RESUMO
GPR37L1 is an orphan receptor that couples through heterotrimeric G-proteins to regulate physiological functions. Since its role in humans is not fully defined, we used an unbiased computational approach to assess the clinical significance of rare G-protein-coupled receptor 37-like 1 (GPR37L1) genetic variants found among 51,289 whole-exome sequences from the DiscovEHR cohort. Rare GPR37L1 coding variants were binned according to predicted pathogenicity and analyzed by sequence kernel association testing to reveal significant associations with disease diagnostic codes for epilepsy and migraine, among others. Since associations do not prove causality, rare GPR37L1 variants were functionally analyzed in SK-N-MC cells to evaluate potential signaling differences and pathogenicity. Notably, receptor variants exhibited varying abilities to reduce cAMP levels, activate mitogen-activated protein kinase (MAPK) signaling, and/or upregulate receptor expression in response to the agonist prosaptide (TX14(A)), as compared with the wild-type receptor. In addition to signaling changes, knock-out (KO) of GPR37L1 or expression of certain rare variants altered cellular cholesterol levels, which were also acutely regulated by administration of the agonist TX14(A) via activation of the MAPK pathway. Finally, to simulate the impact of rare nonsense variants found in the large patient cohort, a KO mouse line lacking Gpr37l1 was generated. Although KO animals did not recapitulate an acute migraine phenotype, the loss of this receptor produced sex-specific changes in anxiety-related disorders often seen in chronic migraineurs. Collectively, these observations define the existence of rare GPR37L1 variants associated with neuropsychiatric conditions in the human population and identify the signaling changes contributing to pathological processes.
Assuntos
Transtornos de Enxaqueca , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/metabolismo , Camundongos , Masculino , Feminino , Camundongos Knockout , Transtornos de Ansiedade/genética , Transtornos de Ansiedade/metabolismo , Camundongos Endogâmicos C57BL , Variação Genética/genéticaRESUMO
The calcium-sensing receptor (CaSR) regulates serum calcium concentrations. CASR loss- or gain-of-function mutations cause familial hypocalciuric hypercalcemia type 1 (FHH1) or autosomal-dominant hypocalcemia type 1 (ADH1), respectively, but the population prevalence of FHH1 or ADH1 is unknown. Rare CASR variants were identified in whole-exome sequences from 51,289 de-identified individuals in the DiscovEHR cohort derived from a single US healthcare system. We integrated bioinformatics pathogenicity triage, mean serum Ca concentrations, and mode of inheritance to identify potential FHH1 or ADH1 variants, and we used a Sequence Kernel Association Test (SKAT) to identify rare variant-associated diseases. We identified predicted heterozygous loss-of-function CASR variants (6 different nonsense/frameshift variants and 12 different missense variants) in 38 unrelated individuals, 21 of whom were hypercalcemic. Missense CASR variants were identified in two unrelated hypocalcemic individuals. Functional studies showed that all hypercalcemia-associated missense variants impaired heterologous expression, plasma membrane targeting, and/or signaling, whereas hypocalcemia-associated missense variants increased expression, plasma membrane targeting, and/or signaling. Thus, 38 individuals with a genetic diagnosis of FHH1 and two individuals with a genetic diagnosis of ADH1 were identified in the 51,289 cohort, giving a prevalence in this population of 74.1 per 100,000 for FHH1 and 3.9 per 100,000 for ADH1. SKAT combining all nonsense, frameshift, and missense loss-of-function variants revealed associations with cardiovascular, neurological, and other diseases. In conclusion, FHH1 is a common cause of hypercalcemia, with prevalence similar to that of primary hyperparathyroidism, and is associated with altered disease risks, whereas ADH1 is a major cause of non-surgical hypoparathyroidism.
