PAC-1 binding and intracellular Ca2+mobilisation in response to activating agonists. Platelet PAR4 expression was measured in platelets by flow cytometry and immunoblot and in transiently transfected HEK293 cells by flow cytometry and fluorescence microscopy.ResultsP1 was a 66 year old male with lifelong mild mucocutaneous bleeding and two gastrointestinal bleeds during aspirin treatment who harboured a heterozygous c.471A>G transition in F2RL3predicting a p.Tyr157Cys (Y157C) substitution in PAR4.Platelet phenotypeCompared to healthy controls, platelets from P1 showed reduced aggregation responses to the PAR4 agonist AYPGKF (60-600 μM; EC50 P1 266 μM vs controls 153 μM (n=11)) and to α-thrombin (5-100 mU.ml-1; EC50 P1 51 mU.ml-1 vs controls 15 mU.ml-1 (n=8)) with the greatest reduction in responses at 150 μM AYPGKF (maximum amplitude (MA) P1 1% vs controls 56%; p<0.0001) and 10 mU.ml-1 α-thrombin (MA P1 3% vs controls 67%; p<0.0001). Platelets from P1 also showed reduced P-selectin exposure, PAC-1 binding and intracellular Ca2+mobilisation in response to AYPGKF (25-1000 μM) compared to healthy controls. Functional responses of platelets from P1 to the PAR1 agonist SFLLRN (0.25-100μM) were similar to controls suggesting a selective defect in PAR4 function. Platelets from P1 showed a mean number of PAR4 receptors/platelet of 13 by flow cytometry, compared to 295 in controls (SEM 98; n=9) indicating markedly reduced surface expression. However, PAR1 surface expression in platelets from P1 was similar to controls. Immunoblots of platelet protein extracts for PAR4 from P1 and controls were similar, suggesting comparable total PAR4 expression.The c.471A>G F2RL3 variant was identified in two sons of P1, who also showed reduced platelet functional responses to AYPGKF and PAR4 surface expression that were similar to P1, indicating co-segregation of platelet phenotype and the variant F2LR3 genotype. However, these subjects did not display abnormal bleeding.Y157C PAR4 in HEK293 cellsVariant PAR4 with a C-terminal CFP tag (Y157C PAR4-CFP) expressed in HEK293 cells showed reduced surface expression by flow cytometry (mean of median fluorescence intensities 28% that of wild-type (WT) PAR4-CFP with SEM 5%; n=3). Consistent with this, fluorescence microscopy revealed predominantly intracellular expression of Y157C PAR4-CFP in the HEK293 cells with limited cell surface expression when compared to WT PAR4-CFP.ConclusionThe Y157 residue is highly conserved within transmembrane domain 3 of PAR4 that, in other G protein coupled receptors, is crucial for maintaining normal receptor structure. Therefore, the Y157C substitution identified in the index case in PAR4 is likely to adversely affect receptor function. Consistent with this, our data from multiple functional assays show reduced platelet responses to α-thrombin and to a PAR4 specific agonist, but not to a PAR1 specific agonist. This platelet phenotype co-segregated with the heterozygous Y157C substitution in the study kindred suggesting a causal association. However, this was not associated with bleeding in all kindred members. In both platelets and HEK293 cells we demonstrated the Y157C substitution was associated with loss of PAR4 function and reduced surface receptor expression.This is the first description of a missense PAR4 variant causing loss of receptor function as a heritable trait. DisclosuresNo relevant conflicts of interest to declare.

