RESUMO
Fukutin-related protein (FKRP, MIM ID 606596) variants cause a range of muscular dystrophies associated with hypo-glycosylation of the matrix receptor, α-dystroglycan. These disorders are almost exclusively caused by homozygous or compound heterozygous missense variants in the FKRP gene that encodes a ribitol phosphotransferase. To understand how seemingly diverse FKRP missense mutations may contribute to disease, we examined the synthesis, intracellular dynamics, and structural consequences of a panel of missense mutations that encompass the disease spectrum. Under non-reducing electrophoresis conditions, wild type FKRP appears to be monomeric whereas disease-causing FKRP mutants migrate as high molecular weight, disulfide-bonded aggregates. These results were recapitulated using cysteine-scanning mutagenesis suggesting that abnormal disulfide bonding may perturb FKRP folding. Using fluorescence recovery after photobleaching, we found that the intracellular mobility of most FKRP mutants in ATP-depleted cells is dramatically reduced but can, in most cases, be rescued with reducing agents. Mass spectrometry showed that wild type and mutant FKRP differentially associate with several endoplasmic reticulum (ER)-resident chaperones. Finally, structural modelling revealed that disease-associated FKRP missense variants affected the local environment of the protein in small but significant ways. These data demonstrate that protein misfolding contributes to the molecular pathophysiology of FKRP-deficient muscular dystrophies and suggest that molecules that rescue this folding defect could be used to treat these disorders.
RESUMO
The Group C G protein-coupled receptors include the metabotropic glutamate receptors (mGluRs), the GABA(B) receptor, the calcium sensor and several taste receptors, most of which are obligate dimers, indeed recent work has shown that dimerization is necessary for the activation of these receptors. Consequently factors that regulate their ability to homo- or heterodimerize are important. The Group 1 mGluRs include mGluR1 and mGluR5 both of which have splice variants with altered C-termini. In this study, we show that mGluR1b is a dimer and that it does not efficiently heterodimerize with mGluR1a, unlike the two splice variants of mGluR5 that can heterodimerize. Mutation of a positively charged motif (RRKK) at the C-terminus of the mGluR1b tail permits mGluR1b to heterodimerize with mGluR1a. Co-expression of mGluR1a and mGluR1b in COS-7 cells results in the accumulation of mGluR1b in intracellular inclusions that do not contain mGluR1a. This behaviour is mimicked by a chimera of the lymphocyte antigen CD2 with the C-terminus of mGluR1b (pCD1b) and depends on the presence of the RRKK motif. These accumulations are immunoreactive for endoplasmic reticulum (ER) markers, but not Golgi and ERGIC markers. This segregation of mGluR1b from other ER proteins may contribute to its failure to dimerize with mGluR1a.
Assuntos
Fragmentos de Peptídeos/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Glutamato Metabotrópico/fisiologia , Animais , Células COS , Cerebelo/fisiologia , Chlorocebus aethiops , Dimerização , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ratos , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genéticaRESUMO
GPCRs (G-protein-coupled receptors) play key roles in many cellular processes, and malfunction may lead to a range of pathologies, including psychiatric and neurological disorders. It is therefore not surprising that this group of receptors supplies a majority of the targets for pharmaceutical drug development. Despite their importance, the mechanisms that regulate their function and signalling still remain only partially understood. Recently, it has become evident that a subset of GPCRs is not homogeneously distributed in the plasma membrane, but localizes instead to specific membrane microdomains known as lipid rafts. Lipid rafts are characterized by their enrichment in cholesterol and sphingolipids, and have been suggested to serve as platforms for a range of cellular signalling complexes. In the present review, we will be discussing the effects of the lipid raft environment on trafficking, signalling and internalization of raft-associated GPCRs.