Assuntos
Atenção à Saúde/estatística & dados numéricos , Hipercalcemia/congênito , Adulto , Idoso , Idoso de 80 Anos ou mais , Cálcio/sangue , Estudos de Coortes , Feminino , Genes Dominantes/genética , Heterozigoto , Humanos , Hipercalcemia/genética , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Prevalência , Receptores de Detecção de Cálcio/genética , Estados UnidosRESUMO
The pace of deorphanization of G protein-coupled receptors (GPCRs) has slowed, and new approaches are required. Small molecule targeting of orphan GPCRs can potentially be of clinical benefit even if the endogenous receptor ligand has not been identified. Many GPCRs lack common variants that lead to reproducible genome-wide disease associations, and rare-variant approaches have emerged as a viable alternative to identify disease associations for such genes. Therefore, our goal was to prioritize orphan GPCRs by determining their associations with human diseases in a large clinical population. We used sequence kernel association tests to assess the disease associations of 85 orphan or understudied GPCRs in an unselected cohort of 51,289 individuals. Using rare loss-of-function variants, missense variants predicted to be pathogenic or likely pathogenic, and a subset of rare synonymous variants that cause large changes in local codon bias as independent data sets, we found strong, phenome-wide disease associations shared by two or more variant categories for 39% of the GPCRs. To validate the bioinformatics and sequence kernel association test analyses, we functionally characterized rare missense and synonymous variants of GPR39, a family A GPCR, revealing altered expression or Zn2+-mediated signaling for members of both variant classes. These results support the utility of rare variant analyses for identifying disease associations for GPCRs that lack impactful common variants. We highlight the importance of rare synonymous variants in human physiology and argue for their routine inclusion in any comprehensive analysis of genomic variants as potential causes of disease.
Assuntos
Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Mutação Silenciosa , Estudo de Associação Genômica Ampla , HumanosRESUMO
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 â¼7.8 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.
Assuntos
Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades sigma do Complexo de Proteínas Adaptadoras/genética , Mutação , Receptores de Detecção de Cálcio/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades sigma do Complexo de Proteínas Adaptadoras/metabolismo , Cinacalcete/farmacologia , Bases de Dados Genéticas , Exoma , Feminino , Humanos , Hipercalcemia/tratamento farmacológico , Hipercalcemia/genética , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Conformação Proteica , Transdução de Sinais , Sequenciamento do ExomaRESUMO
Breast cancer is the second leading cause of cancer mortality in women, estimated at nearly 40,000 deaths and more than 230,000 new cases diagnosed in the U.S. this year alone. One of the defining characteristics of breast cancer is the radiographic presence of microcalcifications. These palpable mineral precipitates are commonly found in the breast after formation of a tumor. Since free Ca(2+) plays a crucial role as a second messenger inside cells, we hypothesize that these chelated precipitates may be a result of dysregulated Ca(2+) secretion associated with tumorigenesis. Transient and sustained elevations of intracellular Ca(2+) regulate cell proliferation, apoptosis and cell migration, and offer numerous therapeutic possibilities in controlling tumor growth and metastasis. During lactation, a developmentally determined program of gene expression controls the massive transcellular mobilization of Ca(2+) from the blood into milk by the coordinated action of calcium transporters, including pumps, channels, sensors and buffers, in a functional module that we term CALTRANS. Here we assess the evidence implicating genes that regulate free and buffered Ca(2+) in normal breast epithelium and cancer cells and discuss mechanisms that are likely to contribute to the pathological characteristics of breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Canais de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Lactação/fisiologia , Receptores de Detecção de Cálcio/metabolismo , Apoptose/fisiologia , Neoplasias da Mama/patologia , Cálcio/metabolismo , Sinalização do Cálcio , Movimento Celular/fisiologia , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Calcificação VascularRESUMO
Calcium sensing receptor (CaSR) mutations or altered expression cause disorders of calcium handling. Recent studies suggest that reduced targeting to the plasma membrane is a feature common to many CaSR loss-of-function mutations. Allosteric agonists (calcimimetics) can rescue signaling of a subset of CaSR mutants. This review evaluates our current understanding of the subcellular site(s) for allosteric modulator rescue of CaSR mutants. Studies to date make a strong case for calcimimetic potentiation of signaling not only at plasma membrane-localized CaSR, but at the endoplasmic reticulum, acting as pharmacoperones to assist in navigation of multiple quality control checkpoints. The possible role of endogenous pharmacoperones, calcium and glutathione, in folding and stabilization of the CaSR extracellular and transmembrane domains are considered. Finally, the possibility that dihydropyridines act as unintended pharmacoperones of CaSR is proposed. While our understanding of pharmacoperone rescue of CaSR requires refinement, promising results to date argue that this may be a fruitful avenue for drug discovery.