5-Hydroxytryptamine (5-HT) Cellular Sequestration during Chronic Exposure Delays 5-HT3 Receptor Resensitization due to Its Subsequent Release

The serotonergic synapse is dynamically regulated by serotonin (5-hydroxytryptamine (5-HT)) with elevated levels leading to the down-regulation of the serotonin transporter and a variety of 5-HT receptors, including the 5-HT type-3 (5-HT3) receptors. We report that recombinantly expressed 5-HT3 receptor binding sites are reduced by chronic exposure to 5-HT (IC50 of 154.0 ± 45.7 μM, t½ = 28.6 min). This is confirmed for 5-HT3 receptor-induced contractions in the guinea pig ileum, which are down-regulated after chronic, but not acute, exposure to 5-HT. The loss of receptor function does not involve endocytosis, and surface receptor levels are unaltered. The rate and extent of down-regulation is potentiated by serotonin transporter function (IC50 of 2.3 ± 1.0 μM, t½ = 3.4 min). Interestingly, the level of 5-HT uptake correlates with the extent of down-regulation. Using TX-114 extraction, we find that accumulated 5-HT remains soluble and not membrane-bound. This cytoplasmically sequestered 5-HT is readily releasable from both COS-7 cells and the guinea pig ileum. Moreover, the 5-HT level released is sufficient to prevent recovery from receptor desensitization in the guinea pig ileum. Together, these findings suggest the existence of a novel mechanism of down-regulation where the chronic release of sequestered 5-HT prolongs receptor desensitization.

Aging of cell communication: loss of receptor function.

Communication between cells is the most important evolutionarily conserved mechanism which enabled the bioconstruction of multicellular organisms. These mechanisms all comprise some general properties such as specific receptors recognized by agonists, molecules capable of activating them as well as the intracellular signalling pathways which activate the effector functions. A large number of such receptors and transmission pathways have been described, and both agonists and antagonists have been identified and are used in medicine. A more recent discovery was the demonstration that several receptor-mediated functions decline with age because either of the loss of receptors or their uncoupling from their specific signalling pathways. The mechanisms and biological as well as pathological consequences of this age-dependent receptor loss and signal transduction changes are described in this chapter.

Molecular defects of the GNRH receptor gene in Chinese patients with idiopathic hypogonadotropic hypogonadism and the severity of hypogonadism

To identify and determine the frequency of mutations in the coding region of the gonadotropin-releasing hormone receptor (GnRHR) gene in forty Chinese patients with normosmic idiopathic hypogonadotropic hypogonadism (IHH) and establish genotype/phenotype correlations where possible. The diagnosis of HH was based on absent or incomplete sexual development after 17 yr of age in girls and 18 yr in boys associated with low or normal levels of LH in both sexes and low levels of testosterone in males and of estradiol in females. All patients presented with a normal sense of smell in an olfactory specific test.40 IHH patients and 40 controls were screened for mutations in the coding sequence of GnRHR gene. The coding region of the GnRHR gene was amplified by PCR and directly sequenced. A missense mutation serine168arginine (S168R) located in the fourth transmembrane domain of the GnRH-R gene was identified in a homozygous state in one male with complete HH, the S168R mutation has been previously Shown to cause in the complete loss of receptor function because hormone binding to the receptor is completely impaired. In another patient, a compound heterozygous mutation (Gln106Arg and Arg262Gln) was identified in a male with partial HH, the Gln106Arg mutation located in the first extracellular loop of GnRH-R, this mutation decrease but not eliminate GnRH binding, while Arg262Gln mutation located in the third extracellular loop of GnRH-R and only decreases signal transduction. A good correlation between genotype and phenotype was found in our patients. The patient, who is homozygous for the completely inactivating S168R mutation, has complete HH. In addition, the affected patient who is compound heterozygotes for the Gln106Arg - Arg262Gln mutations, has partial HH. GnRHR mutations can be classified into partial or complete loss of function mutations. Partially inactivating substitutions of the GnRHR frequently found in familial hypogonadotrophic hypogonadism are Q106R and R262Q. comparison of compound heterozygous with homozygous patients suggests that their phenotype and the response to GnRH is determined by the GnRHR variant with the less severe loss of function.