Assuntos
Microdomínios da Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Transporte Biológico Ativo , Humanos , Microdomínios da Membrana/química , Modelos Moleculares , Estrutura Molecular , Receptor A1 de Adenosina/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores da Bradicinina/metabolismo , Receptores CCR5/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores LHRH/metabolismo , Transdução de SinaisRESUMO
Epitope tagging of a receptor involves introducing a defined amino acid sequence, to which an antibody has already been produced, into the primary amino acid sequence of the receptor. The new sequence can be as short as 10-15 amino acids and the method allows the receptor to be monitored without having to raise an antibody specific to it, and so permits its biochemical characterization and immunolocalization within cells. Other related techniques involve the introduction of functional domains of proteins such as green fluorescence protein or one of its derivatives into receptors to allow their direct visualization. There are a wide range of amino acid tag sequences, and fluorescent proteins, available for use as tags, and choice of tag will depend on several factors including the availability of antisera and cost. The position for the introduction of the tag into the native receptor will depend on precisely what the requirements of the experiments are but it must always be such that the receptor is normally processed, trafficked, and remains functional after tagging. These considerations are discussed fully in this chapter, which also describes examples of strategies for introducing tags, and some general methods for use in the characterization of the tagged proteins.
Assuntos
Epitopos/imunologia , Receptores Citoplasmáticos e Nucleares/imunologia , Coloração e Rotulagem/métodos , Animais , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Glutamato Metabotrópico/imunologia , Receptores de Glutamato Metabotrópico/metabolismoRESUMO
Ionotropic glutamate receptors are functionally diverse but have a common architecture, including the 400-residue amino-terminal domain (ATD). We report a 1.8-A resolution crystal structure of human GluR2-ATD. This dimeric structure provides a mechanism for how the ATDs can drive receptor assembly and subtype-restricted composition. Lattice contacts in a 4.1-A resolution crystal form reveal a tetrameric (dimer-dimer) arrangement consistent with previous cellular and cryo-electron microscopic data for full-length AMPA receptors.
Assuntos
Receptores de AMPA/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
GABAB (gamma-aminobutyric acid, type B) is a heterodimeric G-protein-coupled receptor. The GABAB1 subunit, which contains an endoplasmic reticulum retention sequence, is only transported to the cell surface when it is associated with the GABAB2 subunit. Fluorescence recovery after photobleaching studies in transfected COS-7 cells and hippocampal neurons revealed that GABAB2 diffuses slowly within the plasma membrane whether expressed alone or with the GABAB1 subunit. Treatment of cells with brefeldin A revealed that GABAB2 moves freely within the endoplasmic reticulum, suggesting that slow movement of GABAB2 is a result of its plasma membrane insertion. Disruption of the cytoskeleton did not affect the mobility of GABAB2, indicating that its restricted diffusion is not due to direct interactions with actin or tubulin. To determine whether the C terminus of GABAB2 regulates its diffusion, this region of the subunit was attached to the lymphocyte membrane protein, CD2, which then exhibited a slower rate of lateral diffusion. Furthermore, co-expression of a cytoplasmically expressed soluble form of the GABAB2 C terminus increased movement of the GABAB2 subunit. We constructed forms of GABAB2 with various C-terminal truncations. Truncation of GABAB2 after residue 862, but not residue 886, caused a dramatic increase in its mobility, suggesting that the region between these two residues is critical for restricting GABAB2 diffusion. Finally, we investigated whether activation of GABAB might modulate its movement. Treatment of COS-7 cells with the GABAB receptor agonist baclofen significantly increased its mobile fraction. These data show that the restricted movement of GABAB at the cell surface is regulated by a region within its C terminus.
Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de GABA-B/metabolismo , Actinas/genética , Actinas/metabolismo , Sequência de Aminoácidos/genética , Animais , Antivirais/farmacologia , Baclofeno/farmacologia , Brefeldina A/farmacologia , Antígenos CD2/genética , Antígenos CD2/metabolismo , Células COS , Membrana Celular/genética , Chlorocebus aethiops , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/genética , Citoesqueleto/metabolismo , Agonistas GABAérgicos/farmacologia , Agonistas dos Receptores de GABA-B , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/genética , Deleção de Sequência/genética , Transfecção , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
The time course of the assembly of the N-methyl-D-aspartate receptor was examined in a cell line expressing it under the control of the dexamethasone promoter. These studies suggested a delay between the appearance of the NR1 and NR2A subunits and their stable association as examined by co-immunoprecipitation of NR1 and NR2A. This prompted us to examine the stability and folding of the individual subunits using nonreduced polyacrylamide gels and the sulfhydryl cross-linker BMH. Both studies showed that the NR1 subunit was expressed in a monomer and dimer form, whereas both NR2 and NR3 showed substantial aggregation on both nonreduced gels and after cross-linking. Protein degradation experiments showed that NR1 was relatively stable, whereas NR2 and NR3 were more rapidly degraded. When co-expressed with NR1, NR2 was more stable. Fluorescence recovery after photobleaching experiments showed that, under conditions of reduced ATP, the diffusion rate of NR2 and NR3 in the endoplasmic reticulum was reduced, whereas that of NR1 was unaffected. Together these data show that NR1 folds stably when expressed alone, unlike NR2 and NR3, and provides the substrate for assembly of the N-methyl-D-aspartate receptor.
Assuntos
Retículo Endoplasmático/metabolismo , Dobramento de Proteína , Receptores de N-Metil-D-Aspartato/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Células COS , Chlorocebus aethiops , Dimerização , Expressão Gênica , Humanos , Estrutura Quaternária de Proteína , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, alpha-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu(5b) receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu(5b) receptor and alpha-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of alpha-actinin-1 with mGlu(5b) receptor modulated the cell surface expression of the receptor. This was dependent on the binding of alpha-actinin-1 to the actin cytoskeleton. In addition, the alpha-actinin-1/mGlu(5b) receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an alpha-actinin-1-dependent mGlu(5b) receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.
Assuntos
Actinina/metabolismo , Membrana Celular/metabolismo , Regulação da Expressão Gênica , Receptores de Glutamato Metabotrópico/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Citoesqueleto/metabolismo , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Receptor de Glutamato Metabotrópico 5 , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Myoclonus-dystonia syndrome (MDS) is a genetically heterogeneous disorder characterized by myoclonic jerks often seen in combination with dystonia and psychiatric co-morbidities and epilepsy. Mutations in the gene encoding epsilon-sarcoglycan (SGCE) have been found in some patients with MDS. SGCE is a maternally imprinted gene with the disease being inherited in an autosomal dominant pattern with reduced penetrance upon maternal transmission. In the central nervous system, epsilon-sarcoglycan is widely expressed in neurons of the cerebral cortex, basal ganglia, hippocampus, cerebellum and the olfactory bulb. epsilon-Sarcoglycan is located at the plasma membrane in neurons, muscle and transfected cells. To determine the effect of MDS-associated mutations on the function of epsilon-sarcoglycan we examined the biosynthesis and trafficking of wild-type and mutant proteins in cultured cells. In contrast to the wild-type protein, disease-associated epsilon-sarcoglycan missense mutations (H36P, H36R and L172R) produce proteins that are undetectable at the cell surface and are retained intracellularly. These mutant proteins become polyubiquitinated and are rapidly degraded by the proteasome. Furthermore, torsinA, that is mutated in DYT1 dystonia, a rare type of primary dystonia, binds to and promotes the degradation of epsilon-sarcoglycan mutants when both proteins are co-expressed. These data demonstrate that some MDS-associated mutations in SGCE impair trafficking of the mutant protein to the plasma membrane and suggest a role for torsinA and the ubiquitin proteasome system in the recognition and processing of misfolded epsilon-sarcoglycan.