Assuntos
Descoberta de Drogas , Receptores de Detecção de Cálcio/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Cálcio/metabolismo , Di-Hidropiridinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Mutação , Biossíntese de Proteínas/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores de Detecção de Cálcio/análise , Receptores de Detecção de Cálcio/genéticaRESUMO
GPR37L1 is an orphan receptor that couples through heterotrimeric G-proteins to regulate physiological functions. Since its role in humans is not fully defined, we used an unbiased computational approach to assess the clinical significance of rare GPR37L1 genetic variants found among 51,289 whole exome sequences from the DiscovEHR cohort. Briefly, rare GPR37L1 coding variants were binned according to predicted pathogenicity, and analyzed by Sequence Kernel Association testing to reveal significant associations with disease diagnostic codes for epilepsy and migraine, among others. Since associations do not prove causality, rare GPR37L1 variants were then functionally analyzed in SK-N-MC cells to evaluate potential signaling differences and pathogenicity. Notably, receptor variants exhibited varying abilities to reduce cAMP levels, activate MAPK signaling, and/or upregulate receptor expression in response to the agonist prosaptide (TX14(A)), as compared to the wild-type receptor. In addition to signaling changes, knockout of GPR37L1 or expression of certain rare variants altered cellular cholesterol levels, which were also acutely regulated by administration of the agonist TX14(A) via activation of the MAPK pathway. Finally, to simulate the impact of rare nonsense variants found in the large patient cohort, a knockout (KO) mouse line lacking Gpr37L1 was generated, revealing loss of this receptor produced sex-specific changes implicated in migraine-related disorders. Collectively, these observations define the existence of rare GPR37L1 variants in the human population that are associated with neuropsychiatric conditions and identify the underlying signaling changes that are implicated in the in vivo actions of this receptor in pathological processes leading to anxiety and migraine. SIGNIFICANCE STATEMENT: G-protein coupled receptors (GPCRs) represent a diverse group of membrane receptors that contribute to a wide range of diseases and serve as effective drug targets. However, a number of these receptors have no identified ligands or functions, i.e., orphan receptors. Over the past decade, advances have been made, but there is a need for identifying new strategies to reveal their roles in health and disease. Our results highlight the utility of rare variant analyses of orphan receptors for identifying human disease associations, coupled with functional analyses in relevant cellular and animal systems, to ultimately reveal their roles as novel drug targets for treatment of neurological disorders that lack wide-spread efficacy.
RESUMO
Metabolic labeling with [(35)S]cysteine was used to characterize early events in CaSR biosynthesis. [(35)S]CaSR is relatively stable (half-life approximately 8 h), but maturation to the final glycosylated form is slow and incomplete. Incorporation of [(35)S]cysteine is linear over 60 min, and the rate of [(35)S]CaSR biosynthesis is significantly increased by the membrane-permeant allosteric agonist NPS R-568, which acts as a cotranslational pharmacochaperone. The [(35)S]CaSR biosynthetic rate also varies as a function of conformational bias induced by loss- or gain-of-function mutations. In contrast, [(35)S]CaSR maturation to the plasma membrane was not significantly altered by exposure to the pharmacochaperone NPS R-568, the allosteric agonist neomycin, or the orthosteric agonist Ca(2+) (0.5 or 5 mm), suggesting that CaSR does not control its own release from the endoplasmic reticulum. A CaSR chimera containing the mGluR1alpha carboxyl terminus matures completely (half-time of approximately 8 h) and without a lag period, as does the truncation mutant CaSRDelta868 (half-time of approximately 16 h). CaSRDelta898 exhibits maturation comparable with full-length CaSR, suggesting that the CaSR carboxyl terminus between residues Thr(868) and Arg(898) limits maturation. Overall, these results suggest that CaSR is subject to cotranslational quality control, which includes a pharmacochaperone-sensitive conformational checkpoint. The CaSR carboxyl terminus is the chief determinant of intracellular retention of a significant fraction of total CaSR. Intracellular CaSR may reflect a rapidly mobilizable "storage form" of CaSR and/or may subserve distinct intracellular signaling roles that are sensitive to signaling-dependent changes in endoplasmic reticulum Ca(2+) and/or glutathione.