Maternal Loss of Ube3a Produces an Excitatory/Inhibitory Imbalance through Neuron Type-Specific Synaptic Defects

Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss of the maternally inherited allele of UBE3A. AS model mice, which carry a maternal Ube3a null mutation (Ube3a(m-/p+)), recapitulate major features of AS in humans, including enhanced seizure susceptibility. Excitatory neurotransmission onto neocortical pyramidal neurons is diminished in Ube3a(m-/p+) mice, seemingly at odds with enhanced seizure susceptibility. We show here that inhibitory drive onto neocortical pyramidal neurons is more severely decreased in Ube3a(m-/p+) mice. This inhibitory deficit follows the loss of excitatory inputs and appears to arise from defective presynaptic vesicle cycling in multiple interneuron populations. In contrast, excitatory and inhibitory synaptic inputs onto inhibitory interneurons are largely normal. Our results indicate that there are neuron type-specific synaptic deficits in Ube3a(m-/p+) mice despite the presence of Ube3a in all neurons. These deficits result in excitatory/inhibitory imbalance at cellular and circuit levels and may contribute to seizure susceptibility in AS.

Cross talk between synaptic receptors mediates NMDA-induced suppression of inhibition

Past research has shown that calcium influx through NMDA receptors (NMDARs) depresses GABA(A) currents. We examined upstream triggers of this suppression, including involvement of target synaptic GABA(A) receptors and the NMDARs triggering suppression. In hippocampal neurons, conditioning with 20 μM NMDA for 20 s caused 50% suppression of GABA responses. The suppression was delayed by ≈ 60 s following NMDA application and persisted for at least 5 min following conditioning. Pharmacology experiments suggested a shift in both the sensitivity to GABA and a loss of functional receptors. NMDA conditioning strongly suppressed inhibitory postsynaptic currents and speeded decay kinetics. Synaptic NMDAR conditioning was necessary to suppress GABA current in pyramidal neurons; extrasynaptic NMDAR activation did not suppress, even when matched to synaptic activation. We found no evidence that specific synaptic NMDAR subunits mediate depression of GABA responses. Although physical colocalization of glutamate and GABA(A) receptors is mostly likely in extrasynaptic regions, our evidence suggests that NMDAR-induced suppression of GABA responsiveness prominently affects precise, moment-to-moment signaling from synaptic receptors to synaptic receptors.

A Novel Knock-In Mouse Reveals Mechanistically Distinct Forms of Morphine Tolerance

The role of μ-opioid receptor (MOR) down-regulation in opioid tolerance remains controversial. In this study, we used a novel knock-in mouse to examine how changing the extent of MOR down-regulation alters the development of morphine tolerance. These mice express a mutant MOR, degrading MOR (DMOR), that differs from the wild-type (WT) MOR in two ways: 1) unlike the recycling WT MOR, the mutant DMOR is targeted for degradation after its internalization, thus facilitating down-regulation; and 2) unlike the WT MOR, DMOR is efficiently internalized in response to morphine activation. We found that both WT MOR and DMOR mice develop tolerance to morphine, but DMOR mice exhibit a more rapid onset of tolerance and show receptor down-regulation. WT MOR mice develop morphine tolerance more slowly but even once profoundly tolerant show no receptor down-regulation. Furthermore, WT mice show significantly more morphine dependence than DMOR mice after long-term treatment as indicated by withdrawal. Taken together these data indicate that tolerance mediated by receptor down-regulation manifests differently both at the behavioral and biochemical level than does the actual morphine tolerance that occurs in WT mice and that loss of receptor function is not a major contributor to morphine tolerance in WT MOR mice.