Assuntos
Distúrbios Distônicos/genética , Chaperonas Moleculares/fisiologia , Mutação de Sentido Incorreto , Mioclonia/genética , Processamento de Proteína Pós-Traducional/fisiologia , Sarcoglicanas/genética , Sarcoglicanas/metabolismo , Ubiquitina/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Embrião de Mamíferos , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Transporte Proteico , Ratos , SíndromeRESUMO
Mutations in the gene encoding fukutin-related protein (FKRP) cause a spectrum of diseases including congenital muscular dystrophy type 1C (MDC1C), limb girdle muscular dystrophy 2I (LGMD2I) and congenital muscular dystrophies (CMDs) with brain malformations and mental retardation. Although these diseases are associated with abnormal dystroglycan processing, the cellular consequences of the idiosyncratic FKRP mutations have not been determined. Here we show, in cultured cells, that FKRP mutants associated with the more severe disease phenotypes (S221R, A455D, P448L) are retained in the endoplasmic reticulum (ER), whereas the wild-type protein and the mutant L276I that causes LGMD2I are found predominantly in the Golgi apparatus. The ER-retained proteins have a shorter half-life than the wild-type FKRP and are preferentially degraded by the proteasome. Furthermore, calnexin binds preferentially to the ER-retained mutants suggesting that it may participate in the quality control pathway for FKRP. These data provide the first evidence that the ER-retention of mutant FKRP may play a role in the pathogenesis of CMD and potentially explain why the allelic disorder LGMD2I is milder, because the mutated protein is able to reach the Golgi apparatus.
Assuntos
Retículo Endoplasmático/metabolismo , Distrofias Musculares/genética , Proteínas/genética , Animais , Células COS , Calnexina/metabolismo , Células Cultivadas , Chlorocebus aethiops , Humanos , Microscopia Confocal , Distrofias Musculares/metabolismo , Mutação , Pentosiltransferases , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismoRESUMO
In this study we characterized the heterologous desensitization and internalization of the metabotropic glutamate receptor 1 (mGluR1) splice variants mGluR1a and mGluR1b following activation of endogenous G(q/11)-coupled receptors in HEK293 cells. Agonist activation of M1 muscarinic acetylcholine or P2Y1 purinergic receptors triggered the PKC- and CaMKII-dependent internalization of mGluR1a. In co-immunoprecipitation studies, both glutamate and carbachol increased the association of GRK2 with mGluR1a. Co-addition of the protein kinase C (PKC) inhibitor GF109203X and the Ca(2+) calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 blocked the ability of glutamate and carbachol to increase the association of GRK2 with mGluR1a. Glutamate also increased the association of GRK2 with mGluR1b, whereas carbachol did not. However, unlike mGluR1a, glutamate-stimulated association of GRK2 with mGluR1b was not reduced by PKC/CaMKII inhibition. Pretreatment of cells expressing mGluR1a or mGluR1b with carbachol rapidly desensitized subsequent glutamate-stimulated inositol phosphate accumulation. The carbachol-induced heterologous desensitization and internalization of mGluR1a was blocked by LY367385, an mGluR1a antagonist with inverse agonist activity. Furthermore, LY367385 blocked the ability of carbachol to increase the association of GRK2 with mGluR1a. On the other hand, LY367385 had no effect on the carbachol-induced desensitization and internalization of the nonconstitutively active mGluR1b splice variant. These results demonstrate that the internalization of mGluR1a, triggered homologously by glutamate or heterologously by carbachol, is PKC/CaMKII-, GRK2-, arrestin-, and clathrin-dependent and that PKC/CaMKII activation appears to be necessary for GRK2 to associate with mGluR1a. Furthermore, the heterologous desensitization of mGluR1a is dependent upon the splice variant being in an active conformation.