Assuntos
Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , Receptores de Detecção de Cálcio/metabolismo , Compostos de Anilina/farmacologia , Western Blotting , Cálcio/metabolismo , Cálcio/farmacologia , Linhagem Celular , Membrana Celular/metabolismo , Cisteína/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Leupeptinas/farmacologia , Proteínas Mutantes/biossíntese , Proteínas Mutantes/química , Mutação , Neomicina/farmacologia , Fenetilaminas , Propilaminas , Conformação Proteica , Transporte Proteico/efeitos dos fármacos , Receptores de Detecção de Cálcio/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Radioisótopos de Enxofre , Fatores de Tempo , TransfecçãoRESUMO
Calcium sensing receptor (CaSR) mutations implicated in familial hypocalciuric hypercalcemia, pancreatitis and idiopathic epilepsy syndrome map to an extended arginine-rich region in the proximal carboxyl terminus. Arginine-rich motifs mediate endoplasmic reticulum retention and/or retrieval of multisubunit proteins so we asked whether these mutations, R886P, R896H or R898Q, altered CaSR targeting to the plasma membrane. Targeting was enhanced by all three mutations, and Ca(2+)-stimulated ERK1/2 phosphorylation was increased for R896H and R898Q. To define the role of the extended arginine-rich region in CaSR trafficking, we independently determined the contributions of R890/R891 and/or R896/K897/R898 motifs by mutation to alanine. Disruption of the motif(s) significantly increased surface expression and function relative to wt CaSR. The arginine-rich region is flanked by phosphorylation sites at S892 (protein kinase C) and S899 (protein kinase A). The phosphorylation state of S899 regulated recognition of the arginine-rich region; S899D showed increased surface localization. CaSR assembles in the endoplasmic reticulum as a covalent disulfide-linked dimer and we determined whether retention requires the presence of arginine-rich regions in both subunits. A single arginine-rich region within the dimer was sufficient to confer intracellular retention comparable to wt CaSR. We have identified an extended arginine-rich region in the proximal carboxyl terminus of CaSR (residues R890 - R898) which fosters intracellular retention of CaSR and is regulated by phosphorylation. Mutation(s) identified in chronic pancreatitis and idiopathic epilepsy syndrome therefore increase plasma membrane targeting of CaSR, likely contributing to the altered Ca(2+) signaling characteristic of these diseases.
Assuntos
Epilepsia/metabolismo , Pancreatite/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Proteínas 14-3-3/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sinalização do Cálcio , Linhagem Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Epilepsia/genética , Humanos , Mutagênese Sítio-Dirigida , Pancreatite/genética , Fosforilação , Receptores de Detecção de Cálcio/genéticaRESUMO
The calcium sensing receptor (CaSR) is a Family 3/C G protein-coupled receptor with slow and partial targeting to the plasma membrane in both native and heterologous cells. We identified cargo receptor family member p24A in yeast two-hybrid screens with the CaSR carboxyl terminus. Interactions were confirmed by immunoprecipitation of either p24A or CaSR in transiently transfected HEK293 cells. Only the immaturely glycosylated form of CaSR interacts with p24A. Dissociation likely occurs in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) or cis-Golgi, since only the uncleaved form of a CaSR mutant sensitive to the trans-Golgi enzyme furin was co-immunoprecipitated with p24A. p24A and p24A(DeltaGOLD) significantly increased total and plasma membrane CaSR protein but p24A(FF/AA) did not. The CaSR carboxyl terminus distal to T868 is required for differential sensitivity to p24A and its mutants. Interaction with p24A therefore increases CaSR stability in the ER and enhances plasma membrane targeting. Neither wt Sar1p or the T39N mutant increased CaSR maturation or abundance while the H79G mutant increased abundance but prevented maturation of CaSR. These results suggest that p24A is the limiting factor in CaSR trafficking in the early secretory pathway, and that cycling between the ER and ERGIC protects CaSR from degradation.