A missense mutation in Fgfr1 causes ear and skull defects in hush puppy mice

The hush puppy mouse mutant has been shown previously to have skull and outer, middle, and inner ear defects, and an increase in hearing threshold. The fibroblast growth factor receptor 1 (Fgfr1) gene is located in the region of chromosome 8 containing the mutation. Sequencing of the gene in hush puppy heterozygotes revealed a missense mutation in the kinase domain of the protein (W691R). Homozygotes were found to die during development, at approximately embryonic day 8.5, and displayed a phenotype similar to null mutants. Reverse transcription PCR indicated a decrease in Fgfr1 transcript in heterozygotes and homozygotes. Generation of a construct containing the mutation allowed the function of the mutated receptor to be studied. Immunocytochemistry showed that the mutant receptor protein was present at the cell membrane, suggesting normal expression and trafficking. Measurements of changes in intracellular calcium concentration showed that the mutated receptor could not activate the IP3 pathway, in contrast to the wild-type receptor, nor could it initiate activation of the Ras/MAP kinase pathway. Thus, the hush puppy mutation in fibroblast growth factor receptor 1 appears to cause a loss of receptor function. The mutant protein appears to have a dominant negative effect, which could be due to it dimerising with the wild-type protein and inhibiting its activity, thus further reducing the levels of functional protein. A dominant modifier, Mhspy, which reduces the effect of the hush puppy mutation on pinna and stapes development, has been mapped to the distal end of chromosome 7 and may show imprinting.Electronic supplementary materialThe online version of this article (doi:10.1007/s00335-011-9324-8) contains supplementary material, which is available to authorized users.

Lack of IL-7 Receptor Alpha Chain (CD127) Expression In T Cells Is a Hallmark of T-Cell Immunodeficiency In Schimke Immuno-Osseous Dysplasia (SIOD)

AbstractAbstract 2767Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, incompletely penetrant, childhood disorder associated with biallelic loss-of-function mutations of SMARCAL1 (swi/snf-related matrix-associated actin-dependent regulator of chromatin, subfamily-a-like-1) gene. The SMARCAL1 gene encodes for the DNA annealing helicase although it is unknown why this impairment results in skeletal dysplasia, renal dysfunction, and T-cell lymphopenia. The representation of T-cell subsets in SIOD patients is further characterized by a high proportion of memory (CD45RA−CD45RO+) T cells, however, the etiology of T-cell immunodeficiency has not been elucidated. Here, we demonstrate that T cells in individuals affected by SIOD express markedly reduced surface levels of IL-7 receptor alpha chain (CD127) as compared to their unaffected sibling or normal individuals. In contrast, the alpha-chain component of the IL-2 receptor (CD25) was expressed normally. This reduction in IL-7 receptor alpha chain was observed in all T-cell subsets including naïve T cells. This suggests that the origin of the IL-7 receptor alpha chain deficiency is likely intrathymic, rather than occurring after emigration to the periphery. In addition, T cells from SIOD patients were less responsive to stimulation with IL-7, indicating a loss of functional receptor. From these observations, we propose the lack of functional IL-7 receptor expression in T cells, and possibly their earlier progenitors, may have restricted T-cell development in SIOD patients. The mechanism for reduced IL-7 receptor expression is currently under investigation. Disclosures:No relevant conflicts of interest to declare.

A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis

AbstractWe investigated the cause of mild mucocutaneous bleeding in a 14-year-old male patient (P1). Platelet aggregation and ATP secretion induced by arachidonic acid and the thromboxane A2 receptor (TxA2R) agonist U46619 were reduced in P1 compared with controls, whereas the responses to other platelet agonists were retained. P1 was heterozygous for a transversion within the TBXA2R gene predictive of a D304N substitution in the TxA2R. In Chinese hamster ovary-K1 cells expressing the variant D304N TxA2R, U46619 did not increase cytosolic free Ca2+ concentration, indicating loss of receptor function. The TxA2R antagonist [3H]-SQ29548 showed an approximate 50% decrease in binding to platelets from P1 but absent binding to Chinese hamster ovary-K1 cells expressing variant D304N TxA2R. This is the second naturally occurring TxA2R variant to be associated with platelet dysfunction and the first in which loss of receptor function is associated with reduced ligand binding. D304 lies within a conserved NPXXY motif in transmembrane domain 7 of the TxA2R that is a key structural element in family A G protein-coupled receptors. Our demonstration that the D304N substitution causes clinically significant platelet dysfunction by reducing ligand binding establishes the importance of the NPXXY motif for TxA2R function in vivo.