Assuntos
Regulação para Baixo , Endocitose , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Glicina/análogos & derivados , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Arrestinas/metabolismo , Benzoatos/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Carbacol/farmacologia , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ácido Glutâmico/farmacologia , Glicina/farmacologia , Humanos , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Quinases de Receptores Adrenérgicos betaRESUMO
A Chinese hamster ovary cell line has been established which secretes the N-terminal domain of human mGlu1 receptor. The secreted protein has been modified to contain a C-terminal hexa-histidine tag and can be purified by metal-chelate chromatography to yield a protein with an apparent molecular weight of 130 kDa. Following treatment with dithiothreitol the apparent molecular weight is reduced to 75 kDa showing that the protein is a disulphide-bonded dimer. N-terminal protein sequencing of both the reduced and unreduced forms of the protein yielded identical sequences, confirming that they were derived from the same protein, and identifying the site of signal-peptide cleavage of the receptor as residue 32 in the predicted amino acid sequence. Endoglycosidase treatment of the secreted and intracellular forms of the protein showed that the latter was present as an endoglycosidase H-sensitive dimer, indicating that dimerization is taking place in the endoplasmic reticulum. Characterization of the binding of [3H]quisqualic acid showed that the protein was secreted at levels of up to 2.4 pmol/mL and the secreted protein has a Kd of 5.6 +/- 1.8 nm compared with 10 +/- 1 nm for baby hamster kidney (BHK)-mGlu1alpha receptor-expressing cell membranes. The secreted protein maintained a pharmacological profile similar to that of the native receptor and the binding of glutamate and quisqualate were unaffected by changes in Ca2+ concentration.
Assuntos
Benzoatos , Glicina/análogos & derivados , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células CHO , Cricetinae , Dimerização , Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Glicina/farmacologia , Glicosídeo Hidrolases/farmacologia , Glicosilação , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Ácido Quisquálico/farmacologia , Receptores de Glutamato Metabotrópico/metabolismo , Transfecção , TrítioRESUMO
Lipid raft domains have attracted much recent attention as platforms for plasma membrane signalling complexes. In particular, evidence is emerging that shows them to be key regulators of G protein coupled receptor function. The G protein coupled gamma-aminobutyric acid receptor B (GABA(B) receptor) co-isolates with lipid raft domains from rat brain cerebellum. In the present study, we show that the GABA(B1a,2) receptor was also present in lipid raft domains when expressed ectopically in a Chinese hamster ovary cell line. Lipid raft-associated receptor was functionally active, displaying a concentration-dependent increase in GTPgammaS binding in response to the receptor agonist GABA. Compared with whole cell membranes, lipid raft-associated receptor displayed an increased EC(50) and a reduced magnitude of response to GABA. We conclude that lipid raft association is an intrinsic property of the GABA(B1a,2) receptor and is not cell-type specific. In addition, localisation to lipid raft domains may provide a mechanism to inhibit receptor function.
Assuntos
Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Animais , Células CHO , Cerebelo/metabolismo , Cricetinae , Regulação para Baixo , Agonistas dos Receptores de GABA-B , Expressão Gênica , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Imuno-Histoquímica , Microdomínios da Membrana/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The gamma-aminobutyric acid type B (GABA(B)) receptor is a heterodimeric G-protein-coupled receptor. In humans, three splice variants of the GABA(B) receptor 1 (R1) subunit differ in having one, both, or neither of two putative complement control protein (CCP) modules at the extracellular N terminus, prior to the GABA-binding domain. The in vivo function of these predicted modules remains to be discovered, but a likely association with extracellular matrix proteins is intriguing. The portion of the GABA(B) R1a variant encompassing both of its CCP module-like sequences has been expressed, as have the sequences corresponding to each individual module. Each putative CCP module exhibits the expected pattern of disulfide formation. However, the second module (CCP2) is more compactly folded than the first, and the three-dimensional structure of this more C-terminal module (expressed alone) was solved on the basis of NMR-derived nuclear Overhauser effects. This revealed a strong similarity to previously determined CCP module structures in the regulators of complement activation. The N-terminal module (CCP1) displayed conformational heterogeneity under a wide range of conditions whether expressed alone or together with CCP2. Several lines of evidence indicated the presence of native disorder in CCP1, despite the fact that recombinant CCP1 contributes to binding to the extracellular matrix protein fibulin-2. Thus, we have shown that the two CCP modules of GABA(B) R1a have strikingly different structural properties, reflecting their different functions.