Assuntos
Proteínas de Membrana/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Estabilidade Proteica , Transporte Proteico , Proteínas de Transporte VesicularRESUMO
The past several decades of research into calcium signaling have focused on intracellular calcium (Ca(i)(2+)), revealing both exquisite spatial and dynamic control of this potent second messenger. Our understanding of Ca(i)(2+) signaling has benefited from the evolution of cell culture methods, development of high affinity fluorescent calcium indicators (both membrane-permeant small molecules and genetically encoded proteins), and high-resolution fluorescence microscopy. As our understanding of single cell calcium dynamics has increased, translational efforts have attempted to push calcium signaling studies back into tissues, organs and whole animals. Emerging results from these more complicated, diffusion-limited systems have begun to define a role for extracellular calcium (Ca(o)(2+)) as an agonist, spurred by the cloning and characterization of a G protein-coupled receptor activated by Ca(o)(2+) (the calcium sensing receptor, CaR). Here, we review the current state-of-the art for measurement of Ca(o)(2+) fluctuations, and the evidence that fluctuations in Ca(o)(2+) can act as primary signals regulating cell function. Current results suggest that Ca(o)(2+) in bone and epidermis may act as a chemotactic homing signal, targeting cells to the appropriate tissue locations prior to initiation of the differentiation program. Ca(i)(2+) signaling-mediated Ca(o)(2+) fluctuations in interstitial spaces may integrate cell signaling responses in multicellular networks through activation of CaR. Appreciation of the importance of Ca(o)(2+) fluctuations in coordinating cell function will likely spur identification of additional, niche-specific Ca(2+) sensors, and provide unique insights into the regulation of multicellular signaling networks.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/fisiologia , Animais , Microdomínios da Membrana/fisiologia , Receptores de Detecção de Cálcio/fisiologiaRESUMO
Obesity is a major current public health problem worldwide due to the severe co-morbid conditions that this disease entails. The development of obesity-related cardiometabolic disorders is in direct association with adipose tissue inflammation that leads to its functional impairment. Activation of the Calcium-Sensing Receptor (CaSR) in adipose tissue contributes to inflammation and adipose dysfunction. Autophagy, a process of cell component degradation, is closely related to inflammation in many diseases, however, whether autophagy is associated with CaSR-induced inflammation remains unknown. Using LS14 and SW872 preadipose cell lines as well as primary human preadipocytes, we show that CaSR activation with the allosteric activator cinacalcet induces autophagosome formation. Cinacalcet-induced LC3II content elevation was precluded by knockdown of the CaSR and enhanced by CaSR overexpression, indicating a specific effect. Autophagy inhibition using 3-methyladenine prevented CaSR-induced TNFα production, indicating that autophagy contributes to CaSR-induced inflammation in human preadipocytes. Our results suggest that modulation of CaSR-induced autophagy is an attractive target in obese inflamed adipose tissue, to prevent the development of diseases triggered by adipose dysfunction. We describe a novel mechanism and possible new target to modulate and prevent adipose inflammation and hence the resulting disease-generating adipose tissue dysfunction.