Regulation of An Acquired Inhibitor to the GPVI Platelet Collagen Receptor in a Patient with An Autoimmune Syndrome and Kidney Disease.

GPVI is a major platelet collagen signaling receptor associated with the ITAM-containing FcRγ in the platelet membrane. Autoantibodies to GPVI result in ADAM10-mediated cleavage and loss of receptor function. We now report a young woman with a history of autoimmune and kidney disease associated with systemic lupus erythematosus, and a strong circulating antibody to GPVI. The patient has been followed over a 3-year period. Particularly studied were (i) the platelet aggregation response to collagen (and convulxin (Cvx)), (ii) platelet GPVI levels (flow cytometry and Western blot (WB)), (iii) plasma soluble GPVI levels (GPVIs, dual antibody ELISA with capture and biotinylated detction MoAbs), and (iv) circulating anti-GPVI antibody titres (ELISA using recombinant GPVIs). When first studied in 2006, the patient had a normal platelet count but a virtual absence of platelet aggregation to collagen and Cvx. Nevertheless, her platelets responded to other agonists including cross-linking FcRγIIA by way of the monoclonal antibody, IV.3, showing that the PLCγ2-dependent signaling pathway shared with GPVI-FcRγ was retained. Western blotting with a series of MoAbs to GPVI (extracellular and intracellular domains) and other platelet receptors showed a major and specific deficit of GPVI with appearance of GPVI dimer and a 47 kDa degradation product. Other platelet receptors known to be sensitive to metalloprotease cleavage, GPIbα, P-selectin, TLT-1 were not reduced. However, FcRγ was moderately decreased in WB. Interestingly, the patient's plasma was able to activate normal platelets, a phenomenon blocked by recombinant GPVI-Fc protein but not by IV.3 showing that it was independent of FcRγIIA and probably related to GPVI dimerization. Her plasma also specifically blocked the response of control platelets to Cvx in a time-dependent manner. Studies continued in 2007 when the patient additionally experienced neurological and vascular problems. Platelet expression of GPVI continued to be severely reduced by WB, and ELISA confirmed strong anti-GPVI plasma antibody titers. Plasma GPVIs levels were undetectable compared to control donors. Transfusion of donor platelets prior to a renal biopsy was unsuccessful; they were rapidly cleared and failed to correct the platelet defect. The patient was now subjected to increasingly strict immunosuppressive medication including corticoids (prednisolone), imurel (azathioprine) then endoxan (cyclophosphamide). A large reduction in plasma anti-GPVI levels, the appearance of GPVIs in the patient's plasma (the ELISA results were confirmed by WB of plasma samples), the partial restoration of both platelet GPVI levels and the platelet collagen response accompanied these treatments which was paralleled by an improvement in her nephrotic syndrome. Our results suggest that this patient developed a potent anti-GPVI antibody responsible for the loss of platelet GPVI, with the possible clearance of GPVIs by immune complex formation. The activity of the antibody was much reduced by immunotherapy; further studies are required to know whether this is related to the improvement in her clinical state.

Identification of a Pool of Non-pumping Na/K-ATPase

Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. Here, we present experimental evidence demonstrating that over half of the plasma membrane Na/K-ATPase in LLC-PK1 cells is performing cellular functions other than ion pumping. This "non-pumping" pool of Na/K-ATPase, like the pumping pump, binds ouabain. Depletion of either cholesterol or caveolin-1 moves some of the "non-pumping" Na/K-ATPase into the pumping pool. Graded knock-down of the alpha1 subunit of the Na/K-ATPase eventually results in loss of this "non-pumping" pool while preserving the pumping pool. Our prior studies indicate that a loss of the non-pumping pool is associated with a loss of receptor function as evidenced by the failure of ouabain administration to induce the activation of Src and/or ERK. Therefore, our new findings suggest that a substantial amount of surface-expressed Na/K-ATPase, at least in some types of cells, may function as non-canonical ouabain-binding receptors.