Assuntos
Tecido Adiposo/patologia , Autofagia , Inflamação/patologia , Receptores de Detecção de Cálcio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Calcimiméticos/farmacologia , Linhagem Celular , Cinacalcete/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Inflamação/etiologia , Obesidade/complicações , Obesidade/metabolismo , Cultura Primária de Células , Receptores de Detecção de Cálcio/agonistas , Receptores de Detecção de Cálcio/genéticaRESUMO
Spatial control of G-protein-coupled receptor (GPCR) signaling, which is used by cells to translate complex information into distinct downstream responses, is achieved by using plasma membrane (PM) and endocytic-derived signaling pathways. The roles of the endomembrane in regulating such pleiotropic signaling via multiple G-protein pathways remain unknown. Here, we investigated the effects of disease-causing mutations of the adaptor protein-2σ subunit (AP2σ) on signaling by the class C GPCR calcium-sensing receptor (CaSR). These AP2σ mutations increase CaSR PM expression yet paradoxically reduce CaSR signaling. Hypercalcemia-associated AP2σ mutations reduced CaSR signaling via Gαq/11 and Gαi/o pathways. The mutations also delayed CaSR internalization due to prolonged residency time of CaSR in clathrin structures that impaired or abolished endosomal signaling, which was predominantly mediated by Gαq/11. Thus, compartmental bias for CaSR-mediated Gαq/11 endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling.
Assuntos
Complexo 2 de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Sinalização do Cálcio/genética , Endossomos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores de Detecção de Cálcio/genética , Humanos , MutaçãoRESUMO
The extracellular calcium-sensing receptor (CaR) senses small fluctuations of the extracellular calcium (Ca(2+)(e)) concentration and translates them into potent changes in parathyroid hormone secretion. Dissecting the regulatory mechanisms of CaR-mediated signal transduction may provide insights into the physiology of the receptor and identify new molecules as potential drug targets for the treatment of osteoporosis and/or hyperparathyroidism. CaR can be phosphorylated by protein kinase C (PKC) and G protein-coupled receptor kinases (GRKs), and has been shown to bind to beta-arrestins, potentially contributing to desensitization of CaR, although the mechanisms by which CaR-mediated signal transduction is terminated are not known. We used a PKC phosphorylation site-deficient CaR, GRK and beta-arrestin overexpression or down-regulation to delineate CaR-mediated desensitization. Fluorescence-activated cell sorting was used to determine whether receptor internalization contributed to desensitization. Overexpression of GRK 2 or 3 reduced Ca(2+)(e)-dependent inositol phosphate accumulation by more than 70%, whereas a GRK 2 mutant deficient in G alpha(q) binding (D110A) was without major effect. Overexpression of GRK 4-6 did not reduce Ca(2+)(e)-dependent inositol phosphate accumulation. Overexpression of beta-arrestin 1 or 2 revealed a modest inhibitory effect on Ca(2+)(e)-dependent inositol phosphate production (20-30%), which was not observed for the PKC phosphorylation site-deficient CaR. Agonist-dependent receptor internalization (10-15%) did not account for the described effects. Thus, we conclude that PKC phosphorylation of CaR contributes to beta-arrestin-dependent desensitization of CaR coupling to G proteins. In contrast, GRK 2 predominantly interferes with G protein-mediated inositol-1,4,5-trisphosphate formation by binding to G alpha(q).
Assuntos
Arrestinas/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteína Quinase C/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Quinases de Receptores Adrenérgicos beta/metabolismo , Arrestinas/genética , Linhagem Celular , Quinase 2 de Receptor Acoplado a Proteína G , Expressão Gênica , Humanos , Fosfatos de Inositol/metabolismo , Rim/citologia , Mutagênese , Fosforilação , RNA Interferente Pequeno , Receptores de Detecção de Cálcio/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Quinases de Receptores Adrenérgicos beta/genética , beta-Arrestina 1 , beta-ArrestinasRESUMO
A wide range of patient health data is recorded in Electronic Health Records (EHR). This data includes diagnosis, surgical procedures, clinical laboratory measurements, and medication information. Together this information reflects the patient's medical history. Many studies have efficiently used this data from the EHR to find associations that are clinically relevant, either by utilizing International Classification of Diseases, version 9 (ICD-9) codes or laboratory measurements, or by designing phenotype algorithms to extract case and control status with accuracy from the EHR. Here we developed a strategy to utilize longitudinal quantitative trait data from the EHR at Geisinger Health System focusing on outpatient metabolic and complete blood panel data as a starting point. Comprehensive Metabolic Panel (CMP) as well as Complete Blood Counts (CBC) are parts of routine care and provide a comprehensive picture from high level screening of patients' overall health and disease. We randomly split our data into two datasets to allow for discovery and replication. We first conducted a genome-wide association study (GWAS) with median values of 25 different clinical laboratory measurements to identify variants from Human Omni Express Exome beadchip data that are associated with these measurements. We identified 687 variants that associated and replicated with the tested clinical measurements at p<5×10-08. Since longitudinal data from the EHR provides a record of a patient's medical history, we utilized this information to further investigate the ICD-9 codes that might be associated with differences in variability of the measurements in the longitudinal dataset. We identified low and high variance patients by looking at changes within their individual longitudinal EHR laboratory results for each of the 25 clinical lab values (thus creating 50 groups - a high variance and a low variance for each lab variable). We then performed a PheWAS analysis with ICD-9 diagnosis codes, separately in the high variance group and the low variance group for each lab variable. We found 717 PheWAS associations that replicated at a p-value less than 0.001. Next, we evaluated the results of this study by comparing the association results between the high and low variance groups. For example, we found 39 SNPs (in multiple genes) associated with ICD-9 250.01 (Type-I diabetes) in patients with high variance of plasma glucose levels, but not in patients with low variance in plasma glucose levels. Another example is the association of 4 SNPs in UMOD with chronic kidney disease in patients with high variance for aspartate aminotransferase (discovery p-value: 8.71×10-09 and replication p-value: 2.03×10-06). In general, we see a pattern of many more statistically significant associations from patients with high variance in the quantitative lab variables, in comparison with the low variance group across all of the 25 laboratory measurements. This study is one of the first of its kind to utilize quantitative trait variance from longitudinal laboratory data to find associations among genetic variants and clinical phenotypes obtained from an EHR, integrating laboratory values and diagnosis codes to understand the genetic complexities of common diseases.
Assuntos
Estudo de Associação Genômica Ampla/estatística & dados numéricos , Análise de Variância , Contagem de Células Sanguíneas/estatística & dados numéricos , Análise Química do Sangue/estatística & dados numéricos , Biologia Computacional , Registros Eletrônicos de Saúde/estatística & dados numéricos , Redes Reguladoras de Genes , Humanos , Classificação Internacional de Doenças , Estudos Longitudinais , Polimorfismo de Nucleotídeo Único , Característica Quantitativa HerdávelRESUMO
The cardiovascular system is richly endowed with G protein-coupled receptors (GPCRs), members of the largest family of plasma membrane-localized receptors. During the last 10 years, it has become increasingly clear that many, if not all, GPCRs function in oligomeric complexes, as either homo- or hetero-oligomers. This review explores the mechanistic implications of GPCR dimerization and/or oligomerization on receptor activation and interactions with G proteins. The effects of GPCR oligomerization on receptor pharmacology, GPCR-mediated signaling, and potential contributions to GPCR crosstalk will be considered in the context of receptors important in the cardiovascular system. Our evolving understanding of the structural and functional consequences of GPCR oligomerization may provide novel and more selective sites for pharmacological tuning of cardiovascular function.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Sistema Cardiovascular/metabolismo , Dimerização , Humanos , Modelos BiológicosRESUMO
Calcium sensing receptors (CaSR) interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.
Assuntos
Proteínas 14-3-3/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Proteínas 14-3-3/genética , Membrana Celular/genética , Glicosilação/efeitos dos fármacos , Células HEK293 , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Detecção de Cálcio/genética , Tunicamicina/farmacologiaRESUMO
Calcium sensing receptors are critical to maintenance of organismal Ca2+ homeostasis, translating small changes in serum Ca2+ into changes in PTH secretion by the parathyroid glands and Ca2+ excretion by the kidneys. Calcium sensing receptors are also expressed in many cells and tissues not directly involved in Ca2+ homeostasis where their role(s) are less defined. Recent studies have demonstrated that calcium sensing receptors integrate a variety of metabolic signals, including polyvalent cations, pH, ionic strength, amino acids, and polypeptides, making CaR uniquely capable of generating cell- and tissue-specific responses, sensing not only Ca2+, but the local metabolic environment. The challenge for future studies is to define CaR responsiveness in each varied physiological context.