Role of aromatic and charged ectodomain residues in the P2X4 receptor functions

The localization of ATP binding site(s) at P2X receptors and the molecular rearrangements associated with opening and closing of channels are still not well understood. At P2X(4) receptor, substitution of the K67, F185, K190, F230, R278, D280, R295, and K313 ectodomain residues with alanine generated low or non-responsive mutants, whereas the F294A mutant was functional. The loss of receptor function was also observed in K67R, R295K, and K313R mutants, but not in F185W, K190R, F230W, R278K, and D280E mutants. To examine whether the loss of function reflects decreased sensitivity of mutants for ATP, we treated cells with ivermectin, an antiparasitic agent that enhances responsiveness of P2X(4)R. In the presence of ivermectin, all low or non-responsive mutants responded to ATP in a dose-dependent manner, with the EC(50) values for ATP of about 1, 2, 4, 20, 60, 125, 270, 420, 1000 and 2300 micromol/L at D280A, R278A, F185A, K190A, R295K, K313R, R295A, K313A, K67A and K67R mutants, respectively. These results indicate that lysines 67 and 313 and arginine 295 play a critical role in forming the proper three-dimensional structure of P2X(4)R for agonist binding and/or channel gating.

The calcium-sensing receptor and related diseases

The calcium-sensing receptor (CASR) adjusts the extracellular calcium set point regulating PTH secretion and renal calcium excretion. The receptor is expressed in several tissues and is also involved in other cellular functions such as proliferation, differentiation and other hormonal secretion. High extracellular calcium levels activate the receptor resulting in modulation of several signaling pathways depending on the target tissues. Mutations in the CASR gene can result in gain or loss of receptor function. Gain of function mutations are associated to Autossomal dominant hypocalcemia and Bartter syndrome type V, while loss of function mutations are associated to Familial hypocalciuric hypercalcemia and Neonatal severe hyperparathyroidism. More than one hundred mutations were described in this gene. In addition to calcium, the receptor also interacts with several ions and polyamines. The CASR is a potential therapeutic target to treatment of diseases including hyperparathyroidism and osteoporosis, since its interaction with pharmacological compounds results in modulation of PTH secretion.

Aberrant expression and mutations of TGF-β receptor type II gene in endometrial cancer

Objective. Transforming growth factor β (TGF-β) is a multifunctional cytokine that strongly inhibits epithelial cell growth. Disabling of TGF-β signaling is thought to be involved in development of a variety of tumors in which abnormal expression or function of TGF-β receptor plays critical roles. In the present study, we examined aberrant expression and mutation of the gene TGF-β receptor type II ( TβRII) in endometrial cancers of endometrioid subtype. Methods and results. Real-time PCR analysis using surgical tissue specimens of 27 endometrial cancers and 24 normal endometria revealed that endometrial cancers had significantly decreased levels of TβRII mRNA expression (mean level 2.44 ± 2.65), compared to normal endometria (mean level 7.23 ± 6.07) ( P < 0.001). Methylation status of TβRII promoter containing 30 CpGs was examined by bisulfite sequencing analysis, and 98% (51/52) of the patients were found to have unmethylated TβRII promoter, indicating that promoter hypermethylation is not the major cause of decreased expression of TβRII in endometrial cancers. Mutational analysis revealed that 15.1% (8/53) of endometrial cancers had frameshift mutations at polyadenine repeats in exon 3 of the TβRII gene. Notably, these mutations were preferentially accumulated in patients with MSI-H phenotype (7/19:37%) ( P < 0.001) or with those with methylated MLH1 promoters (6/16:38%) ( P < 0.01). Thus, it appears that the TβRII gene is a target of mismatch repair deficiency. Conclusion. Taken together, we found that the decreased expression of TβRII as well as frameshift mutation of TβRII via mismatch repair deficiency frequently occurs in this tumor type, possibly causing loss of receptor function and unresponsiveness of TGF-β signaling that may lead to endometrial carcinogenesis.