Assuntos
Receptores de Detecção de Cálcio/fisiologia , Transdução de Sinais/fisiologia , Aminoácidos/metabolismo , Animais , Antibacterianos/metabolismo , Sítios de Ligação , Cátions/metabolismo , Espaço Extracelular/metabolismo , Fendilina/química , Fendilina/metabolismo , Homeostase , Humanos , Ligantes , Modelos Biológicos , Concentração Osmolar , Peptídeos/metabolismo , Poliaminas/metabolismo , Receptores de Detecção de Cálcio/agonistas , Receptores de Detecção de Cálcio/antagonistas & inibidoresRESUMO
BACKGROUND: The melanocortin 4 receptor (MC4R) critically regulates feeding and satiety. Rare variants in MC4R are predominantly found in obese individuals. Though some rare variants in MC4R discovered in patients have defects in localization, ligand binding and signaling to cAMP, many have no recognized defects. SUBJECTS/METHODS: In our cohort of 1433 obese subjects that underwent Roux-en-Y Gastric Bypass (RYGB) surgery, we found fifteen variants of MC4R. We matched rare variant carriers to patients with the MC4R reference alleles for gender, age, starting BMI and T2D to determine the variant effect on weight-loss post-RYGB. In vitro, we determined expression of mutant receptors by ELISA and western blot, and cAMP production by microscopy. RESULTS: While carrying a rare MC4R allele is associated with obesity, carriers of rare variants exhibited comparable weight-loss after RYGB to non-carriers. However, subjects carrying three of these variants, V95I, I137T or L250Q, lost less weight after surgery. In vitro, the R305Q mutation caused a defect in cell surface expression while only the I137T and C326R mutations showed impaired cAMP signaling. Despite these apparent differences, there was no correlation between in vitro signaling and pre- or post-surgery clinical phenotype. CONCLUSIONS: These data suggest that subtle differences in receptor signaling conferred by rare MC4R variants combined with additional factors predispose carriers to obesity. In the absence of complete MC4R deficiency, these differences can be overcome by the powerful weight-reducing effects of bariatric surgery. In a complex disorder such as obesity, genetic variants that cause subtle defects that have cumulative effects can be overcome after appropriate clinical intervention.
Assuntos
Derivação Gástrica , Obesidade/genética , Obesidade/cirurgia , Receptor Tipo 4 de Melanocortina/genética , Redução de Peso/genética , Adolescente , Adulto , Idoso , Alelos , Peso Corporal/genética , Estudos de Casos e Controles , Feminino , Derivação Gástrica/estatística & dados numéricos , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/epidemiologia , Polimorfismo de Nucleotídeo Único , Período Pós-Operatório , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
The calcium-sensing receptor (CaSR) must function in the chronic presence of agonist, and recent studies suggest that its ability to signal under such conditions depends upon the unique mechanism(s) regulating its cellular trafficking. This chapter will highlight the evidence supporting an intracellular endoplasmic reticulum-localized pool of CaSR that can be mobilized to the plasma membrane by CaSR signaling, leading to agonist-driven insertional signaling (ADIS). I summarize evidence for the role of small GTP binding proteins (Rabs, Sar1 and ARFs), cargo receptors or chaperones (p24A, RAMPs) and interacting proteins (14-3-3 proteins, calmodulin) in anterograde trafficking of CaSR, and discuss the potential signaling specializations arising from CaSR interactions with caveolins or Filamin A/Rho. Finally, I summarize current knowledge about CaSR endocytosis and degradation by both the proteasome and lysosome, and highlight recent studies indicating that defective trafficking of CaSR or interacting protein mutants contributes to pathology in disorders of calcium homeostasis.